Unity of Mind and Body The Concept of Li

Nanomedicine and
Cancer Therapies
Recent Advances in Nanoscience and Nanotechnology
Volume 2
Nanomedicine and
Cancer Therapies
Edited By
Mathew Sebastian, MD, Neethu Ninan,
and Eldho Elias
Apple Academic Press

TORONTO NEW JERSEY
© 2013 by
Apple Academic Press Inc.
3333 Mistwell Crescent
Oakville, ON L6L 0A2
Canada
Apple Academic Press Inc.

1613 Beaver Dam Road, Suite # 104
Point Pleasant, NJ 08742
USA
Exclusive worldwide distribution by CRC Press, a Taylor & Francis Group
International Standard Book Number: 978-1-926895-18-5 (Hardback)
Printed in the United States of America on acid-free paper

0987654321
Library of Congress Control Number: 2012935668
Library and Archives Canada Cataloguing in Publication
Nanomedicine and cancer therapies / edited by Mathew Sebastian, Neethu Ninan and Eldho Elias.
(Recent advances in nanoscience and nanotechnology; v.2)
Includes bibliographical references and index.
ISBN 978-1-926895-18-5
1. Nanomedicine. 2. Cancer–Treatment. 3. Cancer–Diagnosis.
4. Nanotechnology. I. Sebastian, Mathew II. Ninan, Neethu
III. Elias, Eldho IV. Series: Recent advances in nanoscience and nanotechnology; v.2
R857.N34N36 2012 610.28 C2011-908742-1
Trademark Notice: Registered trademark of products or corporate names are used only for explanation and
identification without intent to infringe.
This book contains information obtained from authentic and highly regarded sources. Reprinted material
is quoted with permission and sources are indicated. A wide variety of references are listed. Reasonable
efforts have been made to publish reliable data and information, but the authors, editors, and the
publisher cannot assume responsibility for the validity of all materials or the consequences of their
use. The authors, editors, and the publisher have attempted to trace the copyright holders of all material
reproduced in this publication and apologize to copyright holders if permission to publish in this form
has not been obtained. If any copyright material has not been acknowledged, please write and let us
know so we may rectify in any future reprint.
All rights reserved. No part of this work covered by the copyright hereon may be reproduced or used in
any form or by any means—graphic, electronic, or mechanical, including photocopying, recording, taping, or
information storage and retrieval systems—without the written permission of the publisher.
Apple Academic Press also publishes its books in a variety of electronic formats. Some content that appears
in print may not be available in electronic format. For information about Apple Academic Press products,
visit our website at www.appleacademicpress.com
Recent Advances in Nanoscience and Nanotechnology
Series Editors-in-Chief
Sabu Thomas, PhD

Dr. Sabu Thomas is the Director of the School of Chemical Sciences, Mahatma Gandhi Uni-
versity, Kottayam, India. He is also a full professor of polymer science and engineering and
Director of the Centre for nanoscience and nanotechnology of the same university. He is a fel-
low of many professional bodies. Professor Thomas has authored or co-authored many papers in
international peer-reviewed journals in the area of polymer processing. He has organized several
international conferences and has more than 420 publications, 11 books and two patents to his
credit. He has been involved in a number of books both as author and editor. He is a reviewer
to many international journals and has received many awards for his excellent work in polymer
processing. His h Index is 42. Professor Thomas is listed as the 5th position in the list of Most
Productive Researchers in India, in 2008.
Mathew Sebastian, MD
Dr. Mathew Sebastian has a degree in surgery (1976) with specialization in Ayurveda. He holds
several diplomas in acupuncture, neural therapy (pain therapy), manual therapy and vascular
diseases. He was a missionary doctor in Mugana Hospital, Bukoba in Tansania, Africa (1976-
1978) and underwent surgical training in different hospitals in Austria, Germany, and India
for more than 10 years. Since 2000 he is the doctor in charge of the Ayurveda and Vein Clinic
in Klagenfurt, Austria. At present he is a Consultant Surgeon at Privatclinic Maria Hilf,
Klagenfurt. He is a member of the scientific advisory committee of the European Academy for
Ayurveda, Birstein, Germany, and the TAM advisory committee (Traditional Asian Medicine,
Sector Ayurveda) of the Austrian Ministry for Health, Vienna. He conducted an International
Ayurveda Congress in Klagenfurt, Austria, in 2010. He has several publications to his name.
Anne George, MD
Anne George, MD, is the Director of the Institute for Holistic Medical Sciences, Kottayam,
Kerala, India. She did her MBBS (Bachelor of Medicine, Bachelor of Surgery) at Trivandrum
Medical College, University of Kerala, India. She acquired a DGO (Diploma in Obstetrics and
Gynaecology) from the University of Vienna, Austria; Diploma Acupuncture from the Uni-
versity of Vienna; and an MD from Kottayam Medical College, Mahatma Gandhi University,
Kerala, India. She has organized several international conferences, is a fellow of the American
Medical Society, and is a member of many international organizations. She has five publications
to her name and has presented 25 papers.
Dr. Yang Weimin

Dr. Yang Weimin is the Taishan Scholar Professor of Quingdao University of Science and Tech-
nology in China. He is a full professor at the Beijing University of Chemical Technology and
a fellow of many professional organizations. Professor Weimin has authored many papers in
international peer-reviewed journals in the area of polymer processing. He has been contributed
to a number of books as author and editor and acts as a reviewer to many international journals.
In addition, he is a consultant to many polymer equipment manufacturers. He has also received
numerous award for his work in polymer processing.
Contents
List of Contributors............................................................................................................ix
List of Abbreviations........................................................................................................ xiii
Preface.............................................................................................................................xvii
1. Nanotechnological Based Systems for Cancer................................................. 1

Jagat R. Kanwar, Ganesh Mahidhara, and Rupinder K. Kanwar
2. In vivo Spectroscopy for Detection and Treatment of GBM with

NPt Implantation.............................................................................................. 19
José de la Rosa, Diego Adrián Fabila, Luis Felipe Hernández, Edgard Moreno,
Suren Stolik, Gabriela de la Rosa, Mayra Álvarez, Alfonso Arellano, Tessy López,
R. Mercado, J. L. Soto, L. Arredondo, J. Bustos, A. Mosqueda, and I. Rivero
3. Nanobiotechnology for Antibacterial Therapy and Diagnosis..................... 31

Jagat R. Kanwar, Kislay Roy, and Rupinder K. Kanwar
4. Chitosan Nanoparticles.................................................................................... 40
Divyen Shah, Vaishali Londhe, and Rima Shah
5. Synthesis and Biomedical Application of Silver Nanoparticles.................... 54

M. Saravanan and V. Anil Kumar
6. Recent Advances in Cancer Therapy Using Phytochemicals....................... 67

Hullathy Subban Ganapathy, Rajakani Senthil Nagarajan, and Hirotaka Ihara
7. Mitochondrial Dysfunction and Cancer: Modulation by Palladium

α-Lipoic Acid Complex.................................................................................... 73
C. V. Krishnan, M. Garnett, and F. Antonawich
8. Unity of Mind and Body: The Concept of Life Purpose Dominant........... 129
Bukhtoyarov Oleg Viktorovich and Samarin Denis Mikhaylovich
9. Thuja occidentalis and Breast Cancer Chemoprevention........................... 141

B. K. Ojeswi, M. Khoobchandani, S. Medhe, D. K. Hazra, and M. M. Srivastava
10. Antioxidants and Combinatorial Therapies in Cancer Treatment............ 155

Arpita Saxena
11. Eruca sativa Inhibits Melanoma Growth: A Scientific Evidence................ 160

M. Khoobchandani, N. Ganesh, L. Valgimigli, and M. M. Srivastava
12. Optical and Mechanical Investigations of Nanostructures for

Biomolecular Detection.................................................................................. 170
G. Malegori, D. Nardi, F. Banfi, C. Giannetti, and G. Ferrini
viii Contents
13. Suffering and Comfort in Portuguese Cancer Patients.............................. 185

João Luís Alves Apóstolo, Rita Susana Soares Capela, and Inês Barata Sá Castro
References........................................................................................................ 195
Index................................................................................................................ 239
List of Contributors
Mayra Álvarez
Laboratorio de Nanotecnología, Instituto Nacional de Neurología y Neurocirugía “MVS”, Insurgentes Sur
3877, La Fama, CP-14269, D.F., México.
F. Antonawich
Garnett McKeen Laboratory, Inc, Bohemia, New York-11716–1735, USA
João Luís Alves Apóstolo

RN, PhD, Health Sciences Research Unit: Nursing, Coimbra Nursing School, Avenida Bissaya Barreto––
Apartado-7001, 3046–851 Coimbra––Portugal
Alfonso Arellano
L. Arredondo
Hospital Civil de Guadalajara, Salvador de Quevedo y Zubieta No. 750 Esq. Sierra Nevada, Independencia,
CP-44340, Guadalajara, Jal., México.
F. Banfi
Dipartimento di Matematica e Fisica, Università Cattolica, I-25121 Brescia, Italy.
J. Bustos
Departamento de Atención a la Salud, Universidad Autónoma Metropolitana Xochimilco, Calzada del Hue-
so 1100, Villa Quietud, CP-04960, D.F., México.
Rita Susana Soares Capela

RN, Master in Palliative Care, Oncology/Haematology Unit of the Portuguese Institute of Oncology of
Porto, Portugal, Rua Dr. António Bernardino de Almeida
Inês Barata Sá Castro
Grant holder, Health Sciences Research Unit: Nursing, Coimbra Nursing School, Avenida Bissaya Bar-
reto––Apartado-7001, 3046-851 Coimbra–Portugal
Diego Adrián Fabila
Instituto Politécnico Nacional, Unidad Profesional “Adolfo López Mateos”, SEPI-ESIME ZAC., Linda-
vista, CP-07738, D.F., México.
G. Ferrini
Hullathy Subban Ganapathy

Priority Organization for Innovation and Excellence, Kumamoto University, 2-39-1 Kurokami, Kumamo-
to-860–8555, Japan
N. Ganesh
Jawaharlal Nehru Cancer Hospital and Research Centre, Bhopal-422001, India
M. Garnett
Garnett McKeen Laboratory, Inc, Bohemia, New York 11716-1735, USA
C. Giannetti
x List of Contributors
D. K. Hazra
S.N Medical College, Agra, India-282002.
Luis Felipe Hernández

Hirotaka Ihara
Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, 2-39-1
Kurokami, Kumamoto-860–8555, Japan
Jagat R. Kanwar
Laboratory of Immunology and Molecular Biomedical Research (LIMBR), Centre for Biotechnology and
Interdisciplinary Biosciences (BioDeakin), Institute for Technology and Research Innovation (ITRI), Gee-
long Technology Precinct (GTP), Deakin University, Pigdons Road, Waurn Ponds, Geelong, Victoria-3217,
Australia.
Rupinder K. Kanwar
Australia.
M. Khoobchandani
Department of Chemistry, Faculty of Science, Dayalbagh Educational Institute, Agra, India-282110.
C. V. Krishnan
Garnett McKeen Laboratory, Inc, Bohemia, New York-11716–1735, USA.
Department of Chemistry, Stony Brook University, New York-11794–3400, USA.
Anil Kumar V.
Department of Biotechnology, Faculty of Science and Humanities, SRM University, SRM Nagar, Kattanku-
lathur, Chennai, Tamilnadu-603203, India.
Vaishali Londhe
School of Pharmacy & Technology Management, SVKM’s NMIMS, Mumbai, Maharashtra-400057, India.
Tessy López
Department of Chemical Engineering, Tulane University, New Orleans, LA-70118.
Ganesh Mahidhara
Interdisciplinary Biosciences (BioDeakin), Institute for Technology Research and Innovation (ITRI), Dea-
kin University, Waurn Ponds, Victoria-3217, Australia.
G. Malegori
S. Medhe
R. Mercado
List of Contributors xi
Samarin Denis Mikhaylovich

Center for Medical and Social Rehabilitation UFSIN Russia of Kaliningrad region
10–103, ul. V. Gakuna-236009, Kaliningrad, Russian Federation.
Edgard Moreno
A. Mosqueda
Rajakani Senthil Nagarajan
Priority Organization for Innovation and Excellence, Kumamoto University, 2-39-1 Kurokami, Kumamo-
to-860–8555, Japan.
D. Nardi
B. K. Ojeswi
I. Rivero
Instituto Nacional de Investigaciones Nucleares, Carretera México-Toluca S/N, La Marquesa, CP 52750,
Ocoyoacac, México, México.
Gabriela de la Rosa
José de la Rosa
Kislay Roy
Australia.
M. Saravanan
Department of Biotechnology, Faculty of Science and Humanities, SRM University, SRM Nagar, Kattanku-
lathur, Chennai, Tamilnadu-603203, India.
Arpita Saxena
Division of Cancer Pharmacology, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu
Tawi-180001, India.
Divyen Shah
School of Pharmacy & Technology Management, SVKM’s NMIMS, Mumbai, Maharashtra-400057, India.
Rima Shah
School of Pharmacy & Technology Management, SVKM’s NMIMS, Shirpur Campus, Shirpur, Maharash-
tra-425405, Ind ia.
J. L. Soto
M. M. Srivastava
xii List of Contributors
Suren Stolik
Instituto Politécnico Nacional, Unidad Profesional “Adolfo López Mateos”, SEPI-ESIME ZAC.,
Lindavista, CP-07738, D.F., México.
L. Valgimigli
R&D Department, University of Bologna, B&C s.r.l, Via C. Monteverdi-49, 47100 Forli Italy
Bukhtoyarov Oleg Viktorovich

Center for Medical and Social Rehabilitation UFSIN Russia of Kaliningrad region
30A-7, ul. S. Razina-236022, Kaliningrad, Russian Federation.
List of Abbreviations
5-ASA 5-Amino salicylic acid

5-ALA 5-Aminolaevullinic acid
ACA Acrocentric association
ATP Adenosine triphosphate
AE Adverse Events
AMD Age-related macular degradation
ATCC American type culture collection
AMPs Antimicrobial peptides
AFM Atomic force microscopy
AISRF Australia-India Strategic Research Fund
CNTs Carbon nanotubes
CPP Cell-penetrating peptides
CR Centric rings
COS Chito oligosaccharides
CS Chitosan
CB Chromatid breaks
CLL Chronic lymphoid leukemia
CPS Chronic psycho-emotional stress
CRC Colorectal cancer
CCS Comfort chemotherapy scale
CPCSEA Control and supervision of experiments on animals
CA1 Cornus ammon’s field 1
CD Current subdominants
CF Cystic fibrosis
DPHP 1-Diphenyl-2-picrylhydrazyl
DSMB Data safety and monitoring board
DSs Dermaseptins
DC Dicentric
DMF Dimethyl formamide
DMSO Dimethylsulphoxide
DNSurR9 Dominant negative survivin R9
DESSTINI Dose escalation safety study in normal individuals
dsRNA Double-stranded ribo nucleic acid
EPR Electron paramagnetic resonance
ESR Electron spin resonance
xiv List of Abbreviations
EPCs Endothelial progenitor cells

EPR Enhanced permeability retention
EtOAc Ethyl acetate
ESF European Science Foundation
ECM Extracellular matrix
FGFs Fibroblast growth factors
FSCP Fish scale collagen peptides
5-FU Five-fluorouracil
FAD Flavin adenine dinucleotide
FRET Fluorescence resonance energy transfer
FAE Follicle associated epithelium
FT Fourier transforms
FTIR Fourier transform infra red
FR Fragment
GBM Glioblastoma Multiforme
GSH Glutathione
GPx Glutathione peroxidase
GRx Glutathione reductase
H-E Hematoxyline-Eosine
HTAB Hexadecyl trimethyl ammonium bromide
HIF-1 Hypoxia inducible factor 1
IgG Immunoglobulin G
IFN-γ Induce interferon-γ
IAP Inhibitor of apoptosis protein
ITRI Institute for Technology Research and Innovation
ICD Intracalary deletion
IP Intraperitoneally
ICDH Isocitrate dehydrogenase
α-KGDH α-Ketoglutarate dehydrogenase
LPD Life purpose dominant
LS Life span
LNCaP Lymph node carcinoma of the prostate
MRI Magnetic resonance imaging
MDH Malate dehydrogenase
MDA Malondialdehyde
MB Methylene Blue
miRNAs Micro ribonucleic acids
MNCEs Micronucleated normochromatic erythrocytes
MPCEs Micronucleated polychromatic erythrocytes
List of Abbreviations xv
MBCs Minimal bactericidal concentrations

MICs Minimal inhibitory concentrations
mtDNA Mitochondrial DNA
MW Molecular weight
MWCNTs Multiwalled carbon nanotubes
NALT Nasal associated lymphoid tissues
NCI National Cancer Institute’s
NDR Negative differential resistance
NADH Nicotinamide adenine dinucleotide
NADPH Nicotinamide adenine dinucleotide phosphate
NOAEL No adverse observed effect level
NC-AFM Non-contact atomic force microscopy
nDNA Nuclear DNA
PILs PEGylated immunoliposomes
PEM Photo-elastic modulator
PCF Photonic-crystal fibers
PVA Poly(vinyl alcohol)
PGA Poly glycolic acid
PLA Poly lactic acid
PLGA Poly lactic-co-glycolic acid
PQA Poly quaternary ammonium
PEG Polyethylene glycol
POMs Polyoxometalates
PUFAH Polyunsaturated fatty acid
Pox Protein oxidation
PDTC Pyrrolidine dithiocarbamate
QCh Quaternised chitosan
RNOS Reactive nitrogen oxygen species
ROS Reactive oxygen species
rHBsAg Recombinant hepatitis B surface antigen
RSV Respiratory syncytial virus
RES Reticulo endothelial system
RNAi Ribo nucleic acid interference
SD Safety dominant
SO Seed oil
SELEX Selective Evaluation of Ligands by Exponential Enrichment
shRNAs Short hairpin ribo nucleic acid
SNPs Single nucleotide polymorphisms
SWNTs Single-walled nanotubes
xvi List of Abbreviations
siRNAs Small interfering ribo nucleic acid

STPP Sodium tri poly phosphate
SDH Succinate dehydrogenase
SPION Superparamagnetic iron oxide nanocrystals
SAWs Surface acoustic waves
SELEX Systematic evolution of ligands by exponential enrichment
TMX Tamoxifen
TS Taxonomic structure
TGA Thermogravity analysis
TLC Thin layer chromatography
TBARS Thiobarbituric acid reacting substance
TEER Transepithelial electrical resistance
TGF β) Transforming growth factor β
TM Transitional meningioma
TEM Transmission electron microscopy
TPP Tri poly phosphate
TNF Tumor necrosis factor
VEGF Vascular endothelial growth factor
VHL Von hippel-lindau
WT Wavelet transform
WHO World Health Organization
ZO-1 Zonula occludens-1
Preface
Cancer is a well-known deadly disease that is caused due to the inability of some un-
controllably growing cells to die. More than 100 types of cancer have been identified
till date, classified on the basis of the type of the initially affected cell. Cancer usually
forms tumors of cells that interfere with the nervous, digestive and circulatory systems
of the body and sometimes alter the functions by releasing unwanted hormones. They
are usually benign and are limited to a region. The more dangerous is the other kind
of tumor, i.e. the malignant ones. It occurs when a cancerous cell manages to travel
throughout the body through the circulatory system and destroy healthy tissues in its
path. Cancer usually develops due to mutation in the genes of the cell making it to
forget to die. The cell goes on multiplying and does not die as it had to. Finally, it starts
forming a mass. These mutations are caused by many different stimuli like X-rays,
radiations, different chemicals etc. The main factor that affects the successful treat-
ment of cancer is based on the timing of detection. An early detection of the deadly
disease greatly improves the odds of successful treatment. Cancer is treated using a
number of techniques that include surgery, chemotherapy, radiation, immunotherapy,
gene therapy, hormone therapy, holistic medicine etc., that come in the category of
oncology. This book discusses how nanomedicine, holistic medicine and other cancer
therapies contribute in the treatment of cancer.
Nanotechnology has the power to radically change the way cancer is diagnosed,
imaged and treated. Currently, there is a lot of research going on to design novel
nanodevices capable of detecting cancer at its earliest stages, pinpointing its loca-
tion within the body and delivering anticancer drugs specifically to malignant cells.
Nanoscale devices smaller than 50 nanometers can easily enter most cells, while those
smaller than 20 nanometers can transit out of blood vessels. As a result, nanoscale
devices can readily interact with biomolecules on both the cell surface and within the
cell. Nanoscale devices are already proving that they can deliver therapeutic agents to
target cells, or even within specific organelles. Yet, despite its small size, a nanoscale
device is capable of holding tens of thousands of small molecules, such as a contrast
agent or drug. The major areas in which nanomedicine is being developed in cancer
include: (a) Prevention and control (b) Early detection and proteomics (c) Imaging
diagnostics and (d) Multifunctional Therapeutics. Earlier detection of cancer means a
better chance of effective treatment.
The holistic approach to cancer involves non-invasive procedures focused upon
restoring the health of the human energy fields, based upon a human energy field
understanding of disease. Holistic care includes the field of study called energy medi-
cine. An element of energy medicine is present in acupuncture, acupressure, home-
opathy, herbal medicine, fresh fruits and vegetables, food supplements, aromatherapy,
music therapy, dance therapy, some massotherapy and chiropractic, physical exercise,
the martial arts, tai chi, qigong, yoga, meditation, compassionate love, emotional re-
lease therapy, touch healing, prayer, and most of the other approaches offered on this
xviii Preface
web site. Each approach acts to restore the human energy fields to health that then
signals the body’s biological cells to become healthy. According to researchers in this
field, when someone is diseased, the person’s human energy field is also diseased,
distorted in specific ways that can cause and maintain the disease. Human energy field
researchers have long referred to cancer as an energy disease. As the cancer cells grow
in number, they begin developing their own energy field system that competes with
the human energy field system for control of body functions. In late stage cancer, the
cancer energy field system has overwhelmed the normal human energy fields, block-
ing therapeutic outcomes of both medical and holistic treatment.
Discover the “REAL” truth about cancer and learn about all of the most potent
cancer healing therapies in this book. The book enlightens how nanomedicne, holistic
medicine and other cancer therapies play their role in treating cancer.
— Mathew Sebastian, MD
Chapter 1
Nanotechnological Based Systems for Cancer
Jagat R. Kanwar, Ganesh Mahidhara, and Rupinder K. Kanwar
INTRODUCTION
Carcinogenesis is a multistep process, caused by combination of environmental and
genetic factors, leading to a series of genetic and epigenetic changes occurring at vari-
ous stages in the development and progression of the disease (Aznavoorian et al.,
1993). Cancer cells arise from a single transformed cell, which has undergone genetic
and epigenetic changes. Thus cancerous state is a result of several sequential events
triggered by various factors including genetic predispositions, transformation by vi-
ruses, radiation and/or by certain chemicals. Many genetic and cytoplasmic events
including de activation of tumor suppressor genes such as P53 or Rb, triggers various
events leading to cancer (Harris and Levine, 2005).
Tumor invasion and metastasis is the one of the major causes of treatment failure
in cancer patients. Also, tumors have a capacity to generate new blood vessels by us-
ing preexisting endothelium and/or endothelial precursor cells, by a process known as
angiogenesis (Cavallaro and Christofori, 2000; Folkman, 1995) which in turn involves
implication of complex and diverse actions such as extra cellular matrix degradation,
proliferation and migration of endothelial cells and morphological differentiation of
endothelial cells to form tubes. A range of factors including various growth factors, cy-
tokines, lipid metabolites and cryptic fragments of homeostatic proteins, are involved
in angiogenesis (Haas et al., 2000).
There are many anticancer, anti-angiogenic drugs approved and in Phase II clini-
cal trials (http://www.cancer.gov/CLINICALTRIALS). But most of them are chemical
and/or fungal derivatives, mainly steroid containing compounds and these have many
side effects, when metabolized that is after producing their secondary metabolites.
So, natural biodegradable compounds are the best alternative for decreasing patient
compliance. In this regard, micro ribonucleic acids (miRNAs) are the ideal candidates
to consider. These non-coding RNAs, reported to have many implications in tempo-
ral and spatial regulation of genes in different organisms. It has been reported that
they have tumor suppressing as well as oncogenic properties. Certain classes of micro
RNAs, for instance Let 7 family of miRNAs have been proved to have antitumor prop-
erties by inhibiting RAS, a factor involved in cell proliferation. So, it is tempting to
use these tiny wonders for antitumor therapies. This chapter discusses about develop-
ment, progression and metastatic stages in the highly interactive process of tumor de-
velopment, angiogenic switch and molecular regulators involved in this process with
specific emphasis on micro RNA, aptamer biology, von Hippel-Lindau (VHL) tumor
suppressor, combinatorial therapy towards eradication of cancer and the possible drug
development for future (Aznavoorian et al., 1993; Cavallaro and Christofori, 2000;
2 Nanomedicine and Cancer Therapies
Folkman, 1995; Haas et al., 2000; Harris and Levine, 2005; Kerr et al., 1972; Knudson,
1971; O’Rourke and Ellem, 2000; Thompson, 1995; Zapata et al., 2001).
APOPTOSIS
The process of cell death was first recognized in the 19th century, when Carl Vogt
first described the process of cell death in the notochord and adjacent cartilage of
metamorphic tads. Later on Fleming in 1885 described the same process of cell death
as “chromalolysis”, in which nuclei of mammalian ovarian follicle broke up and were
cleared off spontaneously (Hockenbery et al., 1990; Krammer, 2000). The term apop-
tosis was first derived from the Greek word describing the process of leaves falling
from trees. Many different types of apoptotic pathways contain a multitude of differ-
ent biochemical components, many of them not yet understood (Wajant, 2002). Any
modification/removal of at least one member in a sequential pathway like apoptosis
causes disastrous effects, often in the form of a disorder.
Mammalian cells undergo apoptosis by two mechanisms; (1) Extrinsic pathway
– mediated by Fas ligand and/or APO1/CD95 and several other proteins such as Fas
associated death domain (Krammer, 2000) and caspases 8–10 (Wajant, 2002) and (2)
Intrinsic pathway- mediated by Bcl-2 family proteins (Hockenbery et al., 1990; Zapata
et al., 2001). These pathways are modulated by definite set of genes, which means
apoptosis is a genetically intervened process. Bcl-2 family members regulate apopto-
sis in response to various death inducing stimuli. Till now, more than 15 members of
this large Bcl-2 protein family have been identified. Death antagonists/anti-apoptotic
proteins including Bcl-2, Bcl-XL, Mcl1, Bcl-W, and A1 which provide protection,
whereas death agonist members including Bax, Bad, Bcl-Xs, Bid, Bim, Bik, and Hrk
increase sensitivity to death inducing signals. The ratio of death agonist to antagonist
signal determines the susceptibility to death stimuli (Scaffidi et al., 1998). In addition,
it is found that members of inhibitor of apoptosis protein (IAP) gene family proteins
including c-IAP1, c-IAP2, XIAP, NAIP, survivin, apollon, MLIAP/livin, and ILP-2
function as endogenous inhibitors of caspases. Among all the IAP members, survivin
and livin are highly expressed in cancer cells and transformed cells but show little
or no expression in normal differentiated tissues (Li, 2003; Li and Ling, 2006). Co-
localization of survivin antibodies and livin antibodies has been reported in sera of
breast cancer patients (Yagihashi et al., 2005). In this regard, we have demonstrated
earlier that, combinational therapy using antisense survivin and B7-1 immunogene
therapy eradicated EL4 thymic lymphoma tumors (Kanwar et al., 2001a). There are a
few reports in using human and/or murine survivin antagonists as anticancer vaccines
(Jiang et al., 2006; Paduano et al., 2006).
ANGIOGENESIS AND ITS MECHANISM

Angiogenesis is the formation of new blood vessels from pre-existing blood vessels
and/or from EPCs. It has implications in many physiological conditions such as em-
bryo development, ovulation, wound healing, and in some pathological conditions
such as arthritis, diabetic retinopathy and of course in metastasis (Figure 1) (Folkman,
1990). It has been observed that some human tumor lines do not form visible tumors
Nanotechnological Based Systems for Cancer 3
when inoculated into immune suppressed mice. Interestingly, when transfected these
cells with some pro-angiogenic factors such as vascular endothelial growth factor
(VEGF), dormancy was found to be overcome by these microscopic tumors. It has
been estimated that more than one third of women between the age 40 and 50 years
found to carry in situ tumors in their breast tissue by observing autopsy data but only
1% of them diagnosed with breast cancer in later days. Same is the case with pros-
tate cancer in men and with thyroid cancer in certain individuals and patients with
Down’s syndrome (Folkman and Kalluri, 2004). These examples clearly support the
phenomenon of “angiogenic switch”. The switch clearly involves more than simple
up-regulation of angiogenic activity and thought to be balance of positive and negative
regulators. The balance between in situ tumor’s total output and an individual’s total
angiogenic balance is what the key factor that determines a tumor to be dormant or
not. Also, this is the main reason why all individuals do not develop tumor metastasis.
Figure 1. Figure showing various stages in the development of malignant metastatic cancer. A
metastatic tumor development is as difficult as development of therapeutic treatments for cancer
cure. Healthy cells, if they escape the process of apoptosis by mutations in one or the other among
various molecular events related to the programmed cell death, will form a group of cells, normally
regarded as a dormant tumor. This tumor then produce various angiogenic factors, balance between
pro and anti-angiogenic factors decide the fate of tumor angiogenesis. Once the blood vessel
formation occurs, the tumor will now be regarded as malignant tumor, which grows independent
of the other tissues, by absorbing nutrients and oxygen required for its growth. Mutated cancer cells
from this tissue can travel to various other healthy tissues via the established angiogenic network,
before establishing themselves as solid tumors.
Angiogenesis, depending on different physiological processes in which it is in-

volved, can be classified into three major types.
1. De novo angiogenesis occurring in embryonic development and in female re-
production.
2. Degenerative angiogenesis in tissue repair and,
3. Pathological angiogenesis occurring in certain disorders such as cancer and
diabetic retinopathies.
ANGIOGENESIS AND TUMOR PROGRESSION

Metastasis continues to be a major hurdle to successful and complete treatment of
malignant tumors, as portrayed in a number of basic research studies. “angiogenic
switch” is the term used to denote the close relationship between angiogenesis and
tumor progression (Folkman, 1996). It has been shown that hypoxia induces angiogen-
esis; high proliferation of tumor cells creates hypoxic areas necessary for the induc-
tion of VEGF expression. Also, the identification of transcription factor HIF1 as the
up regulator of VEGF under low oxygen conditions proved further insights into the
mechanisms of tumor angiogenesis (Maxwell et al., 1997). Hypoxia also has shown
to stimulate other angiogenesis supporting growth factors such as PGDF and FGF
(Kuwabara et al., 1995). It has been reported that succession of micro metastasis to
macro metastasis happens by activation of angiogenic switch. Embryogenic progeni-
tor cells from bone marrow rather than preexisting endothelium were found to be the
critical regulators of this angiogenic switch. Immobilization of endothelial progenitor
cells (EPCs) by blocking the expression of transcription factor id1 leads to the inhibi-
tion of angiogenesis and further reduction in tumor progression (Naumov et al., 2006).
The EPCs, interaction between angiogenic factors, their receptors and the interac-
tion between vasculogenesis and lymphangiogenesis are all on and off buttons to the
switch. As a result of this, tumor vessels tend to break conventional rules of micro cir-
culation by spreading without organization and changing vessel diameter, with some
missing differentiation in arterioles, capillaries and venules.
MODULATORS OF ANGIOGENESIS
Many factors have been found to have implications in the process of angiogenesis
termed as positive or negative regulators depending on their respective roles in stimu-
lating or inhibiting action respectively. Some factors related to modulation of angio-
genesis are as follows.
Angiogenic Factors
Integrins
Integrins are heterodimeric combination consisting one α and one β -sub units each
from 18 different α and β -trans membrane proteins. In one study, blocking integrins
α v β3 and α1 β1 by using specific antibodies showed that antagonists against integrin
α v β3 inhibit pro-angiogenic effects of VEGF and probol esters while antagonists
against that of α1 β1 abolish FGF2 and tumor necrosis factor (TNF) α stimulated
sprouting of new vessels (Smyth and Patterson, 2002). Angiogenic endothelial cells
were shown to undergo apoptosis compared to their resting counterparts, after the α
v β3 antibody administration (Brooks, 1996). Defective vascular network as a conse-
quence of injecting α v β3 antibodies in quail embryos shows the role of α v β3 integ-
rins in endothelial cell proliferation (Clark et al., 1996) and in angiogenesis.
Vascular endothelial growth factor

This is the most important factor among angiogenic factors, which was discovered in
1989 by Ferrara and coworkers (Ferrara et al., 2003). It was found that, by disrupt-
ing a single VEGF allele in mice ~50% reduction in VEGF and ultimately embryonic
death was observed. Six known members of the VEGF family have been discovered
viz. VEGF-A, -B, -C, -D, and -E and the placental growth factor, found to be gener-
ated by alternative splicing of the VEGF gene (Ferrara et al., 2003). The VEGF binds
to VEGF R1, R2, and R3 expressed on endothelial cells/lymphatics. After binding to
their ligand, VEGF receptors undergo dimerization resulting in mitotic, chemotactic
and pro survival signals. Several growth factors such as EGF, TGF α, β FGF, and
PDGF; hormones such as estrogen induce VEGF expression.
Angiopoietins
The angiopoetins (Ang-1–Ang-4) have been implicated in the development of vascu-
lature in a wide variety of tumor types (Plank et al., 2004). Among the four known an-
giopoetins, Ang-1 and Ang-2 are the best characterized cytokines, both exerting their
biologic function through binding to the Tie-2 receptor (Maisonpierre et al., 1997).
The Ang-1 promotes endothelial cell survival and sprouting and stabilizes vascular
networks by recruiting pericytes to immature vessel segments. In contrast, Ang-2, ex-
pressed at sites of vascular remodeling, causes the loss of pericytes and exposes endo-
thelial cells to angiogenic factors.
Fibroblast Growth Factors (FGFs)

The FGFs are a family of heparin binding proteins. The FGF-1 (acidic) and FGF-2 (ba-
sic) are described as inductors of angiogenesis (Szebenyi and Fallon, 1999). All FGFs
bind to heparin sulfate proteoglycans (HLGAGs) of the extracellular matrix (ECM).
The FGFs can bind to four transmembrane specific receptor tyrosine kinases inducing
receptor dimerization and activation. The FGF-1 and FGF-2 induce endothelial cell
proliferation and differentiation of epiblast cells into endothelial cells. Furthermore,
FGF-2 stimulates the release of urinary plasminogen activator and collagenases in
endothelial cells and acts as a chemoattractant for these cells.
Transforming growth factor β (TGF β)

It has been proposed that TGF β has auto and/or paracrine activity on endothelial cells
as well as pericytes (Antonelli-Orlidge et al., 1989). Two isoforms TGF β1 and TGF
β2 have been identified, the former acts as pro-angiogenic and the later act as pro-
angiogenic at low concentrations and oppositely at high concentrations. Dickson and
coworkers found that knockout mice lacking TGF are unable to produce stable vessel
walls as well as inadequate tube formation.
CXC-Chemokines
The CC chemokines, that primarily modulate infiltration of monosite/macrophages
to the sites of inflammation, have been found to associate with modulation of angio-
genesis (Salcedo et al., 2000). Although, underlying mechanism of their involvement
in angiogenesis has not been fully validated, an indirect involvement by stimulating
monocytes and macrophages, which intern directly produce angiogenic factors has
been suggested (Isik et al., 1996). However, recent evidence of endothelial cells ex-
pressing of CCL2 receptor CCR2 shows a direct angiogenic modulation by CXC che-
mokines (Stamatovic et al., 2006; Weber et al., 1999).
Angiostatic factors
Angiostatin
Angiostatin is a 38-kDa plasminogen fragment and systemic injection of angiostatin
has been shown to inhibit tumor neovascularization and metastatic growth by inhibi-
tion of ECM. Our previous reports have shown that combinatorial therapy of plasmids
containing angiostatin gene with B7.1 immunogene therapy have reduced solid EL4
lymphomas in syngenic C57BL/6 mice, supports the use of angiostatin in combinato-
rial therapy (Sun et al., 2001).
Thrombospondin-1
Thrombospondins belong to a family of ECM proteins. The CD36 (also known as
GP88, GP IV, GPIIIb) is an important cellular receptor for TSP-1 on microvascular
endothelium and is necessary for its anti-angiogenic activity (Tonini et al., 2003). The
anti-angiogenic activity of TSP-1 is contained in a structural domain known as the
TSP type I repeat which plays a role in endothelial cell apoptosis (Sargiannidou et al.,
2001). In addition to CD36-mediated anti-angiogenic effects, TSP-1 can potentially
inhibit angiogenesis through an interaction with pro-MMP2/9, MMP-2/9 or induction
of cell cycle arrest.
Endostatin
It inhibits endothelial cell migration and induces endothelial cell apoptosis and cell
cycle arrest and is encoded by COL18A1 gene at 21q22.3. In one interesting study,
the levels of endostatin was observed in the serum in patients with Down’s syndrome
along with normal controls and the results were interesting to see that elevated end-
ostatin levels persist in patients with Down’s syndrome (Down’s syndrome is most
commonly caused by trisomy of 21st chromosome) (Shichiri and Hirata, 2001). It has
been shown that endostatin directly and specifically acts on endothelial cells in culture
to produce intracellular signals such as influx of extracellular calcium, inside the cells
(Sargiannidou et al., 2001; Shichiri and Hirata, 2001). Endostatin’s application down
regulates genes’ expression during endothelial cell growth, and exhibited a potent ant
migratory effect on endothelial cells in vitro, mediated through c-myc down regula-
tion (Shichiri and Hirata, 2001). Inhibitory action by endostatin has been proposed to
involve binding to the receptor α5β1 (Sudhakar et al., 2003). The cDNA and antibody
microarray technology uncovered the full set changes in gene expression following
endostatin administration. Endostatin up regulated anti-angiogeneic thrombospondin,
kininogen, ATIII, and chromogranin A; while down regulating downstream regulators

of anti-apoptotic genes (e.g., Bcl2, COX2, cMyc, and iNOS) such as HIF1α, NFkB,
Ets-1, id1, and STATs, leading to cell cycle arrest (Abdollahi et al., 2004). Therefore,
taking together these evidences, one can articulate that endostatin acts upon one or
more receptors, present on endothelial cells, resulting in intracellular signaling which
decreases their migratory capacity and/or proliferative ability preventing the vascu-
larization.
As discussed earlier in this review, targeting angiogenesis is the best way to fight
with cancer. So, drugs or molecules which can target angiogenic switch are recom-
mended for effective treatment of cancer. Biologically synthesized molecules degrad-
ed biologically by host’s body; a chemically synthesized molecule, considered foreign
to a particular organism, and may cause acute side effects and/or allergic reactions
when it gets converted to a metabolite. Therefore, chemicals and/or modified fungal
derivatives can be avoided and replaced with the molecules which have fewer side ef-
fects. As targeting the specific proteins expressed excessively in tumors and/or angio-
genic blood vessels is the key point in developing an antitumor drug, molecules used
for this purpose should have certain characteristics as listed below:
(1) Capacity to discriminate between oncogenic, non-oncogenic and/or angio-
genic and anti-angiogenic forms of the proteins involved in signaling path-
ways.
(2) Capacity to quantify the level of expression of the oncogenic forms.
(3) Ideally, be usable both for in vitro and in vivo purposes
(4) For therapeutic applications, the capacity to block the activity of the onco-
gene product.
Anti-angiogenic/anticancer peptides, DNA vaccines; oligomers, such as miRNA
and siRNA, which control the synthesis of gene product post transcriptionally and
small oligomeric aptamers are promising agents in this regard. We consider here micro
RNA, aptamers, and certain targeting agents which attracted attention in the recent
past.
MICRO RNAs IN CANCER

It has been speculated over a few decades that only 2% of the animal genome encode
functional protein coding genes and the remaining is junk. However, recent advances
have brought non-protein coding RNAs into spot light. The miRNAs are such kind of
non-coding RNAs found to have implications in cell growth, regulation, differentia-
tion, and apoptosis. Nearly, 400 miRNAs have been identified in humans and the num-
ber is increasing. By using bioinformatics tools, it has been observed that one miRNA
can recognize more than 200 messenger RNAs. Initially miRNAs are transcribed by
RNA-polymerase II, as large precursors (Lee et al., 2002), which are further processed
by Drosha (an Rnase III enzyme and by a double stranded binding protein DGCR8/
Pasha (Gregory and Shiekhattar, 2005; Lee et al., 2003), this processing step pro-
duces stem loop structures of nearly 70 nucleotide length. These precursors then are
processed by another Rnase III enzyme, Dicer is processed into 22 nucleotide double
strand RNA duplex after they got exported from nucleus to cytoplasm by exportin 5 in
a Ran guanosine phosphate dependent manner (Yi et al., 2003). This duplex intern is
incorporated into the miRISC complex. The RISC-miRNA complex then binds to the
corresponding mRNA and represses its translation by blocking translation initiation or
by inducing endonucleolytic cleavage of mRNA.
Cloning of first miRNA, lin4, was achieved by doing genetic analysis of the timing
of development in C. alegans (Ambros, 2001, 2004; Lee et al., 1993) and Reinhart et al.
in 1993 identified its prey, lin14 mRNA while doing heterochronic analysis (Reinhart
et al., 2000). Recently it has been found that carcinogenesis is strongly associated
with inappropriate expression of miRNAs regulating gene expression in translational
level. Based on these observations alone, lin4 and let7 miRNAs are thought to be po-
tential tumor suppressors and it has become more interesting after finding that these
molecules are conserved in mammals (Kanwar et al., 2010; Pasquinelli et al., 2000).
Subsequent reports make it clear that let7 miRNA and miRNAs belong to the family
which act as tumor suppressors by targeting 3′ UTR of RAS mRNA, there by effect-
ing RAS protein (Johnson et al., 2005). On the contrary, miRNAs were shown to
enhance translation as suggested recently in two different studies (Thai et al., 2007;
Tili et al., 2007). It has been observed that miR-155′s over expression resulted in
enhanced translation of Tumor Necrosis Factor α alpha (TNF-α), most probably by
enhancing the stability of its transcript, and mice over expressing miR-155 in B cell
lineage (Eµ-miR-155) which produce more TNF-α alpha when challenged with LPS
(Thai et al., 2007). Further research in a realistic approach is needed to confirm the
role of miRNAs in enhancing gene expression and translation of proteins. The first
evidence for the link between miRNA and cancer came from the work, as reported
by Clain and coworkers; deletion in the region coding for miR 15a and miR 16-1 as
it is highly likely because of over expression of anti-apoptotic protein Bcl-2 in many
chronic lymphoid leukemia (CLL) patients, showing the tumor suppressor functions
of miRNA (Calin et al., 2002). There are reports that important group of miRNAs, the
Let-7 family were found to regulate RAS and/or MYC oncogene expression at the
translational level to be often down regulated in human lung tumors, owing to its growth
repression functions (Akao et al., 2006). Recent evidence suggests that miR-143 and
miR-145 are frequently down regulated in colorectal tumors (Michael et al., 2003).
Down regulation of these miRNAs have also been noted as a common occurrence in
breast carcinomas and breast tumor cell lines (Volinia et al., 2006). On the flip side,
miRNAs acting as oncogenes have also been identified in the recent past, owing to
speculations about their role in tumerogenesis (Tam et al., 2002; Ota et al., 2004). The
miR-21 was demonstrated to be up regulated in glioblastoma (Chan et al., 2005). The
MiR-372 and miR-373 were found to block RAS induced cellular senescence and po-
tentiate RAS mediated cellular transformation (Voorhoeve et al., 2006).
Various studies earlier reported that micro RNAs have implications in the devel-
opment and/or regulation of angiogenesis. The miRNA profiles of endothelial cells
revealed that let7b, miR-16, miR-23a, miR-100, miR-221, and miR-222 are predomi-
nant (Kuehbacher et al., 2007; Poliseno et al., 2006; Suárez et al., 2007), however
there was a little work regarding the abundance of these miRNAs in environments
that imitate tumor microvasculature conditions. Down regulation of anti-angiogenic
genes by miR-130a expressed in HUVECs, in response to addition of serum (FBS)

has been reported recently (Chen and Gorski, 2008). Certain classes of micro RNAs,
for instance miR17-92 have been proved to have pro-angiogenic properties by inhibit-
ing transcription factors involved in anti-angiogenesis (Dews et al., 2006). Hypoxia
is a condition observed in cancer malformation and it has been observed that hypoxia
has implications in the formation of blood vessels. It has been shown that hypoxia
up-regulates VEGF (Plate et al., 1992; Shweiki et al., 1992), owing both to increased
transcription mediated by hypoxia inducing factor-1(HIF-1) and an increase in VEGF
mRNA stability dependent on 3′ region of mRNA (Semenza, 1996). Interestingly, a
recent study (Kulshreshtha et al., 2007) has shown that a specific spectrum of mi-
cro RNAs (including miR-23, -24, -26, -27, -104, -107, -181, -210, and miR-213) is
induced in response to low oxygen concentration. A similar study shows miR-120
(induced in hypoxia), over expression enhanced the formation of capillary like struc-
tures and migration of endothelial cells (Fasanaro et al., 2008). Promotion of cell sur-
vival, tumor growth, and angiogenesis by miRNA-378 was predicted by assaying for
the luciferase activity in constructs synthesized containing 3′UTR of it is possible
downstream effecter molecules, SuFu and Fus-1 (Lee et al., 2007). In another study,
miRNA generation was impaired by silencing the molecules involved in their biogen-
esis, Dicer and Drosha by siRNA and found a reduced angiogenesis in parallel with
decreased spectrum of miRNAs in endothelial cells (Kuehbacher et al., 2007). Simi-
larly, dicer inactivation resulted in altered expression of molecules related to angio-
genesis and endothelial biology, including up regulation of Tie-2, Tie-1 receptors and
eNOS transcription factor (Suárez et al., 2007). However, one cannot generalize these
experiments saying silencing dicer impairs angiogenesis, as it is not about silencing
a particular miRNA and observing for a phenotype and the obtained phenotype is an
overall result of silencing all miRNAs in an endothelial cell. Also, it has already been
demonstrated that dicer itself is required for regulation of angiogenesis (Yang et al.,
2005). The miR-126 was found to regulate many aspects of endothelial cell biology
(Wang et al., 2008). The mir-126 is found to regulate endothelial cells derived from
mouse embryonic stem cells, by promoting VEGF signaling (Fish et al., 2008). It is
interesting to see whether these miRNAs have any effect on regulating HIF dependent
VEGF and/or other pro-angiogenic factor gene up regulation and further implications
in development of metastasis by supporting angiogenesis. In contrast to this, some of
the miRNAs are proposed to have anti-angiogenic properties (Poliseno et al., 2006).
In summary, identification of miRNAs with dual antitumor and anti-angiogenic ef-
fect may lead to discovery of potential anticancer drugs. Also, inhibiting the miRNAs
which have implications in enhancing angiogenesis will be other option. One can use
molecules such as 2’O-methyl anti-sense oligoribonucleotides directed against a par-
ticular pro-angiogenic miRNA. In relation with developing a novel and effective drug
towards prevention of cancer progression and metastasis, we are here with providing
some biomolecules that can be used to in addition to the above given molecules for
increased efficiency, effective targeting and/or for easy penetration of the drug into the
systemic circulation.
APTAMERS
Aptamers (Latin; aptus, to fit+ meros, part or region) are composed of oligonucleic acid
or peptide molecules which can bind to target molecules that are usually expressed on
the surfaces of the membranes. Aptamers are usually created by selecting them from a
large random sequence pool, by using specific techniques such as Selective Evaluation
of Ligands by Exponential Enrichment (SELEX), developed in the laboratory of Larry
Gold in the University of Colorado (Kanwar et al., 2010; Tuerk and Gold, 1990). Natu-
ral aptamers also exist in riboswitches. Aptamers are DNA/RNA aptamers and protein
aptamers depending on their chemical nature and can bind to a range of molecular
targets including nucleic acids, proteins, and small molecules and even to cells. These
can be used for both basic research and clinical purposes as macromolecular drugs.
Pegaptanib, a nuclease-resistant aptamer used for curing age-related macular degrada-
tion (AMD) is one such example. To be able to make these molecules cleaved in the
presence of their target, aptamers can be coupled with catalytic RNA (ribozymes).
This makes these compound molecules have additional research, industrial and clini-
cal applications, including diagnostics, therapeutics, biosensors and tools for probing
fundamental cellular processes (Griffin et al., 1993). Aptamers are more advantageous
than antibodies in the sense that they are easy to synthesize in bulk, rather than us-
ing cell based expression system. During the phosphoramadite chemical synthesis of
aptamers in the lab, fluorescent dyes or chemical modification with functional groups
can be achieved. These modifications can further help in in vivo applications of the
molecules baring the aptamer or for better conjugation of the aptamer on to the other
moiety. Aptamers can also be used to probe cellular processes by binding to specific
functional groups of proteins (Blind et al., 1999) in contrast to other oligonucleotide
agents such as siRNA, RNAi, or ribozymes; aptamers can act on extra cellular targets,
that is same as the other entities that have affinity for proteins. But just like other oligo
peptides, their biggest limitation is bioavailability––upon oral administration. But the
biggest advantage over the peptide and/or antibodies is their low immunogenicity.
When comes to therapeutic application, still the progress is in its infancy, however
one aptameric drug has been approved by FDA. Pegaptanib, as listed above is a RNA
aptamer directed against VEGF, has been implicated for the treatment of all types of
neovascular age related ocular vascular diseases. In one study, adeno viral system was
used to deliver RNA aptamer (AP50), against NFкB, to overcome non-small cell lung
cancer tumor resistance to Doxorubicin, in A539 cells (Mi et al., 2008). However, viral
system for drug delivery has its own side effects. More recently, PEGylated, angiopoi-
etin-2 inhibiting RNA aptamers were shown to inhibit tumor angiogenesis and growth,
by inhibiting Tie-2 phosphorylation (Sarraf-Yazdi et al., 2008).
VHL
The VHL disease is a familial cancer syndrome caused by autosomal dominant trait,
due to mutations in tumor suppressor gene, VHL. It has been observed that, highly
vascular tumors especially, glioma, renal cell carcinoma and pheochromocytomas are
the most common implications of this disease (Kondo and Kaelin, 2001). It has been
shown based on this disease that, application of VHL causes reduced tumor malignancies.
We have previously reported the regression of solid tumors by using a combination

therapy of VHL and antisense HIFα It has been observed that VHL inhibits HIF by
binding to 1α as well as 2α subunits (Sun et al., 2003a). So it is very promising to use
VHL as an antitumor agent in combination therapy.
LACTOFERRIN
Lactoferrin is a natural defense protein, present mainly in milk and bodily secretions.
Besides its main function that is iron absorption in the intestine, lactoferrin also plays
a role in protection against infections, myelopoisis and against autoimmunity. Previ-
ous reports in our lab have shown that 100% iron saturated bovine lactoferrin has
augmented the antitumor cytotoxicity of certain chemotherapeutics, in wide range of
tumors (Kanwar et al., 2003; Kanwar et al., 2008), by increasing cytokines produced
by Th1 family, which includes IL18, IFNg and TNFα, which in turn helps in infiltra-
tion of CTL, CD4+, CD8+, NK, and NKT cells to the tumor tissue. So using lactoferrin,
along with a drug formulation uptake can be increased into systemic circulation and
inhibits angiogenesis.
In summary, aptamers that can bind to specific markers on tumor vasculature can
be used along with certain tumor, HIF and/or angiogenic inhibitors as discussed above,
so as to deliver them to the specified target. However, aptamers are not used that much
in the treatment of tumor angiogenesis, although it has same implications as that of
retinal angiogenesis; pertaining mainly because of their low bioavailability. One can
think of a wonder drug if antitumor peptides and inhibitors of pro-angiogenic factors
delivered orally, along with ligands such as aptamers, VHL and Lactoferrin with some
protecting aids, to protect them from mucosal and gastric environment. Taken togeth-
er, combinational therapy is a better approach to target tumor (Baratchi et al., 2009)
DELIVERING ANTI-ANGIOGENIC/ANTICANCER MOLECULES WITH

NANOPARTICLES
There are many delivery routes devised for drug administration in different diseases
(Baratchi et al., 2009). There are many parenteral and non-parenteral administration
routes of drug delivery that have been developed and devised in the past for therapy
(Kanwar et al., 2009). However, complications such as thromboflebitis or tissue ne-
crosis and poor patient compliance have stimulated the investigation of non-parenteral
routes (Xin Hua, 1994). Among non-parenteral routes, oral administration is usually
preferred because it is noninvasive, most acceptable and convenient for the patient
and required no skilled worker for injecting drugs. But oral bioavailability of these
prodrugs or enzymes and peptides as well as protein drugs is generally very low, ow-
ing to the acidic conditions of the stomach, proteolytic activity of the gastrointestinal
enzymes present in the intestinal tract, and poor permeability across intestinal mucosa
(Zhou and Li, 1991). Various approaches have been proposed to overcome the bio-
pharmaceutical limitations associated with these drugs, such as inhibition of the en-
zymatic degradation (Morimoto et al., 1991), chemical modifications (Conradi et al.,
1992), in situ gel system (Shah and Paradkar, 2005), and the formulation of polymer
or microsphere-based carrier systems (Torchilin et al., 1977). Application of nanocar-
riers for drug delivery, especially RNA, pDNA, prodrugs and bioactive drugs, is an
expanding area of research that provided the design of biomaterials with controlled
rates of drug release (Prokop et al., 2002). Recent advancements in chemotherapy are
using nanomaterials of broad range of chemistry, including PEGylated, hydrophobi-
cated, glycol chitosan moieties and other poly esters. These nano wonders are reported
to protect the drug from the extremities of the pH and enzymatic degradations. Drug
delivery is one of the promising biomedical applications of nanotechnology. Several
nano based cancer drugs for example, Doxil and Abraxane are in clinical trials, to be
able to use in cancer chemotherapy. Ideally speaking, a good nanocarrier will have
minimal side effects and greater hangover time in blood. Nanocarriers made up of
two or more different polymers can spontaneously assume various shapes in specific
solvents and bring several advantages to the nanocore like size, stability, drug loading,
and release efficiencies. Blood vessels in tumor vasculature are found to be leaky and
have pores in the range of 10‑100s of nanometers (Kanwar et al., 2009). This fact can
be exploited for drug delivery to the tumor site, which was being named as enhanced
permeability retention (EPR) effect (Matsumura and Maeda, 1986).
USES OF NANO PARTICLES IN CANCER TREATMENT

A nanocarrier ideally shall have a diameter from 1–100 nm and can carry multiple
drugs. As they will have high surface to volume ratio, one can think of a better drug,
tagging more ligands on their surfaces. Also, nanocarriers can better exploit EPR ef-
fect posed by the tumor vasculature, for improved drug delivery (Peer et al., 2007).
Of late, natural and synthetic polymers are used as drug delivery vectors. Various
nanocarriers used along with polymer conjugates include polymeric nanoparticles,
lipid based carriers such as liposomes and micelles, dendrimers, carbon nanotubes and
inorganic nanoparticles. There has been increased attention towards the development
of biodegradable, polymeric nanoparticles for the diagnosis and treatment of cancer,
in the scientific field. As mentioned above, anticancer biomolecules targeted along
with nanocarriers especially polymeric nanocarriers and dendrimers is being done at
pace. In recent past, some formulations have been tested by using chemotherapeutic
drugs, along with polymeric nanoparticles. A formulation with two distinct layers of
biodegradable polymers with inner nucleus containing antitumor agent, Doxorubicin
and the PEGylated outer envelope with anti-angiogenic agent, has been successfully
tested (Sengupta et al., 2005). In another development, nanoparticles that co-deliver
Paciltaxil and Ceramide to overcome multi drug resistance in tumors, using the same
formulation, developed PEO-PCL nanoparticles with tamoxifen and paciltaxil encap-
sulated with poly ethelene oxide modified poly-ε-caprolactone, to overcome MDR in
breast cancer cell lines (Devalapally et al., 2008). Anticancer drug nutlin-3a has been
actively targeted using EpCAM antibodies, loaded on to poly(lactide-co-glycolide),
has been successfully tested (Das and Sahoo, 2010). Lodamin was the mPEG-PLA
modification of TNP-470, using methylated poly ethelene glycol (PEG) and poly lac-
tic acid (PLA), which have fewer side effects compared to its counterpart, TNP and
was tested successfully in melanoma and LLC cell lines (Benny et al., 2008) and
was reported to be a promising oral administrative, for anti-angiogenic therapy. In
addition, there were some polymer conjugates which are under clinical use such as
Zinostatin, stimalmer, oncaspar, and Neulasta for cancer treatment (Duncan, 2006).
To develop an oral administrative, careful design of the core and exterior coat
is required. Recent developments in bone marrow tissue engineering, dentistry, or-
thopedics, and plastic surgery are making use of these kinds of composites in the
macro scale; utilizing the same principle in nanoscale ensuring more promising drug
development (Kanwar et al., 2009). Ceramic nanocores are being designed in order to
assist protein adsorption on them and various layers of biodegradable polymers are
then coated on their surface, as discussed below, in order to protect them, from lytic
or gastrointestinal enzymes and for obtaining sustained release kinetics of the loaded
drug. Ceramics can be defined as solid compounds that are formed by chemical reac-
tion between a metal and a nonmetallic elemental solid or between a nonmetal and
nonmetallic elemental solids (Benny et al., 2008; Cevc, 2004; Duncan, 2006; Foley
et al., 2002; Martin and Kohli, 2003; Rawat et al., 2006, 2008; Mahidhara et al., 2011).
Some of the ceramic composites are discussed below.
Calcium phosphate ceramics

Various forms of calcium phosphate ceramics are being used for last 3 decades, which
include hydroxyapatite, beta tri calcium phosphate, biphasic calcium phosphate amor-
phous calcium phosphate, carbonated appetite, and calcium deficient hydroxyapatite
(Mahidhara et al., 2011).
Calcium phosphate cements

They consist of powder phase of calcium and/or phosphate salts together with an aque-
ous phase, react at room or body temperature and form a calcium phosphate crystal
that sets by entanglement of crystals.
Bioactive glass
It is produced like a conventional glass, in which basic components are SiO2, Na2O,
CaO, and P2O5. It is commercially available as Bioglass®.
Characteristics of these include high mechanical strength, good body response,
and strong binding ability with proteins, which helps in a sustained release. However,
they are less biodegradable, which may be a draw back and this can be overcome by
adding biopolymers such as chitosan, which not only improve their biodegradability
but also increases tolerance towards variations in pH and protects towards enzyme ac-
tion in the gut. Polymers widely used are listed here.
1. Poly lactic acid/poly glycolic acid polymers such as poly lactic acid (PLA,
PLLA, PDLLA), poly glycolic acid (PGA), poly (lactic-co-glycolic acid)
(PLGA) and poly-caprolactones.
2. Natural proteins such as collagen, gelatin, fibrin, and casein.
3. Carbohydrates and their derivatives such as chitin, chitosan, alginate, cellu-
lose, starch, hyaluronan, and amylopectins.
Recently Saraf et al. (Rawat et al., 2006, 2008) have developed nanoparticles,
which can be used for oral delivery of peptides. However, to the best of our knowledge
no one has used these ACNC carriers for pDNA or RNAi delivery for gene targeting
in any disease in vitro or in vivo in human or animal systems. It is tempting to ex-

pand the utility of nanocarriers for delivery of therapeutic enzymes, because particles
larger than 20 μm are prone to be washed out, being inefficient for mucosal delivery
(Lameiro et al., 2006) and to maintain the native structures (Cleland, 1997). In this
regard, innovative techniques have led to the use of ceramics in high-tech applica-
tions like delivering chemicals and biologicals effectively in vitro and in vivo (Cherian
et al., 2000). We recently for the first time used the covalently cross-linked CPP (R9
and Tat peptides) complexed with siRNA to survivin, an inhibitor of apoptosis which
over expressed in tumors and inflammation (unpublished observations). We designed
covalently cross-linked CPP (R9-siRNA to survivin and Tat peptides-siRNA to sur-
vivin peptides) complexed with siRNA to survivin on to design ACNC for effective
loading and protection of active acid-labile and alkaline-labile large nanocarriers for
oral administration. We tested these nanocarriers loaded targeted nanoparticles in hu-
man colon cancer cells (Caco-2 and HT-29) in transwell assays. As an attempt towards
increasing the bioavailability and conformational stability, we have loaded covalently
complexed CPP with survivin siRNA into ACNC with no effective protection, mu-
coadhesiveness, and sustained release (Mahidhara et al., 2011). The prolonged activ-
ity was obtained due to slow release of the protein or RNA from the ACNC and the
intact structure without denaturation or dehydration during delivery and storage. The
encouraging results obtained in this study could propose ACNC for future in vivo
studies, especially in the delivery of RNA, DNA, protein, peptide and drugs. These
novel nanocarriers were found to be promising for protection of the spatial qualities
for exhibiting better therapeutic effect. But further studies in terms of pharmacoki-
netics, toxicology, and animal studies are required for clinical utility of the formula-
tion. More recently, we were able to load cell permeable dominant negative survivin
R9 (DNSurR9) (Bawa 2009; Baratchi et al., 2010; Cheung et al., 2006) and survivin
and HSP-90 antagonists, “shepherdin” on alginate gel-encapsulated, chitosan ceramic
nanocarriers (ACNC-NPs) and were able to induce apoptosis and disintegrate the mi-
tochondria of colon and breast cancer cell lines (but not normal control cells) more
efficiently in in vitro cell based assays. In this study, we loaded non-covalently cross-
linked CPP (R9-siRNA to survivin and Tat-peptides-siRNA to survivin peptides) com-
plexed with siRNA to survivin and oncogenic antisense microRNA-27a (as-miR-27a)
on ACNC-NPs and transferred to human breast cancer MDA-MB-231 and MCF-7
cell lines by endocytosis, as outlined. Our results show that both R9 and Tat CPP
peptides covalently complexed with as-miR-27a loaded ACNC-NPs exhibits onco-
genic activity. Suppression of miR-27a inhibits breast cancer cell growth and inva-
sion. Simultaneous covalently complexed CPP (R9 and Tat-peptide) with siRNA to
survivin and oncogenic as-miR-27a loaded ACNC-NPs results in down expression of
genes that are important for cell survival and angiogenesis faster and more efficiently
than the monotherapy. In addition, these responses were accompanied by decreased
expression of survivin and angiogenic genes, including survivin isoforms, VEGF, and
VEGF receptor 1(VEGFR1). We also demonstrated the down regulation of survivin
expression in Western blot in the covalently complexed CPP (R9 and Tat-peptide)
with siRNA to survivin and oncogenic as-miR-27a loaded ACNC-NPs treated cells.
The TUNEL assay, caspase activity assay and changes in mitochondrial membrane
potential reveal that cell death was mainly through intrinsic apoptosis pathway. Oral
delivery of siRNA to survivin and oncogenic as-miR-27a loaded ACNC-NPs induced
apoptosis, necrosis, and cytotoxicity in the xenograft breast cancer model. Oral admin-
istration of covalently complexed CPP (R9 and Tat-peptide) with siRNA to survivin
and oncogenic as-miR-27a ACNC-NPs in combination regress tumor growth faster
and inhibits angiogenesis in the xenograft breast cancer mouse model as compared to
monotherapy (Bawa, 2009). We also compared our results with the doxorubicin and
taxol loaded ACNC-NPs (Figure 2). Taken together, our results are highly encourag-
ing for the development of combination nanotherapeutic strategies that combine gene
silencing and drug delivery to provide more potent and targeted therapeutic, especially
in late and metastatic breast cancer.
Figure 2. CPPs (R9 or Tat peptide) were covalently cross linked and complexed with siRNA to survivin
and oncogenic antisense microRNA-27a then loaded on nanoparticles alginate gel-encapsulated,
chitosan ceramic nanocores (ACNCs). These ACNCs were protected from various gastric enzymes
in vitro and in vivo studies. ACNCs were transcytosise in to gut cells and present in circulation and
make available of siRNA to the tumor sites for knockdown of survivin gene. siRNA loaded ACNC
nanoparticles coated with alginate can be endocytosed easily and can be used for oral therapy, as
they can sustain intestinal pH. These nanoparticles can enter blood circulation by transcytosis in
intestinal villi before encountering a cancer tissue via the blood stream.
GENE DELIVERY TO TARGET CELLS

The RNAi-mediated gene-silencing effect is limited in the cells reached by RNAi
effectors, which makes the delivery of RNAi effectors to target cells important in
achieving RNAi-based therapeutic treatment. Lots of in vivo delivery methods of
RNAi effectors have been developed, including those developed and optimized for
the delivery of plasmid DNA (pDNA) for gene therapy for inhibiting angiogenesis
and/or immunomodulation by co-stimulatory pDNA immunogene therapy (Luo et al.,
2006; Sun et al., 2005, 2003b; Kanwar et al., 1999, 2001b; Cheung et al., 2006). Early
studies on in vivo RNAi often used the expression of reporter genes that were ex-
pressed from vectors co-administered with RNAi effectors (Bartlett and Davis, 2006;
Kobayashi et al., 2004; Wooddell et al., 2005). Similarly, suppression of tumor cell
growth by efficient RNAi induction in tumor tissue by intratumoral injection has also
been studied (Zhang et al., 2008). In contrasts to this, a few studies have reported
successful induction of gene silencing in target cells after systemic administration of
shRNA-expressing pDNA. The shRNA-expressing plasmids, which are encapsulated
in the interior of 85 nm PEGylated immunoliposomes (PILs), can suppress the gene
expression in tumor cells intracranialy inoculated into the brain (Maliyekkel et al.,
2006; Zhang et al., 2003). Among the non-viral carriers are cell-penetrating peptides
(CPP), which have the ability to enter cells by crossing the plasma membrane directly
or through uptake by the endocytotic pathway (Meade and Dowdy, 2008). We
(Krissansen et al., 2006) and many other research groups have demonstrated the ef-
fective use of CPP (Meade and Dowdy, 2008; Schaffert and Wagner, 2008; Zhang et al.,
2007; Zuhorn et al., 2007). Here we summarize CPP-based RNA delivery strategies,
and focus on recent improvements that enhance the release of siRNAs trapped in en-
dosomes into the cytosol.
RNAI FOR CANCER THERAPY

Double-stranded RNA (dsRNA) containing a sequence homologous to a specific gene
causes sequence-specific gene silencing, which is termed RNA interference (RNAi).
The RNAi was first discovered in Caenorhabditis elegans, an organism in which gene
expression is down regulated by long dsRNA (Fire et al., 1998). Remarkably, the basic
molecular mechanism of RNAi is conserved in mammalian cells, and the applicability
to mammalian systems was recently discovered using short dsRNAs (19–23 base-
pairs), termed small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs)
(Caplen et al., 2001; Elbashir et al., 2001; Paddison et al., 2002). The RNAi-mediated
gene-silencing is now an essential strategy in analyzing gene functions due to its high
specificity, high efficiency, and great facility. In addition, it offers one of the most
attractive methods for gene therapy for many diseases, including viral infectious dis-
eases and cancers (de Fougerolles et al., 2007; Devi 2006). Many types of diseases
have been demonstrated to be potential targets for RNAi-based therapy in laboratory
experiments.
Appropriate systems that enable safe and efficient RNA delivery to target cells
or organs are required to further expand the use of RNAi to therapeutic applications.
Several kinds of RNA delivery systems have been developed and improved upon,
including viral vectors and non-viral carriers, such as cationic lipids, polymers and
dendrimers. These have been reviewed earlier (Schaffert and Wagner, 2008; Zhang
et al., 2007; Zuhorn et al., 2007). Among the non-viral carriers are CPP, which have
the ability to enter cells by crossing the plasma membrane directly or through up-
take by the endocytotic pathway (Meade and Dowdy, 2008). We (Krissansen et al.,
2006) and many other research groups have demonstrated the effective use of CPP
and observed the efficient down regulation of gene expression by CPP-based siRNA
delivery, although the numbers of reports regarding CPP-based delivery are much
less than those of viral or lipid-based delivery (Schaffert and Wagner, 2008; Zhang
et al., 2007; Zuhorn et al., 2007). However due to inherent problems of viral vectors
and liposome’s here in this review, we summarize CPP-based RNA delivery strate-
gies, and focus on recent improvements that enhance the release of siRNAs trapped
in endosomes into the cytosol. The CPPs are already utilized in many biological and
biotechnological studies to deliver various kinds of biologically active molecules into
cells. The CPPs were first used for the delivery of proteins that were genetically fused
to CPPs. Subsequently, CPPs have been used for the delivery of many types of car-
go molecules, ranging in size from small inorganic or organic molecules (Josephson
et al., 1999; Lewin et al., 2000; Liang and Yang, 2005; Rothbard et al., 2000) to large
liposomes (diameter = 200 μm) (Khalil et al., 2006; Torchilin et al., 2001), by conju-
gating the cargo to the CPP.
CONCLUSION
The ability to precisely and differentially target functionally bio-relevant molecular
signals in patient’s cancers will establish a new paradigm in cancer management; one
which focuses on defining the uniqueness of each patient’s tumor and tumor-host pro-
cesses and interactions following rational target prioritization using computational
systems biology algorithms. This, then, would allow for exploitation of the “attack
vulnerability” of the rewired cancer network by deconstructing essential hubs and
linkages, multiply targeting and eliminating them. Both siRNA and shRNA effectors
are attractive opportunities. The capability of potentiating activity using a bifunctional
design may further enhance safety and efficacy. Though shRNA seems ideal for cancer
related therapeutic development, new technology such as bifunctional RNA interfer-
ence may provide an even greater opportunity for enhancement in potency as well as
heightening safety thereby increasing the opportunities for multiple target therapy.
This, of course, is contingent on optimization of delivery and minimization of off-
target effect which will need to be established through early clinical testing. The RNAi
has rapidly been established as an experimental tool and is expected to be used as
a therapeutic treatment for various diseases beside cancer. Besides siRNA, shRNA-
expressing pDNA is also a promising candidate for RNAi-based therapeutic treatment.
As shRNA-expressing pDNA and siRNA possess advantages and disadvantages, they
should be chosen on a case-by-case basis. There are still difficulties in the success-
ful therapeutic application of RNAi. However, considering the pace of new findings
and developments in the application of RNAi, we believe that these problems will be
solved and that RNAi will become a major therapeutic treatment in the near future.
The RNA delivery for RNAi-mediated gene silencing is a comparatively new area
of CPP-mediated molecular delivery. Endosomal entrapment of RNAs delivered by

CPPs is a problem that must be overcome for practical CPP-based cellular RNA deliv-
ery. A few groups have addressed this problem by using endosomolytic peptides and
reagents, as well as photoinduced endosomal escape strategies. In addition, strategies
targeting the CPP-cargo complex to a specific organ, to cancer, or to virus-infected
cells, will be necessary for therapeutic applications. Recently, there has been a great
advance toward therapeutic applications, in particular through the use of non-covalent
strategies to form cargo-CPP complexes. The photoinduced RNAi strategy may also
be useful as a targeting strategy for therapeutic applications. As RNAi is one of the
most promising strategies for gene therapy, further advances in CPP-based RNA de-
livery with nanocarriers are expected in the near future.
KEYWORDS
•• Angiogenesis
•• Angiostatin
•• Apoptosis
•• Bioactive glass
•• Integrins
•• Micro ribo nucleic acids
ACKNOWLEDGMENT
This work was supported by Department of Innovation Industry Science, Research,
and Commonwealth of Australia (BF030016). We are also gratefully acknowledging
the financial support from Centre for Biotechnology and Interdisciplinary Sciences
(BioDeakin), Institute for Technology Research and Innovation (ITRI), Deakin
University.
Chapter 2
In vivo Spectroscopy for Detection and
Treatment of GBM with NPt Implantation
José de la Rosa, Diego Adrián Fabila, Luis Felipe Hernández,
Edgard Moreno, Suren Stolik, Gabriela de la Rosa, Mayra Álvarez,
Alfonso Arellano, Tessy López, R. Mercado, J. L. Soto, L. Arredondo,
J. Bustos, A. Mosqueda, and I. Rivero
INTRODUCTION
Nowadays, cancer is one of the main human causes of death worldwide. In 2007, 7.9
million deaths were due to this disease. The World Health Organization (WHO) esti-
mates that 84 million people will die in the 10 year period from 2005 to 2015 due to
cancer (IARC, 2008). In Mexico, from 1922 to 2001, the increase in the cancer-related
grew from 0.6 to 13.1% of all the deaths occurring in the overall population (Kuri et al.,
2010). Cancer types vary from one country to another, and specialists estimate that if
current tendencies are maintained, the rate of incidence will rise all over the world.
Since the incidence of most cancers increases with age, these Figures are going to rise
if life expectancy continues to increase.
BRAIN TUMORS
Brain tumors are graded on the basis of their histological characteristics from I to IV;
this Figure which provides an approximate prognostic guide. Grade I grows slowly. It
is circumscribed or encapsulated. Histologically, the tumor cells are well-differentiat-
ed, resembling the origin or native cell. For this category, mitosis is absent or very rare
and blood vessels are scanty and normal. In a remarkable contrast, a Grade IV tumor
grows fast, is highly invasive, destroys the local tissue, and is found surrounded by
edema. In this category, the tumor cells are anaplastic (undifferentiated), and pleomor-
phic (varied in shape, size, and pattern), and mitosis is common and often atypical. In
addition, the blood supply is rich with abnormal vessels, and hemorrhage and necrosis
are common.
Astrocytic constitute the largest group of intracranial tumors. Based on their histo-
logical features, the WHO distinguishes four grades of astrocytic tumors. Glioblastoma
multiformes (GBM) is the most malignant and fastest-growing glioma, and constitutes
50–60% of astrocytic tumors. It most commonly occurs in mid- and late-midlife, with
an average clinical course from 12 to 18 months. This tumor is commonly situated in
the hemispheres, preferentially in the lobes (frontal and temporal) and appears circum-
scribed, though borders are ill-defined.
For GBM-affected tissues, cut surfaces have a multicolored appearance; they con-
tain pinkish-gray viable tissue, yellow necrosis, cystic degenerations, and rusty and
reddish areas associated with old and fresh hemorrhages, respectively. Since GBM is a
fast-growing tumor, extensive edema and mass effects are prominent.
Histologically, GBM is characterized by high cellularity, with a great degree of
anaplasia and pleomorphism. Some cells are small, round to ova or elongated, slen-
der and spindle-shaped. Others are large, with one or multiple hyperchromatic nuclei,
while giant cells are not uncommon. This tumor exhibits high mitotic activity and, a
rich vascular supply; the blood vessels display endothelial and adventitial prolifera-
tions and thrombotic occlusions. A glomeruloid appearance of the proliferated capillar-
ies is also typical. The GBM presents extensive necrotic regions. Smaller occurrences
are surrounded by tumor cells in a pseudopalisading pattern. When GBM reaches the
subarachnoid space or the ventricles, it may disseminate along the CSF pathways,
forming small nodules or diffuse infiltrates. It seldom metastasizes outside the nervous
system to the lymph nodes, lung, liver, and bone marrow (Haberland, 2007).
The GBM is a tumor that has a consistently heterogeneous pattern spreading over
the neighboring cerebral tissue. The solid component is an easily distinguishable mass
but the boundaries between tumor and cerebral tissue are difficult to differentiate be-
cause of the tumoral cells’ infiltration. This non-evident infiltration pattern is also of
great interest in low-grade gliomas because these other tumors have significant pro-
portion of this infiltrative component. Although metastatic or multicentric recurrences
could happen, in up to 90% of the cases tumors typically recur within 2 cm of the
tumor resection bed (Hochberg and Pruitt, 1980; Wallner et al., 1989).
While therapies for high-grade gliomas are helpful, existing treatments cannot
cure these tumors. The two major reasons for this are that tumor cells infiltrate into
surrounding brain tissues and thus cannot be completely removed by the surgeon, and
that most glioma cells are at least partially resistant to radiation and chemotherapy.
The conventional regimen of treatment consists of surgery, followed by chemotherapy
or radiotherapy. The main difficulty is the distinguishing of tumoral tissue and healthy
tissue under normal surgical conditions. During surgical removal of the tumor, func-
tional areas of the brain must be conserved, in order to protect vital functions of the
patient. Nevertheless, in many cases surgical removal is virtually impossible due to the
location of the tumor. No significant increase in survival has been observed in patients
that have received surgery plus chemotherapy/radiotherapy regimens intended to treat
GBM (Burger and Green, 1987).
Many attempts have been made in order to prolong life in patients with GBM. In
the cases where surgery has not been an option, combined coadjuvant chemo/radio-
therapy regimens have been tested in clinical trials with differing results in the treat-
ment of adults (Frappaz et al., 2003). Temozolomide and radiotherapy is perhaps the
most commonly used because Temozolomide is the most effective and well-tolerated
drug for GBM. A random, controlled prospective study showed that the benefit of
this regimen is very poor, with an increase of just 2.5 months of survival, compared
with a radiation only treatment (Stupp et al., 2005). Another trial proposed repeated
surgery and Temozolamide administration; in this study the median survival reached
In vivo Spectroscopy for Detection and Treatment of GBM 21
15.1 months, higher than the 9 months that had initially been estimated (Terasaki
et al., 2007).
Chemotherapeutic agents have several adverse side effects due to the impossibility
of being able to differentiate between healthy and tumoral tissue. Among those effects
are nephotoxicity, headache, vomiting, edema, and alopecia; all of them causing the
patient to lose quality of life. Research regarding this pathology has been focused on
the development of more effective and less toxic chemotherapeutic agents, as well as
on the design of new diagnostic techniques and surgical instruments intended to allow
neurosurgeons a higher degree of accuracy during neurosurgical procedures.
Nanotechnology has become a good alternative for the treatment of this GBM
tumor. López et al. (2008, 2010) have reported on the preparation of novel nanostruc-
tured biocatalysts with antitumor activity. Catalytic nanomedicine has shown that it
is possible to use a nanosized inorganic biocompatible catalyst inside tumors to reach
significant shrinkage (López et al., 2008, 2010). The main advantage of these novel
biocatalysts is the lack of adverse side effects, because nanoparticles are selective and
also because they are locally infiltrated.
Optical biopsy refers to the detection of a cancerous state in a tissue using, optical
methods. This is a new area, offering the potential to use noninvasive or minimally
invasive in vivo optical spectroscopic methods to identify a cancer at its various early
stages and to monitor its progression. The basic principle utilized for the method of
optical biopsy is that absorbance, reflectance, and fluorescence emission are strongly
influenced by the composition and cellular structure of tissues. The change in tissue
from normal state to cancerous state has been shown to alter those properties.
Both auto fluorescence spectroscopy at UVA-Blue excitation (320, 337.1, 360,
366, 370, 405, 410, 440, 470, and 490 nm) and visible emission (400–700 nm) have
been studied for the purpose of demarcating brain tumors (Butte et al., 2005; Chung
et al., 1997; Croce et al., 2003; Lin et al., 2001; Marcu et al., 2004; Saraswathy et al.,
2009 ). After the emission and absorption spectra of biological material in the brain,
(see Figure 1 in (Wagnieres et al., 2003)), NAD(P)H (located principally in the mito-
chondria and throughout the cytosol), FAD (located in the mitochondria), and porphy-
rin (located in the blood) could be considered the main fluorophores responsible for
healthy brain tissue autofluorescence emission at 365 nm excitation (Policard, 1924).
In Table 1, the absorptivity and quantum yield of these components are summarized.
Table 1. Molar absorptivity (e) and fluorescence quantum yield (Q) of principal fluorophores in brain
tissue at 365 nm.
ε
Q
Lmol-1cm-1
NAD(P)H 6.3 x 103 (DaCosta et al., 2003) 0.019 (Scott et al., 1970)
FAD 9.6 x 103 (Ball, 1998) 0.03 (DaCosta et al., 2003)
Porphyrin 1.71 x 104 (Rimington, 1960) 0.1 (Ricchelli, 1995)
Fluorescence measurements excited at 366 nm in GBM have shown that the main
responsible fluorophore for brain autofluorescence emission is the NAD(P)H (Croce
et al., 2003). The measurement of in vivo continuous and time-resolved fluorescence,

requires high intensity levels of light and high-sensitivity detectors due to the poor
fluorescence characteristics of the NAD(P)H and FAD. The reported experiments used
high pressure Hg lamps and spectrographs with OMA systems (Croce et al., 2003),
and N2 lasers with PMT’s (Butte et al., 2005). In the case of time-resolved spectros-
copy high speed electronics are necessary (Butte et al., 2005). Fluorescence in the
red spectral region (>600 nm), which was noted in tumors as early as 1924 (Katz and
Alfano, 1996), was originally attributed to bacteria, but is now thought to be secondary
to endogenous porphyrins. Lin et al. (2001) reported that measurements of UV fluo-
rescence spectroscopy and optical diffuse reflectance from 400 to 800 nm, in a pilot
clinical trial consisting of 26 brain tumor patients, gave a sensitivity and specificity of
100 and 76%, respectively.
Usually, in fluorescence measurements with a fiber optic, the reflected excitation
light is filtered out in order to have a better sensitivity in the fluorescence interval. In
this work, we have reported the spectroscopic reflected scattering and fluorescence
from GBM tumors, captured in the same signal. A portable fluorescence system, based
on a recently-introduced high-power commercial UV-LED, a mini-spectrometer, and
a bifurcated fiber optic was used. The measurements were obtained under continuous
wave excitation at 365 nm. In agreement with previous studies (Lin et al., 2001), it
was observed that the fluorescence intensity is lower in the cancerous tissue than in
the cortex and white matter. Likewise, it was observed that the reflectance is higher
in healthy tissue than at tumor borders, and that it almost disappears in the kern of
the tumor. In order to complement these observations, biopsies were taken for further
histopathological analysis.
EXPERIMENTAL
Nanoparticles
Nanostructured biocatalysts used in the tumors were prepared as previously reported
(López et al., 2008, 2010). The powders were lightly pressed onto a sterile amalgam
setter in such a way that, once inside the tumor bed, the cylindrical device made of
nanoparticles crumbles when it comes into contact with the brain mass and CSF.
Histology
In order to compare the observed fluorescent behavior with pathologic diagnostics,
biopsies of different tumor regions were taken after the in situ fluorescence measure-
ments. The samples were fixed in paraffin wax and sections were stained by conven-
tional Hematoxyline-Eosine (H-E) and Masson methods for microscopic study. The
H-E is the most commonly used microscopical light stain in histology and histopa-
thology. The staining method involves application of a basic dye hematoxylin dye,
which colors basophilic structures with a bluish-purple hue, and alcohol-based acidic
eosin, which colors eosinophilic structures a bright pink. The basophilic structures are
usually the ones containing nucleic acids, such as ribosomes and chromatin-rich cell
nuclei, and the cytoplasmic regions rich in RNA. Masson is a trichromic stain. The
trichromic staining allows selective visualization of muscular fibers, collagen fibers,
fibrin, and erythrocytes by employing three different coloring solutions. The selection
of the employed colorants, which are differentiated by their molecular size, produces
a distinguishable coloration of the different tissue compounds. In this study, H-E and
Masson techniques were used to visualize any morphological change in both normal
and tumor tissue, in order to observe the nanoparticle effect.
Spectrofluorometer
Figure 2 shows the experimental arrangement used to measure the steady-state fluo-
rescence spectra from the brain. The system consists of an excitation light source with
variable radiation power from 2 to 200 mW, a bifurcated optical fiber probe for de-
livery (six 200 µm channels) and collection (one 200 µm central channel) of light, a
mini spectrometer, and a laptop computer. The excitation light is directly coupled to
the bifurcated fiber-optic probe (R200-UV/VIS manufactured by Ocean Optics) with
an efficiency of 18 (± 0.2)%. After sample excitation, part of the emitted fluorescence
light is captured by the central channel of the bifurcated fiber-optic probe and guided
into the entrance slit of a HR4000CG-UV-NIR or USB4000 spectrometer manufac-
tured by Ocean Optics.
Figure 1. Experimental setup.
The electrical output from the spectrometer is sent, via a USB port to laptop for
its analysis. For this study, we used a 200 mW light emitting diode (NCSU033A LED
manufactured by Nichia Co.), which shows a peak emission in the UV-A at 365 nm,
a band width of 9 nm, and a radiation angle of 100º. The LED is powered by a DC
current supply regulated from 10 to 500 mA in order to produce a variable radiation

power from 2 to 200 mW. The radiation power emitted by the LED is displayed on
an LCD (2X16). The emitted power was calibrated with a UV light meter YK-34UV
from Digital Instruments. A 12 V regulated DC power supply was used, which could
be replaced by a battery to have a completely portable spectrofluorometer. The system
is controlled from the laptop by a program written in the graphical language “G” from
LabVIEW, which generates and processes the spectra in real time.
DISCUSSION AND RESULTS

As it was mentioned above, the first therapeutic option for brain tumors is surgery; cra-
niotomy being considered one of the most common surgeries for treating these tumors.
This procedure consists of the surgical removal of a section of bone (bone flap) from
the skull, allowing neurosurgeons access to the brain for tumor removal and surgical
repair. Preoperative MRI, CT, or arteriogram imaging studies is required to identify
the most appropriate site for the procedure (see Figure 2). The head is clamped in place
to avoid any movement during surgery, and the neurosurgeon shaves and marks the
scalp where flap will be cut to expose the skull bone. Several small, interconnected
burr holes are drilled into the skull. A craniotome is used to cut from one hole to the
next to create a removable bone section. The section is then removed, forming a win-
dow that allows access to the tumor. Through that window, neurosurgeons can directly
access and remove the tumor using microsurgical techniques. The bone section is re-
placed after surgery, and the scalp muscle and skin are stitched over the replaced bone.
Although a craniotomy is considered a common procedure, it has some risks like such
as infection, bleeding, seizures and added neurological deficits caused by the tumor re-
moval. During tumor excision many vessels are cut: thus it is important for the surgeon
to avoid hemorrhages. To prevent them, vessels are cauterized (by radiofrequency or
laser) with an electrocautery as the tumor is being removed, sealing the blood vessels.
Figure 2. The RMI of two different patients before surgery. By this technique infiltrated malignant
cells are not visible.
Optical spectra emissions of non-neoplastic and neoplastic tissue were taken

during surgery on patients with GBM that were under clinical trial with NPt (46/09
INNN). The tested regions were the GBM solid portion; the cerebral border infiltrated
by the tumor and apparently healthy brain tissue. Biopsies from pathological tissue
were taken for the purpose of microscopy correlation after each spectral reading. Fol-
lowing Croce et al. (2003), before each measurement, the probe tip was washed with
a saline solution and the investigated site was also rinsed with this solution to remove
blood accumulation at the tissue surface. During measurements, the fiber probe tip was
kept perpendicularly in contact with the tissue surface, without any pressure being ex-
erted, by the neurosurgeon (see Figure 3). The room lights were temporarily turned off
during spectral acquisition. The irradiation power was varied between 6 and 15 mW.
Figure 3. Results of the in vivo fluorescence measurements. The inset shows a portion of extracted
tumor. Note the morphological difference between healthy brain tissue and tumor.
In a patient with a widespread GBM, infiltrating almost half of the brain (see
Figure 4), the neurosurgeon selected three different regions to place the probe: the
cerebral cortex, white matter that was considered healthy tissue, and the tumor region.
The results of the fluorescence and reflection measurements are shown in Figure 5.
Figure 4. Wide-spreading high grade GBM. The cavity size is approximately 7 cm (right image).
(a)
(b)
Figure 5. (a) Brain tissue fluorescence and (b) Diffuse reflectance. In this case, the GBM tumor was
infiltrating a large part of the brain.
Figure 5(a) shows that a fluorescence spectrum in the range of 400–550 nm with a
maximum emission around 490 nm is produced by the cerebral cortex and white mat-
ter. This result can be attributed to the effect of NAD(P)H and FAD. White matter also
shows fluorescence in the range of 600–750 nm; this is principally due to the presence
of porphyrins. Additionally a weaker fluorescence signal was detected in the tumor
region. In Figure 5(b), the excitation light reflection in both the cerebral cortex and in
the white matter was stronger than in the tumor region.
In a well located GBM that reaches the most external part of the brain (see Figure 6)
the excitation light reflection in normal cortex tissue is also stronger than in the tumor
region (see Figure 7). As the measurement region approach the GBM kern, the excita-
tion light decreases reflection until it almost disappears in solid the portion of the tumor.
Figure 6. Typical example of a well located GBM.
Figure 7. Diffuse reflectance spectra in a patient with a well located tumor reaching the most external
part of the brain.
HISTOPATHOLOGY
In Figure 8(a) (40x), a control region of the tumor that corresponds to the surface
temporal region is shown. Here coagulative necrosis was observed, with a “ghostly”
appearance of the cells. In Figure 8(b), we can see the presence of nanoparticle ag-
gregates (in the middle of the picture). These particles divided the tumor into two de-
limited zones; in the left zone viable tumors cells can be clearly observed, whereas on
the right side some differences can be noticed. At higher amplification (Figure 8(c)),
supporting tissue damage is evident and the necrotic process observed in herein liq-
uefactive. This is a type of necrosis which is characteristic of focal bacterial or fungal
infections, in which the affected cell is completely digested by hydrolytic enzymes.
However, in the case of the brain, a hypoxic death of cells within the central nervous
system also results in liquefactive necrosis. This is a process in which lysosomes turn
tissues into a soup, as a result of the lysosomal release of digestive enzymes in the
bacterial-onslaught phase. Loss of tissue architecture means that the tissue is essen-
tially liquefied. Figure 8(d) is a close-up of the right side of Figure 8(c) revealing the
presence of macrophages as well as neutrophiles (basophils). Masson stain (see Figure
8(e) and Figure 8(f)) was used in order to complement the obtained information from
H-E. With this technique, we confirm the damage in supporting tissue as the soft-pink
colored zones indicate.
Figure 8. The H-E images of (a) necrotic tissue from the temporal region (40x), (b) the temporal region
where nanoparticles were administrated (40x), (c) 100x, (d) right side of c (100x); (d), and (e) Masson
stained sections at 40x and 100x, respectively.
The graded anaplasia of the glioma group reaches its extreme in GBM. Most au-
thors consider this lesion to be a neoplasm of astrocytes because (1) the glioblastoma
merges as a clinical entity with the two better-differentiated astrocytomas and anaplas-
tic astrocytomas, (2) pathologically, the gioblastoma sometimes evolves out of a better
differentiated astrocytic tumor, and (3) it often contains neoplastic astrocytes.
One of the main features observed, after NPt treatment, is related to the type of
necrosis. As a result of the tumor growth, commonly the growth is so fast that in some
cases cells die and some necrosis can be observed. However, this common necrosis is
coagulative. In the region where nanoparticle aggregate can be clearly detected, a liq-
uefactive necrosis area appears which can be associated with the action of the NPt. It
is important to note that in this case neither inflammatory cells nor an abscess appears.
These features are common to this type of process. Moreover, the damage caused to
the malignant tissue (as the slimming of the support tissue shows), provides evidence
that the NPt which were previously put inside the tumor, are acting against malignant
cells inducing cell death.
With regard to fluorescence measurements, it was observed that even healthy
white matter has a high absorption coefficient in the range from 300 to 600 nm; as a
result that can be attributed to the porphyrin content in the blood (Roggan et al., 1995).
The high vascularization in GBM tumors could be the reason for low reflection mea-
surements related to the higher absorption in this tissue. So, reflection measurements
can be used to demarcate the GBM border tumors. As the reflection measurements are
more sensitive than those of the fluorescence, they can be used as a better criterion
for finding tumors and cancer infiltrated tissue in the brain. The observed reflectances
for the different brain areas could be an excellent tool for the in situ infiltration of
nanoparticles, improving the odds of the survival after the surgical resectioning of
malignant and infiltrative tumors like GBM.
CONCLUSION
In the present study, in vivo measurements of the reflectance and fluorescence of very-
well- demarcated Glioblastoma tumors at 365 nm UV-light have been presented. As
the measurement region approaches a non-neoplastic zone to the plain tumor, the re-
flectance decreases until it completely vanishes in the tumor. The measurements of
the reflectance at 365 nm could be used to demarcate the resection or nanoparticle-
infiltration zone during a surgical procedure.
The use of nanotechnology for the treatment of malignant tumors is an emerging
research field that may offer more effective treatments that will increase patients’ life
expectancy beyond today’s records. Moreover, the use of tools for in situ demarca-
tion of highly invasive tumors could open new possibilities of more precise surgical
procedures for tumor excision, and, at the same time, allow the precise administration
of local antitumor agents like NPt. The simultaneous use of both detection and treat-
ment tools will enhance the performance and results of surgical procedures, reducing
collateral side effects.
KEYWORDS
•• Brain tumors
•• Fiber-optic probe
•• Glioblastoma multiformes
•• Masson
•• World health organization
ACKNOWLEDGMENT
We acknowledge the FONCICyT-CONACyT 96095 project for their financial sup-
port. Also, the authors are grateful to UAM, H. C. Guadalajara, and INNN for the use
of their facilities.
Chapter 3
Nanobiotechnology for Antibacterial Therapy
and Diagnosis
Jagat R. Kanwar, Kislay Roy, and Rupinder K. Kanwar
INTRODUCTION
The development of structures and devices on a nanometer scale and their application
in several fields is referred to as nanotechnology. One of these fields is medicine, bet-
ter known as nanomedicine. Nanomedicines have many descriptions ranging from,
“the use of materials”, of which at least one of their dimensions that affects their
function is in the scale range 1–100 nm, for a specific diagnostic or therapeutic pur-
pose (Kostarelos, 2006) through to “the science and technology of diagnosing, treating
and preventing disease and traumatic injury, of relieving pain, and of preserving and
improving human health, using molecular tools and molecular knowledge of the hu-
man body” (Hermerén et al., 2007) as described by the European Science Foundation
(ESF).
Due to a constant exposure to any pathogen there is need for an efficient antibacte-
rial therapy. Most of the prevalent methods have proven to be ordinary by the increas-
ing population of multi-drug resistant bacteria (Zampa et al., 2009). Study done by
Matthews et al. (2010) explains the inefficiency of current bacterial diagnostics and
discovery of new and innovative therapeutics and diagnostics. This study highlighted
implementation of nanomaterials and nanotechnology for antibacterial medical thera-
peutics and diagnostics. Introduction of nanomedicine in field of antibacterial thera-
peutics has brought a revelation and the unmet medical need for new treatments has
motivated the drug industry to search for new antimicrobial agents. Single stranded
DNA or RNA known as aptamers have found their way into specifically targeting
microbes (Kanwar et al., 2010). Aptamers have been selected using systematic evolu-
tion of ligands by exponential enrichment (SELEX) specifically for microbes such as
Bacillus anthracis (Sterne strain) to diagnose and target them (Bruno and Kiel, 1999).
This method is much specific when compared to any other technology. The review
discusses some of these pioneering studies in which nanomedicines have been or are
undergoing evaluation as potential therapeutic and diagnostic applications (Yacoby
and Benhar, 2008). This review chapter will be arranged into two main categories;
antibacterial therapeutics and antibacterial therapeutics with diagnostic potential, of
these two main categories there will be sub-categories containing each application
reviewed.
ANTIBACTERIAL THERAPEUTICS
Carbon Nanotubes and Fullerenes
Carbon nanotubes (CNTs) and fullerenes are the most commonly approached methods
in nanotechnology. Yacoby and Benhar (2008) describe an investigation done by Kang
et al. (2007) consisting the interaction of well-characterized, low metal content, nar-
rowly distributed, pristine single-walled nanotubes (SWNTs) with a model bacterium
(Kang et al., 2007). This study claimed to provide the first direct evidence that highly
purified SWNTs can exhibit strong antimicrobial activity. Another carbon-based nano-
moiety evaluated was the fullerene C60. Yacoby and Benhar (2008) explained how
Lyon et al. (2005) investigated the interactions between nano-C60 and two common
laboratory bacteria; Escherichia coli, gram-negative bacterium and, Bacillus subtilis,
a gram-positive bacterium. The results of the tests performed showed both gram-neg-
ative and gram-positive bacteria were affected by nano-C60, with the minimal inhibi-
tory concentrations (MICs) and minimal bactericidal concentrations (MBCs) (Lyon
et al., 2005). The authors proposed three possible hypotheses for the mechanism of
toxicity: the first was that nano-C60 disrupts electron transport, second was that nano-
C60 punctures bacterial membranes and the third being nano-C60 produces radical-
oxygen species that are toxic (Yacoby and Benhar, 2008). Emerging technology has
brought a revelation in the modern trends in nanomedicines. Multiwalled carbon nano-
tubes (MWCNTs) functionalized with covalently bonded lysozyme were prepared and
characterized by thermogravimetery, Raman spectroscopy, transmission electron mi-
croscopy (TEM) and cyclic voltametry (Table 1). It was estimated that per 4000 C
atoms there is binding of 1 lysozyme residue. The MWCNT-lysozyme nanocomposite
showed higher antibacterial activity in the gram positive S. aureus when compared to
the free lysozyme (Merli et al., 2011) (Figure 1). Although this technology has been
refined well and has many applications more research is required to determine its fu-
ture safety and applications.
Table 1. Various antibacterial therapeutics and their mechanism of action.

Antibacterial Examples Developed Mechanism of Reference
therapeutics Against action
carbon nanotubes single walled nano- bacterial model Punctures cell (Kang 2007)
tubes (SWNT) membrane, produce
toxic free radicals,
disruption of electron
transport chain.
fullerenes fullerene C60 E. coli, (Lyon et al. 2005)
B. subtilis
Multi-walled carbon S.aureus hydrolytic action (Merli et al 2011)
nanotubes
(MWCNT)-lysozyme
Bioactive glasses SiO2-Na2O-CaO- E. coli, created high PH in (Yacoby and
P2O5 P. aeruginosa, surrounding Benhar 2008)
S. aureus
Nanobiotechnology for Antibacterial Therapy and Diagnosis 33
Table 1. (Continued)
Antibacterial Examples Developed Mechanism of Reference

therapeutics Against action
Biopolymers quaternised chitosan wide range chelating transition (Ignatova et al.
(QCh) metals and inhibiting 2006a, 2006b)
enzymes
poly vinyl alcohol wide range - (Ignatova et al.
(PVA) 2006a, 2006b)
Fish scale collagen S. aureus, disrupts cell mem- (Wang et al. 2011)
peptides (FSCP)/ E. coli brane
chito oligosaccha-
rides (COS)
Bacteriophages as chloramphenicol- broad range protein synthesis (Yacoby et al.
drug carriers phage-target specific antibiotic inhibitor 2007)
peptides-IgG
aminoglycoside broad range cell membrane (Yacoby 2006)
antibodies-phage- disruption
solubility enhancing
peptides
Nanoemulsions NB-401(oil in water) Burkholderia - (LiPuma et al.
cepacia 2009)
Aptamers DNA aptamer Bacillus an- targets bacterial (Kanwar et al.
thracis (sterne spores 2010)
strain)
Figure 1. The Figure 1 shows antibacterial activity of lysozyme loaded Multiwalled carbon nanotubes
(MWCNT). The nanotubes localize inside the bacteria S.aureus and release the lysozyme which due
to its hydrolytic action destroys the bacteria (Merli et al. 2011).
Bioactive Glasses
The antimicrobial activity of bioactive glasses (SiO2-Na2O-CaO-P2O5 systems) has
been established. Once suspended in aqueous solutions they release their ionic com-
pounds over time which is the cause of the antimicrobial activity. Bioactive glasses
show some promise as dentin disinfectants; however, the antibacterial effectiveness
of calcium hydroxide in human teeth is much superior to that of the bioactive glasses.
Yacoby and Benhar (2008) describe previous attempts to spike bioactive glass with
silver to increase its antimicrobial efficacy using E. coli, Pseudomonas aeruginosa
and Staphylococcus aureus as test microorganisms. Tests showed that concentrations
of silver oxide (Ag2O) bioactive glass (in the range of 0.05 to 0.20 mg/ml) of cultured
medium inhibited the growth of these bacteria. The release of Na+ and Ca2+ ions from,
and the incorporation of H3O+ protons into the corroding bioactive glasses results in
a high pH environment in closed systems (Sepulveda et al., 2002), which is not well
tolerated by micro biota (Allan et al., 2001). The author explained that the introduction
of nanometer scale particles led to more than a 10-fold increase in specific surface area
and, consequently a stronger antimicrobial effect than the currently applied micron-
sized material (Waltimo et al., 2007; Yacoby and Benhar, 2008).
Biopolymers
Nanoparticles of biological origin also exhibit antibacterial activity either in their nat-
ural state or once they are modified. The first example involves an additional applica-
tion of electrospun nanofiber mats spun from polysaccharide chitosan. Polymers with
intrinsic bacteriostatic and bactericidal activity and in particular, polysaccharides that
are considered as promising for wound-healing and wound-dressing applications were
reported. The natural polysaccharide chitosan was reported to possess several biologi-
cal properties, such as hemostatic activity, non-toxicity, biodegradability, intrinsic an-
tibacterial properties and the ability to affect macrophage function which contributes
to a faster wound healing process (Balakrishnan et al., 2005; Muzzarelli et al., 2005).
Yacoby and Benhar (2008) covers a study performed by Ignatova et al. (2006a) on the
antibacterial properties of NPs that were fabricated from quaternised chitosan (QCh)
and poly (vinyl alcohol) (PVA). The QCh derivatives illustrated a higher activity
against bacteria, broader spectrum of activity and higher killing rate when compared
with those of chitosan. The preparation of QCh-containing nanofibers was carried out
by electrospinning of mixed QCh/PVA aqueous solutions and the antibacterial proper-
ties of photo-cross-linked electrospun QCh/PVA mats were studied. It was found that
the antibacterial activity of photo cross-linked electrospun QCh/PVA mats was found
to have a “reduction of bacterial growth by 98% (after 120 min of exposure)” (Yacoby
and Benhar, 2008). In a recent study, nanofibrous membranes of 50–100 nm were
formed using 2:1 ratio of low molecular weight fish scale collagen peptides (FSCP)/
chito oligosaccharides (COS) which had antibacterial activity and were designed to be
used for wound dressing. These nanofibres showed good antibacterial activity against
gram positive S. aureus and gram negative E. coli. Sensitivity for S. aureus was found
to be much higher when compared to E. coli. The antibacterial activity of the nanofiber
was due to its ability to disrupt the cell membranes of the bacterias. However when
tested on human cells, the fibers proved biocompatible and supported the proliferation
of human skin fibroblast cells. Such studies mark the further advance of biopolymers
in the medicinal field (Wang et al., 2011).
Bacteriophage Drug-carrying Platforms

Filamentous bacteriophages (phages) were applied as targeted drug-carrying platforms
aiming to eradicate the pathogenic bacteria and other cells which carry any infection
or disease (Yacoby and Benhar, 2008). Authors describe phage NPs as nanoneedles
with a diameter of ~8 nm, capable of delivering a large dose of a cytotoxic drug to
the target cells. In order to make the procedure successful against pathogenic bacteria
a drug was linked to the genetically modified phage by means of chemical conjuga-
tion through an esterase-cleavable linker subject designed to control release by serum
esterases. The specificity of the drug-carrying phages to target cells was enhanced by
genetic expression of a targeting moiety on the phage coat. This approach may re-
enable the use of drugs that are not in use, owing to their toxicity or low specificity,
and have been excluded from clinical use (Yacoby and Benhar, 2008). The capability
of this approach was demonstrated using chloramphenicol (as it is rarely used to treat
patients systematically due to its toxicity) (Yacoby and Benhar, 2008; Yacoby et al.,
2007). The nanoparticle targeting was accomplished by using two different targeting
moieties: target-specific peptides and antibodies linked to the phage via an immuno-
globulin G (IgG) Fc-binding ZZ domain. The process had drawbacks such as restricted
ability to inhibit bacterial growth due to the limited loading capacity of less than 3,000
drug molecules per phage (Yacoby et al., 2007). To overcome these limitations, a
modified system was developed based on the application of hydrophilic aminoglyco-
side antibiotics as branched, solubility-enhancing linkers using unique drug conjuga-
tion chemistry (Yacoby, 2006); replacing the loading methodology and modifying the
antibody-phage conjugation method, which improved the system for targeting a broad
range of pathogenic bacteria. The new drug-conjugation approach led to an arming
rate of over 40,000 chloramphenicol molecules/phage (Yacoby and Benhar, 2008).
The results obtained were from within an artificial closed system and did not reflect an
in vivo application (Yacoby et al., 2007), although this did offer great possibilities for
the future of in vivo treatments. It was concluded that the drug-carrying phages repre-
sent a therapeutic NPs with wide range of applications that, may become an important
general targeting drug-delivering platform “owing to the chosen coating, by the sim-
plicity of which it can be equipped with a targeting moiety, and massive drug-carrying
capacity”, (Yacoby and Benhar, 2008).
ANTIBACTERIAL NANOEMULSION
Researchers at the University of Michigan (USA), have developed a nanoemulsion with
an immaculate antibacterial activity against ~99% of the resistant bacteria that com-
monly cause respiratory tract infections in patients suffering with cystic fibrosis (CF).
The primary cause of death in persons with cystic fibrosis is respiratory tract infection
involving opportunistic bacteria having broad-spectrum antibiotic resistance (LiPuma
et al., 2009) and treating these patients is made more complicated by the thick sputum
present within the lungs which is caused by this disease. These infections are caused by
bacterial strains highly resistant to common antibacterial agents like the Burkholderia
cepacia complex. A topically administered surfactant-stabilized (oil-in-water) nano-

emulsion, named NB-401 was developed. When this nanoemulsion was tested on 150
bacterial strains, all but two showed sufficient bactericidal activity; irrespective of their
resistance, the growth of all strains was inhibited by NB-401. No decrease in the level
of activity was observed against multi-drug or panresistant strains as well and NB-401
also proved to be effective against planktonic strains growing in human sputum or
against strains that grew in biofilm. The nanoemulsion also overcame difficulties in the
administration of systemic antibiotics. This topical or inhaled (nebulizer) therapy can
achieve high level concentrations of the drug that could never be achieved with an oral
systemic drug because of the systemic toxicity (LiPuma et al., 2009).
Antibacterial Therapeutics with Diagnostic Potential-pluronic Block

Copolymers as Micellar Nanocarriers
This study investigated the specific arrangement of polymeric molecules at the na-
noscale. They enhance the chances for safe and efficient delivery of various drugs,
several biomolecules and genes and can be used for imaging and biosensing as well.
The diameter of Pluronic micelles normally ranges from 10–100 nm (Kabanov et al.,
1995). The core of the micelles consists of hydrophobic poly (propylene oxide) blocks
that are separated from the aqueous exterior by the shell hydrated of hydrophilic poly
(ethylene oxide) chains. The core represents the carrier ship for the incorporation of
various therapeutics and diagnostic reagents (Batrakova and Kabanov, 2008). Pluronic
block polymers with respects to various antibacterial and antifungal drugs showed en-
hanced bioactivity against many microorganisms (Croy and Kwan 2004, 2005; Saski
and Shah, 1965a, 1965b). Much work in this field is still under clinical evaluation and
several fascinating developments in this area are expected in the coming years (Batra-
kova and Kabanov, 2008).
Multifunctional Nanoplatforms
It is very difficult to diagnose and treat a biofilm infection as most of the infections
are resistant to the prevalent treatments and the infectious pathogens stay dormant for
quite some time and infect sporadically producing the symptoms of infections (Costerton
et al., 1999). Multifunctional nanoplatforms such as protein cage architectures can
utilize the infection-specific magnetic resonance imaging (MRI) markers that can be
used to identify and characterize the centers of biofilm infections. It has been estab-
lished that both therapeutics (Flenniken et al., 2005) and imaging agents (Allen et al.,
2005; Flenniken et al., 2006) have been delivered efficiently by protein cages. It is
however important that the size of such nanostructures be in such a range that it can
efficiently pass through the biofilm barriers and yet deliver a sufficient amount of the
desired drug (Suci et al., 2007). More research has to be done to determine the immune
system response and the mechanism of action of these nanostructures before they can
be put to use clinically (Zhang and Falla, 2006).
Dermaseptin 01 Antimicrobial Peptides

A new family of antibiotics namely antimicrobial peptides (AMPs) was investigated
by Zampa et al. for clinical applications (Zampa et al., 2009). Dermaseptins (DSs) is a
member of AMP family isolated from the secretion of frog skin (Phyllomedusa genus)
(Brand et al., 2006; Leite et al., 2008). It consists of cationic molecules 24–34 amino
acids long that can fold into amphiphilic helices when in contact with hydrophobic
media. The DS polypeptide chains are “gene encoded as part of larger precursor mol-
ecule compromising a signal peptide of 22 residues, followed by an acidic pro-pep-
tide, a typical pro-hormones processing signal, and a DS progenitor sequence”. Zampa
et al. (2009) from previous research works explained that DSs has cytolytic action
against numerous microorganisms and are considered as promising agents to fight vi-
ral diseases, drug-resistant bacteria, protozoa, yeast, and filamentous fungi. Adding to
its benefits these peptides do not cause significant cytolysis against mammalian blood
cells. The DS 01, collected from the skin of P. oreades and P. hypochondrialis frogs,
has demonstrated high antibacterial activity against gram-positive and gram-negative
bacteria (Brand et al., 2006). Due to their lack of stability in serum and plasma as
a big drawback the DS peptides are easily degraded by the proteolytic enzymes in
vivo but their efficiency is high in in vtiro. It is however observed that the Multi-
meric forms of the DS peptides are much stable in vivo when compared to the mono-
meric peptides (Pini et al., 2005; Tam et al., 2002). Immobilization of DS 01 within
a nanostructured (nickel tetrasulfonated phthalocyanine nanostructure, film-forming
electroactive molecule) thin film was used to serve as a biosensor. The results showed
that DS 01 appeared to be the most promising irreversible anti-parasitic peptides. The
growth inhibition in accordance with death of Leishmania chagasi, clearly shows the
Leishmanicidal activity of DS 01. Enhanced detection was demonstrated using cy-
clic voltammetry suing the AMP-containing films as electrodes. It was observed that
inclusion of slight modifications in the assay time and the signal to noise ration may
enhance the sensitivity and efficiency of this method. Both high sensitivity and speci-
ficity are reflected by enhanced pathogen detection (Zampa et al., 2009). Nanopar-
ticles are commonly used as delivery systems for various drugs. Most nanoparticles
have sustained drug delivery due to enhanced permeability retention (EPR) effect
and thus the drug can be released over a long period of time reducing the options of
multiple dosages. Various nanoparticle systems have been established as antibacterial
drug carriers such as silver nanoparticles (Sondi and Salopek-sondi, 2004), metal ox-
ide nanoparticles including magnesium oxide (Huang et al., 2005), zinc oxide (Jones
et al., 2008), silicon dioxide (Jia et al., 2008), chitosan nanofiber (Ignatova et al.,
2006b), poly-l-lactide nanoparticles (Salmaso et al., 2004), gold nanoparticles (Faulk
and Taylor, 1971), quantum dots (Edgar et al., 2006), magnetic nanoparticles (Faulk
and Taylor, 1971) and many more. Magnetic nanoparticles consisting of Fe3O4 core
and coated with antibacterial compound poly (quaternary ammonium) (PQA) have
been prepared and well characterized by various methods including Fourier transform
infrared (FTIR), thermogravity analysis (TGA), TEM. These nanoparticles showed
100% biocidal efficiency against E. coli (105 to 106 E.coli/mg nanoparticle) and after
use the nanoparticles could be collected again under influence of external magnetic
field (Dong et al., 2011) (Figure 2). Thus a wide range of work has been done in the
field of nanoparticles based delivery.
Figure 2. The magnetic nanoparticles (Fe₃O₄) loaded with antibacterial compound PQA are shown
in the Figure. These nanoparticles target the bacteria E.coli and kill them due to the antibacterial
activity of PQA. After the use the nanoparticles can be recollected under the influence of external
magnetic field. Thus these nanoparticles are referred to as the recyclable nanoparticles (Dong et al.
2011).
CONCLUSION
With new strains of multi-drug resistant bacteria and viruses always presenting them-
selves, the need for antimicrobial defenses is required on a daily basis. These studies
reviewed were aimed at informing the reader of new and modified antibacterial, ther-
apeutic and diagnostic methods that are being developed. With a lot of these potential
antibacterial nanomedicines still being researched, the possibilities are ever increas-
ing. The applications, such as bacteriophage drug-carrying platforms will soon shift
diagnosis to a pre-symptomatic stage and allow pre-emptive therapeutic measures,
or the treatment of high-resistant pathogens using multifunctional nanoplatforms as
a less invasive method. Pluronic block polymers as micellar nanocarriers and DS 01
antimicrobial peptides would improve diagnostic methods for bacterial infection, as
well as treating them. Introduction of aptamers as nanomedicines has changed the
scenario of drug development and effective targeting. But for these future technolo-
gies to succeed and be of use, they will need to be cost-effective with higher perfor-
mance in terms of resolution, sensitivity, specificity, reliability, reproducibility, and
integration.
KEYWORDS
•• Biopolymers
•• Carbon nanotubes
•• Cystic fibrosis
•• Dermaseptins
•• Diagnostic
•• Nanomedicines
•• Nanotechnology
•• Quaternised chitosan
ACKNOWLEDGMENT
The work was supported by the grants from Institute of Biotechnology, Institute for
Technology & Research Innovation and the Australia-India Strategic Research Fund
(AISRF) Department of Innovation Industry Science, Research, and Commonwealth
of Australia (BF030016), and BioDeakin, ITRI, Deakin University.
Chapter 4
Chitosan Nanoparticles
Divyen Shah, Vaishali Londhe, and Rima Shah
INTRODUCTION
Natural polymers are having advantages like biodegradability and biocompatibility,
which make them favorable for most of the drug delivery system (Schmidt, 2009).
Nanoparticle drug delivery system is used because of its high bioavailability, reduced
dosage and toxicity, targeted delivery, and so on. Chitosan (CS) is one of the com-
monly used naturally obtained polymers with various therapeutic uses. Chitosan
((1→4)-2-amino-2-deoxy-β-D-glucan), a linear polyamine with a high ratio of glu-
cosamine to acetyl-glucosamine units, is a natural mucoadhesive cationic polymer,
which is obtained by partial deacetylation of chitin (Banerjee et al., 2002). Chitosan
(pKa = 6.5) is solubilized in acidic medium (Chen, 2003). The primary amino groups
lead to special properties that render chitosan very interesting for pharmaceutical ap-
plications like development of controlled release drug delivery systems like chitosan
gels, tablets, capsules, microspheres, microcapsules, and nanoparticles for parenteral,
nasal, ophthalmic, transdermal, and implantable delivery of drugs, proteins, peptides,
and gene materials (Chen et al., 2007). The free amino functional group in chito-
san makes it possible to form nanoparticles by cross-linking, emulsion cross-linking,
spray drying, desolvation with cationic salts, ionic complexation/coacervation or ionic
gelation method by interacting with various other reactive groups such as alginates,
dextran sulphate, sodium tri poly phosphate (STPP), polyethylene glycol (PEG), dif-
ferent ligands, antibodies, DNA and pH sensitive moieties, and so on. (Kumar, 2000)
Chitosan can enhance the transmucosal absorption by increasing the paracellular per-
meability of intestinal epithelia. Chitosan nanoparticles are having good potential for
the ocular drug delivery system because of its mucoadhesive nature (Campos, 2001).
It has been extensively used in nasal drug and vaccine delivery (Csaba, 2008). Main
advantage of chitosan nanoparticle is it can be used for hydrophilic drug entrapment.
The hydrophilic nanoparticle remains in circulation for long time without PEGlyation,
by avoiding reticulo endothelial system (RES). The other advantage of using chitosan
nanoparticles is that they do not require any organic solvent or any other extreme con-
ditions mainly in ionotropic gelation or complex coacervation technique which results
in more stabilization of proteins and peptides.
Ionic gelation and complex coacervation are almost same except that, ionic gela-
tion uses electrolyte such as tri poly phosphate (TPP), whereas in complex coacerva-
tion oppositely charged ionic polymer such as alginate are used. Ionic gelation method
using TPP is more common for entrapment of hydrophilic drugs, proteins and plas-
mids. By ionotropic gelation method, we can obtain size from 100 to 1000 nm and zeta
Chitosan Nanoparticles 41
potential between +20 to +60 mv by varying the ratio of chitosan/STPP (Soppimath

et al., 2001). The structure of chitosan and STPP is given in Figure 1.
Figure 1. Structure of (a) Chitosan and (b) Sodium tri poly phosphate.
Size, surface charge, release characteristics and percentage drug entrapment ef-
ficiency of these chitosan/STPP nanoparticles can be modulated by (i) using different
molecular weight chitosan, (ii) by incorporating additional polymers such as polox-
amer, hyaluronic acid, cyclodextrin and so on. or (iii) by using chemically modified
chitosan derivatives, such as N-trimethyl chitosan (Csaba, 2008).
Figure 2 explains chitosan’s application as biomedical material along with per-
centage contribution in various fields (Issa, 2005).
Figure 2. Main application of chitosan as biomedical material (Issa, 2005).

METHODS OF CHITOSAN NANOPARTICLE PREPARATION

Chitosan-based nanoparticles can be prepared by following methods.
1. Ionic gelation
2. Covalent cross-links
3. Complex coacervation
4. Desolvation method
5. Emulsion-droplet coalescence method
6. Reverse micellar method
We will mainly discuss ionotropic gelation method in detail. Other methods are
discussed in brief.
Ionic Cross Linking (Ionic Gelation)

This method is the most common technique to prepare chitosan nanoparticles. In this
method, only aqueous phases are used and no organic solvent is used, and thus the
chances of degradation of peptides are decreased. Chitosan is dissolved in one phase,
and in other phase negative charge generating ployanion like STPP is dissolved. Both
the phases are mixed under magnetic stirring at room temperature which generates gel
like nanoparticles. The nanoparticles are formed due to intra and inter molecular link-
age between chitosan and STPP. The schematic representation of preparation is given
in Figure 3. The pH of both the phases can play important role on particle size and per-
centage entrapment efficiency. Generally the method does not require any stabilizer
or surfactants and the nanoparticles generated are stable in nature (Yang et al., 2009a).
Figure 3. Schematic representation of chitosan/STPP nanoparticle.
To obtain high yield of chitosan-TPP nanoparticles, the chitosan: TPP weight ratio
should be between 3:1 and 6:1. The nanoparticles can also be made by introduction
of other hydrophilic polymer. Even it may increase versatility for association and
delivery of proteins. In nanoparticles of chitosan and diblock copolymer of ethylene

oxide and propylene oxide, PEO-PPO is attached to surface of particle which was
confirmed by transmission electron microscopy (TEM). In general, protein loading
can be obtained as high as 50%. Highest loading efficiency can be obtained when pH
of solution is above isoelectric point of protein, so that it has negative charge. In ad-
dition to ionica crosslinking with TPP, other mechanisms may also work for associa-
tion of macromolecules, because insulin nanoparticles prepared at pH where insulin is
positively charged, showed 30% entrapment efficiency. These mechanisms could be
hydrogen bonding, hydrophobic interaction and other physicochemical forces. Even
the drug release profile of insulin varies with pH medium. Cross-linking induced by
incorporation of TPP will make the nanoparticles compact and resistant to freeze dry-
ing (Janes et al., 2001).
The cross-linking density, crystallinity, and hydrophilicity of cross-linked chito-
san can allow modulation of drug release. Even the swelling behavior of cross-linked
chitosan depends on pH of TPP. Cross-linked chitosan shows higher swelling ability
(Bhumkar, 2006).
There were some studies in which the effects of cationic surfactant like cetyltri-
methylammonium bromide were studied. The addition of cationic surfactant might
reduce the size, while increasing the zeta potential. The decreased size may improve
permeability through extracellular membrane. The increased surface charge density
may facilitate absorption efficiency and may prevent nanoparticles from enzymatic
degradation (Bao, 2008).
Covalent Cross-links
The process involves the precipitation of the polymer followed by chemical cross-
linking by oppositely charged polymer. Precipitation can be done by sodium sulfate
followed by chemical cross-linking using formaldehyde, glutaraldehyde or even using
a natural crosslinking agent such as genepin. Later on the solvent is removed by rotary
evaporator and dry nanoparticles are obtained. In this method, the drug is immobilized
on nanoparticles instead of encapsulation (Hamidi, 2008).
Complex Coacervation
The process is spontaneous phase separation which occurs upon mixing of oppositely
charged polyelectrolyte in aqueous medium. This method is almost similar to ionic
gelation method, but here opposite charge generating polymers like alginate or dextran
sulfate are used. The mechanical strength is comparatively higher than that of ionic
gelation method (Chen et al., 2007).
Desolvation Method
In this method, a more water soluble polymer or water miscible nonsolvent for chito-
san is added drop wise under stirring and then liquid particles are hardened by chemi-
cal cross-linking with glutaraldehyde. These nanoparticles can be prepared by either
o/w or w/o/w emulsion (Chakravarthi, 2007).
Emulsion-droplet Coalescence Method

As chitosan is having solubility in acidic medium (below its pKa= 6.5), it will pre-
cipitate when it will come in contact with other alkali medium. In this method, two
emulsions are prepared. In one emulsion, only chitosan is dissolved along with the
drug while in other emulsion, the chitosan is dissolved along with little quantity of
sodium hydroxide. Finally, both the emulsions are mixed at high speed resulting in
formation of small solid chitosan nanoparticles due to collision between the two emul-
sions (Agnihotri, 2004).
Reverse Micellar Method

In this method, chitosan and drug solutions are added to organic solvent having sur-
factant. The transparency of the solution should be maintained by adding water ad-
ditionally if required. Cross-linking agent is added to above system and is stirred con-
tinuously to evaporate the organic solvent to obtain transparent dry mass. Then, the
obtained transparent material is dispersed in water and then suitable salt is added to
precipitate out the surfactant which was separated by centrifugation. The obtained
supernatant is having drug loaded nanoparticles. By dialyzing the solution, free drug
is removed and the remaining solution is lyophilized (Patel, 2009).
APPLICATIONS OF CHITOSAN NANOPARTICLES

Chitosan nanoparticles are having tremendous application in various fields which are
as follows:
Molecular Imaging
Chitosan-based system such as microbubble, micelles and liposomal nanoparticles are
used in molecular imaging. The incorporation of hard and brittle calcium phosphate
or hydroxyapatite with chitosan yields a bioresorbable composite with favorable me-
chanical property for bone and cartilage tissue engineering. Particles with ferric oxide
can be used in MRI for hepatocyte targeting imaging. The chitosan coated superpara-
magnetic iron oxide nanocrystals (SPION) are used as MRI contrast agent, which can
have high labeling efficiency and high uptake efficiency by stem cells. Water soluble
chitosan-linoleic acid conjugate can be used as contrast agents to target hepatocytes.
Gadolinium-loaded chitosan nanoparticles displayed prolonged retention in tumor tis-
sue after in vivo intratumoral injection (Agrawal, 2010).
Protein Delivery
It can be done via oral route and via nasal route.
Via Oral Route

a. As vaccination
The n-trimethyl chitosan-TPP nanoparticles prepared by ionic gelation method for
protein delivery system by oral route have shown promising results. They have re-
duced the transepithelial electrical resistance (TEER) or have increased the paracel-
lular transport to the cells. They have shown good systemic immune response when
immunized with urease loaded nanoparticles (Chen et al., 2008).
The adsorption of protein on chitosan nanoparticles is giving high loading capac-
ity. Hydrophilic nanoparticle with negative surface charge (excess sodium alginate)
can be uptaken by Rat Peyer’s patches, which can be used as carrier of mucosal vac-
cination (Borges et al., 2006).
Using Bovine serum albumin, the loading efficiency can be obtained as high as
68%. As the deacetylation degree increases, the loading efficiency also increases but
the drug release rate decreases. As the molecular weight of chitosan was increased,
the loading efficiency was increased but the drug release rate was decreased. The PEG
addition can accelerate the drug release (Chun, 2007).
The M cell represents a potential portal for oral delivery of peptides and for mu-
cosal vaccination because of their transcytotic capacity. Alginate modified trimethyl
chitosan nanoparticles were used for loading of urease. Nanoparticles may influence
their ability to enhance drug permeation through paracellular pathway. The systemic
and mucosal immune responses were also good (Prego et al., 2010).
Recombinant hepatitis B surface antigen (rHBsAg) as a model was used to prepare
hepatitis B vaccine, in which chitosan nanoparticles were prepared by chitosan-TPP
ionic gelation method. Normal vaccines prepared by using alum as adjuvant, were un-
stable when there was slight temperature change, and thus stable vaccine was needed.
Chitosan-based nanoparticles were stable, and protected the associated antigen during
storage, either as an aqueous suspension under different temperature conditions (+ 4ºC
and − 20ºC), or as a dried form after freeze-drying the nanoparticles. Even the IgG
level was 9-fold higher than conventional alum adsorbed vaccines (Prego et al., 2010).
b. For Hyperglycemia
Insulin loaded chitosan-TPP nanoparticles were prepared at controlled pH by iono-
tropic gelation method for oral route delivery with 63% entrapment efficiency (Ma,
2002).
Insulin was entrapped by alginate and calcium chloride, and then chitosan formed
polyelectrolyte complex with alginate. They protected insulin from aggressive en-
vironment of GIT, when administered orally. Insulin was released in pH dependent
manner. The glucose level was decreased by more than 40% for 18 hr with 50 IU/kg.
Confocal microscopy study confirms that nanoparticles adhere to intestinal epithelium
(Sarmento et al., 2007).
Via Nasal Route
a. As Vaccination
Chitosan-DNA nanoparticle complexes were prepared for vaccination purpose of flu,
in which hemagglutinin and Influenza A virus were used as plasmids. Even measles
and respiratory syncytial virus (RSV) by nasal route were also prepared (Illum et al.,
2001).
Even they are effective for targeting to nasal associated lymphoid tissues (NALT)
in nasal vaccine delivery (Nagamoto et al., 2004).
Trimethyl chitosan nanoparticles can increase the M cell dependent uptake and
enhance the association of the antigen with dendritic cells (Slutter et al., 2009).
Nanoparticles transport through M cell co-culture model is 5-fold higher than in-
testinal epithelial monolayer, with atleast 80% chitosan-DNA nanoparticles uptake in
30 min (Kadiyala et al., 2010).
Chitosan nanoparticles and chitosan-ethylene oxide- propylene oxide polymer
blocks were used for association and control release of bovine serum albumin, tetanus
and diphtheria toxoid. Protein was released at constant rate which matches with the
intensity of protein loading. Tetanus vaccine was released for atleast 15 days (Calvo
et al., 1997).
b. For Hyperglycemia
As chitosan is having mucoadhesive nature, it will intensify the contact between in-
sulin and nasal mucosa, leading to increased concentration at absorption site. The
nanoparticles were prepared by ionic gelation method, which were having average
size of 300–400 nm and 55% insulin loading efficiency. The molecular weight of chi-
tosan had no impact on drug release profile or on the level of blood glucose level, but
the nanoparticles were able to reduce blood glucose level in rabbit due to increased
nasal absorption (Urrusuno et al., 1999).
The chitosan concentrations, osmolarity, medium and absorption enhancers in chi-
tosan nanoparticles have significant effect on the insulin nasal delivery. As the concen-
tration increases, the insulin transport increases. The permeability will also increase
in hypo or hyper osmotic medium compared to iso-osmatic medium (Yu et al., 2004).
For Anti-fungal Delivery

The amphotericin B is the ideal candidate for many fungal infections. But it is very
less soluble and its bioavailability is poor by oral route. Thus it needs to be given by
parenteral route, which is causing nephrotoxicity. The nanoparticles were prepared by
chitosan-dextran sulfate coacervation method using zinc sulfate as stabilizing agent.
Loading efficiency of 65% was obtained by this method. More importantly, the neph-
rotoxicity was reduced when checked by in vivo renal toxicity study (Tiyaboonchai,
2007).
For Gene Delivery System

The chitosan and DNA interaction is electrostatic, which is strong enough that they do
not dissociate until they have entered in to cell. Even the mucoadhesive and cationic
nature of chitosan play important role in adhesion to cell and lysosomal escape of
DNA. They are divided in two categories depending on mechanism of formation. The
complex can be protected from DNAse to improve bioavailability of plasmid DNA.
a. Chitosan-DNA Complexes
Gentle mixing followed by incubation of chitosan and DNA solution can generate
chitosan-DNA complexes with size from 100 to 600 nm depending on the molecular
weight of chitosan, in which proportion of chitosan is in excess. The stability depends
on positive amino group of chitosan to the negative phosphate group of DNA, and is
also having direct effect on surface charge and particle size. Higher charge ratio can
give better stability along with good transfection efficiency (Janes, 2001).
b. Chitosan-DNA nanosphere
In this method, chitosan and DNA solutions are mixed with controlled speed of mix-
ing and temperature with addition of dilute salt in to DNA solution which works as a
desolvating agent for polymer. They are having size of 200–500 nm with loose rod-
like and toroidal structure. These nanoparticles may be entering to cell via endocytic
pathway (Janes, 2001).
The size of chitosan-DNA complex decreases as the molecular weight (MW) of
chitosan decreases. High MW chitosan are superior to those of low MW chitosan
in enhancing the stability of complex, giving protection to DNA in cellular endo-
somal/lysosomal compartments, but on other side, it restricts release of DNA. Higher
deacetylation of chitosan will result in increase positive charge enabling a greater
DNA binding capacity and cellular uptake. Chitosan in salt form have higher transfec-
tion efficiency than chitosan-base alone. The pH below pKa value of chitosan favors
DNA association and dissociation (Mao, 2010).
Der p 2- a house dust mite’s dermatophagoides pteronyssinus is responsible for
asthma, perennial rhinitis and atopic dermatitis. Thus allergic diseases can be charac-
terized by sensitization of allergen specific Th2 cells and IgE production. The chito-
san pDer p 2 nanoparticles have shown 100% encapsulation efficiency with sufficient
protection of plasmid. They are able to induce interferon-γ (IFN-γ) in serum, and thus
prevent allergic response caused by Th2 sensitization (Li et al., 2009).
Generally chitosan-DNA vaccines are applied through traditional high pressure
gene guns which elicit high titres of protective immunity, but will cause inevitable
pain. To overcome this, a low pressure gene guns were used. The vaccine for Japanese
encephalitis virus was prepared, which have produced specific antibodies, and have
maintained high survival rate (Huang et al., 2009).
Chitosan lactate and chitosan acetate have also been used as carrier of pSV
β-galactosidase plasmid, have shown cell viability of more than 90% (Weecharangsan
et al., 2006).
For Hepatitis Treatment

Glycyrrhetic acid is an aglycone and an active metabolite of glycyrrhizin which is
having anti-inflammatory, anti-hepatotoxic, anti-tumorigenic activity, and is used in
chronic hepatitis. It is having side effect of aldosteronism. Glycerrhetic acid is me-
tabolite of glycyrrhizin and is active in nature. But bioavailability of glycerrhetic is
very less when given as such. Thus the nanoparticles of ammonium glycyrrhizinate
were prepared by PEGlated chitosan-TPP ionic gelation method, which was having
82% entrapment efficiency (Wu et al., 2005).
For Anti-microbial Agents

Chitosan-alginate nanoparticles were prepared for Polymixin B, a potent peptidic an-
tibiotic having effect on gram negative bacteria. They also showed uptake by M cells.
Polymixin B was earlier given by parenteral route because it was absorbed very less
by oral route (Coppi et al., 2006).
Amoxicillin- a broad spectrum antibacterial is having very short half-life suggest-
ing frequent dosing. To overcome this, a controlled drug delivery system of chitosan-
TPP nanoparticle was developed (Singh, 2010).
Chitosan nanoparticles and copper loaded nanoparticles have shown antibacterial
activity against E. coli, S. choleraesuis, S. typhimurium, and S. aureus and the results
state that minimum inhibitory concentration is 0.25 µg/ml, and minimum bactericidal
concentration is 1 µg/ml. Exposure of S. choleraesuis to nanoparticles disrupt the cell
membrane and causes leakage of cytoplasm, which was later on confirmed by atomic
force microscopy (Qi et al., 2004).
For Tumor Targeting

Doxorubicin is one of the potent anti-cancer agents having cardio toxicity. The
nanoparticles prepared by chitosan-dextran have shown reduction in side effects and
improved efficacy in treatment of solid tumor. The size of 100 ± 10 nm was obtained
which favors the enhanced permeability and retention effect observed in solid tumors
(Arhewoh, 2005).
The doxorubicin was not released in cell culture, and it had entered to cell while
remaining associated with nanoparticles, which was later confirmed by confocal mi-
croscopy. The positive charge carrier of nanoparticle will be useful in the treatment of
solid tumor. Even the biodistribution and organ accumulation pattern will be changed
when given by intravenous route (Janes et al., 2001).
The 5-amino salicylic acid (5-ASA), an anti-inflammatory agent used in ulcerative
colitis, Crohn’s disease which may provide protection against development of colorec-
tal cancer in patient suffering from inflammatory bowel disease. It is metabolized in
intestine and eliminated from there. If given orally, adverse effects like hepatitis, blood
dyscrasias, pancreatitis, pleuropericarditis, and intestinal nephritis can be seen. Chi-
tosan-Ca-alginate matrix in which 5-ASA is dispersed can be used for targeting colon
because chitosan is insoluble in pH above 6.5 and is mucoadhesive in nature. In colon,
chitosan is degraded by microflora and free drug is available (Mladenovska et al.,
2007).
Gadolinium neutron capture therapy utilizes photon and electrons emitted in vivo
as a result of nuclear neutron capture reaction with administered gadolinium-157 and
non-radioelement. It is having highest thermal neutron capture cross section, and re-
lease of gamma rays and electron by neutron-capture reaction. Gaopentetic acid load-
ed chitosan nanoparticles were prepared for gadolinium neutron capture therapy and
MRI diagnosis by emulsion droplet coalescence technique. The nanoparticles have not
released gadolinium in phosphate buffer and retained gadolinium in tumor for long
time in vivo after intratumor injection (Tokumitsu, 1999).
Epirubicin is an effective anti-cancer agent. Chitosan was carboxymethylated and
bound covalently on Fe3O4 nanoparticles via carbodiimide activation. Thus, chitosan-
magnetic nanoparticles were prepared for diagnosis and targeted therapy; it could be
used in biomedicine (Chang et al., 2005).
The 5-flourouracil (5-FU) is mainly used in colon cancer. The 5-FU loaded n-
succinyl chitosan nanoparticles were prepared by emulsification solvent diffusion
method. It was having 19% loading capacity with 61% release in 24 hr. They have
shown good anti-tumor activity against sarcoma 180 solid tumor with reduced toxicity
(Yan et al., 2006).
Heparin, a known anticoagulant, is known to interact with diverse groups of pro-
teins having heparin binding domain which regulates cell proliferation, differentiation
and inflammation. It exhibits anti-cancer activity in process of tumor progression and
metastatis. It binds with vascular endothelial growth factor (VEGF) and inhibits an-
giogenesis required for tumor growth. Free heparin molecules within cells interacts
with transcription factors which plays important role in cell survival and growth, ulti-
mately leading to apoptotic cell death via caspase dependent pathway. Chitsan-PEG-
heparin polyelectrolyte complexes were prepared which were having higher toxicity
against B16F10 cells. The cancer cells show higher internalization of complex, so
higher cellular uptake of heparin resulting in dramatic cell death (Bae et al., 2009).
Colorectal cancer is having very short survival time because early detection is
not excellent. The 5-aminolaevullinic acid (5-ALA) loaded chitosan-TPP nanopar-
ticles were prepared to detect colorectal cancer at early stage. The 5-ALA is zwit-
terionic drug, so the entrapment efficiency by this method was greatly affected by pH
of solution. The 5-ALA is degraded to protoporphyrin IX. The protoporphyrin IX is
having different decomposition rate in cancer cell and normal cells, and is a photo-
sensitive flourophore, and thus it can be used for detection of colorectal cancer. Even
nanoparticles were able to escape from bacterial uptake in GIT. Fluorescence micro-
scope showed that nanoparticles are engulfed by caco-2 colon cancer cells (Yang et
al., 2009b).
Arginine rich hexapeptide are blocking the growth and metastasis of VEGF, which
secretes human carcinoma cells. It shows significant inhibition of angiogenesis in-
duced by VEGF in chorioallantoic membrane and rabbit cornea neovascularization.
Thus it is useful in human tumor and angiogenesis dependent diseases which are re-
lated to action of VEGF. The peptide was encapsulated by chitosan-dextran sulfate
nanoparticles by coacervation process, with 75% entrapment efficiency with sustained
release characteristics (Chen, 2003).
Tamoxifen (TMX) is used in breast cancer, is having side effect for vaginal symp-
toms, thrombotic events, stroke, endometrial cancer and drug resistance. The TMX
chitosan nanoparticles prepared shows uptake by Peyer’s patch. The nanoparticles are
transported to breast cells via lymphatic system reducing the toxicity (Coppi, 2009).
Catechin like polyphenolic compound is having anti-oxidant property, by which
heavy metals can be chelated and lipid peroxidation can be prevented, which can re-
duce many side effects in cancer treatment. They undergo high first pass metabolism.
The catechin loaded nanoparticles were prepared by ionic gelation method, which
showed entrapment efficiency of 90% along with 32% drug release in 24 hr (Dudhani,
2008).
For Buccal and Sublingual Delivery System

Chitosan and trimethyl chitosan were even able to increase macromolecule perme-
ation across buccal epithelium, which was later on confirmed by confocal laser mi-
croscopy, histopathology analysis, and immunohistochemistry reaction. The increase
in permeability was quantified by Franz diffusion cell using isolated buccal epithelium
(Sandri et al., 2006).
For Articular Joint Therapy

Macrophages are responsible for increased tissue permeability at inflammation site,
including rheumatoid arthritis. Thus, their selective elimination from such inflam-
matory site may be beneficial. Photodynamic therapy is used in such cases which
involves nontoxic photoactivable dye known as photosensitizer along with harmless
visible light of defined wavelength. When photosensitizers are activated, they form
reactive oxygen species resulting into destruction of macrophages along with cellular
death. The phototoxicity of photosensitizers entrapped in Hyaluronate chitosan nano-
gels was decreased considerably. The nanogel encapsulated photosensitizers were re-
tained in inflamed joint for long time compared to rapid clearance from joints of free
photosensitizers. The treatment showed better result than the standard corticosteroid
therapy for inflammation (Schmitt et al., 2010).
For Ocular Delivery System

Topical application to eye is limited due to protective physiological mechanism which
exists at precorneal area resulting into drug loss, which ultimately results in to less ef-
fective concentration at site of action. The chitosan nanoparticles were stable against
lysozyme and did not affect the mucin viscosity. They also prolonged the retention
time on eye surface. Confocal microscopy study has confirmed that nanoparticles pen-
etrate corneal and conjuctival epithelia with 100% cell survival (Campos et al., 2004).
Pilocarpine is choice of agent in open angle glaucoma. The drop causes frequent
dosing schedule, while gels because blurred vision. Picocarpine loaded chitosan car-
bopol nanoparticles were prepared which improve interaction with negatively charged
biological membrane along with sustained release property (Kao et al., 2006).
Indomethacin reduces post-operative inflammation and decreases intraocular ir-
ritation, cataract extraction and cystoid macular edema with side effects such as burn-
ing sensation, irritation and epithelial keratitis. Using chitosan-TPP nanoparticles, the
residence time of indomethacin was increased on cornea and thus the bioavailability
was also increased. With loading efficiency of 85%, minimum 76% drug release was
observed in 24 hr. The nanoparticles were able to treat chemical ulcer in rabbit eyes
(Badawi et al., 2008).
Cyclosoprin A is effective in extraocular disorders like caratoconjuctivitis sicca
or dry eye disease, with very slow partition rate at corneal epithelium. Nanoparticles
were prepared by ionotropic method having 79% association efficiency. In vivo study
on rabbit cornea suggested that therapeutic concentrations were achieved for 48 hr,
while negligible level was observed in inner ocular structure, blood and plasma (Cam-
pos, 2001).
Gatifloxin is fourth-generation fluoroquinolone antibiotic which inhibits bacterial

enzyme DNA gyrase and topoisomerase IV. A chitosan-sodium alginate nanoparticu-
late reservoir for ocular delivery was developed, which has average size of 205–572
nm and zeta potential from +17.6 mV to 47.8 mV. Nanoparticles have shown sustained
release by non- Fickian diffusion process for ocular delivery. The TEM and Atomic
force microscopy (AFM) confirms that nanoparticles are spherical and dense in nature
(Motwani et al., 2008).
For Brain Delivery

Alzheimer’s disease, a neurodegenerative disease, is becoming public health issue. Ta-
crine- acetylcholinestarase inhibitor, affects by reversible inhibition of cholinesterase,
which increases level of acetylcholine in CNS. It is having high first pass metabolism,
which result in poor oral bioavailability of 17 ± 3% only. Tacrine loaded chitosan
nanoparticles were prepared by spontaneous emulsification method, with good drug
loading capacity. They have shown continuous slow release of drug. They have pro-
vided good result in Alzhemier disease (Wilson et al., 2010).
MECHANISM OF NANOPARTICLE TRANSPORT

With development of nanoparticles system, in vitro model has been performed for the
transport studies which lack the clinical studies. There are mainly two pathway by
which nanoparticle can be transported (Rieux et al., 2006) (Figure 4)
Figure 4. Particle Transport Mechanism (a) Paracellular transport, (b) Enterocytes and (c) M cells.
1. Paracellular pathway
2. Transcellular pathway
Paracellular Transport
The paracellular transport can be enhanced by interaction with negative charge of cell
membrane or by complexing calcium ion involved in structure tight junction. Chitosan is
having effect on depolymerization of cellular F-actin and tight junction protein Zonula
occludens-1 (ZO-1). It can act via activation of protein kinase C.
Chitosan decrease cellular toxicity or damage because its effect on caco-2 cell line
is reversible in nature.
Transcellular Transport
It occurs by transcytosis- particles are taken up by cells, which begins with endocy-
tosis and ends at the time of release at basolateral pole. Absorption occurs by mainly
two kinds of cells: enterocytes and M cells, located on Payer’s patch. The M cells are
able to absorb large range of materials. Some strategies for uptake of nanoparticle is
given below
Non-specific Uptake of Nanoparticles:

a. By epithelial cells
Transcellualr transport can start by one of these endocytosis processes: pinocytosis,
macropinocytosis and clathrin mediated endocytosis. All the activity is active trans-
port, which requires energy. Phagocytosis and clathrin mediated endocytosis are re-
ceptor mediated, while pinocytosis is non-receptor mediated transport process.
Mucoadhesive materials improve transport by increasing residence time in contact
with epithelium, thus increasing the concentration at site of absorption.
Chitosan nanoparticles may enhance oral uptake by crossing the epithelium, or
that chitosan molecules release the drug at the apical pole of epithelial cells, facilitat-
ing somehow their transcytosis. Even it can be due to interaction of chitosan with
tight junctions or adsorptive endocytosis which is saturable, energy and temperature
dependent in nature. They may cause decrease in TEER value.
b. By M cells
Particle transport by M cells is energy dependent and transcellular which occurs by
fluid phase endocytosis, adsorptive endocytosis and phagocytosis. Many factors like
nanoparticle size, hydrophobicity, targeting moiety on surface, and so on can play im-
portant role for nonspecific transcellular uptake. Particles below 1 µm are taken up by
M cells and transported to basal membrane, while particles larger than 5 µm are taken
up by M cells but they remain entrapped in peyer’s patch.
Specific Uptake of Nanoparticles

a. By epithelial cells
The most popular approach is modification of nanoparticle surface. It can be done by
use of lectins, wheat germ agglutinin, Concanavalin A, tomato lactin. The level of tar-
geting and uptake is directly proportional to the amount of targeting agent attached to
particles. Targeting is a specific phenomenon, which is greatly reduced in presence of
free lectin or specific sugar. Chitosan nanoparticles were prepared using glucomanan,
which facilitates interactions of nanoparticles with mannose receptors present in epi-
thelial cells.
b. By M cells
The most popular approach is modification of nanoparticle surface. Most effective
ligand is ulex europaeus agglutinin-1 lectin, which is highly specific for α-L-fucose,
located on apical membrane of M cells. Lectins derived from Sambucus nigra and
Viscum album were able to selectively label the surface of human follicle associated
epithelium (FAE), and therefore could be used as ligands to human drug delivery ap-
plications.
Another strategy can be mimicking some pathogen bacteria, such as Yersinia,
Salmonella, and Shigella species that are able to hijack the mucosal immune system,
by using M cells to invade the intestinal mucosa. These bacteria present microbial
adhesions at their surface, which are responsible for their binding and internalization
by M cells. Immunoglobulins, particularly IgA, can specifically interact with M cell
surface. Gangliosde GM1 could be used for targeting.
CONCLUSION
Chitosan is natural biodegradable, biocompatible, mucoadhesive and water soluble
polymer, which can be used in the form of nanoparticles for ocular, nasal, oral delivery
for drugs, antigens, DNA with improved bioavailability and improved stability. Chito-
san nanoparticles protect material from bacterial uptake and are highly target specific
including their use in diagnosis via MRI, PET, and so on. Main advantage is that they
do not require any stabilizer particularly in ionic gelation method which generates
stable nanoparticles. Still further development is required for commercialization of
chitosan nanoparticles.
KEYWORDS
•• Chitosan
•• Doxorubicin
•• Glycerrhetic acid
•• Nanoparticles
•• Natural polymers
•• Protein
•• Tamoxifen
Chapter 5
Synthesis and Biomedical Application of Silver
Nanoparticles
M. Saravanan and V. Anil Kumar
INTRODUCTION
Nanoscience nowadays is a fast developing field focusing on wide spectrum of syn-
thesis and application of different nanomaterials. This emerging field (Ahmad et al.,
2003) is considered to be a conclusion for solving many stiff-necked problems in multi
disciplinary fields such as pharmaceutical sciences, applied physics, material sciences,
colloidal sciences, device physics, supramolecular chemistry, mechanical, and electri-
cal engineering, and so on. Nanomaterials have received much attention because of
their structure and properties varing from those of atoms, molecules, and bulk materi-
als (Rosei, 2004) and there by having many potential applications (Chen et al., 2009;
Papp et al., 2008). The nanoparticles have significant properties and are used in optical
detectors, laser, sensor, imaging, display, solar cell, photocatalysis, photo chemistry,
biomedicine (Zhang, 2009), pharmaceutical applications (Chan and Nie, 1998), mag-
netic materials and information storage (Gopidas et al., 2003; Jortner and Rao, 2002;
Kamat, 2002; Maxwell et al., 2002; Shon et al., 2003; Walter et al., 2002) and so on.
owing to their vast application an immense demand to obtain these nanoparticles in
non agglomerated, uniform with a well controlled mean size and narrow distribution
has arisen (Brust and Kiely, 2002; Jana et al., 2001; Pileni 2001).
Nanoparticles are referred to those particles with size up to 100 nm (Simi and
Abraham, 2007; Willems and van den Wildenberg, 2005). Silver, gold, copper and so
on are some of the noble metals which are being used for metal nanoparticles synthe-
sis (Leela et al., 2008a). Among all the nanoparticles, silver nanoparticles have great
applications in various fields especially in the fields of biological systems, living or-
ganisms, medicine (Parashar et al., 2009) environmental, and biotechnology (Hussain
et al., 2003; Virender et al., 2009). These nanoparticles have important properties like
catalytic activity, surface enhanced raman scattering effect (Chen et al., 2005; Kearns
et al., 2006; Li et al., 2006; Setua et al., 2007; Smith et al., 2006) good conductiv-
ity, chemical stability, antibacterial activity (Cao et al., 2002; Rosi and Mirkin, 2005;
Tessier et al., 2000), therapeutics (Elechiguerra et al., 2005; Rai et al., 2009), degrad-
ing pesticides (Kuber and D’Souza, 2006), filtration (Cao, 2004), biolabeling (Hayat,
1989) and nanocoated medical devices (Furno et al., 2004). So, a keen viewed atten-
tion has been drawn in synthesis of silver nanoparticles (AgNps) in several ways. Con-
sidering the overall scenario of silver nanoparticles among multidisciplinary fields, the
production of them in bulk amount has a great criterion in this field of nanotechnology.
Synthesis and Biomedical Application of Silver Nanoparticles 55
A number of physical, chemical, and biological approaches are available for the
synthesis of silver nanoparticles (Goia et al., 1998; Panacek et al., 2009). In a broad
view, the physical synthesis procedures involve evaporation, condensation, laser abla-
tion, and the chemical procedures involve reduction of metal ions in the solution to
form small metal clusters or aggregates (Egorova and Revina, 2000; Khomutov and
Gubin, 2002; Oliveira et al., 2005). Based on the reducing agent used, the chemical
methods are subdivided into classical chemical method that involves chemicals like
hydrazine, sodium borohydrate, hydrogen and so on with (Toshima et al., 1993) or
without stabilizing agents (Liz-Marzan and Philipse, 1995) and radiation chemical
method, where the reduction process is initiated by solvated electrons generated by the
ionizing radiation (Joerger et al., 2000).
On the other hand, silver metal nanoparticles are synthesized using a variety of
methods including hard template (Zhou et al., 1999), bioreduction (Canizal et al.,
2001) and solvent phase evaporation (Sun et al., 2003; Yu et al., 1997). In one way the
techniques for synthesis are categorized into top-down and bottom-up methods (Rocio
Balaguera-Gelves, 2006). The top-down approach use silver in bulk form and reduces
its size mechanically to nano scale by lithography and laser ablation (Bae et al., 2002).
In case of bottom-up approach, in other words self-assembly method, involves the
addition of reducing agent into a silver salt solution followed by addition of a stabiliz-
ing agent to prevent agglomeration on synthesized nanoparticles. As these processes
use solvents and reducing agents they show great impact on the physical and mor-
phological characteristics. Other physical and chemical modes of silver nanoparticles
synthesis are a dielectric approach where magnetron sputtering, ion exchange sol-gel
deposition and convective methods are followed. One of the most commonly used fab-
rication methods is the ion implantation (Kishimoto et al., 2004; Stepanov et al., 2000;
Townsend et al., 1994) which provides the controled synthesis of metal nanoparticles,
reduction of metal salts in the solutions (Krishna and Dan, 2009; Pal et al., 2007;
Pillai and Kamat, 2004; Rosemary and Pradeep, 2003; Tripathi et al., 2010b), chemi-
cal and photochemical reactions in the reversible micelles (Maillard et al., 2002; Taleb
et al., 1997; Xie et al., 2006), thermal decomposition of silver compounds (Kim et al.,
2005; Navaladian et al., 2007) as solvothermal synthesis (Starowicz et al., 2006), ra-
diation assisted (Henglein, 2001) like ultra sonic radiation (Salkar et al., 1999) laser
irradiation (Abid et al., 2002) and radio lysis (Ershov et al., 2007; Soroushian et al.,
2005), electrochemical methods (Mazur, 2004; Rodriguez-Sanchez et al 2000; Tang
et al., 2001; Zhu Jian et al., 2005), sonochemical approach (Esau et al., 2010; Zhang
et al., 2004; Zhu et al., 2000), facile methods (Nidhi et al., 2009) and microwave as-
sisted processes (Patel et al., 2005, 2007) are being carried out. Now there is a great
need to develop environmentally safe processes for the synthesis of nanoparticles that
avoid toxic chemicals in the synthesis protocols (Whitesides, 2003). Luoma (2008)
over viewed about silver and the environment including the expected quantities to be
released, source of release, expected pathways, and the toxicity. Biosynthetic meth-
ods employing microorganisms and plant extracts have emerged as a simple and vi-
able alternative to chemical synthetic procedures and physical methods (Revathi and
Prabhu, 2009) and hence a green chemistry route for synthesis of nanoparticles has
started. It has been known for a long time that in nature a variety of nanomaterials
are synthesized by biological processes from microbes such as bacteria (Beveridge

and Muuray, 1980; Brierley, 1990; Golab, 1981), yeast (Huang et al., 1990), fungi
(Frilis and Myers-Keith, 1986; Niu et al., 1993; Volesky, 1990) and algae (Darnall
et al., 1986; Sakaguchi et al., 1979) are able to adsorb and accumulate metals used
in recovery of metals from waste, few examples are the magnetotactic bacteria syn-
thesize intracellular magnetite or greigite nanocrystals (Blakemore, 1982) and some
other diatoms synthesize siliceous materials (Kroger et al., 1999; Mann, 1993). Even
at high concentrations, microorganisms can overcome metal stress and can survive due
to their ability of efflux systems, alteration of solubility and toxicity via reduction or
oxidation, biosorption, bioaccumulation, extracellular complexation or precipitation
of metals and lack of specific metal transport systems (Beveridge et al., 1997; Bruins
et al., 2000,).
METHODS FOR SILVER NANOPARTICLE SYNTHESIS

The synthesis of metallic nanoparticles is an active area of “application research” in
nanotechnology. A variety of chemical and physical procedures could be used for syn-
thesis of metallic nanoparticles. However, these methods are fraught with many prob-
lems including use of toxic solvents, generation of hazardous by products, and high
energy consumption. Accordingly, there is an essential need to develop environmental
friendly procedures for synthesis of metallic bionanoparticles. A promising approach
to achieve this objective is to exploit the array of biological resources in nature. In-
deed, over the past several years, plants, algae, fungi, and bacteria, have been used
for production of low-cost, energy-efficient, and nontoxic metallic bionanoparticles.
Microbial Synthesis of AgNPs

Recently many studies were conducted to explore the synthesis of AgNPs using mi-
croorganisms as a potential bio source; Basavaraja et al. (2007) used Fusarium semi-
tectum for biosynthesis of AgNPs. Sastry et al. (2003) have also reported that when
the fungi Fusarium oxysporium and Verticillium sp. were exposed to Ag+ ions, were
formed AgNPs. New enzymatic approaches using bacteria and fungi in the synthesis
of nanoparticles by both intra and extra cellularly have been expected to play a key
role in many conventional and emerging technologies.
Silver ions and silver based compounds are highly toxic to microorganisms and
hence show strong bactericidal effects in many common species of bacteria including
E.coli (Windt, 2009). The AgNPs were found to accumulate in the bacterial membranes,
in some way interacting with certain building elements of the bacterial membrane thus
causing structural changes, degradation and finally cell death. It is well known that
many organism can provide inorganic materials either intra or extracellularly (Mann,
1996) but it is very important to develop AgNPs in an eco-friendly and easier manner.
Silver bionanoparticles can be produced by physical and chemical methods (Burda et
al., 2005; Kowshik et al., 2003) whereas they can also be produced in biological meth-
ods specifically by bioreduction (Porter et al., 2006). As the physical and chemical
methods use toxic chemicals in their synthesis, it raises great concern for environmen-
tal reasons. Extracellular synthesis of silver nanoparticles from microorganisms has
been proved fruitful because it possesses antimicrobial activity (Ahmad et al., 2003;
Anil Kumar et al., 2007; Bhainsa and D’Souza, 2006; Mukherjee et al., 2002). Another
reason to use AgNPs over bulk silver metals is because of its high specific surface area
and high fraction of surface atoms; hence it has been proved to be a good antimicrobial
agent for pathogenic microorganisms (Cho et al., 2005).
Mechanism for microbial synthesis: Extracellular enzyme shows an excellent re-
dox properties and it can act as an electron shuttle in the metal reduction. Many other
compounds other than extracellular enzymes like hydroquinones, napthoquinones,
and anthroquinones (Baker and Tatum, 1998; Bell et al., 2003; Duran et al., 2002;
Medentsev and Alimenko, 1998) act as electron shuttle in metal reduction (Newman
and Kolter, 2000). Previous studies have indicated that NADH- and NADH-depen-
dent enzymes are important factors in the biosynthesis of metal nanoparticles and
the initiation of metal reduction seemed to be the transfer of electron from NADH
by NADH- dependent reductase as electron carrier (Ahmad et al., 2003; Duran et al.,
2005; Mukherjee et al., 2002). Shankar et al. (2005) reported that Ag+ ion is a highly
active chemical agent which binds strongly to electron donor groups and these bind-
ings with biomolecules like protein could restrict the size of the particle. Gericke and
Pinches (2006) stated that the rate of intracellular particle formation to an extent can
be controlled by altering the parameters such as pH, temperature, substrate concentra-
tion, and exposure time to substrate. Even at high metal ion concentration microorgan-
isms have tolerance against, metal stress by efflux systems, alteration of solubility and
toxicity, biosorption, bio accumulation, extracellular complexation or precipitation of
metals and lack of specific metal transport systems (Beveridge et al., 1997; Bruins et
al., 2000; Fortin and Beveridge, 2000; Rouch et al., 1995; Summers and Silver, 1978).
Table 1. The different fungal strains used for the biosynthesis AgNPs for the last one decade.
Biological entity (Fungal stains) Size (nm) Ref.
Trichoderma reesei 5–50 Vahabi et al. (2011)
Aspergillus clavatus 550–650 Saravanan and Nanda (2010)
Aspergillus flavus 8.92±1.61 Vigneshwaran et al. (2007)
Aspergillus fumigatus 5–25 Bhainsa and D’Souza, (2006)
Cladosporium cladosporioides 10–100 Balaji et al. (2009)
Neurospora crassa 11 Castro-Longoria et al. (2011)
Phoma sp. 71–74 Chen et al. (2003)
Verticillium sp. 25±12 Medentsev and Akimenko (1998) Mukher-
jee et al. (2001a) Senapati et al. (2004)
Nitrate reductases - Fusarium oxysporium 10–25 Kumar et al. (2007)
Penicillium sp. K1 10–100 Maliszewska and Sadowski (2009)
Penicillium sp. K10 18–100 Maliszewska and Sadowski (2009)
Trichoderma asperellum 13–18 Mukherjee and Sadowski (2008)
Trichoderma viride 5–40 Amanulla et al. (2010)
Biological entity (Fungal stains) Size (nm) Ref.

Fusarium oxysporum 5–60 Ahmad et al.(2003)
Duran et al. (2005)
Senapati et al. (2005)
Souza et al. (2004)
Oksanen et al. (2000)
Fusarium oxysporum PTCC 5115 50 Karbasian et al. (2008)
Fusarium acuminatum 5–40 Ingle et al. (2008a)
Fusarium solani (USM-3799) 5–35 Ingle et al.(2008b)
Cladosporium cladosporioides 10–100 Balaji et al. (2009)
Volvariella volvacea 15 Daizy (2009)
Phoma sp. 3.2883 67.6–74.52 Chen et al. (2003)
Verticillium sp. 12–37 Mukherjee et al. (2001a) Sastry et al. (2003)
Pencillium brevibacterium WA 2315 23–105 Nikhil et al. (2009)
Yeast strain MKY3 2–5 Kowshik et al. (2003)
Morganella sp. Parikh et al. (2008)
Table 2. The different bacterial strains used for the synthesis AgNPs for the last one decade.
Biological entity (Bacteria) Size (nm) Ref.
Pseudomonas stutzeri 200 Joerger et al. (2000)
Staphylococcus aureus 160–180 Nanda and Saravanan (2009)
Klebsiella pneumonia 5–32 Shahverdi et al. (2007)
Escherichia coli 50 Gurunathan et al. (2009)
Plectonema boryanum UTEX 485 200 Lengke et al. (2007)
Brevibacterium casei 10–50 Kalishwaralal et al. (2010)
Bacillus subtilis 5–60 Saifuddin et al. (2009)
Bacillus sp. 5–15 Pugazhenthiran et al. (2009)
Bacillus lichenformis 50 Kalimuthu et al. (2008)
Corynebacterium strain SH09 10–15 Haoran et al. (2005)
Pseudomonas stutzeri AG259 100–200 Klaus et al. (1999)
Wei and Qian (2008)
Pseudomonas AG4 60–150 Wei and Qian (2008)
Phytosynthesis of Silver Nanoparticles

Green synthesis of AgNPs follows benign protocols and materials, which is cost effec-
tive and involves bulk synthesis. As the synthesis procedures involve no high pressure,
energy, temperature, and toxic chemicals (David, 2004; Mohanpuria et al., 2008), the
green chemistry methods are aided for the synthesis and for many biomedical applica-
tions.
Usually synthesis of AgNPs refers to synthesis from vascular plants leaf extracts
(Gardea-Torresdey et al., 2002; Parashar et al., 2009). Jain et al. (2009) reported the
synthesis from fruit extracts for the first time. Using plant for the synthesis can be
advantageous over other biological processes by eliminating the elaborate process of
maintaining the cell cultures (Shankar et al., 2004). Phytosynthesis involves prepara-
tion of plant extract and then reduction of silver ions into nanoparticles. Extraction
procedure is usually by boiling of the air dried leaf pieces followed by decantation or
filtration. To this extract, silver nitrate is added and then incubated to produce nanopar-
ticles. Shankar et al. (2004) reported that the terpenoids are believed to be surface
active molecules stabilizing the nanoparticles and the reaction of the metal ions is
possibly facilitated by reducing sugars in neem leaf broth. Li et al. (2007) reported
that proteins which have amine groups played a reducing and controlling role during
the synthesis procedure in Capsicum annum. Ahmad et al. (2011) reported that Des-
modium contain chemically different groups, water soluble scavenging super-oxide
anion radicals and 1, 1-diphenyl-2-picrylhydrazyl (DPHP) radicals present in the plant
extract can be responsible for the reduction of silver and the synthesis of nanopar-
ticles. However, the exact mechanisms about the plant extract and the synthesis of the
nanoparticles are not been concluded.
Leela and Vivekanandan (2008b) reported the synthesis of AgNPs from plants
belonging to basellaceae, asteraceae and poaceae and their rates of reduction of sil-
ver nitrate. Among the plants used, the spectrometric analysis of sunflower reaction
mixture exhibited strong absorption between 400–500 nm and the particles are char-
acterized by XRD. Parashar et al. (2009) reported the advantage of reduction of sil-
ver ions in nanoparticles by using Menthapiperita leaf extract, as it occurs very fast
within 15 min and 90% of Ag ions are reduced to nanoparticles (7–50 nm). Linga
Rao and Savitramma (2011) reported the phytosynthesis of AgNPs from Svensonia
hyderabadensis which belongs to Verbenaceae family and is listed under the rare texa.
Roy and Barik (2010) reported bioreduction property of three aquatic weed leaf
extract such as Ipomoea aquatic (convolvulaceae), enhydrafluctuans (asterceae) and
Ludwigia adscendens (onagraceae) in the synthesis of AgNPs. It was considered to be
the advantageous work due to the use of unwanted plants in synthesizing nanoparticles
and for many biomedical applications. Govindaraju et al. (2010) reported the stability
of synthesized AgNPs from Solanum torvum which have unchanged surface Plasmon
absorbance even after 6 months. Elumalai et al. (2010) described the synthesis of Ag-
NPs using Euphorbia hirta L and stated that the leaf extracts act as a reducing as well
as capping agent. Singh et al. (2010) reported the synthesis of AgNPs using Argemone
Mexicana for the first time. Prabhu et al. (2010) reported the phytosynthesis of AgNPs
from Ayurvedic herbs Oscimum sanctu and Vitex nigundo, and their antibacterial ef-
fect on Proteus vulgaris and Vibrio cholera.
Table 3. The choice of Plant Materials used for the Phytosynthesis of AgNPs.
Biological entity (Plant) Size(nm) Ref.
Azadirachta indica 50–100 Shankar et al. (2004)
Aloe vera 15–20 Chandran et al. (2006)
Biological entity (Plant) Size(nm) Ref.

Emblica officinalis 10–20 Ankamwar et al. (2005a)
Cinnamomum camphora 55–80 Huang et al. (2007)
Tamarind(leaf extract) 20–40 Ankamwar et al. (2005b)
Carcica papaya 25–50 Devendra et al. (2009)
Parthenium hysterophorus L 40–50 Vyom et al. (2009)
Diopyros kaki 15–19 Jae and Beom (2009)
Camellia sinensis 30–40 Alfredo et al. (2008)
Eucalyptus hybrid 50–150 Manish et al. (2009)
Aloe vera 15.2±4.2 Chandran et al. (2006)
Emblica officinalis 10–20 Ankamwar et al. (2005a)
Jatropha curcas(seed) 15–20 Harekrishna et al. (2009b)
Jatropha curcas(latex) 10–20 Harekrishna et al. (2009a)
(dried leaf)
Pine apple (leaf extract) 15–500 Jae and Beom (2009)
Persimmon (leaf extract) 15–500 Jae and Beom (2009)
Ginkgo (leaf extract) 15–500 Jae and Beom (2009)
Magnolia (leaf extract) 15–500 Jae and Beom (2009)
Platanus(leaf extract) 15–500 Jae and Beom (2009)
Azadirachta indica 20 Tripathi et al. (2010a)
Geranium(Pelargonium graveolens) leaf ex- 16–40 Shiv Shankar et al. (2003)
tract
Hibiscus sabdariffa 25 Kamala Nalini (2008)
Phoma glomerata 60–80 Birla et al. (2009)
Chemical Synthesis of AgNPs

The chemical synthesis of colloidal nanoparticles started with the pioneering work
of Farady which paved the way for the synthesis of nanoparticles.Three concepts in-
volved in the “wet” chemical reduction of silver ions. The first one involves the radia-
tion reduction of silver ions with γ-rays (Henglein and Giersig, 1999), UV or visible
light (Huang et al., 1996, Yanagihara et al., 2001, Callegari et al., 2003), microwave
(Gao et al., 2005), or ultra sound irradiation (Xiong et al., 2002). Second concept
refers to the formation of silver colloids with relatively strong reducing agent such
as hydrazine (Taleb et al., 1997). Thirdly it involves reduction of silver by prolonged
refluxing in the presence of weak reducing agents such as glucose (Raveendran et al.,
2003), ascorbic acid (Lee et al., 2004) and polyols (Sun et al., 2002).
A variety of techniques have been developed based on the above mentioned con-
cepts to synthesize metal nanoparticles, they are as follows: chemical reduction (Petit
et al., 1993; Tan et al., 2002; Vorobyova et al., 1999; Yu, 2007), aerosol technique
(Harfenist et al., 1996), electro chemical or sonochemical deposition (Liu et al., 2001;
Zhu et al., 2001,), photochemical reduction (Darroudi et al., 2009), laser irradiation
technique (Abid et al., 2002), chemical vapour deposition (Szłyk et al., 2001), heat
evaporation (Bae et al., 2002, Smetana et al., 2005 ), reversible micelles process
(Maillard et al., 2002; Xie et al., 2006), salt reduction (Pillai and Kamat, 2004), micro-
wave dielectric heating reduction (Patel et al., 2005), ultrasonic irradiation (Salkar et
al., 1999), radiolysis (Ershov et al., 2007; Soroushian et al., 2005,), and solvo thermal
synthesis (Starowicz et al., 2006).
Some of the chemical reductants used are NaBH4, N2H4, ethanol, ethylene gly-
col, sodium citrate, N,N- dimethyl formamide (DMF) and other polyols (He et al.,
2001; Link et al., 1999; Mallin and Murphy, 2002; Pal and Maity, 1986; Petit
et al., 1993; Pastoriza-santos and Liz-Marzan, 2002; Sun and Xia, 2002; Sun et al.,
2003), while the most reduction is by the amines (Chaki et al., 2002). Some of the
surfactants used for chemical synthesis procedure such as sodium dodecyl sulphate
(Kuo and Huang, 2005), cetyl trimethyl ammonium borate (Jana et al., 2001a; Sau and
Murphy, 2004), sodium bis-(2- ethyl hexyl) sulfonyl succinate (Mandal et al., 2005)
and poly-(oxyethylene) iso octeyl-phenyl ether (triton X-100) (Ghosh et al., 2003),
but a majority of these surfactants assisted synthesis of nano sized silver use seed-
mediated growth strategy (Jana et al., 2001b; Sau and Murphy, 2004). After synthesis
of the nanoparticles, they are stabilized by stabilizing agents (quaternary ammonium)
proposed by Bonnemann et al. (1998) typical redox synthesis methods however use
hazardous chemicals as reducing agents or require significant energy input (Cushing
et al., 2004) and hence raises a great concern for environmental reasons (Kowshik
et al., 2003).
Biomemetric Synthesis of Silver Nanoparticles

Rajesh et al. (2002) reported an advanced approach for the synthesis of silver nanopar-
ticles which uses a combinatorial way to identify silver binding peptides from a phage
display library of random peptides instead of using the conventional molecular biology
procedures for isolating silver binding proteins from bacteria. Sequencing was done
by using an ABI 310 automated sequencer of over thirty independent clones, but only
three peptide sequences AG3 AG4 AG5 were confirmed to be silver binding peptides.
Polyoxometalates Method
Weinstock (1998) and Troupis et al. (2002) reported the potential use of polyoxometa-
lates (POMs) for the synthesis of AgNPs as they act as photocatalysts, reducing agent
and as a stabilizer, due to the ability to dissolve in water, undergoing stepwise multi-
electron redox reactions without disturbing their structure. Zhang et al. (2007) have
mentioned a one step synthesis and stabilizing of silver nanostructures with mixed
valence POMs in water at room temperature.
Synthesis of Silver Nanoparticles from Saccharides

Silver nanoparticles that are prepared utilizing water as solvent and polysaccharides
as reducing agent, in some cases serve as both reducing and capping agent. Sreeram
et al. (2008) reported the usage of starch as a template/reducing agent and effect of
synthetic strategy- uncontrolled/controlled heating, microwave synthesis on size and
shape of nanoparticles. Darroudi et al. (2011) reported the usage of D-Glucose as re-
ducing agent and gelatin as a stabilizer for the synthesis of nanoparticles. Vigneshwaran
et al. (2006a) stated the synthesis of stable silver nanoparticles by autoclaving AgNO3
solution with starch at 15 Psi, 121°C for 5 min. Raveendran et al. (2003) reported
starch as a capping agent and β D-glucose as reducing agent for the synthesis of starch
silver nanoparticles in a gently heated system, and then the synthesized particles are
separated from starch at high temperatures. Panacek et al. (2006), mentioned about
the synthesis of silver nanoparticles in controlled sizes with two monosaccharides
(Glucose and Galactose) and disaccharides (Maltose and Lactose). Purwar and
Pokharkar (2011) stated the use of sulphated polysaccharides for the synthesis of Ag-
NPs and confirmed SPR at 404 nm. The reduction mechanism involves a sulphate
group attached to the primary hydroxyl group of sulphated polysaccharide, which dis-
appeared after synthesis of silver nanoparticles.
Irradiation Method
Eutis et al. (2005) reported a photosensitizing technique for the synthesis of AgNPs
using benzoquinone. Sudeep and Kamat (2005) studied the visible light irradiation
for synthesis of AgNPs using thiophene as photosensitizing dye. Zhang et al. (2003)
carried out AgNPs production by illumination of Ag (NH3)+ in ethanol. Microwave
radiation and ionizing radiation can also promote reduction of Ag+ ions in Ag NPs syn-
thesis, and it was reported by Chen et al. (2008) and Long et al. (2007) respectively.
Phong et al. (2009) reported a rapid synthesis of silver colloidal solution by using mi-
crowave irradiation and nontoxic chemistry substances like silver nitrate, oxalic acid,
poly vinyl pyrolidone (PVP; MW = 55000).
Tollen’s Method
It is a one step process for the synthesis of AgNPs with a controlled size (Kvitek
et al 2005; He et al 2006; Sato et al., 2003; Yin Yadong et al., 2002). Tollens reaction
involves the reduction of Ag(NH3)2+, a tollens reagent by an aldehyde
Ag(NH3)2+(aq) + RCHO(aq) → Ag(s) + RCOOH(aq)
Sato et al. (2003) and Kvitek et al. (2005) developed a modified method to re-
duce Ag+ ions to saccharides in the presence of ammonia, yielding silver nanopar-
ticle films in the order 20–50 nm and silver nanoparticles in different shapes. The
AgNPs of different morphologies with less than 10 nm diameter were synthesized
by adjusting the concentrations of N-n hexadecyl trimethyl ammonium bromide
(HTAB) and tollens reagent (Ag(NH3)2+) in water at 120°C (Yu and Yam, 2004; Yu
and Yam, 2005).
BIOMEDICAL APPLICATIONS OF AgNPs

The first recorded medical use of silver was reported during 8th century (Moyer,
1965). Silver vessels were used in ancient times to preserve water and wine whereas
silver powder for beneficial healing and anti disease properties like ulcer treatment
believed by Hippocrates (father of modern medicine). But later the silver compounds
have emerged for medical practice. The ever living facts about silver compounds used
as disinfectant for wound healing in World war-I. In 1884, Crede German obstetrician
introduced 1% silver nitrate as an eye solution for prevention of Gonacoccal opthalmia
neonatorum, which is perhaps the first scientifically documented medical use of silver
(Russell and Hugo, 1994). The disinfectant property of silver is being exploited for hy-
gienic and medical purposes in treatment of mental illness and infectious diseases like
syphilis and gonorrhea (Gulbranson et al., 2000). Other applications include jewels,
utensils, currency, dental alloy, photography, clothing (Chen et al., 2009), and explo-
sives and so on. In higher concentrations, silver is toxic to the human beings whereas
in low concentration it is non-toxic (Pal et al., 2007).
Historically silver compounds and ions were extensively used for both hygienic
and healing purposes (Chen and Sehlueseiner, 2008). Li et al. (2008) reported that
wide ranges of applications were developed in consumer products ranging from disin-
fecting medical devices and home appliances to water treatment. Melaiye et al. (2005)
anticipated that the nano silver particles are found to be a possible antimicrobial agent.
Roy et al. (2008) stated that antimicrobial activity of silver nanoparticles is compa-
rable or better than the broad spectrum of most prominent antibiotics used world wide,
and is dependent on the size of nanoparticle. Silver sulfadiazide is listed by the World
Health Organization as an essential anti-infective topical medicine. Silver has varied
in vivo and in vitro applications (Haes and Van Duyne, 2002; Mc Farland and Richard
P. Van Duyne, 2003) as it has the highest bactericidal activity and bio compatibility
among all the known antibacterial nanoparticles and it also is more advantageous since
it does not require any photocatalytic agent for bactericidal action as required by TiO2,
ZnO, CdSe, ZnS, and so on. (Kloepfer et al., 2005; Qourzal et al., 2006). Dunn and Ed-
wards Jones (2004) stated that some nano silver applications have received approval
from the US food and Drug administration. The mechanism of the antimicrobial ac-
tion of the silver ions is closely related to their interaction with thiol groups. Furr et
al. (1994) mentioned that interaction of silver ions with thiol groups in enzymes and
proteins also plays an essential role in its antimicrobial action although other cellular
components like hydrogen bonding may also be involved.
Silver is known to have effective bactericidal properties for centuries and now
silver nanoparticles are found to have inhibitory and bactericidal effects extending its
application as an antibacterial agent (Atiyeh et al., 2007; Chu et al., 1988; Deitch et al.,
1987; Law et al., 2008; Silver, 2003). The biologically synthesized silver nanoparticles
could be of immense use in medical textiles for their efficient antimicrobial function
(Vigneshwaran et al., 2006b). The sterile cloth and materials play an important role in
hospitals, where often wounds are contaminated with microorganisms; in particular
fungi and bacteria, like S. aureus frequently occur (Lee et al., 2003). Thus, to reduce or
prevent infections, various antibacterial disinfection techniques were developed for all
types of textiles. Silver ions and silver nanoparticles have inhibitory and lethal effect
on bacterial species such as E.coli, S.aureus and even yeast (Gogoi et al., 2006, Kim
et al., 2007). Morones et al. (2005) defined the antibacterial activity of AgNPs against
four types of Gram negative bacteria E. coli, V.cholera, P. aeruginosa and S. typhus.
Antimicrobial resistance is becoming a major factor in virtually all hospitals, since
the acquired infection became untreatable which led to a serious public health problem
(Gad et al., 2004). These concerns have headed to major research effort to discover
alternative strategies for the treatment of bacterial infection (Salata, 2004). Nanobio-
technology is an upcoming and fast developing field with potential application for
human welfare. An important area of nano-biotechnology is to develop a reliable and
eco-friendly process for synthesis of nanoscale particles through biological systems
(Deendayal et al., 2006). Many organisms including unicellular and multi cellular mi-
croorganisms have been explored as potential bio factory for synthesis of metallic
nanoparticles (gold, silver, Cadmium sulfide) either intracellularly or extracellularly
(Ahmad et al., 2003; Klaus et al., 2004; Kowshik et al., 2003; Mukherjee et al., 2001b;
Nair and Pradeep, 2002; and Vigneshwaran et al., 2006a). Recently few studies have
been conducted for characterization and antimicrobial effect of silver nanoparticles.
Souza et al. (2004) showed that the bulk counterparts of AgNPs are an effective anti-
microbial agent against various pathogenic microorganisms. Shrivastava et al. (2007)
has reported that the range of 10–15 nm of AgNPs has increased stability and en-
hanced antimicrobial potency.
Bactericidal effect of AgNPs against multidrug resistant bacteria like Pseudomo-
nas aeroginosa, ampicillin resistant E.coli and erythromycin resistant Streptococcus
pyogenes was mentioned by Lara et al. (2010) and Matsumura et al. (2003). Ahmad et
al. (2007) confirmed about the combination effect of AgNPs with different antibiotics
and proved that the antibacterial activities of penicillin G, amoxicillin, erythromycin,
clindamycin, and vancomycin increases in the presence of AgNPs against S. aureus
and E.coli.
The possible mechanism of antibacterial effect of AgNPs involves the release
of K+ ions from the bacteria and thus the bacteria plasma or cytoplasmic membrane
which is associated with many important enzymes and DNA is an important target site
of silver ions (Miller and McCallan, 1957; Rayman et al., 1972; Schreurs and Rosen-
berg, 1982). Kim et al. (2005) reported the possibility of free radical involvement in
the antibacterial activity of AgNPs but the underlying mechanism and characteristics
remain unclear. Corinne et al. (2000) studied the interaction between relative oxy-
gen species (ROS) and bacterial cell death. The AgNPs when interact with bacterial
DNA or mitochondria releases ROS such as superoxide anion (O2-), hydroxyl radical
(OH) and singlet oxygen (1O2) with subsequent oxidative damage. In many studies,
direct electron microscopic view determined the structural change of bacterial cell,
confirming the cell damage. Lara et al. (2010) suggested that the mode of action of
silver nanoparticles is similar to that of silver ions, which complex with electron do-
nor groups containing sulfur, oxygen or nitrogen atoms normally present as thiols or
phosphates on amino acids and nucleic acids. In addition to their effect on bacterial
enzymes, silver ions cause marked inhibition of bacterial growth and they get depos-
ited in the vacuole and cell wall as granules (Brown and Smith, 1976).
Candida species is one of the most common fungal pathogens causing hospital
acquired sepsis with a mortality rate of about 40% (Patterson, 2007). Prophylaxis with
antifungal may lead to the raise of many drug resistant strains. Just a few studies on
antifungal efficacy of AgNPs were published (Falletta et al., 2008; Roe et al., 2008;
Zeng et al., 2007), but the fungicidal effect and mode of action of silver ions remained
obscure (Kim et al., 2008). By 1980, four classes of antifungal agents—polyenes,
azoles, morpholines and allylamines were identified, but till now only one oral drug
Ketoconazole was introduced for the treatment of systemic fungal infections (Kauffman
and Carver, 1997). Panacek et al. (2009) studies revealed the antifungal activity of
AgNPs against Candida sp. which has no cytotoxicity effect on human fibroblast at a
concentration of 1 mg/l of Ag. It was reported that SDS stabilized nanoparticles pen-
etrated into the cell wall and cytoplasmic membrane to inhibit the activity of various
enzymes such as ATPase activity of P-glycoprotein or lecitin/cholesterol acyltransfer-
ase. Kim et al., 2008 made a study on antimicrobial effect of nano silver on clinical
fungal isolates and ATCC strains of Trichophyton mentagrophytes and Candida spe-
cies and stated that mycelial forms of fungi is responsible for pathogenicity due to the
dimorphic transitions from yeast to mycelia form which is found primarily during the
invasion of host tissue and he also finally concluded that the potential activity of nano
Ag inhibit the dimorphic transition. Young et al. (2009) discussed about the biocidal
effect of AgNPs on phyto pathogenic fungi B. sorokiniana and M. grisea in fields and
other soil borne sterile fungi that rarely produce spores.
CONCLUSION
Nanotechnology is an emerging field in the 21st century which is the root cause for the
next industrial revolution. Synthesis, characterization, manipulation and application
of nanomaterials are being rapidly used in development of nanotechnology. Nanopar-
ticles are the building blocks of nanotechnology as they play vital role in their applica-
tions. The application of nanoscale materials and structures may provide solutions to
technological and environmental challenges in the areas of solar energy conversion,
catalysis, and medicine. With the increased efficiency of pathogenic microorganisms
resistant to multiple antimicrobial agents in this urbanized environment, demands
have increased for better disinfection methods. The use of nanoparticles nowadays
have proved to be a better alternative for antimicrobial properties and although more
experiments have to be done on this, the properties of Ag+ ions were known since an-
cient times and are used widely as bactericide in catheters and wounds.The concept to
eclipse the multidrug resistant pathogens is a great deal in the field of nanomedicine.
The development of multidrug resistant clinical pathogens like MRSA and MRSE
have become a major factor in all hospital acquired infections which are untreatable
and inturn causing severe public health problem. These concerns have led to major
research effort to discover alternative strategies (using Nanobiotechnology tools) for
the treatment of multi drug resistant bacterial infections (Nanda and Saravanan, 2009).
The regular monitoring of antimicrobial susceptibility pattern of pathogens and formu-
lation of a definite antimicrobial policy may be helpful for reducing the incidence of
these infections. The biomedical application of AgNPs on selected synthetic process

was reported in this chapter. In future, nanobiotechnologists will go into the deeper
level of understanding on the biochemical and molecular mechanisms of nanoparticles
formation and achieve better control over size and polydispersity of the nanoparticles.
KEYWORDS
•• Antimicrobial
•• Dimethyl formamide
•• Nanomaterials
•• Nanoparticles
•• Nanoscience nowadays
•• Nanotechnology
•• Polyoxometalates
•• Silver ions
Chapter 6
Recent Advances in Cancer Therapy Using
Phytochemicals
Hullathy Subban Ganapathy, Rajakani Senthil Nagarajan,
and Hirotaka Ihara
INTRODUCTION
Cancer is the most devastating disease with more than 10 million new cases every
year around the world. It is well known that environmental factors and chemical car-
cinogens play a predominant role in the induction of DNA lesions and other genomic
abnormalities which causes the cancer. Currently, several chemotherapeutic agents
are being used in the treatment of cancer, including alkylating agents, antimetabolites
antagonists, anticancer antibiotics, and plant-derived anticancer agents. However, che-
motherapy, being a major treatment method used for the control of advanced stages
of malignancies and metastasis, is known to exhibits severe toxicity (Markowitz and
Bertagnolli, 2009). Because of high death rate associated with cancer and because of
the serious side effects of chemotherapy and radiation therapy, many cancer patients
seek alternative and/or complementary methods of treatment. Phytochemicals are
one of fast growing anticancer agents for such alternative therapies. Basically, phyto-
chemicals are large variety of plant-derived chemical compounds, which are present in
fruits and vegetables that may reduce the risk of cancer, possibly due to dietary fibers,
polyphenol antioxidants, and anti-inflammatory effects. There are many plant-derived
phytochemicals which have been used as anticancer drugs and it has been shown to
inhibit cancer cell growth efficiently. Moreover, they are known to maintain the health
and vitality of individuals, and also cure different diseases, without causing toxicity
(Chung et al., 1995; Tyagi et al., 2010). More than 50% of all modern drugs in clini-
cal use are of natural products, many of which have the ability to control cancer cells
(Chao et al., 2005). A recent survey showed that more than 60% of cancer patients
use vitamins or herbs as therapy. Interestingly, an important and well known cancer
drug, Taxol (paclitaxel), is a phytochemical, initially extracted and purified from the
Pacific yew tree (Taxus brevifolia) (Tyagi et al., 2010). Pharmacologically safe phy-
tochemicals that have been identified from plants or their variant forms can modulate
these molecular targets. These phytochemicals include genistein, resveratrol, dially
sulfide, S-ally cysteine, allicin, lycopene, capsaicin, curcumin, 6-gingerol, ellagic
acid, ursolic acid, betulinic acid, flavopiridol, silymarin, anethol, catechins and eu-
genol (Aggarwal et al., 2004). Because of their pharmacological safety, these agents
can be used alone to prevent cancer and in combination with chemotherapy to treat
cancer. These herbal medicines have been increasingly accepted universally, and they
have an impact on both world health and international trade (Park et al., 1998). The
plant-based traditional medicines are widely used in India and China. Very recently,
for alternative therapy, National Centre for Complementary and Alternative Medicine
has been established in USA. The herbal products have been classified under “dietary
supplements” and are included with vitamins, minerals, amino acids, and “other prod-
ucts intended to supplement the diet”. The National Cancer Institute collected about
35,000 plant samples from 20 countries and has screened around 114,000 extracts for
anticancer activity. Of the 92 anticancer drugs commercially available prior to 1983
in the US and among worldwide approved anticancer drugs between 1983 and 1994,
60% are of natural origin. In this instance, natural origin is defined as natural prod-
ucts, derivatives of natural products, or synthetic pharmaceuticals based on natural
product models (Cragg and Newman, 1997). Other important phytochemicals such as
Allium sativum, Actinidia chinensis, Aloe vera, Ananas comosus, Angelica sinensis,
Annona species, Arctium lappa, Astragalus membranaceus, Betula utilis, Catharan-
thus roseus, Chlorella pyrenoidosa, Colchicum luteum, Combretum caffrum, Curcuma
longa, Echinacea angustifolia, Fagopyrum esculentum, Glycine max, Glycyrrhiza
glabra, Gyrophora esculenta, Lentinus edodes, Panax ginseng, Linum usitatissimum,
Picrorrhiza kurroa, Mentha species, Podophyllum, and Withania somnifer are known
to control the growth of cancer cells (Sakarkar and Deshmukh, 2011). In this article,
we aim to provide an overview the recent advances in the research based on phyto-
chemicals for cancer therapy.
MEDICINAL PLANTS AS CANCER DRUGS

In the recent past, numerous cancer research studies have been conducted using tra-
ditional medicinal plants in an effort to discover new therapeutic agents that lack the
toxic side effects associated with current chemotherapeutic agents. Table 1 shows
list of important phytochemicals studied for different cancers (cell lines and type of
cancer) and the potential bioactive compounds present in these plants. These plant-
derived compounds play an important role in the development of clinically useful
anticancer agents and more than 3,000 plant species have been tested against the can-
cer in vitro and in vivo models (Cragg and Newman, 2004). One of the more versatile
plants used as a source of flavonoids is the root of the traditional Chinese medicinal
herb baikal skullcap (Scutellaria baicalensis), a member of the mint family (Chung et al.,
1995). Traditionally, the dried roots of S. baicalensis were extracted and used in a
Chinese herbal medicine “Huang Qin” to treat a variety of ailments (Maloney, 1998)
and S. baicalensis has remained an important herb in both Chinese and Japanese tra-
ditional prescriptions, such as “Xiao-Chai-Hu-Tang” which is used in the treatment of
viral hepatitis and a variety of tumors (Fei et al., 2002). Various flavonoids isolated
from this traditional Chinese medicinal plant were shown to have antiandrogenic and
growth inhibitory activity against prostate cancer cells in vitro and in vivo (Bonham
et al., 2005; Sandava and Winesburg, 2005). In addition, extracts and isolated flavo-
noids from this herb have been shown to relieve oxidative stress and immune dysfunc-
tion associated with the onset and progress of cancer. Studies have also demonstrated
that flavonoids from S. baicalensis have the ability to arrest the cell cycle of tumor
cell lines that are resistant to multiple chemotherapeutic drugs (Choi et al., 1999) and
act as inhibitors of key steps necessary for the progression of tumor angiogenesis
Recent Advances in Cancer Therapy Using Phytochemicals 69
(Liu et al., 2003). Methanolic extracts from seven Plantago sp. used in traditional
medicine for the treatment of cancer were evaluated for cytotoxic activity against three
human cancer cell lines recommended by the National Cancer Institute (NCI, USA).
The results showed that Plantago sp. exhibited cytotoxic activity, showing a certain
degree of selectivity against the tested cells in culture. Since the flavonoids are able
to strongly inhibit the proliferation of human cancer cell lines, it was identified that
the compound, luteolin-7-O-b-glucoside as major flavonoid present in most of the
Plantago sp. These results could justify the traditional use of the Plantago sp. and
topoisomerase-mediated DNA damage might be a possible mechanism by which fla-
vonoids of Plantago exert their cytotoxicity potential (Gálvez et al., 2003). D. nobile
is an orchid plant, which has compound gigantel that have antimutagenic activity and
the flower extract of D. nobile release the diversified antioxidants, which destroy the
cancer cells. The cytotoxic effect of plant-derived components was tested with DLA
cell lines (Uma Devi et al., 2009).
Botanical name Name of the bioactive Cancer Type Ref.

compound
Type Cell lines
Scutellaria baicalensis Flavanoids Breast MCF7 (Fei et al., 2002)
Prostate PC3
Plantago Flavanoids Breast MCF7 (Gálvez et al., 2003)
Renal TK-10
Melanoma UACC-62
Azadirachta indica Tannin, β-sitosterol, Breast MCF-7/ADR. (Tepsuwan et al., 2002,
(Neem) nimbin, quercetin and Nanduri et al., 2003,
Colon SW620
carotne Arivazhagan et al., 2004,
Lung H522 Subapriya et al., 2005)
Alkaloid and inositol
Melanoma M14
Tannin and phenolic
compounds Ovarian SKOV3
Prostate DU145
Renal A498
Andrographis panicu- Flavonoid, Andro- Breast NCI/ADR-RES (Singh et al., 2001 Kumar
lata graphin and androgra- et al., 2004, Pfisterer et al.,
Colon HT29,SW620
pholide 2010)
Lung H522
Melanoma M14
Ovarian SKOV3
Prostate DU145
Renal A498
CNS U251
Abrus precatorius Methyl ester on N, Antitumor - (Kathiresan et al., 2005)
N-dimethyl tryptophan activity against
metho-cation and Yoshida ascites
picatorine sarcoma
Cajanus cajan Benzophenone and Breast MCF-7 (Ashidi et al., 2010)
β-Sitosterol
Lung COR-L23
amelanotic C32
melanoma

compound
Type Cell lines
Calophyllum inophyl- Polyphenols, Carotene, gastric SGC-7901 (Dai et al., 2010)
lum Epigallo-catechin-
3-gallage
Camellia sinensis Ascorbic acid, Xan- Ovarian SKOV 3 Zhang et al., 2002, Hsu
thine and Inositol and et al., 2002, Su and Arab,
Oral cavity GN56, CAL27,
β-sitosterol , 2002, Lu et al., 2010)
HSG1, HSC-2
Colon RKO
Stomach CNE 2, MGC-803,
Hela
Prostate LNCap, DU-145,
CWR22Rv1, and
PC3
Cassia absus Hydrocyanic acid, Breast MCF-7 (Bingfen et al., 1994)
Cyaniding
Colon HCT-15, SW-620,
COLO 205
HOP-62
Lung
OVCAR-5
Ovarian
PC-3
Prostate
SiHa
cervix
Careya arborea Methanol extract solid tumor DLA (Natesan et al., 2007)
Cissus quadrangularis Flavonoid, flowers, GastricUlcer - (Jainu and Shyamala Devi,
Limooid, Tangeretin 2003)
Ceiba pentandra Tetracyclic triterpenoid liver - (Bairwa et al., 2010)
and β-sitosterol
Citrus limon Resin, Essential oils, Breast - (Hirano et al., 1995, Heber,
Coumarins 2004)
Prostate -
Lung -
Leukemia HL60
Eugenia caryophyllata Dimethlsulfone, caffeic Cervical Hela (Kouidhi et al., 2010)
acid
Colon HT 29
Lung A549, MRC-5
Glycerrhiza glabra C-oxidase, Catalase, Colon - (Lee et al., 2007)
Caffeic acid, Oleic,
Lauric, and Palmitic
acid
Ipomoea batatas Essential oils, Lung, colon - (Konczak-Islam et al.,
(menthol, menthone, 2003, Konczak et al., 2004)
limonene)
Mallotus philippensis Vitamins (A,C) Colon HT 29 (Sharma, 2011)
Mentha arvensis Quercetin, β-Sitosterol, Cervical HeLa, (Janin et al., 2011)

saponin and Glucoside
Breast MCF-7,
Leukemia Jurkat,
colon HT-29
Recent Advances in Cancer Therapy Using Phytochemicals 71

compound
Type Cell lines
Moringa olifera Essential oil, crystal- Leukemia HL60,HCT-8, (Costa-Lotufo et al., 2005)
line Furocoumarin CEM
Piper sps Ca, Fe and Vitamins Breast MCF-7 (Sakpakdeejaroen and
(A,B,C) Itharat, 2009)
colon
Tetragonia tetragoni- β-Sitosterol , Flucoside Liver carcinoma HepG2 (Kyung-A et al., 2011)
oides
Thespesia populnea Lupeol, and Oral - (Dhanarasu et al., 2010)
β-Sitosterol
colon
Vaccinium flavonols, proanthocy- Breast MCF-7 (Seeram et al., 2004, Sun
anidin, oligomers, and and Hai Liu, 2006, Neto
macrocarpon Colon HT-29, HCT116,
triterpenoids et al., 2007, Dhanamani et
SW480, SW620
al., 2011)
Prostate RWPE-1, RWPE-
2, 22Rv1
Oral KB, CAL27
Vernonia cinerea Colon (Dhanamani et al., 2011)
Many of the naturally derived anticancer agents originally discovered using such
assays, have been shown to exert their cytotoxic action through interaction with cancer
cells. Additional phytochemicals with such anticancer capabilities on different cancer
cells with the literature citation are listed in the Table 1.
Carcinogenesis is a multistep process consisting of tumor initiation, promotion,
and progression. Phytochemicals can act at any of the stages. Initiation is the conver-
sion of a normal cell to a cancer cell after exposure or uptake of a carcinogen and its
interaction with the cellular DNA. Cancer-blocking agents prevent carcinogens from
reaching the cell, or prevent the carcinogen from interacting with cellular compo-
nents. Examples of phytochemicals that block initiation include ellagic acid, indole-
3-carbinol, sulphoraphane, and flavonoids. Cancer-suppressing agents, on the other
hand, block the promotion stage, which is the slow multiplication of cancer cells to
pre-neoplastic cells, or to the progression stage, which is the conversion to neoplastic
cells that can invade tissues and metastasize (spread). Examples of cancer-suppressing
phytochemicals include β-carotene, curcumin, epigallocatechin gallate, genistein, res-
veratrol, gingerol, and capsaicin (Joon Surh, 2003). Most of these cancer-suppressing
phytochemicals act on signaling molecules that have been abnormally activated or
silenced. These signaling molecules, which are kinase enzymes, are responsible for
the activation of genes that regulate cell growth, differentiation, and apoptosis.
CONCLUSION
The use of alternative medicine and natural approaches and the value of natural heal-
ing substances have been largely acknowledged and gained popularity in the recent
years. However, though there is increasing evidence from several model systems from
cell lines to animal models in demonstrating an anticancer role for herbal drugs, the
underlying mechanisms and molecular players in this field still remain largely unknown,
and therefore further research is needed to address these questions. Although, the clini-
cal trials showed that herbs were helpful against cancer, these outcomes require further
confirmation with rigorously controlled clinical trials. Though many of researchers
have demonstrated that herbs are helpful against cancer, especially useful in improv-
ing survival and quality of life in patients suffering from advanced cancer, the lack of
controls and reporting bias have been severe flaws. In certain cases, excessive dosage
or inappropriate administration of certain herbs could result in severe toxicity as well.
Hence, it is very important that scientists pay attention to the scientific basis of such
studies using herbal drugs in the future, so as to improve the status, because plants
could actually be sometimes dangerous to the human body if not used correctly. Fur-
thermore, special emphasis should be given to understanding herb-drug interactions, if
being prescribed at the same time (Das, 2002; Romero-Jimenez et al., 2005; Sakarkar
and Deshmukh, 2011).
KEYWORDS
•• Cancer
•• Cancer-suppressing
•• Carcinogenesis
•• Flavonoids
•• Phytochemicals
ACKNOWLEDGMENT
Funding was supported by a Grant-in-Aid for Scientific Research from the Ministry of
Education, Culture, Science and Technology of Japan (KAKENHI: No. 2324501801).
Chapter 7
Mitochondrial Dysfunction and Cancer: Modulation
by Palladium α-Lipoic Acid Complex
C. V. Krishnan, M. Garnett, and F. Antonawich
INTRODUCTION
Cancer is the uncontrolled multiplication of subtly modified or mutated normal hu-
man cells. While surgery, radiation, and chemotherapy are all commonly used to treat
cancer, chemotherapy is the only option if the cancer has metastasized and spread
through the body. To minimize side effects, one need cancer-specific cytotoxic drugs,
including DNA-binding agents, alkylating agents, and antimetabolites that interfere
with DNA replication.
The numerous side effects as well as the cost associated with modern medicine
have driven cancer patients to look for other treatments, collectively termed as al-
ternative or holistic medicine. Holistic medicine’s ostensible aims include address-
ing the physical, mental, emotional, and spiritual problems of the body. The medical
systems that make up holistic medicine include herbal medicine including ayurveda,
homeopathy, acupuncture, yoga, and others. There are different philosophies driving
each of these diverging systems, with the tenets of each often conflicting. There is a
general reluctance on the part of practitioners of modern medicine to accept the tenets
of holistic medicine, partly due to lack of rigorous clinical investigation. But there is
a modern trend to accept some of these treatments as a complementary or adjuvant
therapy to attain a feeling of maximum wellness, one that goes beyond recovery. In
that sense a symbiotic relationship is slowly developing between modern medicine
and its less tested brethren.
Our research is based on a holistic approach of a different kind. It addresses the
need for modern medicine to include the role played by the mitochondria in optimal
cellular function. It is different from traditional holistic medicine in that the results pre-
sented in this chapter are based on substantial investigations of mitochondrial effects.
Mitochondria are involved in energy metabolism, calcium regulation and apoptosis-
signaling pathways. The number of mitochondria in a cell is decided by its energy
requirements (Alberts et al., 1989; Beattie, 2002; Voet and Voet, 1995). Cells that is
metabolically more active, such as those in cardiac and skeletal muscles, the brain and
the liver have the most mitochondria. All human cells, other than mature erythrocytes,
have mitochondria. We believe strongly that medications targeting the mitochondria
address the same issues that holistic medicine focuses on, because healthy mitochon-
dria contribute substantially to the physical, mental, and emotional elements needed
to complement the allopathic or modern medicine. Mitochondria are ubiquitous, and
taking care of mitochondria is similar to taking care of all the parts leading to greater
achievements than the sum of the parts. This is essentially the true slogan of holistic
medicine.
A recent focus of research has been on a group of agents with anticancer activity,
mitocans that induce apoptosis (Galluzzi et al., 2006) by way of mitochondrial desta-
bilization (Biasutto et al., 2010). Natural compounds including fruits and vegetables
that preferentially kill cancer cells with mitochondrial dysfunction are receiving closer
scrutiny to understand the underlying mechanisms and therapeutic implications for
cancer treatment and prevention (Aggarwal and Shishodia, 2006; Chen et al., 2010;
Sarkar et al., 2009).
We have tried to establish a symbiotic relationship with modern medicine as well
as holistic medicine by selecting a metal complex, palladium α-lipoic acid, which is
active in mitochondrial cellular metabolism as well as in cancer cell death.
A substantial fraction of the cytoplasm in almost all eukaryotic cells is occupied by
mitochondria (Alberts et al., 1989). Mitochondria were first identified at the end of the
19th century. The energy-converting organelles of eukaryotes were generally believed
to be evolved from prokaryotes that were engulfed by primitive eukaryotic cells or
aerobic bacteria. This symbiotic relationship started more than 1.5 billion years ago
allowed the evolution of multicellular organisms with aerobic respiration (Alberts et
al., 1989). Since, the development of procedures for isolation of intact mitochondria
in 1948, extensive studies have been carried out to understand their role in energy
metabolism (Voet and Voet, 1995).
An animal cell without mitochondria would be dependent on anaerobic glycolysis
to make adenosine triphosphate (ATP). The conversion of glucose to pyruvate by gly-
colysis produces only two molecules of ATP compared to 36 molecules of ATP pro-
duced by glucose oxidation. The pyruvate produced in the cytosol by glycolysis and
the fatty acids are selectively transported into the mitochondrial matrix where they are
broken down into the acetyl group on acetyl coenzyme A (acetyl-CoA or acetyl-SCoA)
before being fed into the tricarboxylic acid cycle or citric acid cycle or Krebs cycle.
The ATP in a cell is being continuously hydrolyzed and regenerated, with a half-
life from seconds to minutes depending on the cell. An average person at rest consumes
and regenerates ATP at a rate of ~ 3 mol (1.5 kg) hr–1and as much as an order of mag-
nitude faster during strenuous activity (Voet and Voet, 1995). The rapid deterioration
of brain tissue by oxygen deprivation is due to the fact that brain cells have only a few
seconds of ATP available (Voet and Voet, 1995). It is clear that the cellular role of ATP
is as a free energy transmitter rather than as a free energy reservoir. Phosphocreatine
acts as a reservoir of ATP in muscles and nerve cells that have high ATP turnover rates.
ATP + Creatine = Phosphocreatine + ADP (1)
Even though this is an endergonic reaction under standard conditions, it is close to
equilibrium due to the prevailing intracellular concentrations of its reactants and prod-
ucts. This allows the equilibrium to shift to the right at resting state because of high
ATP concentration and shift to the left at high metabolic activity because of low ATP.
The glycolytic product pyruvate is the immediate precursor to acetyl-CoA from
carbohydrate sources.
Mitochondrial Dysfunction and Cancer 75
Glucose + 2NAD+ + 2ADP + 2Pi → 2NADH + 2Pyruvate + 2ATP + 2H2O + 4H+ (2)
For glycolysis to continue, the NADH (nicotinamide adenine dinucleotide) pro-
duced must be reoxidized to NAD+ because of its limited availability in cells. Under
anaerobic conditions, this is achieved by oxidation of NADH by pyruvate to yield
NAD+ and lactate with the aid of lactate dehydrogenase. Under aerobic conditions,
each NADH oxidized by the mitochondrial electron transport chain produces one
NAD+ and 3ATP. The pyruvate, under aerobic conditions, undergoes a series of five
sequential reactions and produces NADH and acetyl-CoA with the aid of the enzyme,
pyruvate dehydrogenase.
Pyruvate + CoA + NAD+ → acetyl-CoA + CO2 + NADH (3)
The acetyl group of the common intermediate acetyl-CoA, obtained from the
breakdown of carbohydrates, lipids, and proteins, is then converted to CO2 and H2O
through a series of consecutive enzymatic reactions of Krebs cycle, the electron trans-
port chain, and oxidative phosphorylation.
Even though the role of mitochondrial defects had been recognized in the develop-
ment of cancer, more than 80 years ago, mitochondrial dysfunction and its restoration
have started gaining momentum only recently. Our emphasis is on restoration from
mitochondrial dysfunction. To minimize toxic side effects or to avoid toxic effects
completely, we have selected a ligand, α-lipoic acid that plays a key role in the mito-
chondria. The α-Lipoic acid is part of the multi enzyme complexes, pyruvate dehydro-
genase as well as α-ketogluarate dehydrogenase, the latter being involved in the Krebs
cycle. A third enzyme, also containing α-lipoic acid, is the branched-chain α-keto acid
dehydrogenase. This enzyme participates in the degradation of isoleucine, leucine,
and valine. The other unique properties of this ligand are discussed in a later section.
After selecting a ligand, α-lipoic acid, that plays a critical role in biological en-
ergy metabolism, we wanted to tweak the properties of the ligand by complexing it
with a metal that has very high catalytic and electronic properties. After numerous
investigations with a variety of metals, the final selection was made to use palladium,
a transition metal. The properties of the resulting complex were remarkable in many
ways. After a series of investigations over numerous years, we have established that
palladium α-lipoic acid complex formulation
1. Has practically no toxic effects and its aqueous solution is safe up to at least 40
ml (0.037 M) per day.
2. Repairs DNA damage resulting from radiation.
3. Scavenges free radicals and lowers lipid peroxidation.
4. Increases the levels of glutathione and glutathione peroxidase(GPx).
5. Increases the levels of manganese superoxide dismutase, and catalase.
6. Enhances the Krebs cycle enzymes: isocitrate dehydrogenase, α-ketoglutarate
dehydrogenase, succinate dehydrogenase, and malate dehydrogenase.
7. Enhances mitochondrial respiratory enzymes, complex I, complex II, complex
III, and complex IV.
8. Promotes cell death in a variety of cancer cell lines such as skin melanoma, hu-
man (SKMel-5); liver, hepatocellular carcinoma, human (Hep G2); lung, ma-
lignant melanoma, human (malme-3M); mammary gland, ductal carcinoma,
human (MDA-MB 435); prostate, left supraclavicular lymph node carcinoma,
human (LNCaP); colon, colorectal adenocarcinoma, human (HT-29); human
brain, glioblastoma; astrocytoma (U87); and glioblastoma (U251MG).
9. Acts as a prophylactic for neuronal protection from transient ischemic attack.
10. Acts as a prophylactic for protection from radiation.
11. Exhibits unique electronic properties corresponding to diode or tunnel diode
behavior.
The influence of protein structure on the rate of electron transfer is beyond the
scope of this chapter. It is known that reduced hemes in the mitochondria can transfer
electrons at physiologically significant rates over a distance of 100–200 nm (Voet and
Voet, 1995). The electron transfer taking place through space or through bonds and
the role of protein structure are all active areas of current research. To understand
mitochondria related electron transfer, we have taken a few, but new, small steps to
elucidate the electronic character of some components of the proteins involved in the
electron transfer process. Apart from our ligand and our complex we have also inves-
tigated cysteine, lysine, histidine, and flavin adenine dinucleotide (FAD). Since, reac-
tive oxygen species (ROS) is also a source of mitochondrial dysfunction, we have also
looked at the electronic properties of H2O2.
The technique we have utilized to investigate the electronic properties of these
small molecules is impedance spectroscopy, a field with high potential for drug dis-
covery, but used only to a limited extent by researchers in the pharmaceutical industry.
To obtain a fairly complete picture of this complex, we have included, briefly at
least, other topics such as platinum(II) complexes, mitochondria and its dysfunction
or oxidative stress, free radicals, common antioxidants, diode or tunnel diode behavior
of some enzymes, and electron spin coupling.
PLATINUM(II) AND PALLADIUM(II) COMPLEXES

Active platinum(II) compounds such as cisplatin, carboplatin, and oxaliplatin are
the cornerstones of solid tumor chemotherapy. Cis-platin, cis-diamminedichloro
platinum(II), is a square planar d8 platinum(II) complex. Since, its approval in 1978
for clinical use, cisplatin has made significant contributions to the treatment of tes-
ticular and ovarian cancer (Sherman and Lippard, 1987). However, cisplatin and other
platinum drugs suffer from serious side effects such as tissue toxicity, and resistance
to the treatment (Dabrowiak, 2009; Fricker, 2007; Sherman and Lippard, 1987). Cis-
platin is highly toxic to kidneys, limiting its dose. The therapy gets complicated due
to the nauseas and intense vomits, indicating gastrointestinal toxicity. Cisplatin is also
not orally bioavailable. The molecular target for the platinum drugs is DNA. Recent
advances have identified other molecular targets such as thiol-containing proteins and
growth factor receptors (Fricker, 2007).
Cisplatin reacts with water to give several aqua species, replacing the chloride
ligands. The rate constants for the hydrolysis of the first chloride from cis or trans-plat-
ins at 25°C are 2.5 × 10–5 and 9.8 × 10–5 s–1, respectively (Sherman and Lippard, 1987).
[Pt(NH3)2Cl2] + H2O = [Pt(NH3)2Cl(OH2)]+ + Cl– (4)
[Pt(NH3)2Cl(OH2)]+ = [Pt(NH3)2Cl(OH)] + H+ (5)
[Pt(NH3)2Cl(OH2)]+ + H2O = [Pt(NH3)2(OH2)2]2+ + Cl– (6)
[Pt(NH3)2(OH2)2]2+ = [Pt(NH3)2(OH2)(OH)]+ + H+ (7)
[Pt(NH3)2(OH2)(OH)]+ = [Pt(NH3)2(OH)2] + H+ (8)
These reactions are dependent on pH and chloride concentrations. The higher
(~0.1 M) Cl– concentration in the plasma facilitates the passage across cell membranes
as the neutral cisplatin. The lower (~0.004 M) Cl– concentration inside the cell facili-
tates its hydrolysis. At the pH of the blood of 7.4, most of cisplatin is in the monohy-
droxo form. The pKa for the deprotonation reaction of the monoaqua species is 6.41
(Dabrowiak, 2009).
Cisplatin interacts with DNA to form inter- and intra-strand cross-links. The intra-
strand cross-link between adjacent guanine bases on the DNA strand causes cancer
cell death.
The coordination chemistry of palladium(II) and platinum(II) compounds being
similar, the antitumor activity of several palladium(II) complexes had been explored
(Abu-Surrah et al., 2008; Caires, 2007; Gao et al., 2009; Rau et al., 1998). Mono-
nuclear palladium(II) complexes with aromatic N-containing ligands, amino acid li-
gands, S-donor ligands, and P-containing ligands have respective qualities and prop-
erties due to the different structures as well as properties of the ligands (Gao et al.,
2009). It is interesting to note that the cisplatin analogue of palladium did not exhibit
any antitumor activity probably due to its high reactivity and consequent inability to
reach the DNA. Palladium(II) analogues of platinum(II) complexes are about 104–105
times more reactive (Gao et al., 2009). To minimize the high lability and fast hydro-
lysis of palladium(II) complexes in biological environments, chelating ligands were
used to synthesize the antitumor agents. An interesting observation was that the trans
palladium(II) complexes had better activity than the cispalladium(II) or cisplatinum(II)
complexes (Caires, 2007). Advances involving palladium complexes mainly for can-
cer therapy have recently been reviewed (Abu-Surrah et al., 2008; Caires, 2007; Gao
et al., 2009). Even though there are structural and thermodynamic similarities between
platinum(II) and palladium(II) complexes, palladium(II) complexes seem to exhibit
biological action very different from those of the toxic platinum complexes. While the
main target of platinum based drugs is DNA, palladium based drugs show preferential
targets such as enzymes and lysosomes (Caires, 2007).
It has been found that the antitumor activity of a ligand or metal was much less
than the metal-ligand complex because the binding affinity of metals to proteins or
enzymes will change their interaction process with DNA thereby affecting the DNA
replication and cell proliferation (Maloň et al., 2001). These conclusions were drawn
from studies using cell lines, human malignant melanoma G-361, human osteogenic
sarcoma HOS, human chronic myelogenous leukemia K-562, and human breast
adenocarcinoma MCF7 and iron(III) and copper(II) complexes of N6-benzylamino-

purine derivatives. Similar results were also observed for palladium(II)–benzyl bis
(thiosemicarbazonate) against cell lines, cervix epithelial human carcinoma (HeLa),
transformed monkey kidney fibroblasts (Vero), normal murine keratinocytes (Pam
212), and murine keratinocytes transformed with the H-ras oncogene and resistant to
cisplatin (Pam-ras) (Matesanz et al., 1999).
A recent review on palladium(II) complexes for the cancer therapy has lamented
the lack of progress of palladium-based drugs. “In addition, it is important to note
that, to the best of our knowledge, the palladium-based complex had not yet been
tested in human beings due to the following factors: the success of platinum-based
complexes in the cancer therapy, the enormous quantity of these complexes described
in the literature, the high costs of the developmental phases in human clinical trials,
the legal difficulties involving the drug assays in human beings and for the novelty of
this subject” (Caires, 2007). We must add that the palladium α-lipoic acid complex
formulation has been in the market for more than 15 years, without specifying any
potential benefits for any cancer even though the cell line data presented here indicate
its potential applications for a variety of cases.
GENE-BASED THERAPY
Still in its infancy, serious attempts are being made in drug discovery based on phar-
macogenomics. It is based on the proteins, enzymes, and RNA molecules associated
with genes and specific diseases and based on a patient’s genetic profile. Sorting out a
few single nucleotide polymorphisms (SNPs) responsible for the disease from the mil-
lions of SNPs and their response for each specific drug remain a Herculean challenge.
For example, a recent sequencing of 20,661 protein coding genes in 22 human glio-
blastoma multiforme (grade IV astrocytoma) samples revealed recurrent mutations
in the active site of isocitrate dehydrogenase 1 (IDH1 gene on chromosome 2q33)
(Parsons et al., 2008). The oxidative carboxylation of isocitrate to α-ketoglutarate re-
sulting in the production of nicotinamide adenine dinucleotide phosphate (NADPH) is
catalyzed by isocitrate dehydrogenase 1. These IDH1 mutations were found to occur
preferentially in younger patients compared to the older patients with wild-type muta-
tions in IDH1. A similar study of 20,661 protein-coding genes in 24 pancreatic cancers
revealed 63 genetic alterations defining a core set of 12 cellular signaling pathways
(Jones et al., 2008). The complexity of gene-based therapy can be recognized easily
by knowing the enormous number of mutated genes in some tumors such as pancreas
(1007), brain (685), and breast (1026) (Jones et al., 2008).
HALLMARKS OF CANCER
Self-sufficiency in growth signals, insensitivity to anti-growth signals, evading pro-
grammed cell death or apoptosis, limitless replicative potential, sustained angiogen-
esis, and tissue invasion and metastasis seem to characterize the cancer cells (Hanahan
and Weinberg, 2000). The need for adding mitochondrial dysfunction to this list is sub-
stantiated by a growing list of compelling evidence. Normal differentiated cells gener-
ate their energy for cellular processes by mitochondrial oxidative phosphorylation. Cancer
cells on the other hand produce their energy by glycolysis even under aerobic condi-
tions (Heiden et al., 2009; McKnight, 2010; Vazquez et al., 2010; Warburg, 1956). The
number of moles of ATP per mole of glucose produced by oxidative phosphorylation
is ~ 36 and by glycolysis are 2. In a proliferative tissue, the number of moles of ATP
produced per mole of glucose is ~4 (Heiden et al., 2009). The cancer cells’ preference
for the less efficient glycolytic pathway for energy production is still on a hot pursuit.
A correlation is observed between glycolytic ATP production and aggressiveness of
tumor cells (Simonnet et al., 2002). Numerous investigations have been carried out to
understand this “Warburg effect” and the link between cellular metabolism and growth
control. Several signaling pathways involved in cell proliferation have been linked to
anabolic metabolism. These signaling pathways also regulate metabolic pathways that
incorporate nutrients into production of nucleotides, amino acids, and lipids for accel-
erating cell proliferation instead of efficient energy production (Heiden et al., 2009).
The recent special section of “Science” on “Metabolism” has highlighted “the con-
trol of the metabolic switch in cancers by oncogenes and tumor suppressor genes”
(Levine and Puzio-Kuter, 2010), “circadian integration of metabolism and energetics”
and its consequences for treatment of obesity and diabetes (Bass and Takahashi, 2010),
the role of “autophagy in metabolism” and its implications for treatment of cancer and
degenerative diseases (Rabinowitz and White, 2010), and manufacturing molecules
through metabolic engineering (Keasling, 2010).
Most anticancer drug development strategies are based on recent advances in the
discovery of oncogenes and tumor suppressor genes. Recent studies on the role of mi-
tochondria in signaling pathways for cell death and regulation of calcium homeostasis
have generated renewed interest in investigating the role of antioxidants and nutraceu-
tical supplements for induction of apoptosis and anticancer treatment (Aggarwal and
Shishodia, 2006; Biasutto et al., 2010; Chen et al., 2010; Kroemer et al., 2007; Singh,
2006; Sarkar et al., 2009).
MITOCHONDRIA
Mitochondria or the “powerhouse of the cell”, are about 0.5–1 µm in diameter and 7
µm in length (Voet and Voet, 1995). Mitochondria exist in a variety of different shapes
depending on the source from which they are derived. In electron micrographs they
appear as spheres, rods or filamentous bodies. Twenty percent by volume of a typical
eukaryotic cell is occupied by about 2,000 mitochondria. These semi-autonomous,
highly dynamic organelles, containing a double membrane structure, are involved in
cellular respiration (aerobic metabolism), regulation of calcium homeostasis and cell
death. Two membranes that have different sets of enzymes and biochemical functions
surround the internal matrix. The matrix contains the enzymes of the Krebs cycle
except succinate dehydrogenase (SDH). The SDH is bound to the inner membrane.
To increase the surface area, nature has cleverly convoluted or invaginated the inner
membrane into the matrix of the mitochondrion to form cristae, its number varying
with the respiratory activity of the type of cell. The components of the respiratory
chain and the mechanism for ATP synthesis are part of the inner membrane. While the
outer membrane is relatively permeable or porous to metabolites and solutes smaller
than ~5 kD, the inner membrane is highly selective. This makes the contents of the
intermembrane space and the matrix different. The enzymatic composition of the vari-
ous mitochondrial sub compartments, outer membrane, intermembrane space, inner
membrane and matrix, are easily available (Alberts et al., 1989; Beattie, 2002; Voet
and Voet, 1995).
The transport of electrons through respiratory chain complexes I–IV in the in-
ner mitochondrial membrane requires a series of coupled redox reactions. Within the
inner mitochondrial membrane, these complexes are all laterally mobile. The redox
enzymes involved in the electron transfer process play a major part in the bioenergetic
metabolism. The fate of the reducing equivalents from catabolic processes entering
the respiratory chain as NADH and FADH2 is briefly described (Beattie, 2002; Voet
and Voet, 1995).
Complex I (NADH-ubiquinone oxidoreductase or NADH-CoQ reductase) cata-
lyzes oxidation of NADH by coenzyme Q (also known as CoQ10 in mammals).
NADH + oxidized CoQ → NAD+ + reduced CoQ or CoQH2, ∆Eo′ = 0.360V (9)
Complex II (succinate-ubiquinone oxidoreductase or succinate-CoQ reductase)
catalyzes oxidation of FADH2 by coenzyme Q. This reaction does not produce enough
energy to synthesize ATP. It serves as a conduit to inject electrons from FADH2 to the
electron transport chain.
FADH2 + oxidized CoQ → FAD + CoQH2, ∆Eo′ = 0.015V (10)
Complex III (coenzyme Q-cytochrome c reductase) catalyzes oxidation of CoQH2
by cytochrome c
CoQH2 + oxidized cytochrome c → oxidized CoQ + reduced cytochrome c, ∆Eo′ =

0.190V (11)
Complex IV (cytochrome c oxidase) catalyzes oxidation of reduced cytochrome
c by O2.
Reduced cytochrome c + ½ O2 → oxidized cytochrome c + H2O. ∆Eo′ = 0.580V (12)
Complex V (proton translocating ATP synthase) carries out energy coupling or
energy transduction. The electron transport and ATP synthesis are coupled.
2H+ + ½ O2 →H2O, ∆Eo′ = 0.815V (13)
Even though NADH can participate in a 2e transfer process only, the coenzymes
FMN and CoQ of complex I are capable of receiving or donating one or 2e because of
their stable semiquinone radical forms. Cytochromes, on the other hand, allow passage
of only 1e.
Part of the energy released during this passage of “high energy” electrons along a
series of electron carriers embedded in the ion-impermeable membrane is harnessed
to pump protons from one side to the other side of the membrane. Complexes I, III,
and IV pump protons. This movement of protons from the mitochondrial matrix to
the intermembrane space creates an electrochemical gradient. The redox enzymes in
the respiratory chain build this proton gradient or pH gradient in a stepwise fashion.
The ATP-synthase, a non-redox enzyme, is responsible for keeping the ATP ratio far
from equilibrium by catalyzing the phosphorylation of ADP. The ATP-synthase uses
the gradient of the electrochemical potential of the proton as a source of free energy.
The electronic properties (bias or rectifier or “ratchet”) of some of these redox en-
zymes and the group responsible for these activities are described later in this chapter.
Research in molecular biology during the 1950s and early 1960s focused on find-
ing out the reasons for the deviations from Mendelian rules for the transmission of
some mitochondrial characteristics and instead following the cytoplasmic inheritance
patterns led to the discovery of mitochondrial DNA. Mitochondria contain their own
genome with their own transcription, translation, and machinery for protein synthesis.
Two genetic systems, the mitochondrial DNA (mtDNA) and the nuclear DNA (nDNA)
encode the mitochondrial electron transport chain complexes. The mtDNA codes for
13 different complexes are given in Table 1. The codes for nDNA are 36, 4, 10, 10, and
14 protein subunits for Complexes I, II, III, IV, and V respectively (Carew and Huang,
2002). Complex II is encoded by nDNA only. Mitochondrial DNA also contains genes
encoding 2 ribosomal RNAs (12S rRNA and 16S rRNA) and all the necessary 22
transfer RNAs that are required for protein synthesis in mitochondria. The human
mtDNA is a supercoiled, double-stranded molecule containing 16,569 base pairs. It
is also known that the frequency of mtDNA migration to nDNA is much greater than
nDNA migration to mtDNA (Carew and Huang, 2002).
Table 1. Subunits of Electron Transport Chain Complexes Encoded by Human Mitochondrial DNA
(Carew and Huang, 2002).
Complex Subunits Number of Subunits Encoded by mtDNA
I NADH dehydrogenase 7
II Succinate dehydrogenase 0
III Cytochrome b 1
IV Cytochrome c oxidase 3
V ATP synthase 2
Mitochondria are not self-replicating organelles in spite of their ability to tran-

scribe their own DNA and translate the resulting mRNAs. Most of the mitochondrial
proteins (more than 90%), synthesized in cytosol and imported into mitochondria, are
encoded in nDNA.
Apart from respiration, a second crucial function of mitochondria is the control
of apoptosis or programmed cell death. An early activation of pro-apoptotic protein
disrupts the mitochondrial membrane permeabilization and results in the formation of
pores. Consequently, the electron transport chain protein, cytochrome c, is released
into the cytosol, along with the release of pro-caspase 9.
Another important function of mitochondrion is the distribution/redistribution of
Ca2+ pools within cells. Uptake of high concentrations of Ca2+ into mitochondria opens
the pore of the outer membrane and consequently releases cytochrome c.
MITOCHONDRIAL DYSFUNCTION
Apart from ATP production, mitochondria also produce ROS. Some electrons escap-
ing or leaking from the electron transport complexes, mainly from complexes I and III,
during respiration react with oxygen to form superoxide radicals. Oxygen undergoes a
series of progressive reduction reactions producing superoxide anion (O2–), hydrogen
peroxide (H2O2), and finally hydroxyl radical (HO) along with hydroxide ion (OH–).
The cause of this leakage of electrons is not clearly understood. However, it may be
possible for mtDNA mutations to disrupt the normal electron flow and seriously affect
energy production. Oxidative damage and the resulting serious consequences have
been extensively reviewed recently (Singh, 2006). Compared to nDNA, mtDNA is far
more susceptible to mutations due to a lack of histone protection and limited repair
capacity (Carew and Huang, 2002; Singh, 2006).
During the production of ATP in the cell, about 85% of oxygen is consumed by the
mitochondria. Superoxide radical, O2–, may be produced from about 4% of all oxy-
gen consumed (Singh, 2006). Enzymes such as NADPH oxidases, xanthine oxidase,
cyclooxygenases, and lipooxygenases also produce ROS. The iron-sulfur cluster in
the aconitase enzyme, localized to the matrix space of mitochondria, is oxidized by
superoxide and the exposed iron reacts with the peroxide to produce hydroxyl radicals
(Singh, 2006). Also the NO produced within mitochondria by mitochondrial NO syn-
thase produces peroxynitrite radical (ONOO–) by reaction with O2–. Superoxide and
peroxinitrite radicals contribute to substantial mitochondrial damage.
Enzymes such as super oxide dismutase, GPx, catalase, peroxoredoxin, and thio-
redoxin can inactivate some of the ROS. Manganese superoxide dismutase or copper/
zinc superoxide dismutase converts the superoxide radical into hydrogen peroxide.
The active site of cytosolic and extracelllar forms of superoxide dismutase contain
copper/zinc and the mitochondrial form contains manganese (Beattie, 2002). Oxida-
tive damage is due to the inadequacy of these detoxifying processes.
Lipid peroxidation by the hydroxyl radical can alter the structural integrity of
membranes. In patients with Parkinson’s disease, the excess Fe2+ can reduce peroxide
and produce HO. These radicals and their reactions cause oxidative stress and conse-
quent mitochondrial damage resulting in mutations and probably cancer.
Table 2. Mutated Genes in Different Cancers (Carew and Huang, 2002).
Type of Cancer Mutated Gene/Region Affected Respiratory Complex

Breast 16S rRNA None
ND1 Complex I
ND2 Complex I
ND4 Complex I
ND5 Complex I
Cyt b Complex III
ATPase 6 Complex V
Ovarian 12S rRNA None
Type of Cancer Mutated Gene/Region Affected Respiratory Complex

16S rRNA None
Cyt b Complex III
Pancreatic 12S rRNA None
16S rRNA None
ND1 Complex I
ND2 Complex I
ND4 Complex I
ND4L Complex I
ND5 Complex I
ND6 Complex I
Cyt b Complex III
COXI Complex IV
COXII Complex IV
COXIII Complex IV
ATPase 6 Complex V
Prostate 16S rRNA None
ND4 Complex I
ND4L Complex I
Since, each cell contains many mitochondria with multiple copies of mtDNA, it
is possible for wild-type and mutant mtDNA to coexist in a state called heteroplasmy.
After numerous cell divisions over time, this status may change to predominantly
wild-type or mutant, a state called homoplasmy (Chatterjee et al., 2006). Since, tu-
mors have mostly homoplasmic mtDNA mutations their cells carry the same mtDNA
mutation (Carew and Huang, 2002).
Mitochondrial DNA depletion and deletion to cancer progression has been re-
viewed recently (Higuchi, 2007). The mitochondrial function and energy metabolism
in cancer cells as well as mitochondrial genetics have also been reviewed (Kroemer
and Pouyssegur, 2008; Mayevsky, 2009; Wallace and Fan, 2010).
The Warburg effect or aerobic glycolysis in cancer cells and its potential benefits
for cancer cells have been examined in detail (Carew and Huang, 2002; Frezza and
Gottlieb, 2009; Gogvadze et al., 2008; Hsu and Sabatini, 2008; Kroemer and Pouysse-
gur, 2008). Both glucose and glutamine are consumed heavily by cancer cells and may
be the source of increased lactate production in cancer cells. In most cancer cells about
60% of ATP is produced by glycolysis (Frezza and Gottlieb, 2009). The linkage be-
tween aerobic glycolysis and apoptosis resistance remains higly elusive. The link be-
tween aerobic glycolysis and mitochondrial outermembrane permeabilization (which
mediates the intrinsic pathway of apoptosis) resistance, is also not clearly established.
Attempts are being made to counteract aerobic glycolysis and to induce mitochondrial
outermembrane permeabilization and consequent apoptosis for therapeutic purposes

(Kroemer, 2006).
Direct evidence of tumorogenesis from mitochondrial dysfunction was obtained
when mutations in succinate dehydrogenase or fumarate hydratase were found to initi-
ate familial paraganglioma and of papillary renal cell cancer respectively. A general
feature of malignant cells seems to be alterations in respiratory chain activity and
mtDNA abnormalities. Many tumors have somatic mutations in mtDNA (Chatterjee
et al., 2006). Gene mutations observed in some common cancers are briefly included
in Table 2.
Glioblastoma Multiforme (GBM) and Type I Meningioma (transitional

meningioma, TM)
It has been found that mtDNA was highly amplified in most of the malignant glioma
specimens (Carew and Huang, 2002). The comparative amplification of nDNA was
very low. The membrane phospholipids of the brain containing high amounts of un-
saturated fatty acids are likely to be damaged by oxidation by oxygen radicals. The
formation of these lipid peroxides affects the integrity and function of the membranes
proteins and DNA The natural defense mechanism against this adverse effect is by
GPx, glutathione reductase (GRx), and superoxide dismutase.
Table 3. Comparison of glutathione peroxidase, glutathione reductase and proteinoxidation levels in

Glioblastoma, transitional meningioma, and normal brain tissues (Tanriverdi et al., 2007).
Parameters GPx (U/g wet tissue) GRx (U/g wet tissue) Pox (nmol/g wet tissue)
Glioblastoma 17.72±3.9 5.11±0.9 599.6±56
Transitional
Meningioma 19.04±2.0 5.66±0.9 588.3±49
Normal
Brain tissue 45.26±6.9 10.08±1.2 439.1±31
Superoxide dismutase converts O2– into H2O2, which is eliminated by the actions of
catalases and peroxidases. An analysis, given in Table 3, of 48 brain tumors obtained
during surgery and 15 normal brain tissues collected during autopsy for GPx, GRx,
and protein oxidation (POx) revealed that GPx and GRx activities were significantly
lower in GBM and TM when compared to the controls (Tanriverdi et al., 2007). The
decrease in GPx and GRx were more obvious in GBM than in TM. Also the POx levels
were much higher in both GBM and TM compared to controls.
Breast Cancer
The bulk of the mutations were identified in the D-loop region, the main non-coding
area of the mtDNA. The others are given in Table 2. Lipid peroxidation, coenzyme
Q10 levels and antioxidant status of breast cancer patients have been investigated
(Portakal et al., 2000; Punnonen et al., 1994; Rajneesh et al., 2008; Sinha et al., 2009).
It was observed that coenzyme Q10 concentrations were significantly less in tumor
tissues compared to normal surrounding tissues (Portakal et al., 2000). Also higher
levels of malondialdehyde (MDA) (a measure of lipid peroxidation) were observed in
tumor tissues.
Table 4. Comparison of lipid peroxidation, catalase and superoxide dismutase levels innormal and
breast cancer blood and tissue (Sinha et al., 2009).
Parameters in blood Control Patients
Malondialdehyde
(nmol/ml plasma) 2.27±0.36 4.52±0.78
Catalase (U/ml red blood cell) 15.42±0.59 10.60±0.99
Superoxide dismutase (U/ml red blood cell) 10.52±0.37 7.36±0.55
Parameters in tissue
Malondialdehyde
(nmol/ml plasma) 2.20±0.22 4.73±0.69
Catalase (U/ml red blood cell) 15.61±0.72 10.37±1.16
Superoxide dismutase (U/ml red blood cell) 10.61±0.36 7.24±0.26
Table 5. Comparison of lipid peroxidation, catalase and superoxide dismutase levels in different
stages of breast carcinoma in blood and tissue (Sinha et al., 2009).
Parameters in blood plasma Stage I Stage II Stage III Stage IV
Malondialdehyde
(nmol/ml plasma) 3.38±0.44 3.99±0.60 4.52±0.58 5.12±0.49
Catalase
(U/ml red blood cell) 10.76±0.95 11.23±1.24 11.03±0.55 9.90±0.65
Superoxide dismutase
(U/ml red blood cell) 8.15 ±0.90 7.58±0.55 7.22 ±0.22 7.13±0.39
Parameters in tissue
Malondialdehyde
(nmol/ml plasma) 3.86±0.63 4.60±0.71 4.57±0.41 5.12±0.63
Catalase
(U/ml red blood cell) 12.21±1.02 10.85±1.07 9.82±0.72 9.99±0.97
(U/ml red blood cell) 7.08 ±0.32 7.43±0.26 7.23 ±0.21 7.19±0.32
However, the activities of manganese superoxide dismutase, total superoxide dis-

mutase, glutathione peroxidase, and catalase levels were also higher in tumor tissues
compared to normal tissues (Portakal et al., 2000; Rajneesh et al., 2008). On the other
hand, the superoxide dismutase and catalase levels, given in Table 4 and Table 5,
reported in another recent study (Sinha et al., 2009) indicate a trend that is consistent
with data for other cancers. This discrepancy is attributed probably to the differences
in the stage of the tumor selected for different studies. It was suggested, from a study
of free radicals and antioxidants in stages I–IV of carcinoma breast in blood and tis-
sue that at the early stages of cancer, the antioxidant levels may be higher to meet the
challenge of carcinogenesis (Sinha et al., 2009).
Ovarian Cancer
Somatic mutations were mostly observed in 4 regions of the mitochondrial ge-
nome (Table 2), D-loop, 12S rRNA, 16S rRNA, and cytochrome b (Carew and
Huang, 2002). The levels of lipid peroxidation and conjugated dienes were much
higher in ovarian cancer patients compared to controls (Senthil et al., 2004). The
catalase and superoxide dismutase levels were lower in the ovarian cancer pa-
tients. The levels of antioxidant vitamins C and E were also lower in the ovarian
cancer patients.
Table 6. Comparison of lipid peroxidation, conjugated dienes, catalase, superoxide dismutase,

vitamin C and vitamin E levels in blood in normal and ovarian cancer patients (Senthil et al., 2004).
Parameters in blood Control Patients
Malondialdehyde
(nmol/ml plasma) 2.13±0.18 5.64±0.52
Conjugated dienes
(μmol/ml plasma) 0.71±0.06 1.71±0.13
Catalase (U/mg hemoglobin) 5.71±0.61 4.4±0.39
(U/mg hemoglobin) 1.91 ±0.29 0.9±0.11
Vitamin C (mg/dl of plasma) 1.05 ±0.09 0.40±0.03
Vitamin E (mg/dl of plasma) 2.82 ±0.20 1.36±0.10
Prostate Cancer
Incidence of mutations in mitochondrial DNA of prostate cancer was infrequent
(Carew and Huang, 2002). A recent study of lipid peroxidation and antioxidant status
in prostate cancer patients, shown in Table 7, revealed elevated levels of lipid peroxi-
dation and decreased levels of vitamin C, vitamin E, reduced glutathione, glutathione
peroxides, and superoxide dismutase in plasma, erythrocytes and erythrocyte mem-
branes when compared with normal patients (Sandhya et al., 2010).
Table 7. Comparison of lipid peroxidation, catalase, reduced glutathione, glutathione peroxidase,

vitamin C and vitamin E levels in plasma, in erythrocytes and erythrocyte membranes of normal and
prostate cancer patients (Sandhya et al., 2010).
Parameters in plasma Control Patients

Malondialdehyde
(nmol/ml plasma) 3.8±0.2 6.9±0.52
Conjugated dienes
(μmol/ml plasma) 0.71±0.06 1.71±0.13
Catalase (U/ml plasma) 0.76±0.07 0.56±0.04
Vitamin C (mg/dl of plasma) 1.39 ±0.007 1.29±0.06
Vitamin E (mg/dl of plasma) 1.4 ±0.06 1.28±0.09
Reduced glutathione
(mg/dl plasma) 52.7 ±4.2 42.8±2.9
Glutathione peroxidase
(U/l) 189.8±23.4 160.1±12.7
Parameters in Erythrocyte
Malondialdehyde
(nmol/mg protein) 0.33±0.04 5.7±0.42
Reduced glutathione
(mg/dl plasma) 54.9 ±3.8 44.8±2.7
Vitamin E (μg/mg protein) 2.31 ±0.09 1.76±0.09
Parameters in Erythrocyte Mem-
brane
(U/mg hemoglobin) 4.71±0.52 4.32±0.34
Catalase (U/mg hemoglobin) 1.7±0.09 1.3±0.07
Glutathione peroxidase
(U/g hemoglobin) 22.2 ±1.7 20.6±1.7
It is obvious from the examples given above that the main gateway for electrons to
enter the respiratory chain, complex I, is affected in all these cancers.
FREE RADICALS AND ANTIOXIDANTS

The appearance of oxygen in the atmosphere is associated with a great expansion of
the varieties and numbers of higher living forms. Oxygen is the source for the emer-
gence of respiratory metabolism and energy efficiency. It is also the source of free
radicals such as hydroxyl and superoxide.
A free radical is a highly reactive species with an unpaired electron. It can be a
neutral species such as hydroxyl, HO, or a charged negative ion (anion) such as su-
peroxide, O2–, or a charged positive ion (cation) such as guanine radical. An unpaired
electron is shown as a dot after the symbol (example: HO). Free radicals are, in general,
good oxidizing agents. They can remove an electron from other materials and in that
process the unpaired electron gets paired. They can also participate in chain reactions
to produce new free radicals.
The free radicals produced during phagocytosis are beneficial (Singh, 2006; Voet
and Voet, 1995). The primary purpose of leukocytes is phagocytosis, which is the
engulfing and destruction of particulate matter and bacteria. Leukocytes contain the
enzymes of the hexose-monophosphate shunt, glycolysis, citric acid cycle, and respi-
ratory enzymes. Phagocytosis requires a lot of energy, which is obtained from glucose
by glycolysis and also by the hexose-monophosphate shunt. The role of this shunt
is to produce hydrogen peroxide from superoxide free radical, which is used in the
phagocytotic process.
The beneficial aspect of hydrogen peroxide in cell signaling is emerging. Neurons
and brain macrophages produce superoxide ions in pathological situations and the hy-
drogen peroxide produced from superoxide increases gap junctional communication
in astrocytes (Rouach et al., 2004). Examples of signaling processes include the over
oxidation of the cysteine in peroxiredoxins from the cysteine sulfenic acid to cysteine
sulfinic acid, and the over oxidation of methionine residues in proteins to methionine
sulfoxide (Wood et al., 2003).
The need for a certain amount of oxidative stress and the role of redox for embry-
onic and fetal growth have been exemplified recently (Dennery, 2010). It details the
level of oxygen levels and antioxidant status at the first, second and third trimester
of pregnancy. It is also interesting to note that low levels of H2O2 and superoxide
produced by human sperm are important for the capacitation process that allows the
sperm to penetrate the zona pellucida of the ovum. It has also been observed that at
low, moderate, and highly oxidative state, proliferation, differentiation, and apoptosis
or necrosis respectively are favored.
Free radicals are also a liability because they produce DNA damage by easily oxi-
dizing the guanine base in DNA. The altered form of guanine, 8-oxoguanine, has been
the subject of much study. Another liability of free radicals is that oxyradicals allow
lipid peroxidation.
Antioxidants are physiologic reducing agents. They donate electrons to free radi-
cals and in that process become oxidized. Their specific reactions are a function of
their redox potential, measured in volts.
Reduction or redox potentials predict the direction of a reaction. They cannot
predict how fast the reaction will take place. Each oxidized species of a redox couple
having a higher positive voltage is capable of extracting an electron from a reduced
species of a redox couple having a less positive or higher negative voltage. Some
examples of 1e reduction potentials and 2e reduction potentials of reactions of bio-
logical interest are given in Table 8 (Buettner, 1993) and Table 9 (Voet and Voet,
1995). Depending on the position of the redox couple in the redox table of poten-
tials, free radicals can act as both oxidizing and reducing agents and produce other
free radicals.
Table 8. 1e reduction potentials at pH 7.0, 1 atm, and 1.0 M (Buettner, 1993).
Couple E/mV
HO, H+/H2O 2310
O3–, 2H+/H2O + O2 1800
HOO, H+/H2O2 1060
PUFA, H+/PUFAH 600
(Polyunsaturated fatty acid, bis-allylic-H)
α-Tocopheroxyl, H+/α-tocopherol 500
(Vitamin E)
H2O2, H+/H2O, HO 320
Ascorbate–, H+/ascorbate monoanion 282
(Vitamin C)
Ferricytochrome c/Ferrocytochrome c 260
Semiubiquinone, H /ubiquinol
+
200
(CoQ–, 2H+/CoQH2)
Ubiquinone, H+/semiubiquinone –36
(CoQ/CoQ ) –
Dehydroascorbic/ascorbate– –174
O2/O2– –330
O2, H /HO2
+
–460
GSSG/GSSG– (Glutathione disulfide and its radical ion) –1500
Enzymes such as superoxide dismutase, catalase, and glutathione peroxidase are

known as preventive antioxidants because they eliminate the species involved in the
initiation of free radical chain reactions. Hydrogen peroxide and organic hydroperox-
ides in the cytosol are destroyed by a selenium (a cofactor) containing metalloenzyme
glutathione peroxidase. Superoxide dismutases exist in several varieties with copper,
zinc, and manganese in the active center. The dismutation yields hydrogen peroxide,
which can be removed by catalase and glutathione peroxidase to oxygen and water by
different mechanisms. The highest concentration of catalase is present in peroxisomes
and to a lesser extent in cytosol and mitochondria (Beattie, 2002).
Table 9. 2e reduction potentials at pH 7.0, 1 atm, and 1.0 M (Voet and Voet, 1995).
Reaction E/mV
2H+ + 2e– = H2 –421
Cystine + 2H + 2e = Cysteine
+ –
–340
NAD+ + H+ + 2e– = NADH –315
NADP+ + H+ + 2e– = NADPH –320
Lipoic acid + 2H+ + 2e– = Dihydrolipoic acid –290
FAD + 2H+ + 2e– = FADH2 (free coenzyme) –219
Pyruvate + 2H+ + 2e– = Lactate –190

FAD + 2H+ + 2e– = FADH2 (in flavoproteins) ~ 0.
Ubiquinone + 2H + 2e = Ubiquinol
+ –
45
½ O2 + 2H+ + 2e– = H2O 820
Food industry uses butylated hydroxyl toluene and butylated hydroxyl anisole for
preservation. However their metabolic fate is not clearly understood.
Small molecules such as ascorbate (vitamin C), tocopherols (vitamin E), reduced
coenzyme Q10 (CoQH2), glutathione (γ-glutamylcysteinylglycine), and α-lipoic acid
“repair” oxidizing radicals directly and are known as chain breaking antioxidants.
The criteria often used to evaluate the antioxidant potential as well as preventive
or therapeutic applications of a compound are (1) specificity of free radical quench-
ing, (2) metal chelating ability, (3) interaction with other antioxidants, (4) effects on
gene expression, (5) absorption and bioavailability, (6) concentration in tissues, cells,
and extracellular fluid, and (7) location (in aqueous or membrane domains or in both)
(Packer et.al., 1995).
FREE RADICAL PRODUCTION AND CHAIN REACTIONS

Normal (triplet state) oxygen, O2, has two single parallel (spin) electrons in separate
orbitals. A 2e interaction is not possible because it will result in parallel spins in the
same orbital, which is not allowed. Thus the preferable interaction is reduction of oxy-
gen by addition of 1e at a time. This process leads to the production of oxygen radicals
that can cause cellular damage. The high energy singlet oxygen with 2e and opposite
spins in the two orbitals is capable of 2e interactions.
Progressive 1e reduction of O2 produces O2–, H2O2, and finally HO. along with
OH–.
O2 + e– → O2– (superoxide anion) (14)
O2– + e– + 2H+ → H2O2 (hydrogen peroxide) (15)
H2O2 + e– → OH– + HO (hydroxyl radical) (16)
HO + e– + H+ → H2O (17)
The hydroxyl radical is undoubtedly the most dangerous. It is involved in lipid per-
oxidation and generation of other toxic radicals. While there is no enzyme to destroy
it, there is enzymatic transfer of hydroxyl to the proline in procollagen. Because of its
high reactivity, hydroxyl radical has a short life.
The superoxide ion can act both as a reductant (for Fe3+) and as an oxidant for
catecholamines. Free iron and copper present under physiological conditions are se-
questered by proteins (iron as transferrin and ferritin and copper by ceruloplasmin) to
minimize production of free radical chain reactions such as reactions (18) and (19).
O2– + Fe3+ → O2 + Fe2+ (18)
H2O2 + Fe2+ + H+ → HO.+ Fe3+ + H2O (classical Fenton reaction) (19)

Adding reactions (18) and (19) gives the Haber‑Weiss reaction (20) which is
catalyzed by metal ions.
H2O2 + O2– + H+ → HO + O2 + H2O (20)
The superoxide anion shown in reaction (14) is often released by mitochondria.
Dismutation of superoxide anion, shown in reaction (21) produces hydrogen peroxide.
2H+ + O2– + O2– → H2O2 + O2 (21)
Damage to both mtDNA and nDNA may result in mutations. Nonspecific binding
of Fe2+ to DNA may result in the formation of HO.(Reaction 19) that attack individual
bases and cause strand breaks.
An example of a polyunsaturated fatty acid (PUFAH) peroxidation chain reaction
is the following.
Initiation reaction: PUFAH → PUFA + H. (22)
Propagation reaction: PUFA + O2 → PUFAOO (23)
Propagation reaction: PUFAOO + PUFAH → PUFAOOH + PUFA (24)
Tocopherol (vitamin E) can break the propagation chain reaction by reacting with
lipid peroxyl radical, PUFAOO.
The most reactive hydroxyl radical, HO., is produced in biological systems by re-
ductive cleavage of H2O2 by a reduced metal complex. The source of iron (II) complex
may be ferrocytochrome c, iron(II) citrate, iron(II) transferrin, and iron(II) ADP. The
source of H2O2 may be a direct 2e reduction of O2 or a 1e reduction of O2 to produce
superoxide ion.
Iron (II) complex + H2O2 → Iron (III) complex + OH– + HO (25)
HO2 (the perhydroxyl radical) + O2 (+ H ) → H2O2 + O2 (26)
– +
The dangerous superoxide ion converts the iron (III) complex directly to the iron
(II) complex.
O2– + iron(III) complex → iron(II) complex + O2 (27)
Production of O2– is from the use of stronger reductants. Thus O2– can produce both
hydrogen peroxide and the iron (II) complex needed for the Fenton reaction.
VITAMIN C, L-ASCORBIC ACID

Ascorbic acid is in the form of ascorbate anion at biological pH because it has pK
values of 4.17 and 11.57. It is a cofactor in several biosynthetic pathways. It is also
an antioxidant. Humans do not synthesize it due to lack of the enzyme L-gulono-
γ-lactone oxidase. This enzyme mediates the last step in the ascorbate biosynthetic
pathway originating from glucose.
Ascorbic acid is a cofactor for the enzyme prolylhydroxylase, which modifies the
polypeptide collagen precursor to facilitate the formation of collagen fibers. It also
plays important roles in carnitine synthesis, catabolism of tyrosine, synthesis of

norepinephrine by dopamine β-monooxygenase or dopamine β-hydroxylase, and the
amidation of peptides with C-terminal glycine to activate hormone precursors.
Due to resonance, the ascorbate radical has a long half life of 1 second. Ascorbate
can be oxidized in two successive 1e steps to ascorbate free radical and dehydroascor-
bic acid respectively.
Ascorbate anion–e– → Ascorbate free radical (28)
Ascorbate free radical–e– → dehydroascorbic acid (29)
The unpaired electron in the ascorbate free radical is distributed over its ring
structure. This electron distribution stabilizes the molecule. Ascorbate radical dispro-
portionates to give ascorbate anion and dehydroascorbic acid. This acid is a strained
molecule and is not stable. The strain is relieved by hydration and subsequent bicyclic
structure formation, which is hydrolyzed to form a linear molecule 2,3-diketo-L-gulo-
nic acid. This ring opening reaction is biologically irreversible and results in the loss
of the vitamin. However the oxidation of ascorbate to the radical and dehydroascorbic
acid can easily be reversed. Both ascorbate radical anion and dehydroascorbic acid
can be reduced by enzyme systems that use NADH or NADPH as sources of reducing
equivalents.
The redox chemistry of ascorbate is pH dependent. The ascorbate radical has pK
values of 1.10 and 4.25 and is an anion at physiological pH.
Dehydroascorbic acid imported into the mitochondria via facilitative glucose
transporter 1, GLUT1, is reduced by glutathione and protects the mitochondrial ge-
nome and membrane (Nualart et al., 2003).
VITAMIN E
Vitamin E includes eight different related homologues. Of these, α-tocopherol is the
most abundant and active form in vivo. The dynamics of antioxidant action of vita-
min E have been recently reviewed (Niki and Noguchi, 2004). Vitamin E acts only in
membrane or lipid domains. It quenches lipid peroxyl radicals. It has no activity in the
aqueous phase.
Vitamin E, the primary lipid soluble small molecule antioxidant and vitamin C,
the terminal water soluble small molecule antioxidant cooperate to protect lipids and
lipid structures against peroxidation. Although vitamin E is located in membranes and
vitamin C is located in aqueous phases, vitamin C is able to recycle vitamin E (See
Table 8 and reaction (31). That is, vitamin C repairs the tocopheroxyl (chromanoxyl)
radical of vitamin E thereby permitting vitamin E to function again as a free radical
chain breaking antioxidant.
α-Tocopheroxyl.+ ascorbate monoanion → ascorbate– + α-tocopherol,
E = + 218 mV (30)
(Vitamin E) (Vitamin C)
The concentration of vitamin E is less than 0.1 nmol per mg of membrane protein
which corresponds to about one molecule for every 1000–2000 membrane phospholip-
ids molecules that are the target of oxidation (Buettner, 1993). Lipid peroxyl radicals
are generated at the rate 1–5 nmol per mg of membrane protein per minute (Packer
et.al., 1995). Since, vitamin E is recycled by other antioxidants such as vitamin C, ubi-
quinols and glutathione, the membrane is not degraded and the vitamin E levels stay
nearly the same. Recycling of vitamin E by dihydrolipoic acid seems weak. However,
dihydrolipoic acid prevents lipid peroxidation by regenerating glutathione. Dihydro-
lipoic acid can recycle vitamin E via glutathione, vitamin C, ubiquinol, NADPH and
NADH (Packer et al., 1995).
Each α-tocopherol (vitamin E) can donate 2e as a chain breaking antioxidant.
GLUTATHIONE
Glutathione or γ-glutamylcysteinylglycine, GSH, one of the body’s major antioxi-
dants, can react with various highly oxidizing species such as HO, RO,.or ROO.and
make, H2O, ROH, or ROOH and GS.which is glutathiyl radical. This is less oxidizing.
However, GS can react rapidly with GSH, most efficiently via GS– to make GSSG–,
which is a very strong reducing species. It can produce O2– and glutathione disulfide,
GSSG, by reaction with oxygen.
GSSG– + O2 → O2– + GSSG (31)

Selenium (as selenocysteine) is a cofactor of glutathione peroxidase (Beattie,
2002). One can see that superoxide dismutase and glutathione providing an excel-
lent natural combination for cellular antioxidant defense by removing O2– and HO.
respectively.
The GS reacts with oxygen to form GSOO and other free radicals such as GS– sul-
fonylperoxyl radical (GSO2OO), GS-sulfonyl radical (GSO2), and GS-sulfinyl radical
(GSO). The stable end products of glutathione oxidation are glutathione disulfide, glu-
tathione sulfinic acid (GSOOH), and glutathione sulfonic acid (GSO3H).
The intracellular concentration of GSH is about 1 mM while the mitochondrial
respiration keeps O2 about 0–10 µM in the cell. Therefore, 99% of GS.formed should
react with GSH to make GSSG and O2–. Thus the importance of superoxide dismutase
is obvious. The normal GSH to GSSG ratio in erythrocytes is 100:1 (Beattie, 2002).
Glutathione is involved in reactions such as peroxide detoxification by glutathione
peroxidase, NADPH dependent reduction of GSSG to GSH by GRx, thiol transferase
modulation of protein disulfide balance and leukotriene biosynthesis by glutathione-
S-transferase (Voet and Voet, 1995). It is also involved in the transport of amino acids
across cell membranes. GRx contains an electron-transfer prosthetic group FAD. The
electronic properties of the cysteine and FAD groups are discussed in a later section.
HYDROGEN PEROXIDE (H2O2)

Pure hydrogen peroxide is a pale blue syrupy liquid with a boiling point of 152.1oC
and a freezing point of –0.89oC. The dielectric constant of 93 at 25oC for the pure
liquid increases to 120 for a 65% aqueous solution (Cotton and Wilkinson, 1972). In
the pure liquid state, the hydrogen peroxide is more strongly associated by hydrogen
bonding than pure water. The dipole moment of hydrogen peroxide is 2.1 Debye units
compared to 1.84 Debye units for water. Thus ion-dipole interactions are stronger with
hydrogen peroxide than with water. Its influence on ion solvation is discussed in a
later section.
Hydrogen peroxide has a skew, chain structure. The O-H bond distance is 97 pm
and O-O bond distance is 149 pm. A dilute aqueous solution of hydrogen peroxide is
more acidic than water (Cotton and Wilkinson, 1972).
H2O2 = H+ + HO2– K20oC = 1.5 × 10–12 (32)

The potential given by the following equations indicate that hydrogen peroxide is
a strong oxidizing agent in both acidic and basic solutions.
H2O2 + 2H+ + 2e– = 2H2O, Eo = 1.77V (33)
O2 + 2H+ + 2e– = H2O2, Eo = 0.68V (34)
HO2– + H2O + 2e– = 3OH–, Eo = 0.87V (35)
Hydrogen peroxide behaves as a reducing agent only in the presence of stronger
oxidizing agents such as permanganate.
The enzymes, monoamine oxidases, located in the outer mitochondrial membrane
of mammalian tissues catalyze the oxidation of biogenic amines and produce H2O2.
Hydrogen peroxide plays a dual role (Lázaro, 2007). Cancer cells produce high
amounts of H2O2. These increased levels of H2O2 result in DNA alterations, cell pro-
liferation, apoptosis resistance, metastasis, angiogenesis, and hypoxia inducible factor
1(HIF-1) activation. Activation of HIF-1 plays crucial roles in apoptosis resistance,
invasion/metastasis and angiogenesis. Many human cancers have over expressed HIF-
1. On the other hand, hydrogen peroxide also induces apoptosis in cancer cells selec-
tively and the activity of many anticancer drugs is mediated, at least in part by H2O2.
With increasing concentrations of cellular H2O2, its function gradually changes
from cell signaling to cell malignant transformation to cell death (Lázaro, 2007). The
mystery surrounding the different roles of hydrogen peroxide may be solved, at least
partially, by looking at its electronic properties, which is described in a later section.
Our impedance data suggest that the electronic properties (and consequent circuits) of
H2O2 are dependent on its concentration. Our data also suggest that one has to take a
serious look at another important contribution of H2O2, the preferential solvation of
ions such as sodium and its consequences.
The direct administration of H2O2 for cancer treatment is controversial. However
treatments using H2O2 generating systems such as high-dose intravenous vitamin C
are less controversial. Since, with increasing concentrations of H2O2, its effect changes
gradually from chemopreventive effects to carcinogenic effects to chemotherapeutic
effects, the choice of antioxidant/prooxidant agents and their concentrations should
be evaluated very carefully for use as chemopreventive and chemotherapeutic agents
(Lázaro, 2007).
Oxyradical reactions catalyze the mitochondrial electron transfer chain to oxygen.
While the energy advantage of oxygen metabolism favors selection of such develop-
ments, we have studied the contribution of periodic oscillation which peroxide brings
to hydrated physico-chemical systems. Hydrogen peroxide exhibits a variety of oscil-
lations (Koper, 1996; Mukouyama et al., 2001) under potentiostatic conditions.
Spatiotemporal oscillation in a system allows the far reaching electronics of alternat-

ing current circuits, long range signals, and oscillation amplifier behavior. Peroxide
may allow these reactions to occur in the thin film hydration double layers throughout
the cell and organism.
To highlight the importance of mitochondrial dysfunction in other major diseases,
other than cancer, we have included two brief sections, Parkinson’s disease and Al-
zheimer’s disease,
Parkinson’s Disease
Oxidative stress is implicated in the pathogenesis of Parkinson’s disease (Olanow and
Lieberman, 1992; Weiner et al., 2007). Reduced activity of Complex I of the electron
transport chain and the gene mutations in Parkinson’s disease have been discussed in
great detail (Greenamyre et al., 1999; Parker et al., 1989; Schapira, 2004; Winklhofer
and Haass, 2010). The hydroxyl radical can lead to lipid peroxidation and alter the
structural integrity of neural membranes. Excess Fe2+, also found in patients with Par-
kinson’s disease, can reduce peroxide and produce HO. Dopamine undergoes autooxi-
dation, producing HO, H2O2, semiquinone radical and finally a quinone (Olanow and
Lieberman, 1992). Enzymatic metabolism of dopamine also produces H2O2.
Dopamine + O2 → Semiquinone + O2– + H+ (36)
Dopamine + O2– + 2H+ → Semiquinone + H2O2 (37)
Semiquinone + O2 → Quinone + O2– + H+ (38)
Hydrogen peroxide is also produced when dopamine is metabolized enzymatically
by monoamine oxidase (Olanow and Lieberman, 1992).
Dopamine + O2 + H2O ® 3,4 Dihydroxyphenylacetaldehyde + NH3 + H2O2 (39)
Alzheimer’s Disease
Oxidative damage to both mtDNA and nDNA has been examined in several studies
(Bubber et al., 2005; Castellani et al., 2002; Gibson et al., 2000; Mecocci et al., 1994).
Significant 3-fold increase in the amount of 8-hydroxy-2’-deoxyguanosine in parietal
cortex of Alzheimer’s patients in mtDNA and a small significant increase in oxidative
damage to nDNA have been observed (Mecocci et al., 1994). A deficiency in cyto-
chrome c oxidase has been reported in Alzheimer’s disease (Castellani et al., 2002).
Significant decreases were observed in the activities of pyruvate dehydrogenase com-
plex (–41%), isocitrate dehydrogenase (–27%), α-ketoglutarate dehydrogenase com-
plex (–57%). There were good correlations between the diminished activity of these en-
zymes and the Clinical Dementia Rating (Bubber et al., 2005; Gibson et al., 2000). On
the other hand the activities of succinate dehdrogenase (complex II) (+44%) and malate
dehydrogenase (+54%) were increased. The activities of the other 4 Krebs cycle en-
zymes, citrate synthase, aconitase, succinate thiokinase, and fumarase were unchanged.
α-LIPOIC ACID
Lipoic acid, shown in Figure 1, is a very unique biological molecule. It has a carbox-
ylic acid (pKa 4.7) which is ionized at biological pH, and it has a cyclic disulfide or
dithiolane ring (Baumgartner et al., 1996; Patel and Packer, 2008). It exists intracel-
lularly as the reduced form, dihydrolipoic acid. The redox property, the antioxidant
capacity and the fatty acid properties of lipoic acid account for its biological effects.
We will describe its electronic contributions later in this chapter.
The dihydrolipoic acid can regenerate or recycle the antioxidants CoQ (ubiquinol),
vitamins C and E, and glutathione. Both lipoic acid and its reduced form are known
to scavenge reactive oxygen and nitrogen species such as H2O2, HO., hypochlorous
acid (HOCl), and peroxynitrite (ONOO–) (Packer et al., 1995; Patel and Packer, 2008).
Figure 1. Alpha lipoic acid in its oxidized and reduced forms.
Compared to the inefficient transport of disulfides such as cystine that is needed in

modulating GSH levels in cells, the efficient transport of lipoic acid and dihydrolipoic
acid in and out of the both mitochondria and cells as well as mitochondrial β-oxidation
have been attributed to its fatty acid properties, similar to that of octanoic acid (Patel
and Packer, 2008). Lipoic acid can also cross the blood-brain barrier. The β-oxidation
products of lipoic acid, the oxidized and reduced forms of bisnorlipoic acid and tetra-
norlipoic acid may also have important redox and antioxidant biological effects.
The α-lipoic acid/dihydrolipoic acid couple is called a “universal antioxidant” be-
cause it fulfills all the criteria mentioned earlier (Packer et al., 1995). The α-lipoic acid
absorbed from the diet is readily converted into dihydrolipoic acid in many tissues.
There is ample evidence to indicate the usefulness of this redox couple as a therapeu-
tic agent for diabetes, ischemia-reperfusion injury, and heavy metal poisoning. The
α-lipoic acid was found to protect hematopoietic tissues in mice from radiation dam-
age (Ramakrishnan et al., 1992). It was also found that α-lipoic acid offered protection
from radiation for children affected by the Chernobyl nuclear accident (Korkina et al.,
1993), neurodegeneration, and HIV infection (packer et al., 1995; Patel and Packer,
2008). The α-lipoic acid scavenges hydroxyl radicals but is not effective against hy-
drogen peroxide and superoxide radical. The reduction potential for the α-lipoic acid/
dihydrolipoic acid couple of –320 mV (Packer et al., 1995) or –290 mV (Voet and
Voet, 1995) and the GSSG/GSH) couple of –240 mV indicate that dihydrolipoic acid
can react with glutathione disulfide and regenerate glutathione (Packer et al., 1995).
Thus lipoic acid helps to maintain GSH/GSSG ratio (about 100–10,000 times greater
than other redox couples such as NAD+/NADH, and NADP+/NADPH), an estimate of
redox state, in cells (Patel and Packer, 2008).
Treatment with lipoic acid increases the GSH levels in cells. This is explained by
(1) facile transport of lipoic acid into cells, where it is reduced by NADH or NADPH
dependent pathways to dihydrolipoic acid. (2) Dihydrolipoic acid is transported back
into the extracellular media where it is oxidized by cystine regenerating lipoic acid and
producing cysteine. (3) Compared to cystine, cysteine is more easily transported into
the cell which aids the synthesis of GSH (Patel and Packer, 2008).
The pharmacokinetics of R-lipoic acid, reviewed recently, revealed a plasma level
concentration, Cmax, of 1.154 μg/ml from 1 g R-lipoic acid compared to the proposed
therapeutic range of 10–20 μg/ml or 50–100 μM (Carlson et al., 2008). A dose of 600–
800 mg sodium R-lipoate gave plasma levels of 8–18 μg/ml, which is within the thera-
peutic range. The upper limit suggested for therapeutic action of 45 μg/ml or 225 µM is
reached by a dose of about 1.2 g of racemic-α-lipoic acid. The no adverse observed ef-
fect level (NOAEL) of racemic lipoic acid is considered to be 60 mg/kg body mass/day.
The oxidized form α-lipoic acid can undergo further oxidation at sulfur or get
reduced. Therapeutic and energy production applications of this powerful antioxidant
have also been explored extensively (Patel and Packer, 2008).
Located within the mitochondrial matrix is lipoic acid requiring enzymes: three
α-keto acid dehydrogenase complexes, that catalyze the oxidative decarboxylation of
α-keto acids such as pyruvate, α-ketoglutarate, and branched chain α-ketoacids (Voet
and Voet, 1995). In organisms, hydrogen atom transfer and acyl group transfer take
place in the oxidative decarboxylation of α-ketoacids with the aid of α-lipoic acid.
The reversible redox reaction between α-lipoic acid and dihydrolipoic acid is thus a
very important biochemical reaction. The reversible reduction to dihydrolipoic acid
is favored by the presence of the ring strain in the 1,2-dithiolane ring of about 15–25
kJmol–1 (Patel and Packer, 2008).
The multienzyme complex, pyruvate dehydrogenase, consists of three enzymes,
pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl
dehydrogenase (E3) (Voet and Voet, 1995). This enzyme complex participates in five
sequential reactions during the conversion of pyruvate to acetyl-CoA. The lipoic acid
is covalently linked to a ε-amino group of lysine residue via an amide linkage. These
lipoic acid containing enzymes participate in four out of the five reactions.
The multienzyme complex, α-ketoglutarate dehydrogenase, also consists of three
enzymes, α-ketoglutarate dehydrogenase (E1), dihydrolipoyl transsuccinylase (E2),
and dihydrolipoyl dehydrogenase (E3) (Voet and Voet, 1995).
The branched chain α-ketoacid dehydrogenase is also a multienzyme complex re-
sembling the other two enzymes mentioned above. These three enzymes have the same
dihydrolipoyl dehydrogenase and employ the coenzymes thiamine pyrophosphate, li-
poamide, FAD and the terminal oxidizing agent NAD+ (Voet and Voet, 1995). The im-
portance of lipoic acid in the energy metabolism is illustrated by these three enzymes.
SYNTHESIS OF PALLADIUM α-LIPOIC ACID COMPLEX

The synthesis of copper, zinc, and arsenic complexes of α-lipoic acid have been re-
ported (Baumgartner et al., 1996; Strasdeit et al., 1995). Palladium α-lipoic acid com-
plexes with (1:2) (Strasdeit et al., 1995) and 1:1 (Garnett, 1995a) stoichiometry have
also been reported. Details of synthesis of palladium α-lipoic acid complex (1:1) using
alkaline sodium lipoate and H2PdCl4 in basic conditions and its possible applications
for treatment of tumors and psoriasis are also available (Garnett, 1995b; 1997; 1998).
INVESTIGATIONS USING PALLADIUM α-LIPOIC ACID COMPLEX

FORMULATION
The different characteristics of this complex in comparison with that of the ligand are
described in this section. These include in vitro cell lines studies, animal mitochondria,
radiation protection, animal glioblastoma studies, human safety studies, voltammetry,
and impedance spectroscopic studies.
Voltammetric Studies of Palladium α-Lipoic Acid Complex Formulation

The anodic oxidation of α-lipoic acid at a glassy carbon electrode and palladium
α-lipoic acid complex interaction with double stranded DNA have been investigated
using atomic force microscopy and voltammetry at highly oriented pyrolytic graphite
electrode (Corduneanu et al., 2007; 2009). An important observation was the disso-
ciation of the palladium α-lipoic acid complex at negative potentials and deposition
of Pd(0) nanoparticle deposition. The application of a positive potential induced the
oxidation of the palladium α-lipoic acid complex and the formation of a mixed layer
of lipoic acid and palladium oxides.
Oxygen Radical Absorbance Capacity or ORAC analysis of Palladium α-Lipoic

Acid Complex Formulation
The ORAC assay measures the oxygen radical absorbance capacity of a compound as
compared to Trolox (vitamin E). These analyses carried out by Brunswick Labs, Inc.,
Wareham, Massachusetts gave the following normalized values as Trolox equivalent
per gram: Vitamin A 1.6; Vitamin C 1.12; Vitamin E 1.0; α-Lipoic acid 1.4; and Pal-
ladium α-lipoic acid complex formulation 5.65. Thus the superior free radical scav-
enging capacity of the palladium α-lipoic acid complex formulation is obvious. This
radical scavenging superiority of the metal complex compared to that of the ligand is
similar to the antitumor activities observed for metal complexes (Maloň et al., 2001;
Matesanz et al., 1999).
In vitro Cell line Studies using Palladium α-Lipoic Acid Complex Formulation
The effects of the palladium α-lipoic acid complex formulation on the following 8 dif-
ferent cell lines were examined at K.G.K. Synergize Inc, Canada. (1) Skin melanoma,
human (SKMel-5); (2) Liver, hepatocellular carcinoma, human (Hep G2); (3) Lung,
malignant melanoma, human (malme-3M); (4) Mammary gland, ductal carcinoma,
human (MDA-MB 435); (5) Prostate, left supraclavicular lymph node carcinoma, hu-
man (LNCaP or lymph node carcinoma of the prostate): (6) Colon, colorectal adeno-
carcinoma, human (HT-29); (7) Human brain, glioblastoma; astrocytoma (U87); (8)
Glioblastoma (U-251MG). Palladium α-lipoic acid formulation was administered at
3 different dosages and the cell growth was measured using [3H] thymidine uptake
after 24, 48, and 72 hr of culture. The data shown in Figure 2 is 48 hr after exposure.
Palladium α-lipoic acid formulation was effective to varying degrees of cell death
(statistically significant level of cell death), on the entire group of cell lines tested. The
varying effectiveness appears to be a consequence of the particular cell lines used and
their associated degree of anaplasia.
Figure 2. Effect of palladium α-lipoic acid complex formulation, after 48 hr, on (1) Skin melanoma,
human (SKMel-5); (2) Liver, hepatocellular carcinoma, human (Hep G2); (3) Lung, malignant
melanoma, human (malme-3M); (4) Mammary gland, ductal carcinoma, human (MDA-MB 435);
(5) Prostate, left supraclavicular lymph node carcinoma, human (LNCaP): (6) Colon, colorectal
adenocarcinoma, human (HT-29); (7) Human brain, glioblastoma; astrocytoma (U87); (8)
Glioblastoma (U-251MG).
The effect of palladium lipoic acid complex formulation on the growth of canine
osteosarcoma (CCL-183, D17) cells was also examined in vitro by a similar proce-
dure. While the lowest dose did not have any significant effect, the higher doses of 100
and 1000 µg/ml inhibited the growth of the cells after 48 and 72 hr of culture.
We have examined the effects of palladium α-lipoic acid complex formulation on
different cell lines from National Cancer Institute’s (NCI) repository, breast cancer
(adenocarcinoma, MCF-7), brain tumor (stage IV glioblastoma multiform, U-251Mg),
lung (non-small cell carcinoma, A-549), and brain (astrocytoma, H-4), ovarian cancer
(OVCAR-5) using NCI’s cell screening protocol, sulforhodamine B assay. The results
shown in Figure 3 indicate significant cell death.
Figure 3. Effect of palladium α-lipoic acid complex formulation on (1) Breast cancer (adenocarcinoma,
MCF-7), (2) brain tumor (stage IV glioblastoma multiform, U-251MG), (3) lung (non-small cell
carcinoma, A-549), (4) brain (astrocytoma, H-4), and (5) ovarian cancer (OVCAR-5).
Whether we use [3H] thymidine uptake assay or suforhodamine B assay, signifi-

cant reduction in cell growth is observed in a variety of cell lines.
In vivo Studies of Palladium α-Lipoic Acid Complex Formulation

These studies were carried out at Calvert Labs (previously known as Pharmakon
USA), PA. The Ames/Salmonella Plate incorporation assay confirmed that the com-
plex formulation is free of mutagenicity. Also acute oral toxicological studies showed
no accumulation in or damage to any tissues. The median lethal dose, LD50, in mice
was found to be greater than the highest dosage tested, 5000 mg kg–1.
Figure 4. Glioblastoma tumor volume in nude mice, oral dose of vehicle (0.9% saline), 0.5 mg, 1
mg, and 2 mg palladium lipoic acid formulation per mouse daily for 4 weeks. To reduce clutter only
one half of the error bar is shown.
The effectiveness of palladium lipoic acid formulation in halting the growth of

glioblastoma cells in vivo was studied using nude mice. On day zero, 10 million
U-87 MG tumor cells (glioblastoma-astrocytoma, human) from American Type
Culture Collection (ATCC) were injected subcutaneously in the scruff of the neck
of female Swiss nude mice (11 weeks old). When the tumors reached 200–400
mm3 in volume, the mice were divided into 8 groups of 10 mice. Four groups
of mice were given daily intravenous (i.v.) doses of this formulation or placebo
(0.9% saline); four groups were given orally by gavage (p.o.) doses of 0.5, 1.0,
or 2.0 mg palladium lipoic acid complex per mouse for a total of 4 weeks or until
the tumors became too large for the viability of the animal. Tumor volume was
measured throughout the study, twice per week. The results are given in Figures
4 and 5.
Figure 5. Glioblastoma tumor volume in nude mice, intravenous dose of vehicle (0.9% saline), 0.5
mg, 1 mg, and 2 mg palladium lipoic acid formulation per mouse daily for 4 weeks. To reduce clutter
only one half of the error bar is shown.
A reduction in tumor size compared to the placebo treated group was seen in all
groups of mice treated orally with the palladium lipoic acid complex. However the
only statistically significant reduction was in the group treated with 1 mg/mouse.
When mice were treated intravenously with the palladium lipoic acid complex, a
statistically significant reduction was observed in all treatment groups compared to the
placebo group.
Clinical Veterinary Studies

The largest integrative cancer investigation of palladium lipoic acid complex formu-
lation was an open-label, veterinary oncology program at CVS Angel Care Cancer
Center, San Diego, CA, USA, with over 900 dogs enrolled. The dogs received pal-
ladium lipoic acid complex formulation as part of their chemotherapy, radiation and/
or surgical protocol at a dosage of 1ml/2.3kg. p.o. twice daily (equivalent human dose
of approximately 40 ml per 70 kg.). The palladium lipoic acid formulation seemed
most effective in the cases of solid tumors (such as soft tissue sarcoma, hemangiosar-
coma, mast cell, transition cell carcinoma, lung, anal sac carcinoma, renal carcinoma,
squamous cell carcinoma, fibrosarcoma, melanoma, meningioma, neuroblastoma, and
mammary adenocarcinoma). Some of the most effective findings were apparent in
the dogs suffering from osteosarcoma. The etiology of osteosarcoma in large dogs is
considered identical to the disease progression in humans. While in canines the “stan-
dard of care” is limb amputation followed by chemotherapy, in human patients, limb–
sparing surgery following tumor excision is performed (Ogilvie and Moore, 2000).
The results summarized in Table 10 suggest the following. In this open labeled study,
integrative palladium lipoic acid complex formulation support improved the animals’
median survival time 62% (103 days more) compared to surgery alone (n = 11 and 162
respectively).
Table 10. Open label veterinary oncology study using Palladium α-Lipoic acid complex formulation.
Study Median Survival, days

Amputation alone (n = 162) 165
Amputation with palladium
lipoic acid complex formulation
(n = 11) 268
Amputation with chemotherapy
(n = 32) 288
Amputation with chemotherapy +
palladium lipoic acid complex
formulation (n = 17) 367
When the palladium lipoic acid complex formulation was added to the chemother-
apeutic regimen (carboplatin + doxorubicin) the dogs exhibited a 27% longer median
survival (79 days more). Furthermore, there was no significant difference (p = 0.30)
in median survival time between dogs treated with amputation + palladium lipoic acid
complex formulation versus those that were treated with amputation + the “standard
of care” chemotherapy.
It was observed that following palladium α-lipoic acid complex formulation com-
plementary support, chemotherapeutic animals’ demonstrated improvements in vari-
ous objective parameters (i.e., weight, anemia, liver and kidney function). In addition
to these enhanced clinical parameters, a subjective owner quality of life survey result-
ed in an 86% improvement following the addition of palladium α-lipoic acid complex
formulation adjunctive support.
Transient Ischemia Studies with Gerbils

Animal studies, carried out at Stony Brook University using adult male Mongolian
gerbils (Charles River, Inc., New York), used as controls or treatment group, demon-
strated that acute, post ischemic and prophylactic administration of palladium α-lipoic
acid complex formulation limits ischemic damage. The animals were sacrificed after
72 hr after transient ischemia surgery (n = 6 per surgical group; n = 6 per sham group,
each trial in triplicate) (Antonawich et al., 2004). The palladium α-lipoic acid complex
formulation was administered intraperitoneally (IP) immediately after surgery, then
once daily for 3 days. The control group received saline while the treatment group
received 30, 50 or 70 mg/kg of palladium lipoic acid formulation.
Selective neural damage to hippocampal cornus ammon’s field 1 (CA1) of the
hippocampus neurons takes place after transient ischemic attack. An activation of
the pro-apoptotic protein, bax, results in a shift in the dimerization ratio between the
anti-apoptotic protein bc1-x1 and bax. The increase in bax results in the formation
of pores in the mitochondrial membrane and these pores facilitate the passage of the
electron transport chain protein, cytochrome c, into the cytosol along with the release
of the pro-caspase 9. The initiator caspase 9 activates the caspase family of cysteine
proteases and results in the destruction of the cell.
Following bilateral carotid artery occlusion in the Mongolian gerbil, palladium
lipoic acid complex formulation treatment significantly protected CA1 hippocampal
pyramidal cells from transient global ischemia at 30 (p < 0.05), 50 (p < 0.01), and 70
(p < 0.05) mg/kg per 24 hr.
A delayed application of the palladium α-lipoic acid complex formulation after 48
hr of ischemic attack had no significant effect in protecting CA1 cells. On the other
hand, a delayed administration of palladium α-lipoic acid complex formulation after
6 hr of ischemic attack was as good as giving it immediately after ischemic attack in
minimizing cell death.
Nesting behavior is an inherent behavior in Mongolian gerbils. Five minutes of ca-
rotid artery occlusion was sufficient to hinder or impair nesting behavior for approxi-
mately 3 days. The nesting behavior of gerbils was observed to improve significantly
after treatment with palladium lipoic acid complex formulation (50 mg/kg every 24 hr
(P < 0.05) and 30 mg/kg/24 hr at 24 and 72 hr after ischemia. There were no significant
differences after the 70 mg/kg/24 hr treatment (n = 6 per group, each experiment was
conducted in triplicate). The 70 mg/kg treated animals demonstrated excessive energy,
thus ignoring the nesting material.
It was observed that preventive or prophylactic treatment with 10 mg/kg gerbil
(based on allometric scaling from rodent to human, 10 ml human dosage) offered sig-
nificant behavioral and morphological improvement from transient global ischemia.
While behavioral improvement was apparent with 3 days of pretreatment, approxi-
mately one week of pre-treatment was necessary for morphological rescue.
In summary, treatment with palladium α-lipoic acid complex formulation after a
transient ischemic attack offers behavioral improvement as well as morphological pro-
tection of CA1 hippocampal pyramidal cells.
Clinical Human Studies

A Phase I, palladium α-lipoic acid complex formulation “dose escalation safety study
in normal individuals” (DESSTINI) was carried out at Stony Brook University, New
York, USA. This study was divided into three tiers, each consisting of five subjects.
Tier I, Tier II, and Tier III received oral dosages of the formulation, 10, 20, and 40 ml/
day respectively for a period of 6 weeks. Subjects were monitored for the washout of
palladium by examining the concentration of palladium in both blood serum and urine.
Washout periods ranged from three to 17 weeks after cessation of the formulation.
However, the washout period did not appear to be related to dosage.
No serious adverse effects occurred. The 15 subjects experienced a total of 24
AE (Adverse Events) during the study that was considered potentially, possibly or
probably related to the study formulation. The events included: fatigue after cessation
of oral dosage, diarrhea, worsening leg cramps, headache, increased urination, light-
headedness, difficulty sleeping, and increased excitement. All AEs were adjudicated
by the Data Safety and Monitoring Board (DSMB), with approximately 66% being
anticipated or considered mild. Overall, the tolerability of all three tiers was 93.3%
and the DSMB deemed the formulation to be safe. In addition, DSMB gave consent to
continue with a subsequent, ongoing glioblastoma trial.
Mitochondrial Studies using Palladium α-Lipoic Acid Complex Formulation

The results with the transient ischemia studies with gerbils prompted us to investigate
the influence of palladium lipoic acid complex on the activities of enzymes involved
in energy production. In eukaryotes and prokaryotes, the most common mode of oxi-
dative degradation of carbohydrates, fatty acids, and amino acids is by the citric acid
cycle or the tricarboxylic acid cycle or Krebs cycle. The net reaction is
3NAD+ + FAD + GDP + Pi + acetyl-CoA → 3NADH +FADH2
+GTP + CoA +2CO2 (40)
The liberated energy is used for ATP generation. The influence of palladium li-
poic acid complex formulation has been investigated on the activities of four Krebs
cycle enzymes, isocitrate dehydrogenase (ICDH), α-ketoglutarate dehydrogenase
(α-KGDH), 6) succinate dehydrogenase (SDH), and 8) malate dehydrogenase (MDH).
The other 4 enzymes of the Krebs cycle, citrate synthase, aconitase, succinyl-CoA
synthetase, and fumarase as well as the enzyme, pyruvate dehydrogenase were not
investigated. These investigations were carried out Amala Cancer Research Centre,
Kerala, India (Sudheesh et al., 2009; 2010).
Table 11. Effect of Palladium Lipoic Acid Complex on the Activity of Krebs cycle Enzymes (Sudheesh
et al., 2009).
Groups ICDH α-KGDH SDH MDH
Aged control 702.8±133.4 63.0±15.1 42.4±14.2 260.9±26.1
DL-α-lipoic acid 3428.2±348.9 189.0±50.4 73.4±21.2 386.1±265.5
(5 mg/kg body mass)
Palladium lipoic acid complex, 3483.1±388.9 145.0±50.6 98.6±7.4 1305.7±56.4
(0.38 mg/kg body mass)
Units: ICDH–µ moles of NAD + reduced/min/mg protein; α-KGDH–µ moles of NAD + reduced /min/mg protein;
SDH–µ moles of 2,6-dichlorophenol indophenol sodium salt (DCPIP) reduced /min/mg protein; MDH–µ moles of
NADH oxidized/min/mg protein
Table 12. Effect of Palladium Lipoic Acid Complex on the Activity of Respiratory Complexes
(Sudheesh et al., 2009).
Groups Complex I Complex II Complex III Complex IV
Aged control 23.34±2.12 26.75±2.09 13.57±3.89 30.85±1.31
DL-α-lipoic acid 62.04±11.90 45.55±28.25 21.16±8.36 47.36±7.54
(5 mg/kg body mass)
Palladium lipoic acid complex, 58.76±31.11 83.37±28.46 21.34 ±3.31 48.13±7.32
Units: Complex I–µ moles of DCPIP reduced/min/mg protein; complex II–µ moles of DCPIP reduced/min/mg protein;
Complex III–µ moles of ferricytochrome-C reduced/min/mg protein; Complex IV–µ moles of ferrocytochrome-C
oxidized/min/mg protein.
Male albino rats of Wistar strain and 24–26 months old were used to study their
hearts. Each group had six rats and the animals were sacrificed at the end of 30 days
of oral administration. The results for the Krebs cycle enzymes are given in Table 11.
The activities of ICDH, α-KGDH, SDH, and MDH, when compared to the aged
control animals, indicate that administration of palladium lipoic acid complex formu-
lation significantly increased the Krebs cycle enzyme activities. Both the α-lipoic acid
and palladium lipoic acid complex increased the activities of the enzymes.
The results for the respiratory complexes I, II, III, and IV in aged rats is given
in Table 11. The enhanced activities of complexes I, III, and IV were very similar
for both the palladium α-lipoic acid formulation and the α-lipoic acid administered
groups. The average values indicate a ~2.5-, 1.6-, and 1.6-fold increase for the ac-
tivities of the complexes I, III, and IV when compared to the aged control group. In
the case of complex II, the palladium α-lipoic acid complex formulation group had
~1.8-fold increases in the activity compared to the α-lipoic acid administered group.
Sice the actual α-lipoic acid equivalent in the metal complex used for the oral admin-
istration is 13.2 times less than that of the ligand, we are tempted to conclude that the
palladium α-lipoic acid complex is far superior to α-lipoic acid.
The major question which is a still an unsolved puzzle is the source of the superior-
ity of the metal complex compared to that of the ligand. Since, we know from the phar-
macokinetics of α-lipoic acid that the oral dose and the available plasma concentra-
tion are completely different, it is possible that the available plasma concentration for
therapeutic effect in the presence of the palladium α-lipoic acid complex may be much
higher than that of the α-lipoic acid only. Or somehow the chemistry of the transition
metal is playing a dominant role in the enzymatic activity. It is interesting to note that
the 2010 Noble Prize in chemistry was awarded to three palladium chemists, Richard
F. Heck, Ei-ichi Negishi, and Akira Suzuki for “palladium-catalyzed cross-couplings
in organic synthesis”. While their methods are used by pharmaceutical industry for the
synthesis of at least 25% of drugs, it is ironic that no palladium-based drugs are avail-
able in the market today. However, a palladium α-lipoic acid complex formulation had
been available in the market for more than 15 years as a dietary supplement. The start-
ing material in the synthesis of the palladium α-lipoic acid complex is palladium(II).
If the final complex is also palladium(II), the chances are it has no paramagnetism
because almost all palladium(II) complexes are diamagnetic. An ESR/EPR spectrum
would help solve this puzzle. It is also possible, that under physiological conditions,
if dihydrolipoic acid is produced through 1e reduction processes, then it is possible to
have reactive intermediates with unpaired electrons. In such a case the electron spin
may be involved in the enzymatic process. The impedance characteristics of the pal-
ladium α-lipoic acid as well as that of the α-lipoic acid, described later in this review,
strongly suggest this possibility.
Male albino Wistar strain rats, both young and old, were also used to examine the
declined mitochondrial antioxidant status in the myocardium of aged rats. The animals
were administered orally for 30 days 0.38 mg lipoic acid and an equivalent dose of
lipoic acid from the palladium lipoic acid complex formulation. The results for Mn
SOD, CAT, and GPx are given in Table 13. As expected the young group had higher
levels of all the three enzymes than that of the aged control group. Also the levels of
Mn SOD, CAT, and GPx were higher with the palladium lipoic acid treated group than
with the α-lipoic acid group.
Table 13. Effect of Palladium Lipoic Acid Complex on the Activities of Enzymes in the Heart
Mitochondria of Rats (Sudheesh et al., 2010).
Groups Mn SOD, CAT GPX
U/mg protein U/mg protein U/mg protein
Aged control 12.23±2.33 4.05±0.82 22.70±4.24
Young control 16.34±1.17 9.61±1.17 73.24±20.65
DL-α-lipoic acid 15.59±5.31 8.26±1.48 168.58±63.74
(0.38mg/kg body mass)
Palladium lipoic acid complex, 12.72±5.94 4.81±1.34 64.19±15.50
0.38 mg/kg body mass)
Mn SOD = manganese superoxide dismutase; CAT = catalase; and GPx = glutathione peroxidase
The results of the lipid peroxidation levels measured as thiobarbituric acid reacting
substance (TBARS) and expressed as equivalents of MDA and the glutathione levels
are given in Table 14.
Table 14. Lipid Peroxidation and GSH Level in the Heart Mitochondria of Aged Rats (Sudheesh
et al., 2009).
Groups Lipid peroxidation, GSH (moles/mg protein)

n moles of MDA formed/mg protein
Aged control 1.94±0.27 5.08±0.39
Young control 0.88±0.06 6.78±0.45
DL-α-lipoic acid 1.68±0.19 5.20±0.18
(0.38mg/kg body mass)
Palladium lipoic acid complex, 1.17±0.09 6.42±0.35
As expected, the lipid peroxidation level was less and the glutathione level was
higher in the young control compared to that of the aged control. There was no signifi-
cant different difference between the aged control group and the α-lipoic acid group
for both the lipoic peroxidation level and glutathione level. However, the lipid per-
oxidation was less and glutathione levels were higher with the palladium lipoic acid
complex formulation group when compared to the aged control group.
The Krebs cycle and mitochondrial respiratory chain enzymatic studies data also
indicate that the palladium lipoic acid complex is catalytically more active than that
of the ligand, α-lipoic acid. This is similar to the observations of antitumor activity of
iron (III) and copper (II) complexes of N6-benzylaminopurine derivatives and palladium
(II) –benzyl bis (thiosemicarbazonate) where the complex had more activity than the
ligand (Maloň et al., 2001; Matesanz et al., 1999).
Figure 6. Influence of palladium α-lipoic acid complex formulation on Krebs cycle enzymes and
mitochondrial electron transport chain complexes.
The influence of palladium α-lipoic acid complex formulation on the activities of

some of the Krebs cycle enzymes and mitochondrial respiratory enzymes are summa-
rized in Figure 6. The percentage increase in enzymatic activities are indicated by an
upward arrow. The CoQ is pictorially shown twice for convenience to show that the
electron transfer from complex I and complex II are to CoQ and then to complex III.
It is not clear at this time whether the antioxidant properties of the palladium
α-lipoic acid complex formulation has anything to do with its enhancement of Krebs
cycle and mitochondrial enzyme activities. Since, α-lipoic acid also enhances the
activities of these enzymes but to a much less extent than palladium α-lipoic acid
complex formulation, we are tempted to assume an interconnection between the two.
It suggests that scavenging some free radicals by either α-lipoic acid or palladium
α-lipoic acid complex formulation results in better performance of Krebs cycle and
mitochondrial enzymatic activities.
It should be mentioned that succinate dehydrogenase which is part of the Krebs
cycle as well as Complex II is known to be a tumor suppressor (Frezza and Gottlieb,
2009). The enhanced activity of this enzyme in the present studies correlates well
with the inhibition of the growth of tumor cell lines studied. It is also suggested that
the inhibitory effects of increased succinate due to mutated succinate dehydrogenase
can be overcome by increased α-ketoglutarate. The activity of α-ketoglutarate is also
increased substantially by palladium α-lipoic acid formulation.
In cancer cells lactate dehydrogenase and pyruvate dehydrogenase kinase are over
expressed. Inhibition of lactate dehydrogenase by oxamate as well as suppression of
pyruvate dehydrogenase kinase by dichloroacetate have been found to stimulate mi-
tochondrial ATP production. Stimulation of mitochondrial oxidative metabolism has
also been found to inhibit growth of cancer cell lines (Gogvadze et al., 2008). Even
though the upregulation of rate limiting steps of glycolysis, the accumulation of muta-
tions in in the mitochondrial genome, the hypoxia induced switch from mitochondrial
respiration to glycolysis and the metabolic reprogramming resulting from the loss of
function of fumarate hydratase, succinate and isocitrate dehydrogenases (Kroemer,
2006; McKnight, 2010) are not yet well understood, our Krebs cycle and mitochon-
drial enzyme activities data as well as the in vitro and in vivo cancer cell death data
with palladium α-lipoic acid complex formulation support the notion that promotion
of Krebs cycle and mitochondrial oxidative phosphorylation is a good approach for
inhibiting cancer cell growth promoting wellness.
Protection from Radiation using Palladium α-Lipoic Acid Complex

Formulation
These studies were also carried out at Amala Cancer Research Centre, Kerala,
India (Menon et al., 2009; Ramachandran et al., 2010). In vivo radioprotection of
cellular DNA was investigated using 6–8 weeks old Swiss albino mice exposed
to 8 Gy radiation from 60Co at a dose rate of 1.88 Gy per min. The palladium
lipoic acid complex formulation dose (oral), administered 1 hr prior to radiation
exposure, was 1ml/kg and 2 ml/kg body mass for two different groups. After 1
hr, the animals were sacrificed and their blood leukocytes and bone marrow were
examined for DNA damage using alkaline single cell gel electrophoresis (alkaline
comet assay) and compared with those with sham irradiation and with distilled
water as control. The comet parameters, DNA in tail, tail length, tail moment and
olive tail moment were analyzed. All the comet parameters were significantly re-
duced in animals administered with the palladium lipoic acid complex formulation
1 hr prior to the radiation exposure. This significant reduction in DNA damage in
mice receiving palladium lipoic acid complex formulation with doses of 1ml/kg
and 2 ml/kg body mass demonstrates the in vivo radioprotection ability of the pal-
ladium lipoic acid complex.
Antioxidant Activity, Prophylactic Effects of Palladium α-Lipoic Acid Complex

Formulation Determined from Radiation Experiments
The antioxidant activity of palladium lipoic acid formulation was examined using 4
groups of Swiss albino mice, 6–8 weeks old, one group receiving distilled water and
the other group palladium lipoic acid complex formulation, 2 ml/kg body mass. Two
other groups similar to the earlier ones received radiation exposure, 6 Gy at the rate of
1.88 Gy per minute. The animals were sacrificed after 7 days of administration, and
then radiation to two groups. Liver, kidney and brain were examined for lipid peroxi-
dation, glutathione, super oxide dismutase, and glutathione peroxidase.
Table 15. Antioxidant Activity of Palladium α-Lipoic Acid Complex Formulation in Liver, Kidney and
Brain of Mice (Menon et al., 2009).
Groups Lipid peroxidation, GSH (nanomoles/ Super oxide Gutathione
nano moles of MDA mg protein dismutase Peroxidase
formed/mg protein U/mg protein U/mg protein
(1) Liver
With radiation 4.65±0.86 15.28± 1.73 8.36±0.63 14.07±2.87
With complex and 1.82± 0.36 21.46±3.30 10.90±0.50 22.44± 2.40
with radiation
(2) Kidney
With radiation 8.62±0.76 21.19±7.25 0.45±0.09 24.48±2.30
With complex and 5.44±0.98 42.61±4.61 0.98±0.29 39.28±10.28
with radiation
(3) Brain
With radiation 16.94±2.04 55.60±14.58 0.68±0.10 32.68±2.90
With complex and 11.42±0.79 126.81±9.43 1.08±0.09 44.10±2.90
with radiation
6 Gy radiation, 7 days palladium lipoic acid complex formulation dose before irradiation, 2 ml/kg
body mass
The differences between the distilled water group and the palladium lipoic acid
treated group were not statistically significant for all the enzymes studied. On the
other hand the data given in table for the group that received water and the group that
received 2ml/kg body mass palladium lipoic acid complex formulation for 7 days prior
to 6 Gy irradiation indicate remarkable differences in each case. The glutathione, glu-
tathione peroxidase, and superoxide dismutase levels were higher and the lipid peroxi-
dation levels measured as TBARS and expressed as equivalents of MDA were lower
in liver, kidney and brain for the group that received palladium lipoic acid complex
formulation for 7 days prior to 6 Gy irradiation. This clearly indicates the prophylactic
protective effect of palladium lipoic acid complex formulation from radiation. It was
also observed that 6 Gy radiation significantly reduced GSH, GPx, and SOD levels
and increased the lipid peroxidation levels compared to the controls that received no
radiation.
It should also be mentioned that the over expression of superoxide dismutases
in tumor cells has been found to reduce malignant features of cancer cells such as
tumor cell growth and metastasis (Lázaro, 2007). It is not clear whether these antican-
cer effects are due to the catalytic conversion of O2– to H2O2 or due to the increased
concentration of H2O2. The reversal of this effect by over expression of catalase and
glutathione peroxidase supports the concept of increased levels of H2O2.
Radiation induced significant lowering of antioxidant levels. Administration of
palladium lipoic acid complex formulation for 7 days, at a dose rate of 2 ml/kg body
mass, kept nearly the same levels of antioxidants as the ones that received no radiation.
It is not clear at this time whether this observation is the due to the ability of the tissues
to counteract the ROS generated from radiation injury or the ability of the tissue to
regenerate the cellular antioxidants in response to radiation injury.
Oral administration of palladium lipoic acid complex formulation to male Balb/C
mice, 6–8 weeks old, exposed to sub lethal 6 Gy γ-radiation enhanced endogenous
spleen colony formation. Also alkaline comet assay showed that nuclear DNA comet
parameters such as percent DNA tail, tail length, tail moment, and olive tail moment
of the bone marrow and spleen cells increased after the whole body radiation of 8 Gy.
These DNA damages as well as mortality rates were reduced by the administration of
palladium lipoic acid complex formulation. Also it aided in the recovery from radia-
tion induced weight loss in mice surviving after 8 Gy radiations.
These studies, carried out at Amala Cancer Centre coupled with the toxicity and
cell line studies suggest the following. Unlike toxic platinum chemotherapy agents,
the commercially available palladium α-lipoic acid complex formulation is safe and
nontoxic. Its oral administration can be continued indefinitely. It is specially designed
to provide energy for compromised body systems and promote overall health. It fa-
cilitates aerobic metabolism much more than that of α-lipoic acid, by significantly
enhancing enzymatic activity of isocitrate dehydrogenase, α-ketoglutarate dehydro-
genase, succinate dehydrogenase and malate dehydrogenase at the Krebs cycle and
mitochondrial complexes I, II, III, and IV of the electron transport chain in the heart
of aged rats (Sudheesh et al., 2009). It also enhances the activities of catalase and glu-
tathione peroxidase more than that of α-lipoic acid. The level of glutathione also was
significantly improved and the level of lipid peroxidation was decreased in the heart
mitochondria of aged rats (Sudheesh et al., 2010). It also protects DNA from radiation.
It must be pointed out that preventive or prophylactic effects observed in these
radiation experiments are consistent with the observations of similar effects in gerbils
after induction of transient global ischemia (Antonawich et al., 2004).
DIODE OR TUNNEL-DIODE BEHAVIOR IN BIOLOGICAL SYSTEMS

To gain an understanding of the electronic aspects of biological functions such as cell
signaling, long-range electron transfer, and biochemical oscillations in ATP produc-
tion (Field and Gyorgyi, 1993), one needs at least a rudimentary knowledge of the
solid state electronics. A very brief attempt is made here to introduce the concepts.
A diode material is normally doped with one impurity atom per 10 million semi-
conductor atoms. This results in a relatively wide depletion region. When the potential
applied is large enough to overcome the potential barrier of the junction, conduction
takes place (Fink, 1975). In a tunnel diode, on the other hand, the doping level is
about thousand impurity atoms per ten million semiconductor atoms. This results in an
extremely narrow depletion region. Compared to a normal junction diode, the tunnel
diode exhibits an unusual current-voltage characteristic curve, a negative differential
resistance (NDR) region (Fink, 1975).
Protein film voltammetry data (Ackrell et al., 1993; Elliot et al., 2002; Gwyer et
al., 2005; Hurst et al., 1996; Léger and Bertrand, 2008; Pershad et al., 1999; Sucheta
et al., 1992; Van Hellemond et al., 1995) have indicated that the catalytic activity of
enzymes, especially electron transport enzymes involved in the respiratory chains,
may be optimized at certain electrochemical potentials as well as pH. This technique

allows the rate of catalysis to be measured accurately as a function of the applied
potential (the driving force). Interesting current-potential curves were observed for
several enzymes in which the optimum rate occurs at a particular potential and the rate
thereafter drops in spite of the increase in the thermodynamic driving force. We must
keep in mind that the active site, such as in flavins and Mo-bismolybdopterin guanine
dinucleotide cofactors, may be the oxidized state, intermediate state or reduced state
and these states may have their own characteristic affinities for the substrate before the
reaction and for the product after the reaction. This offers the possibility for catalytic
pathway by several different routes (Léger and Bertrand, 2008). The electrochemical
potential controls the rate and thermodynamics of electron supply for the different
states. A unique current-potential curve, often observed for respiratory enzymes such
as succinate:ubiquinone oxidoreductase (complex II of mitochondria, with active site
FAD and three Fe-S clusters)) and molybdoenzyme nitrate reductases, is interpreted in
terms of a “potential dependent gate that bars catalysis of the reverse process” (Léger
and Bertrand, 2008). This intrinsic property of the enzyme is similar to the behavior
of a tunnel diode with a characteristic NDR region observed in the current potential
curves. In SDH, the group responsible for this behavior is attributed to the active site
FAD. Similar to the electrochemical potential, the membrane provides a variable po-
tential in the form of the ratio of quinone to hydroquinol (Q/QH2). This potential can
be further tuned depending on the nature of the quinone present. At least three differ-
ent quinones (ubiquinone being the dominant redox carrier during aerobic growth and
menaquinone and demethylmenaquinone dominating under anaerobic conditions) are
synthesized depending on the aerobicity.
An attenuation of the reductive activity at low potential was also observed for the
mitochondrial enzyme, the “Fp” subcomplex of Complex I. This is understandable
because, mitochondrial complex I (NADH-Ubiquinone oxidoreductase) also houses a
flavin at the site of NADH oxidation and nine iron-sulfur clusters.
Our reason for including this section on tunnel diode behavior in biological sys-
tems is to indicate the “ratchet” or biased nature of enzymes systems depending on the
potential and pH allowing possible feedback fine control of respiratory rates. We have
demonstrated the ability of an enzyme to “rectify” electron flow at potentials close
to electrochemical reversibility by studying impedance characteristics of simple bio-
logical molecules that are an integral part of the enzyme. For example, some of these
enzymes have FAD, and Mo and we had shown in impedance studies the NDR char-
acteristics of FAD as well as Mo-peroxo complexes (Krishnan and Garnett, 2006).
IMPEDANCE SPECTROSCOPY
We have utilized the technique of impedance spectroscopy for understanding solute-
solvent interactions, ‘π–way’ conduction, ion pair formation, water-structure enforced
ion pair formation, potential induced and solvent mediated ion pair formation at the
double layer, and semiconduction characteristics of simple biological molecules
(Krishnan and Garnett, 2006; Krishnan et al., 2007a, 2007b; 2008a, 2008b, 2008c,
2008d; 2009a, 2009b). Simple molecules such as arginine, histidine, lysine, FAD,
riboflavin, cysteine, lidocaine hydrochloride, α-lipoic acid, and hydrogen peroxide

exhibit negative differential resistance, which is a characteristic of diode or tunnel
diode behavior. This technique has not been utilized extensively for discovery of drug
molecules. Our technique of exploring drug discovery is based on impedance char-
acteristics and self assembly and differs from the conventional drug discovery tech-
niques. A brief outline of this technique (Lasia, 1999; Macdonald and Johnson, 2005)
is given below.
In impedance measurements, a perturbing sinusoidal voltage E = E0sin(wt ) is ap-
plied at angular frequency w (2π f, where f in the conventional frequency in Hz) to
the electrode system. The response is analyzed in terms of the resultant current I =
I0sin(wt + Ф), where Ф represents a characteristic phase angle shift. The correspond-
ing complex impedance spectrum Z(w ), obtained by varying the signal frequency w,
is expressed in terms of the displacement of the vector Z(w). In the plane of Cartesian
coordinates, an impedance is expressed by its real (Z′) and imaginary (Z′′) parts, that
is Z(w) = Z′ – jZ′′. The modulus │Z│and phase angle Ф of Z(w) can be obtained from
│Z│= [Z′2 + Z′′2]1/2 and Ф = tan–1 [Z′′/Z′], respectively. Over a frequency bandwidth of
interest, the impedance spectrum can be represented in various ways; typically in the
well known Nyquist or Cole-Cole plot (Z′′ as the Y-axis and Z′ as the X-axis for the
range of frequencies explored at a fixed potential) or Bode plots (│Z│and Ф vs. logw).
The impedance spectrum reflects dialectic behavior, oxidation-reduction reactions and
mass migration across the electrochemical interfaces, that are determined by the elec-
trical and chemical properties of the corrosive medium, and the electrode materials.
The impedance spectrum can also be considered as a “fingerprint”, which is related
to the transient behavior of a specific electrochemical interface. In simple terms, im-
pedance is like a frequency dependent generalized resistance. In electrochemistry, the
imaginary impedance is almost always capacitive and therefore negative. Phase angle
is a balance between capacitive and resistive components. For a pure resistance Ф = 0
and for pure capacitance Ф = π/2.
The electrochemical impedance measurements reported in this chapter were made
using and EG & G PARC Model 303A SMDE trielectrode system (mercury working
electrode, platinum counter electrode and Ag/AgCl saturated KCl reference electrode)
along with Autolab ecochemie. The measurements were carried out in the range 1,000
Hz to 30 mHz. The amplitude of the sinusoidal perturbation was 10 mV.
In our present studies we have explored the behavior of mercury in both the nega-
tive and positive range of potentials because in natural biochemical systems we have
both positively and negatively charged surfaces at close distances where water mole-
cules will be subjected to competing influences from the electric field of these charged
centers as well as the charges from the electrolytes. We have used mercury as the
working electrode because it allows us to get reproducible surface for our studies by
using a fresh drop each time. Compared to using any other metal, fresh mercury drops
allow repetitions and reproducibility better and easy. For example if we corrode a
metal, it is not easy to clean and get the noncorroded surface again and again for each
experiment.
Electronic Properties of H2O2

All living systems exhibit dynamical spatio-temporal periodicities (Field and Gyor-
gyi, 1993). The dynamical oscillations observed in the electrochemical passivation
of metals and used as models for biological oscillations are attributed to the negative
Faradaic impedance of the electrode (Koper, 1996).
Figure 7. Nyquist plot for 0.10 M KCl, 88 mM hydrogen peroxide at pH 4.5.
Potential or current oscillations of different types have been observed in hy-

drogen peroxide systems at high concentrations and high acidities (Mukouyama
et al., 1999; 2001). The coupling of two or more chemical oscillations occurring at
different locations, an important aspect for signal transport or communication in
biological systems has also been duplicated in an electrochemical system involv-
ing hydrogen peroxide (300–400 mM in 0.5 M H2SO4) (Fukushima et al., 2005). To
simulate biological systems, we had focused our studies on oscillatory behavior at
low concentrations of peroxide and at low acidities. We had established and reported
(Krishnan et al., 2008d) the concentration range needed to exhibit oscillations in the
presence of NaCl.
Figure 8. Admittance comparison of 0.10 M NaCl, pH 5.25 1) 500 Hz 2) 250 Hz 3) 100 Hz and 0.10
M NaCl and 88 mM H2O2, pH ~ 6.0 4) 500 Hz 5) 250 Hz 6) 100 Hz.
Here we report, as an example, the impedance behavior of 88 mM H2O2 in 0.10 M

KCl at pH 4.5. It is obvious that negative differential resistance (NDR), a character-
istic of tunnel diode behavior is observed at the potentials shown in Figure 7. It was
observed that NDR occurred at 3.72 Hz, 2.90 Hz, and 2.24 Hz at potentials of –0.36
V, –0.37 V, and –0.38 V.
We had carried out detailed cyclic voltammetric and impedance investigations of
aqueous solutions of H2O2 in the presence of NaCl to understand Parkinson’s disease
(Krishnan et al., 2008c, 2008d). Some relevant points from these studies of hydrogen
peroxide in aqueous sodium chloride are illustrated here
The negative impedance observed in Figure 7 is also seen at hydrogen peroxide
concentrations as small as 10 mM and is sensitive to pH. At higher acidities, the po-
tential at which NDR occurred shifted to more anodic potentials. Also, as the hydrogen
peroxide concentration is decreased, the potential at which NDR occurred shifted to
more anodic potentials. The frequency at which NDR occurred also depended on the
potential and concentration of hydrogen peroxide. We have also observed sensitivity
for chloride.
The importance of impedance measurements in understanding solute-solvent in-
teractions is vividly illustrated in Figure 8. By comparing the admittance measurements
of NaCl in the presence and absence of H2O2 at 3 different frequencies, it is obvious

that the sodium and chloride ions are forming ion pairs under the influence of H2O2 at
potentials in the range –0.25 to 0.0V. This results in a decrease in admittance.
To understand the admittance behavior of small amounts of H2O2 in 100 mM
NaCl, it is worthwhile to look at its relevant aqueous solution properties. The activ-
ity coefficient of hydrogen peroxide in sodium chloride and sodium sulfate solutions,
determined using partition experiments with iso-amyl alcohol (Livingston, 1928),
was found to be less than unity. This salting in effect was later confirmed for several
electrolytes (Gorin, 1935). These results suggest that water molecules surrounding
the sodium ions were displaced by peroxide. This was attributed to the higher dipole
moment of hydrogen peroxide compared to that of water. On the other hand, it was
concluded from solubility measurements in hydrogen peroxide-water mixtures that
smaller ions such as Li+ and Na+ were solvated by water and K+, Rb+, and Cs+ were
preferentially solvated by hydrogen peroxide (Everhard et al., 1962).
The properties such as (1) deviations from Raoult’s law, (2) finite heat of mixing
and (3) finite volume changes on mixing for hydrogen peroxide-water mixtures indi-
cate an enhancement of either the number or the force of attractions between the two
molecules on forming the solutions (Everhard et al., 1962). The decrease in conduc-
tance of alkali chlorides in hydrogen peroxide-water mixtures correlated well with the
changes in viscosity. These results could not give any indication as to when the solva-
tion of an ion changes from water to hydrogen peroxide (Thomas and Maass, 1958).
It should be mentioned that the studies mentioned above were not at low hydrogen
peroxide concentrations. The low concentrations employed in admittance measure-
ments are comparatively low and closer to biological concentrations. However, the
data are more complicated because of the double layer and changing potentials as
well as frequencies. The results do suggest an ion-dipole interaction with peroxide
preferentially, especially when the applied potential is about to change from negative
to positive. We had explained these results using the concept of “potential induced and
peroxide mediated ion pair formation” (Krishnan et al., 2008d).
These indicate the role of peroxide not only in neuron degeneration but also in
controlling of electronic circuits involved in neuronal communications. The role of
the stimulator implants seems to be to counteract the role of the new circuits caused
by neuronal degeneration. Also the variability of the electronic circuit or signaling
produced by hydrogen peroxide depending on the concentration, the ion-dipole inter-
action with water and/or peroxide, and the potential available may partially explain the
multiple roles of hydrogen peroxide in biological systems.
Before closing this section, we want to add a comment on another related topic,
“mechanisms for DNA charge transport”. This hot subject has been investigated exten-
sively during the last 15–20 years (Genereux and Barton, 2010). Numerous arguments
regarding the nature of DNA as to whether it is a wire, a semiconductor or an insulator
have been proposed and discussed. The role of the π-stacked base pairs in DNA has
also been investigated in detail. In our impedance measurements with aqueous lido-
caine hydrochloride, we could see the influence of “π-way” conduction (Krishnan et
al., 2009a). This technique has not been explored in detail in the case of DNA. More
importantly our results with H2O2 indicate its unique electronic properties. These prop-
erties are very sensitive to its concentration, electrolyte, frequency and available po-
tential. Our results along with that of salting in effect of peroxide by electrolytes sug-
gest a mechanism by which an electrolyte can pass though membranes as an ion pair
solvated by peroxide. The solvation effect of peroxide on DNA bases and the DNA
charge transport in the presence of small amounts of peroxide need to be investigated
to get a deeper understanding of this important field. Unfortunately the varying and
tremendous influences of peroxide on these processes such as ion pair formation, pref-
erential solvation by peroxide and its unique electronic properties have been ignored
or neglected so far. We hope physicists, chemists and biologists will take a serious look
at these properties of hydrogen peroxide in relation to their investigations.
Modulation of Electronic Properties of Simple Molecules by Transition Metals

The target of most metal based drugs is DNA (Dabrowiak, 2009; Sherman and Lip-
pard, 1987). Targets other than DNA that have recently been reviewed include a
gold(I) carbene complex interacting with mitochondrial membrane (Fricker, 2007).
However, the focus of our work was to improve mitochondrial enzyme activity by
selecting a simple molecule involved heavily in mitochondrial enzyme activities and
modulate its enzymatic activity by complexing with the metal palladium. Palladium
(II), Platinum(II), and gold(III) have the same number of d8 electrons. We believe
strongly that by improving the mitochondrial enzyme activities, we can improve
the quality of life, can induce apoptosis, and thus ward off many diseases including
cancer.
The technique we have utilized to investigate the modulation of properties of
simple molecules by transition metals is the electrochemical impedance technique.
While we have investigated many molecules using this technique (Krishnan and
Garnett, 2006; Krishnan et al., 2007a, 2007b; 2008a, 2008b, 2008c, 2008d; 2009a,
2008b) the electronic properties of hydrogen peroxide are briefly included in this
chapter because of its unique electronic properties and importance in biological
systems.
Figure 9(a) gives the Nyquist plot for differing concentrations of H2O2 in 0.10
M NaCl at –0.28 V. This may be compared with that in Figure 7 for 88 mM H2O2
in 0.10 M KCl. The results are similar in that they both are characterized by NDR.
However, the electronic circuits are subtly different because of their own unique
shapes. The data for 88 mM H2O2 in 0.10 M NaCl shown as almost a dot in Fig-
ure 9(a) are shown in the expanded scale in Figure 9(b). Also the frequencies at
which the NDR takes place as well the potentials are slightly different. The data at
for 8.8 mM H2O2 indicate double capacitance and are different from the NDR for
other concentrations. Even 8.8 mM H2O2 exhibits NDR behavior by changing the
potential. It is shown here to show the sensivity of NDR to the applied potential
as well as to the concentration of H2O2. The modulation by molybdenum in the
peroxo complex at a higher acidity is impressive (Figure 9(c)). We had discussed
earlier the diode like behavior of the molybdoenzyme nitrate reductases (Sucheta
et al., 1992).
Figure 9. (a) Nyquist plot in 0.10M NaCl, pH 5.2, for different H2O2 concentrations, mM (1) 8.8 (2)
26.4 (3) 35.2 (4) 88 and (b) Expanded curve 4 for 88mM H2O2 in 0.10M NaCl; Potential applied is
–0.28 V for and b. (c) Modulation of peroxide impedance by molybdenum. Nyquist plot for 0.005M
H2Mo2O3(O2)4 (obtained by dissolving Mo metal in peroxide), pH 1.87, 0.15V.
Similarly the impedance spectra for α-lipoic acid and its modulation by complexing
with palladium are shown in Figure 10. While α-lipoic acid exhibits NDR and shows
impedance in only 3 quadrants, it is extended to 4 quadrants and much more smoothly
by complexation with palladium. Of course the NDR behavior can be optimized by
slightly tweaking the applied potential. This enhancement in NDR behavior may be
compared to the similar enhanced Krebs cycle and mitochondrial respiratory chain en-
zymatic activities of the palladium α-lipoic acid compared to that of the ligand.
Figure 10. (a) Nyquist plot for 0.0373 M sodium lipoate, –1.15V, pH 7.79, NDR at 4.81Hz. (b)
Modulation of lipoate impedance by palladium in 0.0373M palladium α-lipoic acid (1:1 complex)
in 0.1792 M NaCl, –1.18V, pH 7.78, NDR at 66Hz.
Before concluding this chapter, we want to include the electronic properties of a

few other simple biological molecules such as lysine, FAD, cysteine, and histidine.
Their involvement in the mitochondrial electron transport chain is included in a later
section of this chapter. The data in Figures 9–12 are intended to demonstrate the use-
fulness of the impedance technique to understand the electronic character of simple
biological molecules. These also illustrate the sensitivity of the electronic character
for concentration, pH, surface area, applied potential, and the frequency at which the
NDR is observed.
The Nyquist plots for L-lysine as well its modulation by molybdenum are shown
in Figure 11.
L-lysine, a dibasic amino acid with a butyl ammonium side chain has pK1(a-
COOH), pK2(a-NH3+, and pK3(e-NH3+) values of 2.16, 9.06, and 10.54 respectively
so that it is positively charged at physiological pH. Elevated levels of lysine in blood
and urine have been linked to mental and physical retardation. Histones have a large
number of lysine residues and its positive charge promotes interaction with negatively
charged phosphodiester linkages of DNA. Acetylation of lysine weakens this electro-
static interaction and loosens the chromatin structure allowing gene expression. The
reversible histone acetylation and deacetylation reactions control the activation and
inactivation of gene expression.
Another important aspect of this system is the fact α-lipoic acid is linked to ly-
sine by an amide bond in the multienzyme complexes of pyruvate dehydrogenase,
α-ketoglutarate dehydrogenase and branched chain α-ketoglutarate dehydrogenase.
Thus both α-lipoic acid and lysine have heavy involvement in the electronic aspects
of the enzymatic process.
Figure 11. (Continued)

Figure 11. Nyquist plot for (a) 0.10 M lysine, 0.021 M HCl, pH 9.6; (b) 0.095 M Na2MoO4, 0.19M
lysine, 0.12 M HCl, pH 8.9.
The impedance data on the interaction between sodium molybdate and FAD are
shown in Figure 12. The FAD, a cofactor in a number of enzymes, when bound to a
protein can exist as it’s fully oxidized flavoquinone form, its 1e reduced flavosemiqui-
none form or its 2e reduced flavohydroquinone form.
It is well known that the orientation of the flavin and adenine groups at the elec-
trode surface depends on the concentration of FAD (Roscoe, 1996). The cyclic voltam-
mogram of FAD is highly concentration dependent. The same behavior is reflected
in the impedance data shown in Figure 12(a). The data in Figure 12(b) indicate the
potential dependence of its electronic character. The NDR is observed only at select
potentials.
Another important aspect of this system is the fact FAD is also an integral part of
the multienzyme complexes of pyruvate dehydrogenase, α-ketoglutarate dehydroge-
nase and branched chain α-ketoglutarate dehydrogenase.
An important group present in complex I, II, and III is the cysteine group. We have
carried out extensive investigation of this in the presence and absence of molybdate
(Krishnan et al., 2008a, 2008b). A typical example is shown in Figure 13. The pH
dependence on the observed NDR is vividly demonstrated in Figure 13(a). The data in
Figure 13(b) demonstrate that either the adsorbed molecule is regenerated for a repeat
cycle or that the double layer near the electrode surface remains intact. In all our ex-
periments a fresh mercury drop is used at the start of every impedance measurement.
One of our major concerns was that the applied potential was very near the passivation
of mercury. Repeat use of mercury giving nearly identical impedance curve seems to
validate the procedure.
Figure 12. Nyquist plot for (a) molybdate-FAD system, 0.02 M molybdate, pH 6.5 and (b) 0.02 M
Na2MoO4, 0.01 M FAD, (1) –0.7 V, (2) –0.8V.
Figure 13. Nyquist plot for 0.1M cysteine-sodium molybdate, (a) 0.3 V, pH (1) 5.22 (2) 5.79 (3) 6.91
(4) 9.34 and (b) 0.25 V (1) Fresh Hg drop (2) Repeat with the used Hg drop (3) Repeat second time
with the used Hg drop.
Figure 14. Nyquist plot for 0.177 M histidine-sodium molybdate, 0.067 M NaOH, pH 9.60, (a) (1)
–0.1 V (2) 0.0V (3) 0.03 V (4) 0.05 V (5) 0.07 V (6) 0.09 V and (b) expanded scale for (1) –0.1 V (2)
0.09 V
L-Histidine, an essential amino acid, has an aromatic nitrogen-heterocyclic im-

idazole side chain with pK1(a-COOH), pK2(a-NH3+), and pK3(imidazole) values of
1.78, 8.97, and 5.97 respectively. Its isoelectric point (pH) is 7.47. Decarboxylation
of histidine yields the neurotransmitter histamine. Histamine occurs in mast cells and
basophils of blood. Histamine binds to H1 receptors of the smooth muscle of bronchi
which contracts leading to breathing difficulties (as in Asthma). Histidine is also an
important component of enzymes such as carbonic anhydrase. The histidine residues
in cytochromes a, b, c stretch both on the cytosolic side as well as on the matrix side.
The electronic character of this important molecule is exemplified in Figures 14 and
15. The influence of the surface is exemplified in Figure 15. It is clear the NDR or
diode or tunnel diode characterisitic is sensitive to potential as well as surface area.
Figure 15. Nyquist plot for 0.177 M histidine-sodium molybdate, 0.067 M NaOH, pH 9.60, 0.05 V,
influence of surface area of mercury drop, mm2 (1) 0.011 (2) 0.017 (3) 0.022 (4) 0.031.
These impedance data provide information on the instabilities or bifurcations and

distinguish between saddle-node and Hopf bifurcations. However these aspects are not
included in this chapter. Only the importance of the electronic properties of these mol-
ecules and its modulation by molybdenum to demonstrate the diode or tunnel diode
characteristics of this system. The unique impedance behavior reflects periodicities
and suggest global electronic coupling. Our data suggest that we have to include the
electronic properties of these molecules to complement the enzymatic process.
Thus we have made a brief attempt to demonstrate the resourcefulness of the im-
pedance technique as illustrated in Figures 7–15, to understand the electronic properties
of biological molecules. The negative differential resistance characteristics are dem-

onstrated vividly and support the conclusions drawn from protein film voltammetry
regarding the tunnel diode characteristics of some enzymes. Our data presented here
raise the possibility that some of these simple molecules that are present in the en-
zymes may be the ones that are exhibiting the unique electronic properties in biologi-
cal systems.
SELF-ASSEMBLY OF PALLADIUM α-LIPOIC ACID COMPLEX

The phase microscopy pictures (300X) of 1.34 × 10–2 M and 2.7 × 10–4 M palladium
α-lipoic acid complex (1:1) are shown in Figure 16. The self-assembly of the complex
even at very dilute solutions is remarkable. No self-assembly was observed for sodium
lipoate even at concentrations as high as 0.20 M.
Figure 16. Phase microscopy 300X (a) 1.34 x 10–2 M and (b) 2.7 x 10–4 M palladium α-lipoic acid
complex.
A long flexible arm that can oscillate a distance of ~200 Å produced by the binding
of a lysine residue in the protein to the lipoyl group of E2 in 2-oxoacid dehydrogenases
is utilized during the catalytic cycle (Patel and Packer, 2008). It is obvious that the self
assembled palladium α-lipoic acid complex can make this process more facile.
It must be pointed out that in homogeneous systems, autocatalytic reactions and
diffusion resulting from chemical instabilities lead to the formation of spiral waves
and other concentration patterns of spatiotemporal phenomena. Our data suggest that
the propagation of electrical signaling among the packing units and extending to long
distances is viable by such self-assembled systems. Thus the self-assembly of bio-
logical molecules facilitates local disturbances to be felt at long distances by global
coupling.
The physics and chemistry of non-equilibrium systems have been utilized to un-
derstand some of the spatial patterns and temporal patterning observed in biologi-
cal processes such as bacterial colonies shaped by diffusive instabilities and calcium
waves governed by nonlinear amplification during intracellular signaling (Levine and
Jacob, 2004). We believe that the self assembled patterns of palladium lipoic acid may
help electron transfer processes by extending it into the bulk from the membrane. In
other words it may provide a spatial extension of the membrane with much more sur-
face area with much less need for a bulky multi enzyme complex.
This is similar to the coupling that takes places in large multi-enzyme systems
such as complex I where the Fe-S clusters help the signaling process during electron
transfer.
SPIN COUPLING IN ELECTRON TRANSFER

Numerous electron paramagnetic resonance (EPR) or electron spin resonance (ESR)
measurements have been carried out in biological systems (Berliner et al., 2000). This
technique allows detection of unpaired electrons in any phase and the large magnetic
dipole of the electron results in very-long range effects on the line shapes on responses
to pulses and electron-electron spin relaxation times. It also allows to measure dis-
tances between spins based on the dipolar interactions.
The most complicated mitochondrial complex I, with a molar mass greater than
850 kD and at least 26 subunits, has been studied thoroughly by this technique to un-
derstand its structural aspects, the location of the NADH binding site, flavin, and most
of the iron-sulfur clusters located in the hydrophilic electron entry domain of complex
I. Also from spin-spin coupling interactions, it has been possible to identify the loca-
tions of cluster [4Fe-4S]N2 and two complexes I associated species of semiquinone
(Ohnishi, 1998). The nature of spin and orbitals states in [4Fe-4S] complexes have
been reviewed recently (Noodleman et al., 1995). Vectorial translocation of 4 or 5
protons across the mitochondrial inner membrane is coupled to this electron transfer
process from NADH to quinone.
NADH + CoQ + H+ + n(H+)matrix → NAD+ + CoQH2 + n(H+)intermembrane space (41)
The electron transfer takes place from NADH to Complex I by passing through
flavin mononucleotide, FMN, to a series of redox active iron-sulfur clusters such as
[2Fe-2S]N1a, [2Fe-2S]N1b, [4Fe-4S]N3, [4Fe-4S]N4, [4Fe-4S]N5, and [4Fe-4S]N2, and two
protein bound species of quinone, QNf and QNs.
Complex II, which is much less complicated than Complex I has a molar mass of
about 127 kD and 5 subunits. The electrons pass through FAD to iron-sulfur clusters,
[2Fe-2S]S1, [4Fe-4S]S2, and [3Fe-4S]S3 as well as cytochrome b560.
Complex III has a molar mass of about 280 kD and 10 subunits. The electrons pass
through cytochrome bL (b566), cytochrome bH (b562), [2Fe-2S], and cytochrome c1.
Complex IV with a molar mass of about 200 kD has 6–13 subunits. The electrons
pass through cytochrome a, CuA, CuB and cytochrome a3.
The iron-sulfur clusters as well as semiquinone radicals in complex I are all EPR
detectable. The oxidized forms of these clusters are diamagnetic and reduced forms
are paramagnetic. The iron atoms are bridged by acid-labile inorganic sulfides. Each
iron-sulfur cluster has four protein cysteinyl sulfur bonds. The iron, with oxidation
state varying between +2 and +3, in each cluster is tetrahedrally bonded to the sulfur.
Cytochromes a, b, and c have heme proteins compared to the nonheme proteins in
the above mentioned iron-sulfur clusters. The heme group of c cytochromes has added
cysteinyl sulfhydryl groups across their double bonds to forming thioether linkages
to the protein. The heme iron of the cytochromes a, b, and c also have one or two
histidine residues as axial ligands. The histidine residues are on the cytoplasmic side
as well as the matrix side. Cytochrome c also has several invariant lysine residues that
lie in a ring around the exposed edge of it’s otherwise buried heme group (Voet and
Voet, 1995).
Apart from understanding the electron transfer pathways, topology of iron-sulfur
clusters, and site of coupling in NADH-ubiquinone reductase, Complex I investiga-
tions have also been instrumental in understanding the mechanism of superoxide gen-
eration at the flavin site of Complex I (Berrisford and Sazanov, 2009; Galkin and
Brandt, 2005; Kussmaul and Hirst, 2006).
Since, complex I dysfunction is implicated in many human neurodegenerative dis-
eases as well as cancer, it is critical to understand its function thoroughly. We must
point out a missing link here. We have briefly indicated the contributions from the
electronic character of cysteine from our impedance studies. Since, it is bonded to the
iron in the iron-sulfur clusters, it is important to investigate its electronic contributions
to the electron transfer process. A recent X-ray investigation confirms our sugges-
tion. “Cluster N2 is the electron donor to quinone and is coordinated by unique mo-
tif involving two consecutive (tandem) cysteines. An unprecedented “on/off switch”
(disconnection) of coordinating bonds between the tandem cysteines was observed
upon reduction” (Noodelman et al., 1995). This is also true of FAD and histidine in
Complex II. Since, the palladium lipoic acid complex formulation enhances the Com-
plex I and Complex II activities by 151 and 212% more than that α-lipoic acid, we
have reason to believe that the self-assembled structure of the complex, by providing
a spatial extension of the membrane with much more surface area, may be catalyzing
the electron transfer process by enhancing the spin coupling.
Some supporting evidence for the probable electron spin coupling, even though
not directly from the data of palladium α-lipoic acid complex, is given by the free
radical reaction mechanism for the reaction of dihydrolipoyl dehydrogenase (Massey
et al., 1960), studies on the1e reduction of the disulfide linkages (Hoffman and Hayon,
1972) and studies on the lipoic acid free radical (Chan et al., 1974). Sulfhydryl free
radicals of monothiol compounds tend to interact with their parent compounds.
R-S + RS– → [R-S-S-R]– (42)
There was no similar reaction between the lipoic acid radical and dihydrolipoic
acid. The pKa of lipoyl radical (RS.S(H)R of 5.85 compared to 4.7 of the carboxyl
group implied that the negative charge is on the sulfur of the radical (Chan et al., 1974;
Hoffman and Hayon, 1972). A direct electron transfer from the lipoic acid radical to
FAD forming FADH was suggested (Massey et al., 1960) and confirmed by pulse ra-
diolysis studies (Chan et al., 1974).
[RS-SR]– + FAD + H+ → RS-SR + FADH. (43)

The FADH radicals disproportionate eventually forming FAD and FADH2. Similar
radical formation with the lone electron in the sulfur or palladium or an oscillation
between the two can couple the electron transfer process and enhance the catalytic
process.
OXIDATIVE STRESS IN HIV INFECTION

The HIV infected patients were found to have lower level of intracellular glutathione,
plasma cystine, and cysteine (Dröge, 2006). Increased lipid peroxidation was also evi-
dent from the increased plasma MDA and plasma lipid peroxides. Plasma concentra-
tions of vitamin C and β-carotene/vitamin A were also significantly reduced. Increased
levels of oxidized 8-hydroxyguanine in DNA as well as TBARS along with decreases
in the levels of superoxide dismutase and catalase in HIV infected patients have also
been reported (Dröge, 2006; Packer et al., 1995). Supplementation for 6 months with
vitamin A or vitamin C or vitamin E decreased the level of DNA damage, along with
a reduction of TBARS and restoration of the activity of the enzymes (Jaruga et al.,
2002). The α-lipoic acid supplementation study, 150 mg of lipoate three times daily
for a period of 14 days, of HIV positive (classified CDC IV) patients showed increased
levels of plasma ascorbate and glutathione and decreased plasma MDA in most pa-
tients. Also in a majority of patients, the T-helper cells increased and the T-helper/T-
suppressor ratio improved (Packer et al., 1995).
A small study to probe the effectiveness of palladium α-lipoic acid complex formu-
lation was also carried out with 5 HIV/AIDS patients suffering from chronic fatigue.
This work was done at CIRCLE Medical LLC, Norwalk, CT, USA. It was observed
that the palladium α-lipoic acid complex formulation was generally well-tolerated in
all subjects; 4/5 patients reported sustained improvements in energy/fatigue through
week 4 (1/5 patients noted improvements through week 2 with “decrease” by week 4);
all subjects reported decrease energy/increased fatigue during the 2 week “wash-out”
period; mean MOS-HIV Energy/Fatigue scores increased significantly through week
4, with a significant decrease in scores during the “wash-out” period; decreases were
observed in TC, TC/HDL, and TG throughout the study period; and increased CD4%,
decreased CD8%, and increased CD4:CD8 were observed. Of course these results are
only preliminary and a comparative study with α-lipoic acid and many patients have
to be carried out to arrive at meaningful conclusions. But the preliminary results are
very promising.
AMELIORATION OF DRUG INDUCED TOXICITY

A recent review has detailed the medication-induced mitochondrial damage and dis-
ease and suggested mitochondrial toxicity testing as part of the pre-approval process
for medications to protect the public by identifying the most toxic medications before
they are allowed to reach the market (Neustadt and Pieczenik, 2008). Recent stud-
ies indicate effectiveness of, natural antioxidants against adriamycin-induced toxicity
in cancer patients (Principal et al., 2010), lipoic acid against methotrexate-induced
oxidative stress when treating leukemia and autoimmune diseases (Tabassum et al.,
2010), and lipoic acid against isoniazid-rifampicin-induced hepatotoxicity when treat-
ing tuberculosis (Saad et al., 2010). Since, all studies with palladium lipoic acid for-
mulation have indicated that it is much more effective than lipoic acid, there is no
reason to doubt its effectiveness in using it as an adjuvant for treating tuberculosis,
leukemia, other cancers and autoimmune diseases. Of course confirmation of these
statements needs experimental verification.
CONCLUSION
We have suggested a new way of looking at the holistic medicine or alternative medi-
cine. Mitochondria are ubiquitous. By developing new ways of treating mitochondrial
dysfunction, a symbiotic or bridging relationship between modern medicine and a
generally neglected part in modern medicine, the mitochondria, can be generated for
improving the mental, emotional, and spiritual elements of the body. Medications tar-
geting the mitochondria are much closer to a real holistic medicine because if you have
healthy mitochondria, they will contribute substantially to the physical, mental, and
emotional elements needed to complement the modern medicine.
Unlike toxic platinum chemotherapy agents, palladium α-lipoic acid complex for-
mulation is safe and nontoxic. Its oral administration can be continued indefinitely.
Palladium α-lipoic acid complex formulation is designed to provide energy for com-
promised body systems and promote overall health. It facilitates aerobic metabolism
much more than that of α-lipoic acid, by significantly enhancing the enzymatic activ-
ity of isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydroge-
nase, and malate dehydrogenase at the Krebs cycle and mitochondrial complexes I, II,
III, and IV of the electron transport chain. Of course further investigations are needed
to understand the mechanism of action of palladium α-lipoic acid complex formula-
tion on some of these activities because the enzymes containing lipoamide are not
direct participants in some of these activities.
Prior ischemia studies in gerbils demonstrated this energy benefit provided by pal-
ladium α-lipoic acid complex formulation to maintain the integrity of the electron
transport chain following an ischemic insult.
Preliminary studies of HIV/AIDS patients under various cocktail treatment proto-
cols demonstrated an almost immediate improvement in patient quality of life. Ben-
efits included less depression and lethargy, more daily energy and increased appetite.
Patients demonstrated significantly improved MOS-HIV Energy/Fatigue scores, in-
creases in CD4, decreases in CD8, as well as improved lipid levels.
The unique electronic properties of palladium modulating the properties of
α-lipoic acid appear to be a key to this physiological effectiveness. This is exemplified
in our electrochemical impedance spectroscopic studies of α-lipoic acid and palladium
α-lipoic acid.
The electronic properties of palladium also appear to modulate the antioxidant
properties of α-lipoic acid in that palladium α-lipoic acid complex formulation en-
hances the activities of catalase and glutathione peroxidase more than that of α-lipoic
acid. The level of glutathione also was significantly improved and the level of lipid
peroxidation was decreased in the heart mitochondria of aged rats. Oral administration
of palladium α-lipoic acid complex formulation showed an increase in glutathione and
glutathione peroxidase levels and a decrease in MDA (a secondary product of lipid
peroxidation) in the kidney and liver. Also palladium α-lipoic acid complex formula-
tion offered protection to cellular DNA from whole body radiation (8 Gy). It decreased
the radiation–induced hematopoietic injury as revealed by the bone marrow cellular-
ity, hemoglobin level, and endogenous spleen colony formation in irradiated animals.
Palladium α-lipoic acid complex formulation is similar to a multi-spectrum drug in

that it carries out several functions such as combating age related as well as disease as-
sociated fatigue, and minimizes the effects of ischemic injury. It acts as a prophylactic
for neuronal regeneration from transient ischemic attack and also for protection from
radiation. Apart from being a powerful free radical scavenger, it is also highly effec-
tive against various cancer cells such as glioblastoma, breast, ovarian, osteosarcoma,
and lung.
Finally, we believe that the aim of the slogan for holistic medicine or alternative
medicine, “the whole is much more than the sum of the parts”, is accomplished readily
by targeting mitochondria, a ubiquitous organelle in the human body. This is achieved
with the judicious choice of a naturally present ligand in the human body, α-lipoic
acid, that plays a crucial role in the mitochondrial energy metabolism because of its
unique chemical structure and consequent redox properties, and complexing it with
a metal with very high catalytic and electronic properties, palladium. What we lack
in its continuing saga is many more challenging investigations to probe more deeply
to understand the underlying mechanisms of some of the impressive or remarkable
observations.
KEYWORDS
•• Adenosine triphosphate
•• Antioxidants
•• Chemotherapy
•• Cisplatin
•• Flavin adenine dinucleotide
•• Glutathione
•• Glutathione peroxidase
•• Glutathione reductase
•• Hypoxia inducible factor 1
•• Malondialdehyde
•• Mitochondria
•• Polyunsaturated fatty acid
•• Reactive oxygen species
•• Succinate dehydrogenase
ACKNOWLEDGMENT
The authors wish to express their sincere thanks and gratitude for many fruitful dis-
cussions and collaborations in various aspects of the work presented in this chapter:
B.Chu, C. J. Perkins, G. Blick, G.K. Ogilvie, P. Valane, K. K. Janardhanan, T.A. Ajith,
C.K.K. Nair, N.P. Sudheesh, A. Menon, L. Ramachandran, Calvert Labs, Brunswick
Labs, Inc., and K.G.K. Synergize Inc.
Chapter 8
Unity of Mind and Body: The Concept of Life
Purpose Dominant
Bukhtoyarov Oleg Viktorovich and Samarin Denis Mikhaylovich
INTRODUCTION
Problems of links between mind and body, ideal and material always attracted atten-
tion of the scientists and philosophers and within the framework of medicine there has
always been a clear understanding of necessity in holistic perception of the patients,
however actual approach to the patients appears to be determinative. Unobviousness
of influence of mind on body and lack of a system view on psychosomatic links con-
tinuity have made modern practical medicine somatically focused both in diagnostics
of diseases and in their treatment and preventive maintenance. The scientific search is
also mainly focused on study of somatic parameters of organism without the account
of mind influences on them. The appearance of new research technology still carries
scientists even more in depths of organism. The huge piles of fragmented facts are
taken on a surface which are difficult to give the system analysis to. At the same time,
the huge amount of scientific data has kept showing extensive damaging influences of
chronic psycho-emotional stress (CPS) on organism of animals in experiment and on
human being in daily life (Gidron et. al., 2006; McEwen, 2007; Ostrander et. al., 2006;
Simon et al., 2006; Spinelli et al., 2009). It is possible to state that CPS is an important
an etiological and pathogenetic factor in development of many somatic diseases in-
cluding “diseases of civilization”: atherosclerosis, cardiovascular disease (Dimsdale,
2008; Nemeroff, 2008; Knox, 2001; Roy-Byrne et al., 2008; Shpagina et. al., 2008)
and cancer (Adamekova et al., 2003; Mravec et. al., 2008; Reiche et. al., 2005) about
that has been stated earlier in a hypothesis of psychogenic carcinogenesis. Psychogen-
ic factor has always been and still remains essential component which mainly defines
occurrence, development and outcome of diseases in human beings, however in view
of its idealness and unobviousness it is latent behind a facade of a clinical disease
picture and as a rule is left untouched by pathogenetic treatment. In connection with
above stated, there is one large, difficult and, at first sight, unsolved question: “How to
see mind, biological, personal and social aspects of a healthy human and a patient in
dynamic unity instead of considering only separate pathological process?”
THE CONCEPT OF LIFE PURPOSE DOMINANT

The answer to this raised question would allow bringing in the proved and purposeful
corrective amendments to scientific researches, diagnostic, medical and preventive
measures in work with the patients. We offer the concept of life purpose dominant
(LPD) which opens a system view on health of a human and process of any serious
chronic disease formation with participation of mind and shows a possibility to con-
trol this pathological condition. On the basis of this offer by us, LPDs concept lays
inter-subject doctrine of a dominant as universal, biological principle of work of the
nervous centers and vital functions of all living systems, general law of the intercentral
relations in living organism (Ukhtomsky, 1927; 1966). The doctrine of a dominant
was created by the academician А. А. Ukhtomsky (1884, 1942), who is the largest
thinker and ingenious scientist of the twentieth century. However, his doctrine has not
received a due estimation and recognition neither during life of the author nor after
his death. His scientific school existed simultaneously and in parallel with a school of
the Nobel winner academician I. P. Pavlov that was recognized by the Soviet power as
“sole correct scientific idea”, therefore discovery of the ingenious scientist remained
unnoticed for a long time. For the sake of justice it is necessary to tell that the basic
rules of the doctrine of a dominant and a term “dominant” used in the works of scien-
tists which have created a lot of the well-known theories: theory of human being mo-
tivation (Maslow, 1943), theory of installation (Uznadze, 1997), psychological theory
of activity (Leont’ev, 1978), theory of movement behavior (Bernstein, 1967), theory
of dynamic localization of mental functions (Luria, 1970), theory of the functional
system (Anokhin, 1970), search activity concept (Rotenberg, 2009) and even a lot in
Pavlov’s doctrine of conditioned reflex appear to be component of the doctrine of a
dominant. Really, uncountable set of reflexes in complete sense would blow up organ-
ism in the first instant of the existence if submission to their principle of a dominant
when all reflexes work under the slogan “everybody for one, one for everybody”. By
the way, the formation of each conditional reflex under influence of conditional irritant
is nothing else as the process of a dominant formation in which preservation directly
depends on supporting influences of conditional irritant.
BRIEFLY ABOUT THE DOCTRINE OF A DOMINANT OF THE ACADEMICIAN

A. A. UKHTOMSKY
Dominant, according to А. А. Ukhtomsky, is not any one topographic certain center
of excitation in the central nervous system. It is a certain constellation of the ner-
vous centers with increased excitability in various departments of a brain and spinal
marrow, in vegetative nervous system as well as it is a temporary association of the
nervous centers for the solution of the certain task (Ukhtomsky, 1966). Spiral marrow
and brain stem, conditional reflexes, processes of association, integrated images are
equally subordinate to a principle of work of dominant reflexes of a spinal marrow
where the environment as well as high nervous activity is perceived. The dominant
is characterized with the following four features: (1) high excitability, (2) stability of
the excitation, (3) ability to sum (accumulate) coming excitations and also (4) inertia
(the dominant “insists on itself”). The condition of a dominant is not super excitation
which would by all means be finished by braking and more or less long persistence
of excitation “in one place and connected braking in the other”. The dominant is ca-
pable to pull external irritants together that are not related to it, and do not prevent its
development but strengthen it. The dominant represents prevailing need, motivation,
and aim and is the powerful activator of activity. However, any dominant is always
temporary and stops in the following cases: complete spontaneous end of dominant
Unity of Mind and Body: The Concept of Life Purpose Dominant 131
condition (for example, any of the biological acts), complete termination of reinforce-
ment by adequate irritant and suppression by a more powerful competing dominant.
It is necessary to pay special attention that at incomplete cancellation of adequate ir-
ritant, the dominant amplifies, aspires to keep itself. We shall return to this situation
while considering treatment of human diseases.
А. А. Ukhtomsky paid special attention to cortical dominant––dominants of the
high order which are the latent factors of psychological activity. All vital functions of
a human being are dominant in their sense; they consist of a set of uncountable func-
tional conditions of organism consistently changing each other––current dominants.
However, there is the main dominant of a human to which all current dominants (more
precisely subdominants) are subordinated, which holds in its power a whole field of
spiritual life, defines “spiritual anatomy” and a vector of human existence. We dared
to name it the LPD.
LIFE PURPOSE DOMINANT IN A HUMAN

The LPD is a non-material construction with material expression, which is formed in
mental sphere and is shown by the maximal integration of mental and somatic process-
es, subordination of the current subdominants of a human being, maximal sanogenetic
and adaptive possibilities of organism that allows him to resist to constant pressure
of the environmental factors successfully. The LPD is formed extremely under influ-
ence of a complex of verbal and not verbal suggestive irritants (processes of educa-
tion and training, skills development, models of other people behavior etc.) which
defines the life purpose that a human being aspires to achieve. A vivid example of an
exclusive role suggestive irritants play in formation of life aims and personality is the
well known phenomenon of Homo ferus (“Mowgli Syndrome”) (Yousef, 2008) when
children who have been brought up by animals completely acquire all behavior ste-
reotypes of animals. In view of suggestive basis of LPD, life purpose and its loss can
not be clearly realized by a human. For achievement of long-term, instead of momen-
tary goal, whole organism appears in subordination to its main conductor––LPD. The
LPD provides coordination of asynchronous work of organs and systems, mental and
somatic processes, defines a vector of apparent chaos of numerous reflexes of organ-
ism, current subdominants (biological, mental, social etc.) and trajectory of everyday
behavior of a human being. The LPD has certain similarity to work of ants carrying
construction material in an anthill when the vectors of movement of separate ants are
multidirectional and even opposite, but the resulting vector of their movement allows
moving construction material in an anthill (Perelman, 2008). The LPD defines not
only functional condition of the central nervous system, high nervous activity and vec-
tor of behavior of a human being, it defines a functional condition of a whole organ-
ism at all its levels––from subcellular up to organismic. Let us notice, that at the adult
human LPD has the most various contents but in a fetus, neonatus and a child, LPD
is shown by aspiration to safety. However, in process of development of a personality
which represents a set of already holding suggestions, LPD is filled with other sugges-
tive by the contents, that stability of LPD depends on.
INTERRELATIONS BETWEEN A LIFE PURPOSE DOMINANT AND CURRENT

SUBDOMINANTS OF A HUMAN
The LPD has supporting influences from numerous current subdominants, which
do not have any direct attitude to it at all. However, there are basic subdominants
among numerous LPD subdominants––“subdominants of health”, its reinforcement
and strengthening, which are actively created only by human being despite of constant
action of external irritants (unfavorable environmental factors), competing subdomi-
nants, menacing formation and capable to occupy a place of LPD or even to destroy it.
For example, a scientist is overcoming inconceivable number of obstacles in search of
the truth or an actor is constantly aspiring to improve himself to be in demand, to feel
love of the spectators and to receive the worthy fee. If these people terminate to create
basic subdominants, their dominants of life purpose by all means will disappear that
threatens with heavy mental and somatic consequences, we shall speak below about.
Restriction of possibilities to create supporting basic subdominants is observed among
refugees, disabled, prisoners and other people who lost life prospect. At the same time,
use of the minimal possibilities to reinforce LPD allows a human to keep his/her health
even in conditions of massive chronic psycho emotional stress. For example, dur-
ing the Second World war, some war prisoners in concentration camps died quickly
and others planned their lives after concentration camps, they washed, had a shave,
cared for others every day and being in inhuman conditions of existence they did not
even catch colds at all (Rotenberg and Arshavsky, 1984). There is a great variety of
examples of huge LPD force in a world history, in daily life and in clinical practice.
We have to mention numerous situations connected with achievement of life pur-
pose, the termination of basic subdominants formation and natural LPD loss. A good
example is the people with the most favorable financial, economic and social status
who have achieved the life purpose and any possible well-being but imperceptibly
appeared in “without dominant” condition––condition of CPS with the subsequent
development of heavy diseases.
LIFE PURPOSE DOMINANT AT AN ANIMAL

Proceeding from universality of the doctrine about a dominant for all living systems
LPD should exist at an animal too. We consider that unlike a human being, LPD at an
animal is biologically predetermined, formed in the central nervous system, constant at
any age and is the dominant of safety––filled with aspiration to safety. Unlike human
being, the animal practically is unable to show own activity in creation of strengthen-
ing basic LPDs subdominants. At wild animals, the strengthening LPD occurs by a
natural image under action of short-term subdominants––functional condition of an
organism arising as a result of reactions on acute stressful irritants. At domestication
and training of an animal, human being becomes main irritant in formation of a unique
basic subdominant strengthening an animal dominant of safety. This understanding
is important, as it allows in experimental models on animals to simulate loss of LPD,
similar to loss of LPD at a human.
ROLE OF SUGGESTIONS IN OCCURRENCE AND LOSS OF A HUMAN’S LIFE

PURPOSE DOMINANT
From above stated, there is a clear exclusive role of suggestions in life of a human, as
LPD and personality are a product of systematic suggestive influences. From all vari-
ety of irritants influencing a human being during his life suggestive influences are ca-
pable to destroy LPD directly and to become a lead though invisible pathogenic part in
development of many diseases. In contrast to animals at which the acute stress always
strengthens LPD, at a human depends on various results of intrapsychic processing of
suggestive information of acute stress. For example, if the threat to life of an animal
is finished with flight and LPD reinforcement, the threat to life of a human can both
support LPD and be finished with its loss and development of disease, for example,
post traumatic stress disorder. Besides, suggestive influence can in some minutes de-
prive a human being of life purpose and result in his death; how an academician V. М.
Bekhterev informed in the work describing experiment on a criminal sentenced to a
death penalty (Bekhterev, 1998).
DISEASES AS DOMINANT CONDITIONS

Any disease of a human being contains all features of a dominant therefore it can be
considered as pathological dominant condition which is formed under influence of
somatogenic and/or psychogenic irritants–etiological factors. The dominant dies away
and disappears according to the doctrine of dominant after termination of adequate
irritant. However, human diseases as pathological dominant condition do not disap-
pear at complete termination of etiological irritants, but become chronic, as are sup-
ported by others, already pathogenic irritants. In this connection, chronic diseases, as
pathological dominant condition, have the supporting influences on the part of numer-
ous current subdominants. Please note that there are pathological basic subdominants
among them––“subdominants of disease” which are formed under the influence of
hetero- and auto suggestive irritants. Actually, they are pathological reflexes, for ex-
ample, bronchial asthma attack, spasm of colon, arrhythmia attack or more complex
cascade of reflex disorders at a relapse of a multiple sclerosis or cancer generated in a
result of psycho-emotional shocks. These basic pathological subdominants (pathologi-
cal reflexes) become a basis psychogenic component of chronic diseases.
PSYCHOGENIC COMPONENT OF DISEASE AS THE BASIC PART OF

PATHOGENESIS
Psychogenic component of disease is an indispensable reaction of the person to dis-
ease with a complex of emotional, intellectual and volitional disorders connected to
comprehension, experience and attitude of the patient to the condition and also with
vegetative component which naturally interweaves with a structure of clinical displays
of disease that gives it qualitatively new features. We consider that namely psycho-
genic component in human being defines development and outcome of human dis-
eases as its basic pathogenic role consists of a distortion or blocking of sanogenesis
mechanisms. There are no human diseases without psychogenic component and this
is the cardinal difference of human diseases from diseases of animals. It is possible
to state that the body does not suffer at influence of the unfavorable environmental
factors but the mind is always injured, that is psychogenic factor cannot be etiological
but it becomes pathogenic. Psychogenic component cannot be missed, deliberately
ignored and waved away from it. On the contrary, it is necessary to see psychogenic
component of disease to reveal pathological basic subdominants that is to understand
and control it to use successfully during treatment of the patients.
Thus, utter elimination of pathological dominant condition, that is the patient’s
recovery, assumes elimination of not only a set of known etiological and pathogenic
irritants supporting a pathological dominant but also requires indispensable elimina-
tion of a psychogenic component of disease. Otherwise, the incomplete elimination
of irritants will indeed strengthen a pathological dominant of disease, which becomes
more active, progressing and/or resistant therapy. Unfortunately, this phenomenon is
quite often observed in clinical practice.
THE CHARACTERISTIC OF BASIC INTEGRATED HUMAN FUNCTIONAL

CONDITIONS
The basic integrated functional conditions of a human organism are defined by the
contents of his main dominant that allows marking out 5 integrated functional condi-
tions which replace each other during a whole life of a human in direct and opposite
directions as a result of constant pressure of the various factors (irritants) of an envi-
ronment:
1. “Ideal health dominant” is a functional organism condition which is character-
ized by LPD presence with its basic subdominants, maximum integration of
psychosomatic processes, maximum and adaptive organism possibilities and
lack of any chronic diseases.
2. “Relative health dominant” is a functional condition of organism which is
characterized by LPD presence with basic subdominants, sufficient integra-
tion of psychosomatic processes, sufficient sanogenetic and adaptive organism
resources, compensating any available chronic diseases.
3. “Without dominant condition” is a transitive functional condition of organism
deprived of basic subdominants and LPD which is characterized by disinte-
gration of brain systems, psychosomatic processes, progressing reduction of
sanogenetic and adaptive organism resources, formation of any psychosomatic
pathology or decompensation of already available chronic diseases.
4. “Disease dominant” is a pathological functional condition of organism which
is characterized with occurrence of a dominant of any psychosomatic or soma
psychic disease instead of LPD with formation of pathological basic subdomi-
nants supporting disease.
5. “Self-destruction dominant” is a pathological functional condition of organ-
ism which is characterized by occurrence of a dominant condition instead of
LPD described by aspiration to death––a dominant of death with numerous
pathological basic subdominants, maximal disintegration of brain systems and
psychosomatic processes, failure of sanogenetic and adaptive of processes
conducting to organism destruction.
DYNAMICS OF THE INTEGRATED HUMAN FUNCTIONAL CONDITIONS

WITHIN A LIFE SPAN
The dynamic links and change of the basic integrated functional condition of a human
organism on a background of constant pressure of the environmental factors (irritants)
are presented in Figure 2. To visualize the dynamic presentation of complex psycho-
somatic processes, each integrated condition of a human being is shown as “iceberg of
psychosomatics” where the surface part––soma, underwater part––mind (psychogenic
component) and central place in each condition is occupied with predominant domi-
nant subordinating numerous current subdominants. All subdominants are formed in
the central nervous system, in mental sphere, but have obligatory manifestations in
soma and the speed of these manifestations depends on lag effect of somatic processes
(nervous reactions, vascular reaction, hormone reaction, exchange processes in bones
etc.). In Figure 1 the small black circles are various current subdominants (meals,
dream, walking etc.), black triangles––basic LPD subdominants, black squares––path-
ological basic subdominants of disease.
“Ideal health dominant” (see Figure 2, I) which is met in a smaller part of the
population and more often among young people, turns into “relative health dominant”
condition (see Figure 2, II) under influence of the unfavorable (pathogenic) factors
of an environment (trauma, infections, stresses etc.). Thus, pathogenic factors (irri-
tants) do not destroy predominant dominant––LPD but result in occurrence of some
chronic diseases (rather serious ones) which appear to be compensated because they
become the current subdominants subordinate to LPD, and psychogenic component
of these diseases carries out no pathogenic and sanogenic role. The people with dis-
abilities participating in Paralympic Games or keen people living with HIV/AIDS,
elderly people conducting an active lifestyle, that is actively creating and supporting
basic “subdominants of health” can serve as an example. The reverse transition from
condition II in a condition I seem to be difficult. In case of LPD (sense, life purpose),
loss under pressure of the environmental factors, a human being appears in transitive
“without dominant condition”, in power of daily subdominants (see Figure 2, III) that
is characterized by a condition of chronic consumptive psycho-emotional stress with
its mental and somatic manifestations. For example, loss of the close person with
whom the plans for the future are connected or loss of any life prospects as a result of
social shocks (war, terrorism, financial and economic crisis, acts of nature etc.) and
also others psycho-traumatic situations. A person can stay in a condition “without
dominant” from several minutes up to several years. Under favorable conditions (the
life purpose appearance), a person comes back in a condition of “relative health domi-
nant” (see Figure 2, II) otherwise he/she stays in power of “disease dominant” (see
Figure 2, IV) or “self-destruction dominant” (see Figure 2, V).
“Dominant of serious chronic disease” is represented in some serious chronic
disease which has arisen in the period of “without dominant condition” (CPS), with
formation of pathological basic subdominants which make a basis psychogenic com-
ponent of disease. They are manifested both in mental sphere (anxiety, depression,
phobias etc.), and in somatic sphere (vegetative, neuroendocrinal, neuroimmune dis-
orders, etc.) deforming psychosomatic relation and actively participating in patho-
genesis of disease. There are numerous examples of occurrence of the most various
diseases on a CPS background including development of malignant tumors (Levav et
al., 2000) or multiple sclerosis (Li et al., 2004) after loss of the close person.
Figure 1. “Iceberg psychosomatics”: dynamics of the basic integrated conditions of the human
organism within the life span under constant pressure of the environmental factors. - dominant
of life purpose (LPD); • - current subdominants; ▲- basic subdominants LPD; ■ - pathological basic
subdominants of illness; - dominant of serious chronic disease; - dominant of death. (I) Condition
“ideal health dominant”: LPD presence and its basic subdominants, orderliness of psychosomatic
processes, absence of illnesses; (II) Condition “relative health dominant”: LPD presence and its
basic subdominants, equilibrium of psychosomatic processes ensuring remission of any chronic
diseases; (III) “without dominant condition”: LPD absence, disorder of subdominants, disintegration
of psychosomatic processes, chronic psycho-emotional stress, possibility of transition into condition
II; (IV) Condition “disease dominant”: Occurrence instead of LPD dominant of serious chronic disease
supported by pathological basic subdominants, deep disintegration of psychosomatic processes,
opportunity of return into condition III. (V) Condition “self-destruction dominant”: occurrence of death
dominant instead of LPD with numerous pathological basic subdominants, practically irreversible
disintegration of psychosomatic of processes, failure of sanogenesis mechanisms leading to death.
“Self-destruction dominant” (see Figure 2, V) always occurs from “without domi-

nant condition” (see Figure 2, III) which in turn can arise from “disease dominant”
(see Figure 2, transition IV in III). Psychogenic component “self-destruction domi-
nant” contains a significant number of pathological basic subdominants that makes
“self-destruction dominant” very strong and complicates reverse transition in “with-
out dominant condition”. Psychogenic component of “self-destruction dominant” is
always brightly painted and clinically is shown through depressive symptomatology,
phenomena of feebleness, hopelessness, down to catatonoid state with complete re-
fusal of a human of the further life prospects and from the life itself, it is psychological
capitulation (phenomenon “given-up/giving-up”) under pressure of the environmental
factors with active or passive aspiration to death. Somatogenic component “self-de-
struction dominant” is frequently shown through expressed somatic disorders connect-
ed mainly to heavy frustration, intimate––of vascular system (cardiac arrhythmias,
weakness of cardiac activity etc.). A vivid example of “self-destruction dominant” is
a known phenomenon “voodoo death” or psychogenic death (Lester, 2009), which
was studied by us in oncological practice (Bukhtoyarov and Arkhangelsky, 2006).
Psychological capitulation on a background of depression results in suicides precedes
and accelerates approach of death of the patients with diseases of heart (Seymour and
Benning, 2009; Surtees et al., 2008), cancer patients (Lloyd-Williams et al., 2009; Ro-
din et al., 2009) and patients with other diseases (Grossardt et al., 2009). By the way,
self-liquidating behavior of some fans, sectarians or suicide attacker also is caused by
“self-destruction dominant” arisen under influence of the external unfavorable mainly
suggestive factors.
VIEW OF AN ANIMAL FROM POSITIONS OF A LIFE PURPOSE DOMINANT

CONCEPT
The main differences between a human and an animal are the second signal system
(speech) and ability to abstract thinking, which defines existence of psychogenic
component at a human only. The set of integrated functional condition of animal’s
organism within a life span is sharply narrowed because of absence of a psychogenic
component (see Figure 3).
Figure 2. Dynamics of the basic integrated functional conditions of an animal organism within a
life span on a background of constant pressure of the environmental factors. — safety dominant”
(SD), • — current subdominants (CD). (a) Condition “dominant of life”: presence SD, supported
CD, integration nervous-somatic processes; (b) Condition “chronic stress dominant”: presence of
SD but CD mismatch, increasing but convertible disintegration of nervous-somatic processes; (c)
Disappearance of SD on a background of irreversible disintegration nervous-somatic processes
conducting to inevitable animal destruction.
A unique (sole) integrated functional condition with the maximum integration of

nervous and somatic processes, maximum adaptive and sanogenetic resources is the
condition when an animal LPD (safety dominant) is kept. We named this integrated
condition of an animal organism “life dominant” where all current subdominants are
subordinated to an animal LPD (see Figure 3a). For a wild animal, the acute stresses
and for a pet, human being is the irritants, which strengthen their LPD. In situations
of long influence of unfavorable irritants (chemical, physical, biological), there is a
threat of LPD loss with chaos of the current subdominants, increasing disintegration
of nervous and somatic processes, reduction of adaptive and sanogenic resources of
organism. We named this integrated condition of an animal organism “chronic stress
dominant” (see Figure 3b) that has a certain similarity with “without dominant condi-
tion” of a human described also by a CPS condition. The LPD does not disappear in
this condition only at an animal as its disappearance is equivalent to death. “Chronic
stress dominant” is supported only by external irritants and is capable to reverse tran-
sition in “a life dominant” only after the termination of exogenous influences. Other-
wise, there will be irreversible disorders in organism with LPD loss and subsequent
destruction of an animal (see Figure 2c).
The stated representations show that the experimental models on animals can not
precisely correspond to real events in human organism and require very careful prepa-
ration of experiments. For example, it is incorrect to run experimental approbation of
anti-cancer drugs on animals that are in an integrated condition of “life dominant” and/
or exposed to acute stress as organism of such animals has greatest sanogenetic and
adaptive resources, which can not be basically present in cancer patients. It would be
more correct to put animals into chronic stress condition on which background to test
action of anti-cancer drugs.
EXAMPLE OF A SYSTEM VIEW OF CHRONIC DISEASE FROM POSITIONS OF

THE CONCEPT LIFE PURPOSE DOMINANT‑‑CANCER DISEASE
In the hypothesis of psychogenic carcinogenesis was shown the scheme of forma-
tion of basic pathogenic parts of carcinogenesis under CPS influence. Thus, term
“CPS” has remained only general concept not reflecting all depth of its origin but
from positions of the LPD concept CPS genesis with its known damaging influences
on organism becomes clear. Our long-term researches have shown that up to an ac-
tual making out a cancer diagnosis, majority of the patients were in a condition of
feebleness, hopelessness, helplessness despair (frequently not realized by the patients)
which characterize LPD loss, appeared CPS. Statistic data prove dramatic increase in
possibility of malignant tumors diseases with age (Jemal et al., 2008). It may be con-
nected with loss of life prospects and plans for the future of an elderly person, in fact
this is LPD loss with all ensuing negative psychosomatic consequences. Making out
diagnosis of a cancer is in turn a powerful iatrogenic (suggestive) influence closing a
vicious circle of carcinogenesis with formation of new additional pathological basic
subdominants, supporting and strengthening the main pathological dominant––cancer
dominant, which strongly occupies a LPD place. Modern somatic focused therapy of
a cancer does not take into account and does not pay due influence to psychogenic
component of cancer disease which defines development and outcome of a cancer in

human. Somatic approach to cancer therapy is not capable to explain uncontrollability
of carcinogenesis. The reasons for uncontrollability of cancer process during somati-
cally focused therapy become clear from positions of the LPD concept (see Figure 3).
Figure 3. The “Iceberg psychosomatics”: schema of psychogenic reproduction of a cancer disease

(cancer relapse). –– cancer dominant (CD), • —current subdominants, —pathological basic
subdominant CD. (a) Influence of standard anticancer treatment on somatic component CD and
in general “disease dominant”; (b) “Reconstructed soma” with previous psychogenic component
(c) Complete reflex reproduction of CD and in general “disease dominant” (cancer relapse) during
repeated reminding CPS.
The modern complex therapy of cancer (surgery, chemotherapy, radiotherapy) re-

sults in incomplete elimination of irritants supporting cancer dominant and on the
whole pathologically integrated condition of organism “disease dominant”, that is
carries out correction of its somatic component only (see Figure 4b) that can even
strengthen manifestations of cancer disease. Besides, maintenance of psychogenic
component of cancer dominant and “disease dominant” on the whole creates sufficient
conditions for complete reflex restoration of its somatic component that is occurrence
of a recurrent cancer or occurrence of cancer of other tissue localizations at repeated
reminding influence of psychogenic irritants (see Figure 4c) even after many years
of the first cancer incident. In this connection, in pathogenically proved complex ap-
proach to cancer treatment the effective influences on psychogenic component of can-
cer disease should be stipulated.
CONCEPT IMPLICATIONS
1. The presented concept allows seeing close interaction of mind and body, to
generate holistic view of vital functions of a human.
2. The concept proves complex pathogenetic approaches to prophylaxis and
treatment of human diseases.
3. The concept states a special role of social medium as a source of suggestive
flows of information in the personality development of a human and his/her
life aims.
4. On the basis of this concept the processes occurring not only in separate human
being but also in various people communities can be clarified.
5. From positions of this concept an opportunity appears to develop purposeful
experimental models on animals that will become maximum adequate to the
research problems.
CONCLUSION
The concept of a LPD gives an opportunity of new vision of interrelations between
ideal and material, mind and body, dynamic unity of psychosomatic processes and also
allows to understand and to predict the phenomena of human vital functions of healthy
person and a patient.
KEYWORDS
•• Chronic psycho-emotional stress
•• Ideal health dominant
•• Life purpose dominant
•• Psychogenic component
•• Sanogenetic and adaptive
Chapter 9
Thuja occidentalis and Breast Cancer
Chemoprevention
B. K. Ojeswi, M. Khoobchandani, S. Medhe, D. K. Hazra,
and M. M. Srivastava
INTRODUCTION
Breast cancer is the most common cancer affecting women throughout the world
(Bryle et al., 2003). It accounts highest morbidity and mortality worldwide. Globally,
1.9 million new cases of breast cancer were diagnosed and 0.6 million deaths were
caused in the year 2009 from this disease (DeSantis et al., 2009, 2010). In India, breast
cancer is the second most common cancer, where 0.07 million new cases and 0.035
million deaths are reported every year (Ghumare and Cunningham, 2007). The present
trend in the management of cancer development involves either reduction of the expo-
sure of an individual to known carcinogen to the extent possible or seeking advantage
of the inhibitors of carcinogenesis for their eventual application as anticancer agents.
Since, exposure to the environmental carcinogens is often unavoidable, the latter field
has been widely explored.
Owing to recently observed side effects and toxicity in various commonly used
therapies for breast cancer, it has now become the need of the day to develop second
generation drugs which are safe, effective, and non-resistant (Ojeswi et al., 2009).
This search has brought about newly emerging term like come back to nature, grey
to green chemistry, and various eco-friendly therapeutic remediation involving green
chemicals from natural products (Khoobchandani et al., 2009; Werneke et al., 2004).
Phytochemical prevention for severe health problems has recently gained scientific
recognition worldwide. Studies on the pharmacological mechanisms and search for
chemical structures of herbal extracts responsible for anticancer activity caught great
interest. The present piece of work explains protective effects of the plant against 7, 12
dimethylbenz (a)anthracene (DMBA) induced mammary tumor in ICRC mice.
Thuja occidentalis Linn. is a plant of the family Cupressaceae, commonly known
as Arbor-Vitae and a native tree of Europe. It has coniferous pyramidal features with
flattened branches and twigs in one plane, bearing small scale-like leaves. The plant
leaves were first identified as a remedy by native Indians in Canada during sixteenth
century for the treatment of scurvy (Millspaugh, 1974). In folk medicine, T. occiden-
talis worked as an abortificiant, contraceptive, migraine remedy, antidiarrhoel, and
hepatoprotective (Deb et al., 2007; Naser et al., 2009). As mother tincture, it is used
to treat fever, warts, and piles (Dubey and Batra, 2008; Gupta, 2002). T. occidentalis
leaves and twigs mainly contain flavonoids, terpenoids, steroids, and polysaccharides.
Thuja occidentalis plant
EXPERIMENTS AND PROTOCOL

The shade dried powdered leaves of the plant T. occidentalis were subjected to extrac-
tion, successively with solvents of increasing polarity (petroleum ether (Pt. ether),
ethyl acetate (EtOAc), and methanol (MeOH)) to recover the wide range of com-
pounds. The residual portions, obtained after removing the respective solvent (vacuum
distillation Rota vapor) were dried by purging nitrogen, weighed, and refrigerated un-
til further use.
The MCF-7 and MDA-MB-468 human breast cancer cell lines were procured from
National Centre for Cell Sciences, Pune, India. Cells were grown in Nutrient mixture
F-12, 82.5% supplemented with 2.5% FBS, 0.2% sodium bicarbonate, antibiotic, and
antimycotic solution. The cells were grown in the following conditions: 5% CO2, 95%
atmosphere in high humidity at 37°C in a CO2 incubator. Each batch of cells was as-
sessed for cell cytotoxicity by Trypan blue exclusion (Frieauff et al., 2001) and Methyl
thiazole tetrazolium cell viability assay (Lee et al., 2002). Cells for passage number
between 18 and 25 were used in the study. The ICRC mice are inbred line of albino
mouse of high breast-tumor incidence produce at Indian Cancer Research Centre,
Thuja occidentalis and Breast Cancer Chemoprevention 143
India. This strain has high susceptibility for spontaneous mammary tumors (Kanekar,
1962). Female ICRC mice (20 ± 5 g body weight) were maintained in ventilated ani-
mal house at temperature 24 ± 2°C with a 12 hr light/dark cycle and 60 ± 5% humidity.
They were provided with standard pellet diet and water ad libitum. The experiment
was carried out as per the guidelines of Ethical Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA), New Delhi, India.
All ICRC mice were divided into seven groups of eight mice each. Group I: normal
control animals were administered with 2% Dimethylsulphoxide (DMSO) (Yao et al.,
2002). Group II: tumor induced cancerous control animals received a single dose of
DMBA dissolved in olive oil. Group III and IV: animals received two doses of EtOAc
extract (5 and 10 mg/kg body weight) after DMBA administration on 0 day. Group V
and VI: animals received two doses of MeOH extract (5 and 10 mg/kg body weight)
after DMBA administration on 0 day. Group VII: received doxorubicin (standard drug
5 mg/kg body weight) (In vivo cancer model (1976–1982)) after DMBA administra-
tion on 0 day. The DMBA is a carcinogen which induces mice mammary carcinoma
from the ductal elements of the mammary gland by increasing substantial oxidative
stress. Tumor was induced (Barros et al., 2004) using DMBA as a carcinogen by a
single dose of 20 mg/kg body weight, dissolved in olive oil (1 ml) given through an
oral gavages. The test samples of the extracts were given daily through an oral gavage.
During the experimental period, animals were weighed weekly. Palpation of mam-
mary tumors began 4 week after animals received DMBA. Animals were observed
daily to assess their general health. The volume of individual tumor was measured
weekly. Tumor volume was calculated using the formula: Tumor volume (cc) = 4/3
πr3. On 120th day, mice were sacrificed and tumors were removed from the animals
and weighed. Each tumor was fixed in 10% buffered formal saline and processed for
routine histological examination. Haematoxylin and eosin stained slides were studied.
FREE RADICAL SCAVENGING ACTIVITY

Different solvent extracts (Pt. ether, EtOAc, and MeOH) of the plant T. occidentalis
(leaves) were tested for hydroxyl (Halliwell et al., 1992) and DPPH (Shimada et al.,
1992) radical scavenging capacity using α-tocoferol as a positive control with ten
increasing concentrations (10–100 µg/µl). Different test samples show percent inhi-
bition of OH˙ as follows: Pt. ether (6.42–63.04%), EtOAc (28.02–83.31%), MeOH
(20.06–78.87%), and α-tocoferol (28.08–96.22%). The DPPH radical scavenging ef-
fect of different solvent extracts were as follows: Pt. ether (7.21–64.44%), EtOAc
(28.06–82.31%), and MeOH (10.02–78.17%) with ten increasing concentrations
(10–100 µg/µl). Standard antioxidant (α-tocoferol) inhibits DPPH radical formation
(45.05–96.22%) at the same concentration range. A gradual increase in percent inhibi-
tion of DPPH with the increasing concentrations of test samples indicating its dose
dependent nature. In vitro antioxidant assay revealed that among the various extracts
studied, EtOAc extract was found to be the most potent antioxidant. The overall order
of potency was: α-tocoferol (96%) > EtOAc (83%) > MeOH (79%) > Pt. ether (63%)
at the concentration of 90 µg/µl (Table 1(a) and (b)).
Table 1. (a) Effect of various solvent extracts of the plant Thuja occidentalis on percent inhibition of
hydroxyl radical against reference (α-tocoferol).
Concentration (µg/ml) % Inhibition
Pt. ether EtOAc MeOH Toco
10 6.42 ± 1.32 10.02 ± 1.31 20.21 ± 1.30 28.08 ± 1.28
20 16.11 ± 1.34 20.02 ± 1.36 29.92 ± 1.35 37.32 ± 1.30
30 20.05 ± 1.40 26.71 ± 1.39 43.32 ± 1.41 46.04 ± 1.33
40 28.64 ± 1.52 34.27 ± 1.48 51.01 ± 1.47 57.71 ± 1.39
50 37.91 ± 1.59 47.08 ± 1.51 56.46 ± 1.53 72.82 ± 1.42
60 44.02 ± 1.60 55.42 ± 1.58 67.09 ± 1.32 80.32 ± 1.46
70 52.08 ± 1.62 61.21 ± 1.60 78.82 ± 1.32 84.04 ± 1.51
80 58.26 ± 1.71 65.04 ± 1.67 86.72 ± 1.32 95.32 ± 1.59
90 62.55 ± 1.76 69.95 ± 1.72 78.75 ± 1.32 96.22 ± 1.64
100 63.04 ± 1.81 78.31 ± 1.75 80.87 ± 1.32 96.02 ± 1.69
Each value is mean ± SD (n = 3). P < 0.05 (petroleum ether extract) vs. α-tocoferol; p > 0.05 (ethyl
acetate and methanol extract) vs. α-tocoferol (Khoobchandani et al., 2009a, 2009b).
Table 1. (b) Effect of various solvent extracts of the plant Thuja occidentalis on percent inhibition of
DPPH radicals against reference (α-tocoferol).
Concentration (µg/ml) % Inhibition
Pt. ether EtOAc MeOH Toco
10 7.21 ± 1.24 10.02 ± 1.22 28.06 ± 1.24 45.05 ± 1.25
20 17.01 ± 1.28 15.52 ± 1.25 30.08 ± 1.25 48.09 ± 1.28
30 19.03 ± 1.31 20.41 ± 1.29 37.07 ± 1.29 52.09 ± 1.31
40 27.94 ± 1.35 32.26 ± 1.32 45.87 ± 1.31 58.43 ± 1.34
50 37.07 ± 1.38 47.08 ± 1.36 56.87 ± 1.37 65.52 ± 1.38
60 45.02 ± 1.42 50.04 ± 1.39 62.21 ± 1.40 72.65 ± 1.42
70 51.04 ± 1.44 61.21 ± 1.41 67.87 ± 1.44 88.05 ± 1.46
80 59.26 ± 1.47 64.48 ± 1.44 73.26 ± 1.47 94.42 ± 1.49
90 63.45 ± 1.50 69.95 ± 1.48 78.75 ± 1.50 96.22 ± 1.52
100 63.44 ± 1.54 72.31 ± 1.50 80.87 ± 1.52 96.02 ± 1.54
Each value is mean ± SD (n = 3). P < 0.05 (petroleum ether extract) vs. α-tocoferol; p > 0.05 ethyl
acetate and methanol extract) vs. α-tocoferol (Khoobchandani et al., 2009a; 2009b).
In vivo antioxidative effect of solvent extracts (EtOAc and MeOH) of the plant
leaves was estimated in terms of percentage of down regulation of reduced glutathione
(GSH) in DMBA induced oxidative stress in female ICRC mice liver (Ojeswi et al.,
2010). As Pt. ether extract did not show any marked antioxidative activity in earlier in
vitro experiments, was not considered for in vivo studies. The EtOAc and MeOH ex-
tracts of the plant T. occidentalis (leaves) were considered in test concentration 5 and
10 mg/kg body weight of experimental mice separately and percentage of down regulation
of reduced GSH was recorded as a function of days (20, 40, 60, and 120 days) against
normal and cancerous control experimental mice. In normal, no DMBA and extracts
were administered and in cancerous control, no extract was given while all other ex-
perimental conditions remained same as that of the treatments. An increased level of
down regulation of reduced GSH in cancerous control animals was observed when
compared with EtOAc extract (10 mg/kg body weight) treated animals. The percent-
age of down regulation of reduced GSH in doxorubicin drug treated group was near to
EtOAc extract (10 mg/kg body weight) treated mice which indicate its protective role
against DMBA induced oxidative stress (Figure 1).
The DMBA toxicity is associated with its oxidative metabolism leading to the
formation of free radicals, which bind covalently to nucleophillic sites on cellular
macromolecules eliciting cancerous responses. Free radicals and their biochemical
reactions in each stage of the metabolic process are involved in cancer development
(Kun-Young et al., 2003). Antioxidants act as the primary line of defense against re-
active oxygen species and suggest their usefulness in estimating the risk of oxida-
tive damage induced during carcinogenesis. The GSH is non-protein cellular thiol
which in conjunction with GPx has a regulatory role in cell proliferation (Anbuselvam
et al., 2007). The GSH and its dependent enzymes scavenge the electrophilic moi-
eties involved in the cancer initiation (Sunde and Hoekstra, 1980) and serves as
marker for the evaluation of oxidative stress (Comporti, 1989; Nam and Kang, 2008).
We observed decreased down regulation of reduced GSH in EtOAc extract of the
plant T. occidentalis treated animals which suggest the antioxidative properties of T.
occidentalis.
Figure 1. Effect of EtOAc extract of Thuja occidentalis (leaves) on reduced GSH in liver of
experimental mice. Values are expressed as mean ± SD (n = 8); p < 0.05 vs. cancerous control
(Ojeswi et al., 2010).
ANTIBREAST CANCER ACTIVITY

In vitro antibreast cancer activity of Pt. ether, EtOAc, and MeOH extracts of T.
occidentalis (leaves) were screened against human breast cancer cell lines MCF-7 and
MDA-MB-468 with ten increasing concentrations (10–100 µg/µl) for 24 hr by the
TBE and MTT bioassays (Ojeswi et al., 2009). The Pt. ether extract did not show any
marked cytotoxic activity. In case of EtOAc extract, maximum inhibition 51.42 and
51.21% of MDA-MB-468 cell lines was achieved at the concentration level of 90 µg/
µl, by TBE and MTT assays respectively while in MeOH extract, the growth of MDA-
MB-468 cells was inhibited up to 49.02 and 49.02% at the concentration level of 90
µg/µl by the above assays (Table 2(a) and (b)). The growth of MCF-7 cell line was in-
hibited by the EtOAc extract up to the maximum level of 91.42 and 94.06% at the con-
centration level of 90 µg/µl in TBE and MTT assays, respectively. In case of MeOH
extract, maximum inhibition 88.02 and 92.04% of MCF-7 cell line was observed at
the same concentration using respective assays (Table 3(a) and (b)). Percent inhibition
resulting from in vitro bioassays demonstrated that both the extracts exhibit cytotoxic-
ity (antibreast cancer activity) against the cell lines MDA-MB-468 and MCF-7. These
extracts showed more pronounced efficacy on MCF-7 compared to MDA-MB-468
cell line (p < 0.05). It was also inferred that EtOAc extract is more significant an-
tibreast cancer agent against cell line MCF-7 showing maximum inhibition 94.06%
at the concentration 90 µg/µl at 24 hr. IC50 values calculated from MTT assay using
probit analysis were as follows: Pt. ether (25.70), EtOAc (21.08), and MeOH (22.97).
Table 2(a). Percentage growth inhibitory activity of various solvent extracts of the plant. Thuja
occidentalis on MDA-MB-468 Cell line by Trypan blue exclusion assay.
Concentration (µg/µl) % Inhibition
Pt. ether EtOAc MeOH
10 7.52 ± 1.21 9.01 ± 1.23 11.01 ± 1.23
20 17.41 ± 1.24 19.02 ± 1.26 20.22 ± 1.27
30 22.07 ± 1.28 24.12 ± 1.29 31.06 ± 1.30
40 26.71 ± 1.31 31.88 ± 1.32 37.12 ± 1.33
50 30.09 ± 1.33 37.75 ± 1.35 40.44 ± 1.35
60 35.02 ± 1.35 42.16 ± 1.37 44.83 ± 1.38
70 37.24 ± 1.38 43.75 ± 1.39 46.43 ± 1.41
80 40.41 ± 1.39 48.02 ± 1.42 49.08 ± 1.44
90 42.5 ± 1.42 48.42 ± 1.45 49.42 ± 1.46
100 23.41 ± 1.45 49.2 ± 1.46 50.02 ± 1.48
Each value is mean ± SD (n = 3). P < 0.05 (Ojeswi et al., 2009)

Table 2. (b) Percentage growth inhibitory activity of various solvent extracts of the plant Thuja
occidentalis on MDA-MB-468 Cell line by MTT assay.
10 8.52 ± 1.12 10.11 ± 1.14 12.01 ± 1.42
20 18.41 ± 1.15 20.02 ± 1.21 22.22 ± 1.37
30 23.67 ± 1.18 27.12 ± 1.25 32.66 ± 1.31
40 28.37 ± 1.21 33.88 ± 1.32 39.11 ± 1.33
50 31.09 ± 1.23 38.75 ± 1.36 42.44 ± 1.37
60 36.02 ± 1.26 42.16 ± 1.38 45.83 ± 1.42
70 39.24 ± 1.29 44.75 ± 1.44 47.43 ± 1.44
80 42.41 ± 1.33 49.02 ± 1.48 50.08 ± 1.47
90 44.05 ± 1.35 49.01 ± 1.53 50.4 ± 1.49
100 45.08 ± 1.37 50.2 ± 1.61 51.02 ± 1.52
Each value is mean ± SD (n = 3), P < 0.05 (Ojeswi et al., 2009).
Table 3. (a) Percentage growth inhibitory activity of various solvent extracts of the plant. Thuja
occidentalis on MCF-7 Cell line by Trypan blue exclusion assay
10 15.02 ± 1.30 19.13 ± 1.27 20.42 ± 1.32
20 34.41 ± 1.33 38.24 ± 1.30 39.22 ± 1.34
30 46.12 ± 1.35 54.66 ± 1.33 59.66 ± 1.35
40 49.07 ± 1.39 70.08 ± 1.37 75.32 ± 1.39
50 60.01 ± 1.42 73.15 ±1.39 79.04 ±1.40
60 66.32 ± 1.44 70.06 ± 1.43 82.83 ± 1.44
70 73.21 ± 1.47 84.75 ± 1.45 86.43 ± 1.46
80 80.41 ± 1.49 86.02 ± 1.47 88.08 ± 1.50
90 84.05 ± 1.51 89.01 ± 1.49 88.9 ± 1.52
100 86.08 ± 1.58 88.42 ± 1.50 89.02 ± 1.54
Each value is mean ± SD (n = 3), P < 0.05 (Ojeswi et al., 2009).

Table 3. (b) Percentage growth inhibitory activity of various solvent extracts of the plant Thuja
occidentalis on MCF-7 Cell line by MTT assay.

10 18.52 ± 1.20 20.11 ± 1.22 21.01 ± 1.24
20 38.41 ± 1.23 40.02 ± 1.24 41.22 ± 1.26
30 46.67 ± 1.25 57.12 ± 1.27 61.66 ± 1.27
40 51.37 ± 1.28 73.88 ± 1.29 75.11 ± 1.29
50 61.09 ± 1.30 76.75 ± 1.31 82.44 ± 1.30
60 69.02 ± 1.33 82.16 ± 1.33 85.83 ± 1.32
70 76.24 ± 1.35 86.75 ± 1.35 87.43 ± 1.34
80 83.41 ± 1.37 89.02 ± 1.37 90.08 ± 1.38
90 85.05 ± 1.39 90.01 ± 1.39 94.06 ± 1.39
100 85.08 ± 1.40 92.42 ± 1.41 94.4 ± 1.41
Each value is mean ± SD (n = 3). P < 0.05 (Ojeswi et al., 2009).
In vivo experiment has been conducted to observe the preventive role of EtOAc
and MeOH extracts of T. occidentalis (leaves) against DMBA induced mammary can-
cer (Ojeswi et al., 2010). As Pt. ether extract did not show any marked cytotoxic activ-
ity, therefore, was not considered for the present study. The EtOAc and MeOH extracts
in two doses (5 and 10 mg/kg body weight) of the plant were tested for DMBA induced
ICRC mice mammary carcinoma in terms of tumor weight, volume, increase in sur-
vival rate, body weight, histological variation, and mutagenicity against the standard
drug doxorubicin. The effect of test samples on mean tumor volume was measured. It
was found that tumor volume in the extracts treated was smaller than cancerous con-
trol group. The EtOAc extract (10 mg/kg body weight) showed significant reduction
of tumor volume compared to cancerous control (p < 0.05). However, smallest tumor
volume was observed in case of doxorubicin drug treated group. Thus, EtOAc extract
(10 mg/kg body weight) administered in ICRC mice appeared to reduce tumor volume
up to 50% compared to cancerous control group (Figure 2). The effect of doses (5
and 10 mg/kg body weight) of EtOAc and MeOH extracts of the plant T. occidentalis
(leaves) on tumor weight was observed in cancerous control, EtOAc and MeOH ex-
tract treated, and doxorubicin drug treated groups. The EtOAc extract (10 mg/kg body
weight) exhibited significant (p < 0.05) reduction in tumor weight (39%) compared to
cancerous control group at 120th day. However, smallest tumor weight was observed
in case of doxorubicin drug treated group. The tumor weight of mice in other groups
was higher than EtOAc extract (10 mg/kg body weight) and lower than cancerous con-
trol groups (Table 4). A gradual increase in body weight was observed in all animals up
to 48th day of experiment. The body weight of the mice in cancerous control group did
not show any increase after 48th day and then started decreasing constantly. The body
weight of the normal control group increased up to 120th day, demonstrating a normal
growth pattern. There was a sharp and significant difference in body weight between
the normal and cancerous control group and cancerous control and EtOAc extract (10
mg/kg body weight) group at the end of 120th day (p < 0.05). The body weight of
doxorubicin drug treated group was near to cancerous control group. The body weight
of mice in other groups was between cancerous control and EtOAc extract (10 mg/kg
body weight) group. (Table 4)
The effect of test samples on percentage of survival was measured. It was concluded
that in EtOAc extract (5 and 10 mg/kg body weight) and MeOH extract (10 mg/kg body
weight) treated groups, 90% survival was observed at final day of experimentation. In
case of MeOH extract (5 mg/kg body weight) treated, 80% survival while in doxorubi-
cin drug treated group 66% survival were observed at the final 120th day. The lowest
survival rate, 40% was observed in cancerous control group. Thus, EtOAc extract (10
mg/kg body weight) administered in ICRC mice appeared to increase life span of ani-
mals compared to cancerous control and doxorubicin drug treated groups (Figure 3).
Figure 2. Effect of EtOAc and MeOH extracts of Thuja occidentalis (leaves) on palpable tumor
volume. Each value is mean ± SD (n = 8). P > 0.05 vs. cancerous control. Th.: Thuja occidentalis;
EtOAc: ethylacetate; MeOH: methanol; Doxo: doxorubicin. (Ojeswi et al., 2010).
Table 4. Effect of Thuja occidentalis extracts on body weight and tumor weight.
Animals Parameters
Body weight (g) Tumor weight (g)
Normal Control 62.25 ± 8.30 ----
Cancerous Control 30.45 ± 6.902 5.20 ± 0.29
EtOAc extract (5 mg/Kg body weight) 48.06 ± 3.71 2,3 2.19 ± 0.125
EtOAc extract (10 mg/Kg body weight) 51.24 ± 8.01 1;4
2.01 ± 0.346
MeOH extract (5 mg/Kg body weight ) 40.02 ± 2.80 2,3
4.02 ± 0.315
MeOH extract (10 mg/Kg body weight ) 42.31 ± 2.82 2,3 3.76 ± 0.145
Doxorubicin (5 mg/Kg body weight ) 28.43 ± 2.21 2
1.42 ± 0.026
Values are expressed as mean ± SD (n = 8); 1p > 0.05 vs. normal control. 2p < 0.05 vs. normal
control. 3p > 0.05 vs. cancerous control and doxorubicin. 4p < 0.05 vs. cancerous control and
doxorubicin. 5p > 0.05 vs. cancerous control. 6p < 0.05 vs. cancerous control. (Ojeswi et al., 2010).
Figure 3. Effect of EtOAc and MeOH extracts of Thuja occidentalis (leaves) on percent survival. Each
value is mean ± SD (n = 8). P < 0.05: EtOAc (5 and 10 mg/kg body weight) and MeOH (10 mg/kg body
weight) extracts vs. cancerous control; p < 0.05: EtOAc (10 mg/kg body weight) vs. oxorubicin. Th:
Thuja occidentalis; EtOAc: ethyl acetate; MeOH: methanol; Doxo: doxorubicin. (Ojeswi et al., 2010).
HISTOPATHOLOGICAL STUDY
Mammary tumors were excised from control and test samples treated animals for their
histological evaluation at the 120th day. Histological evaluation revealed that all tumors
from the cancerous control group were highly malignant cells and none of the tumors
showed necrosis. In tumors excised from animals receiving EtOAc extract (10 mg/kg
body weight), significant areas of necrosis were present compared to MeOH (10 mg/kg
body weight) treated group while in case of doxorubicin treated tumors, large foci of ne-
crosis areas were present which were distinctly appeared in Slide 1 (Ojeswi et al., 2010).
Slide 1. Microscopic view of Haematoxylin and eosin stained slides of cancerous control, doxorubicin
treated, EtoAc extract (10 mg/kg body weight) and MeOH extract (10 mg/kg body weight) of T.
occidentalis-treated tumors. Dark colored areas show well-developed tumor cells while light colored
areas show necrosis. (Ojeswi et al., 2010).
ANTIMUTAGENIC ACTIVITY
Antimutagenic activity (Savage, 1993) of various solvent extracts of the plant T.
occidentalis (leaves) was evaluated against DMBA induced cytogenetic damage in
female ICRC mice bone marrow. The effect of test samples on percent of chromo-
somal aberration was measured in terms of chromatid breaks (CB), centric rings (CR),
exchanges, acrocentric association (ACA), acentric fragments (FR), intracalary dele-
tion (ICD), and total abnormal metaphases. Percent of aberrant metaphase in vari-
ous groups were as follows: cancerous control–92.34%, doxorubicin treated (5 mg/kg
body weight)–73.56%, EtOAc extract (5 mg/kg body weight)–53.34%, EtOAc extract
(10 mg/kg body weight–45.25%, MeOH extract (5 mg/kg body weight)–51.90%, and
MeOH extract (10 mg/kg body weight–57.74% (Table 5). The extracts under study
reduce the chromosomal aberration like CB, CR, ACA, FR, ICD, and pulverization
in bone marrow cells compared to cancerous control and standard drug doxorubicin
treated groups. The EtOAc extract (10 mg/kg body weight) exhibited maximum reduc-
tion in chromosomal aberration in bone marrow of cancerous mice (p <0.05) while,
doxorubicin was not found to exhibit the reduction in abnormal metaphases (Slide 2).
Table 5. Effect of various solvent extracts of the plant Thuja occidentalis on Percent aberrant
metaphases in the bone marrow of experimental ICRC mice.
Abnormal
Group CB CR FR ACA Pulvi
metaphase
Normal Control 0.20 ± 0.40 0.0 ± 0 0.60 ± 0.54 0.0 ± 0 0.0 ± 0 0.8 ± 0.84
Cancerous Control 16.80 ± 1.28 2
6.70 ± 1.83 2
43.80 ± 3.49 2
57.20 ± 1.92 2
5.20 ± 1.45 2
92.34 ± 4.49 2
Doxorubicin
10.60 ± 3.362 5.60 ± 1.232 27.40 ± 2.302 46.60 ± 2.382 3.67 ± 2.962 73.56 ± 3.252
(5mg/Kg body weight )
EtOAc extract (5 mg/Kg
8.20 ± 2.483 3.12 ± 2.183 11.25 ± 2.363 22.4 6± 3.463 0.82 ± 0.453 53.34 ± 4.673
body weight)
EtOAc extract (10 mg/
6.34±3.343 2.04±2.183 10.36±3.163 25.25±2.413 0.0±01 43.25±6.563
Kg body weight)
MeOH extract (5 mg/
9.23±2.093 4.03±1.703 12.84±2.933 38.84±3.733 0.34±0.283 57.74±2.943
Kg) body weight)
MeOH extract (10 mg/
8.46±1.593 3.23±2.143 11.98±2.513 34.92±3.563 0.0±01 51.90±5.853
Kg body weight)
Mean ± SE (n = 8). Where; CB: Chromatid Break, CR: Centric Ring, DC: Dicentric, FR: Fragment, ACA: Acrocentric
Association, Pulvi: Pulvirization, P value: 1p > 0.05 vs. Normal Control; 2p < 0.001 vs. Normal Control; 3p < 0.01
vs. Normal control and Tumor Control.
The antibreast cancer bioassay of the extracts (EtOAc and MeOH) indicates that
EtOAc extract of T. occidentalis (leaves) showed significant antibreast cancer activity
against DMBA induced mammary carcinoma in ICRC mice. The EtOAc extract of the
plant exhibit reduction of tumor volume (50%), tumor weight (39%) and chromosom-
al aberration (54%) compared to cancerous control group with the increase in body
weight and life span in comparison with cancerous control and doxorubicin treated
group. Therefore, EtOAc extract appears to be most effective for antibreast cancer ac-
tivity. The observed results in all the bioassay parameters indicate the presence of some
chemical moiety in the leaves of the plant T. occidentalis, responsible for its antibreast
cancer activity. The EtOAc extract being potent antibreast cancer agent against DMBA
induced mammary carcinoma in ICRC mice and is considered for characterization of
bioactive principle.
Slide 2. Chromosomal aberration in bone marrow of experimental mice (a) Normal Control, (b)
Tumor Control, (c) Doxorubicin treated, (d) EtOAc extract (10 mg/kg body weight) treated.
ACTIVITY GUIDED CHROMATOGRAPHIC FRACTIONATION OF THE

ETHYL ACETATE EXTRACT
The EtOAc extract was refluxed with Pt. ether for 20 hr to remove fat content. Defat-
ted EtOAc soluble mass was subjected to chromatographic separation using a column
(120 cm long and 4 cm diameter with stationary phase of 125 g of silica gel) eluted
with CH3OH: water (H2O) (1:1). After the removal of solvent, a brown mass was
obtained which was monitored by thin layer chromatography (TLC) using solvent
system EtOAc (CH3COOC2H5): formic acid (HCOOH): H2O (8:1:1). The develop-
ment of chromatogram in iodine chamber showed six spots. The brown mass was re-
chromatographed using 150 cm long and 3 cm diameter column with stationary phase
of 80 g silica gel and eluted with different composition of solvent mixture chloroform
(CHCl3): MeOH. Different fractions of 25 ml each were collected and subjected to
TLC to ensure their purity using (EtOAc: CHCl3: CH3OH; 7:1:2) mobile phase. Frac-
tions of same Rf values, fraction 13‑24; Rf = 0.44 labeled as LEA1 (first fraction of
EtOAc extract; fraction 25‑38; Rf = 0.66 labeled as LEA2 (second fraction of EtOAc
extract, fraction 39‑53; Rf = 0.81 labeled as LEA3 (third fraction of EtOAc extract,
fraction 63‑77; Rf = 0.85 labeled as LEA4 (fourth fraction of EtOAc extract, fraction
78‑99; Rf = 0.94 labeled as LEA5 (fifth fraction of EtOAc extract and fraction 109‑116;
Rf = 0.96 labeled as LEA6 (sixth fraction of EtOAc extract were mixed (Table 6).
Removal of solvent furnished a white (LEA2), pale yellow (LEA3), and light yellow
(LEA5) compounds. Compound LEA1, LEA4,, and LEA6 were found in trace amount.
Table 6. Details of chromatographic fractionation of ethyl acetate extract of Thuja occidentalis

(leaves) (Ojeswi et al., 2010).
Fraction No. Solvent System TLC Rf Remarks
1–12 CHCl3:MeOH (9:1) Nil - -
13–24 CHCl3:MeOH (9:1) One Spot 0.04 LEA1
54–62 CHCl3:MeOH (7:3) Nil - -

100–108 CHCl3:MeOH (4:6) Nil - -

117–124 CHCl3:MeOH (2:8) Nil - -
125–135 CHCl3:MeOH (1:9) Nil - -
ANTI BREAST CANCER BIOASSAY OF THE EtOAc FRACTIONS

The major compounds (LEA2, LEA3, and LEA5) recovered from EtOAc extract were
screened for antibreast cancer activity in terms of cytotoxic activity against MCF-
7 cell line. Compound LEA2 and LEA3 did not show noticeable cytotoxic activity,
30.04 and 32.86% inhibition of MCF-7 cell line at 24 hr. In case of LEA5, maximum
inhibition 93.88% of MCF-7 cells was observed at the concentration of 90 µg/µl.
The compound LEA5 was further assessed for antibreast cancer activity in terms of
tumor weight and volume in ICRC mice. Improvement in the bioefficacy in terms of
decrease in the treatment concentration from 10 mg/kg body weight to 4.5 mg/kg body
weight has been observed in the compound (LEA5) exhibiting tumor weight: 1.99 ±
0.34 g; tumor volume: 0.40 ± 0.15 cc (Ojeswi et al., 2010). The compound (LEA5)
also exhibited large necrosis area at the concentration of 4.5 mg/kg body weight in the
histological slide of mammary tumor as equivalent to EtOAc extract (10 mg/kg body
weight) treated tumors (Ojeswi et al., 2010). The compound LEA5 was, therefore,
considered for its chemical characterization which is in progress.
CONCLUSION
The present piece of work demonstrates that EtOAc extract of the plant T. occidentalis
exhibited decreased tumor weight and volume compared to cancerous control with
enhanced body weight and longevity compared to cancerous control and doxorubicin
drug treated group. The chromatographic fractionation of the EtOAc extract to its
derived compound LEA5A (aglycone) has resulted into the lowering of effective test
concentration from 10 mg/ml to 4.5 mg/ml and 5 mg/ml to 2.6 mg/ml with equivalent
bioefficacy.
KEYWORDS
•• CHARGE syndrome
•• Chromatin-remodeling enzyme KISMET (KIS)
•• Circadian rhythmicity
•• Constant darkness (DD)
•• Cryptochrome (CRY)
•• Day:night cycle
•• Drosophila
•• Jetlag (JET)
•• Light-dependent degradation
ACKNOWLEDGMENT
The authors gratefully acknowledge Prof. V. G Dass, Director, Dayalbagh Educational
Institute, Dayalbagh, Agra, for providing necessary research facilities. Authors ac-
knowledge Board of Research in Nuclear Science, Mumbai for providing Financial
Assistant.
Chapter 10
Antioxidants and Combinatorial Therapies in
Cancer Treatment
Arpita Saxena
INTRODUCTION
Cancer has been posed as a major threat to humans not because there are no medi-
cations available, but because all available therapies have many side effects (Sax-
ena et al., 2010). All the current chemotherapeutic agents cause a lot of damage to
non-cancerous cells along with the cancerous cells. Plant derived anticancer drugs act
through multi-targets simultaneously and/or synergistically. Many of these drugs are
also chemo preventive, which prevent the both primary and secondary recurrence of
the disease. Many cancer patients, who are undergoing the therapy, take antioxidant
supplements in an effort to alleviate treatment toxicity and improve the long-term
outcomes. The modulating effects of antioxidants in treatment depend on a wide range
of factors, including the metabolic state of the patient, the stage and site of the dis-
ease, and the modality being used (Carmia, 2004). Agents used in chemotherapy dam-
age a plethora of cellular molecules, increase lipid peroxidation of molecules, reduce
antioxidant levels, and enhance oxidative stress (Sangeetha et al., 1990). Therefore,
combination of antioxidants with conventional anticancer drug will be beneficial.
Dietary and endogenous antioxidants prevent cellular damage by reacting with and
eliminating oxidizing free radicals. Considerable laboratory evidence from chemical,
cell culture, and animal studies indicate that antioxidants may slow or possibly prevent
the development of cancer. Studies show that a high intake of antioxidant rich foods
is inversely related to cancer risk. While clinical studies on the effect of antioxidants
in modulating cancer treatment are limited in number and size. Experimental studies
show that antioxidant vitamins and some phytochemicals selectively induce apoptosis
in cancer cells but not in normal cells and prevent the angiogenesis and metastatic
spread, suggesting a potential role for antioxidants as adjuvants in cancer therapy.
Henceforth, this synergistic approach can lead to minimized side effects and effective
dose of conventional chemotherapeutics.
Many studies target towards the lowering of the dose of known anticancer drugs or
potent anti-neoplastic leads with higher efficacy (apoptotic potential) is using various
antioxidants. Such studies assume that antioxidants may be helpful in the existing can-
cer therapies (Borek, 2004; Lee et al., 1999). The idea that drives them usually is that
if a combination of antioxidants can reduce the dose, then the side-effects of conven-
tional anticancer drugs like inflammation of adjoining tissues, interference with proper
metabolism and hindering the normal activities can be avoided to a large extent. The
consideration of whether to use antioxidants concomitantly with chemotherapy and
radiation therapy has evolved into a heated debate. Great debates have sometimes
spawned great breakthroughs in medical treatment, improving patient outcomes and
saving lives. There are two groups of scientists who have asserted two different opin-
ions about using antioxidants in the cancer therapy. One camp holds that taking an-
tioxidants during cancer treatment could interfere with the way chemo and radiation
work and diminish their benefits to the patient (Block, 2004). This is because radiation
and some chemotherapy agents work by generating free radicals, which kill rapidly
dividing cancer cells. Since, antioxidants scavenge free radicals, they might interfere
with the therapeutic effects of these treatments. The opposing argument is that oxida-
tion supports the proliferation of cancer cells and may itself interfere with treatment
(Duthie et al., 1996). People who hold this view maintain that antioxidants may coun-
ter the harmful effects of oxidation in the malignant process and thereby increase the
effects of drugs or radiation therapy in the benefit of the patient. Moreover, they note
that some evidence suggests that antioxidant supplements to offer patient protection
from the toxic effects of therapy and increase the efficacy (Lamson and Brignall, 1999,
2000; Prasad, 2004).
SYNERGISTIC ENHANCEMENT OF ANTICANCER POTENTIAL OF A LIGNIN

COMPOSITION FROM CEDRUS DEODARA BY NATURAL ANTIOXIDANTS
In a recent chapter published by us, we tried to experiment with the synergy of two dif-
ferent antioxidant entities distinctly known for their anticancer properties. However,
results showed the same extent of activities at one third the concentration of them by
combining those (Saxena et al., 2010). The study involved AP9-cd, a standardized
lignan from Cedrus deodar, which showed cytotoxicity and antitumor activity in vari-
ous human cancer cell lines and different murine cancer models (Singh et al., 2007)
and three different natural antioxidants namely Curcumin, Acteoside, and Silymarin.
The AP9-cd has an optimum cytotoxic and growth inhibitory potential of 30 µg/ml in
human leukaemia HL-60 and Molt-4 cells. This means that the apoptotic potential of
AP9-cd was synergised by these antioxidants by three times and more.
The conclusion was that the cytotoxic and apoptotic potential of AP9-cd was sig-
nificantly synergized by three natural antioxidants curcumin, acteoside, and silymarin
in HL-60 cells. The mechanism of synergy involves the strong antioxidant effect of
these antioxidants on HL-60 cells. All the three antioxidants reduce the reactive ni-
trogen oxygen species (RNOS) burst in HL-60 cells and inhibit the activation and
translocation of NF-κB in the nucleus of HL-60 cells. Curcumin showed maximum
synergy with AP9-cd in terms of cytotoxic and apoptotic potential than silymarin and
acteoside in HL-60 cells. The combinations of antioxidants with AP9-cd provided an
effective approach for cancer therapy that overcomes chemo-resistance and possible
side effects.
SUPPLEMENTATION OF ANTIOXIDANT NUTRIENTS MAY PROTECT

AGAINST CISPLATIN-INDUCED OXIDATIVE DAMAGE WHILE RETAINING
THE ANTITUMOR EFFICACY
A study performed by Weij et al. (1998) states that cisplatin chemotherapy induces
acute and more gradually occurring decreases in several major plasma antioxidants.
Antioxidants and Combinatorial Therapies in Cancer Treatment 157
The observed fall in plasma antioxidant concentrations is probably determined by

more than one mechanism, namely oxidative stress-induced consumption of antioxi-
dants and renal loss of water-soluble low molecular weight antioxidants due to hyper
filtration in combination with a specific cisplatin-related renal tubular defect. This is
an undesirable situation as it may lead to diminished protection from chemotherapy
induced oxidative stress and increased oxidative damage to normal tissues such as
renal tubular cells. All in all, the results and findings of the authors suggested that
supplementation of antioxidant nutrients may protect against cisplatin-induced oxida-
tive damage while retaining the antitumor efficacy. It is the opinion of the authors of
this chapter that the role of pro-oxidative metals, for example, copper and iron, and of
antioxidants such as ceruloplasmin in the pathogenesis amelioration of chemotherapy-
induced toxicity is further studied.
ENHANCEMENT OF CYTOTOXIC POTENTIAL OF CHEMOTHERAPEUTIC

AGENTS IN COLORECTAL CANCER BY ANTIOXIDANTS
A study published by Chinery et al. (1997), showed that the antioxidants pyrrolidine
dithiocarbamate (PDTC) and vitamin E induce apoptosis in CRC Cells. They further
proved that this effect is mediated by induction of p21WAF1/CIP1, a powerful in-
hibitor of the cell cycle, through a mechanism involving C/EBP β (a member of the
CCAAT/enhancer binding protein family of transcription factors), independent of p53.
Despite a response rate of only 20%, five-fluorouracil (5FU) remains the single most
effective treatment for advanced colorectal cancer (CRC), which is the second-leading
cause of cancer deaths in the United States. Antioxidants significantly enhanced CRC
tumor growth inhibition by cytotoxic chemotherapy in vitro (5FU and doxorubicin)
and in vivo (5FU). Thus the authors insist that chemotherapeutic agents administered
in the presence of antioxidants may provide a novel therapy for colorectal cancer.
ENHANCEMENT OF EFFECT OF DOXORUBICIN BY ANTIOXIDANTS

Another study by Liu and Tan published in 2002 states the fish oil and vitamin E ap-
peared to enhance the antitumor effect of optimal doses of doxorubicin. In this study,
four kinds of rodent diets, CO, FO, CVe, and FVe, were used by addition of canola oil,
oil mixture (fish oil + canola oil), canola oil plus vitamin E, and oil mixture plus vi-
tamin E, respectively, to a basic diet, AIN-93G, to investigate the influence of dietary
fish oil and vitamin E on doxorubicin treatment in P388 ascitic mice. Animal life span
(LS) and heart damage were recorded in mice fed the four different diets and treated
with distinct doses of doxorubicin. The optimal doses of doxorubicin for antitumor
effect as manifested by increased LS were 6.0 and 9.0 mg/kg. The work comprehen-
sively investigated the influence of dietary fish oil and vitamin E, individually and in
combination, on the therapeutic efficacy of doxorubicin and has shown their additively
enhancing effects on optimal doses of doxorubicin in P388 ascitic mice. The authors,
however, were honest to state that increasing doxorubicin dose led to severe heart
damage, which was exacerbated by fish oil and vitamin E. Thus overall, it appeared to
them that both fish oil and vitamin E modulate the effects of doxorubicin in the labora-
tory mouse like a double-edged sword, on the one hand, enhancing its antitumor effect
and on the other, aggravating its cardiotoxicity.
CANCER PREVENTIVE AND CURATIVE ABILITY OF SILYMARIN

Silymarin is a naturally occurring polyphenolic antioxidantflavonoid extracted from
the milk thistleplant [Silybum marianum (L.) Gaertneri]. Silymarin is the collective
name for the active compounds derived from the plant, and silibinin is the most active
and abundant constituent. Silymarin is one such agent, which has been extensively
used since ages for the treatment of liver conditions, and thus has possibly the greatest
patient acceptability. A study published in Hogan et al. (2007) opinions that silibinin
significantly inhibits proliferation through cell-cycle arrest via inhibition of cyclin-
CDK promoter activity. Despite its antioxidant profile, there is no effect on COX-2
expression. Apoptosis does not appear to be greatly increased in human colon cancer
cell lines Fet, Geo, and HCT116. Rather, inhibition of cell cycle regulatory proteins
play a fundamental role in silibinin’s mechanism of action, and this may serve as a
basis for combined use with conventional chemotherapeutics.
Another study published in the same year by Kaur and Agarwal (2007) reiterates
that silymarin has cancer protective effects against skin, prostrate, breast, bladder,
hepatocellular, lung, colon, and ovarian carcinomas. They have also performed clini-
cal trials on cancer patients where patients were administered silymarin orally. Their
observations have significant relevance for translating the basic research to clinical
settings, as two major hurdles in this transition that is bioavailability and toxicity, have
been somewhat defined for silymarin and silibinin. As they suggest, hepatoprotective
effects of silymarin and silibinin confer added advantage of using them in adjuvant
therapy, not limiting only to their cancer chemopreventive efficacy.
HEATED DEBATES OVER THE USE OF ANTIOXIDANTS IN COMBINATION

WITH CANCER DRUGS
There is an Irish saying“Hope is the physician of each misery” and that “There is no
hope unmingled with fear, and no fear unmingled with hope”Baruch Spinoza.
Numerous articles and several reviews have been published on the role of an-
tioxidants, and diet and lifestyle modifications in cancer prevention. However, the
potential role of these factors in the management of human cancer has been largely
ignored (Kedar et al., 1999). Extensive in vitro studies and limited in vivo studies
have revealed that individual antioxidants such as vitamin A (retinoids), vitamin E
(primarily α-tocopheryl succinate), vitamin C (primarily sodium ascorbate), and carot-
enoids (primarily polar carotenoids) induce cell differentiation and growth inhibition
to various degrees in rodent and human cancer cells by complex mechanisms. The
proposed mechanisms for these effects include inhibition of protein kinase C activ-
ity, prostaglandin E1-stimulated adenylate cyclase activity, expression of c-myc, H-
ras, and a transcription factor (E2F), and induction of transforming growth factor-β
and p21genes. Furthermore, antioxidant vitamins seperately or in combination enhance
the growth-inhibitory effects of x-irradiation, chemotherapeutic agents, hyperthermia,
and biological response modifiers on tumor cells, primarily in vitro. These vitamins,
individually, also reduce the toxicity of several standard tumor therapeutic agents on
normal cells. Low fat and high fiber diets can further enhance the efficacy of standard
cancer therapeutic agents; the proposed mechanisms for these effects include the pro-
Antioxidants and Combinatorial Therapies in Cancer Treatment 159
duction of increased levels of butyric acid and binding of potential mutagens in the
gastrointestinal tract by high fiber and reduced levels of growth promoting agents such
as prostaglandins, certain fatty acids, and estrogen by low fat.
It was suggested in a recent publication that no supplementary antioxidants be
given concurrently with chemotherapy agents who employ a free-radical mechanism
(Labriola and Livingston, 1999). The present authors are by no means recommending
any lack of caution about the use of antioxidants. On the contrary, published research
indicates the cautious and judicious use of a number of antioxidants can be helpful
in the treatment of cancer; as sole agents and as adjuncts to standard radiation and
chemotherapy protocols (Lamson and Brignall, 1999). It is the opinion of the authors
of this chapter that interactions between antioxidants and chemotherapeutics cannot
be predicted solely based on the presumed mechanisms of action. The fact remains
that physicians must be aware of the available research to help their patients take
advantage of positive interactions existing between antioxidants and chemotherapy or
radiation. Additionally, physicians need to remain aware of the large body of evidence
showing a positive effect of antioxidants in the period following chemotherapy admin-
istration. The general protocol with standard oncologic therapies is to follow a watch-
and-wait strategy after therapeutic administration is concluded. This is a period when
supplemental therapies are highly indicated and have been demonstrated to result in a
higher percentage of successful outcomes (Lamm et al., 1994; Whelan et al., 1999). In
words of Derek (2009) reducing complicated interactions to a single sentence can be
an oversimplification.
KEYWORDS
•• Antioxidants
•• Colorectal cancer
•• Cytotoxic
•• Five-fluorouracil
•• Life span
•• Silymarin
Chapter 11
Eruca sativa Inhibits Melanoma Growth:
A Scientific Evidence
M. Khoobchandani, N. Ganesh, L. Valgimigli, and M. M. Srivastava
INTRODUCTION
Cancer is the abnormal growth of cells usually invades and destroys normal cells in
our bodies. These cells are born due to imbalance in the body viz. metabolic disorder
in cellular system and reactive oxygen species formation, triggering the morbidity, and
mortality in living organisms. An overproduction of ROS from disrupted metabolism
referred as oxidative stress may cause damage through mutations terminating into can-
cer (Nascimento et al., 2007; Shureiqi et al., 2000). Mutations are changes to the base
pair sequence of genetic material and cause genetics and other degenerative disorders.
The worldwide new incidence of cancer is about 6 million cases per year (Greenlee et
al., 2001). Among various cancer forms, melanoma is a malignant neoplasm of mela-
nocytes, most frequently arising from the skin. Melanoma is accounted for 2·6% of the
global cancer incidence and 1·1% of cancer-related deaths (Hoey et al., 2007). Even if
these data rank melanoma eighth or ninth in incidence, its doubling rate every 10–20
years is more worrying (Diepgen and Mahler, 2002). It is estimated that 68,130 men
and women (38,870 men and 29,260 women) will be diagnosed with and 8,700 men
and women will die of melanoma of the skin in 2010 (Altekruse et al., 2010). Mul-
tidisciplinary scientific investigations are making best efforts to combat this disease.
The curative surgical treatment of melanoma remains a significant clinical challenge
(Balch, 1992) and trials of post-surgical adjuvant therapy have proved largely unsuc-
cessful with the majority inducing severe side effects at therapeutically effective doses
(Balch et al., 2001).
An emphasis, recently, has been given towards the researches on complementary
and alternative medicine that deals with cancer management. Epidemiological data
indicates a beneficial effect of the “Mediterranean diet” on human health, on several
degenerative diseases, including cancer (Cassileth, 2009). Encouraging intervention
studies are now available (Tseng et al., 2008), however most investigations focus on
main food products, such as olive oil (Pauwels and Covas, 2009), tomato (Tang et al.,
2009), and red wine (Guerrero et al., 2009), while relatively little is known on food
products consumed on less regular basis. Among the latter, Eruca sativa (rocket) cer-
tainly deserves attention. E. sativa, (Miller) Thell (Figure 1) belongs to the Crucifer-
ous family and is originated in the Mediterranean region (Zeven and de Wet, 1982)
but widely distributed all over the world (Warwick, 1994). The seeds are used for the
production of spicy (taramira) oil while leaves are consumed as salads in India and
European countries (Bianco, 1995). Investigations have been carried out to provide
Eruca sativa Inhibits Melanoma Growth: A Scientific Evidence 161
evidence that higher intakes of Cruciferous vegetables are associated with decreased
cancer risk in humans (Higdon et al., 2007; Verhoeven et al., 1996). Glucosinolates
and their derived products isothiocyanate found in Cruciferous vegetables have been
reported to inhibit growth of melanoma cells (Melchini et al., 2009).
Figure 1. Eruca sativa.
The present chapter explains anti-melanoma activity of E. sativa plant (seed oil),
demonstrating that the isothiocyanates found in seed oil play important role in inhibi-
tion of proliferation of cancerous cells. The intention behind this communication is
to raise awareness and encourage implementation of herbalism for combating cancer.
FREE RADICAL SCAVENGING

Seed oil of plant E. sativa was tested for their free radical scavenging effect by us-
ing Fenton (Halliwell et al., 1992) and DPPH (Shimada et al., 1992) assays. Seed oil
showed percent inhibition 95.55% against hydroxyl radicals at the concentration 100
µg/ml. Standard (α-tocoferol) antioxidant inhibited 99.46% of hydroxyl radicals for
the same concentration. A gradual increase in percent inhibition of DPPH˙ with ten in-
creasing concentrations of test samples was also found to be dose dependent (Figure 2).
Figure 2. Percentage inhibition of hydroxyl and DPPH radicals, concentration dependency of seed
oil against standard α-tocoferol. Each value is mean ± SD (n = 3). P > 0.05 (SO) vs. α-tocoferol.
Hydroxyl free radical is known to have damaging effect to almost every biological
molecule found in living cells. In vitro Fenton assay involves hydroxyl radical gen-
eration through the incubation of Fe2+-EDTA chelate at pH 7.4 which in turn degrade
deoxyribose sugar (Khoobchandani et al., 2009; Ojeswi et al., 2009). The rate of deg-
radation of deoxyribose sugar in test samples are compared with control in terms of
appearance of pink chromogen with thiobarbituric acid. Seed oil exhibited maximum
antioxidant activity as that of standard antioxidant α-tocoferol at the concentration of
90 µg/ml. A similar trend of free radical scavenging was found in DPPH assay.
Seed oil increased the level of down-regulation of reduced glutathione in in vivo
study. Free radicals and their biochemical reactions in each stage of the metabolic pro-
cess are involved in cancer development (Kun-Young et al., 2003). Antioxidants act as
the primary line of defense against reactive oxygen species and suggest their useful-
ness in estimating the risk of oxidative damage induced during carcinogenesis. The
GSH is non-protein cellular thiol which in conjunction with glutathione peroxydase
has a regulatory role in cell proliferation. The GSH and its dependent enzymes scav-
enge the electrophilic moieties involved in the cancer initiation and serves as marker
for the evaluation of oxidative stress (Comporti, 1989; Nam and Kang, 2008). Seed oil
rendered significant protection against oxidative stress induced by melanoma in liver
tissues in a dose dependent manner. Our observation supports the fact that melanoma
cells induced oxidative stress is related to depletion of antioxidant system.
ANTITUMOR ACTIVITY
Seed oil inhibited the cell proliferation in a dose dependent manner. The inhibitions
recorded with the two assays Trypan blue exclusion (Frieauff et al., 2001) and Methyl
thiazole tetrazolium cell viability assay (Lee et al., 2002). Percent inhibition resulting
from in vitro cell viability bioassays demonstrates that seed oil (IC50 24.78 µg/ml)
is the most efficient candidate as cytotoxic bioagent. The percent inhibition against
B16F10 cells was significantly (p < 0.05) more pronounced for seed oil, therefore it
was further studied for in vivo anti-melanoma activity. Melanoma cells injected sub-
cutaneously into mice grew to an average size of tumor volume 2,000 mm3 in the
control group. Seed oil produced significant inhibition of tumor growth in animals as
compared to tumor control group. Seed oil at a dose of 1 and 2 mg/kg body weight in-
hibited 19.79 and 29.48% of melanoma growth respectively and doxorubicin reduced
37% of tumor growth at 21st day (Figure 3). The intraperitoneal route of seed oil was
found to be effective in inhibiting melanoma growth. Both SO and reference doxorubi-
cin reduced significantly (p < 0.01) tumor growth as compared to control tumor group.
It is interesting to note that neither life threatening toxicity nor a loss of body weight
during the seed oil treatment was observed as that of normal control animals. The
finding is significant in comparison with side effects (loss of body weight) normally
observed in adjuvant therapy (Balch, 1992), highlighting the ability of naturally oc-
curring (seed oil) to inhibit melanoma growth with the view to develop new antitumor
substances with low toxic potential.
Figure 3. Effect of the Eruca sativa seed oil (every other day, from day 5th to 21st) on the melanoma
growth. Mean ± SE (n = 5). DOXO: Doxorubicin, SO: Seed Oil.
ANTIMUTAGENIC ACTIVITY
Antimutagenic activity of seed oil was observed in terms of chromosomal aberration
(Savage, 1993) and micronucleus assay (Schmid, 1975) by the induction of mela-
noma cells. The effect of the test samples on percent of chromosomal aberration was
measured in terms of chromatid breaks, centric rings, acrocentric association, acen-
tric fragments, intercalary deletion, and total abnormal metaphases (Slide 1). Percent
of aberrant metaphase in various groups were found as: cancerous control (82.60%),
doxorubicin treated (77.80%; 1 mg/kg) while for seed oil treated (51.20 and 47.50%)
at two doses 1 and 2 mg/kg, respectively (Table 1). Seed oil exhibited significantly (p
< 0.01) reduction in chromosomal aberration like CB, CR, ACA, FR, ICD, and pul-
verization in bone marrow cells compared to tumor control and standard doxorubicin
drug. The order of chromosomal aberration was found as: seed oil > doxorubicin drug
> tumor control group.
Slide 1. Chromosomal aberration in (A) Normal control animal and (B) Tumor control animal (C) E.
sativa seed oil treated animal (D) Doxorubicin treated animal after induction of B16F10 melanoma
cells.
Table 1. Effect of Eruca sativa seed oil on aberrant metaphases in the bone marrow of melanoma
tumor induced mice.
Abnormal
Group CB CR FR ACA Pulvi ICD
metaphase
Grp I 0.20 ± 0.40 0.0 ± 0 0.60 ± 0.54 0.0 ± 0 0.0 ± 0 0.0 ± 0 0.8 ± 0.84
Grp II 12.80 ± 2.28b 6.20 ± 0.83b 23.80 ± 2.49b 34.20 ± 1.92b 4.20 ± 1.09b 4.40 ± 3.20b 82.60 ± 2.49 a
Grp III 10.60 ± 3.36b 5.60 ± 1.14b 17.40 ± 2.30b 33.60 ± 2.88b 3.20 ± 1.92b 5.40 ± 1.94b 77.80 ± 3.76 a
Grp IV 8.60 ± 1.82C 2.60 ± 1.14C 11.24 ± 1.51C 28.92 ± 3.12C 0.0 ± 0a 2.60 ± 1.14C 51.20 ± 5.32 b
Grp V 8.20 ± 1.09C 2.40 ± 0.70C 10.30 ± 1.93C 23.35 ± 3.42C 0.0 ± 0a 3.60 ± 1.14C 47.50 ± 2.95 b
Mean ± SE (n = 5). ap > 0.05, bp < 0.001 vs. Normal Control; cp < 0.01 vs. Normal and Tumor
Control.
Group I: Normal; Group II: Tumor control; Group III: Doxorubicin, Group IV: Seed oil (1 mg
dose); Group V: Seed oil (2 mg dose). CB: Chromatid Break, CR: Centric Ring, FR: Fragment, ACA:
Acrocentric Association, Pulvi: Pulvirization, ICD: Intracalary Deletion.
The effect of seed oil on melanoma induced mice was determined in terms of mi-
cronucleated polychromatic erythrocytes (MPCEs) and normochromatic erythrocytes
(MPCEs) per 1,000 cells. Percent of MPCE and MNCE in various groups were found
as: cancerous control (100%), doxorubicin treated (84‑89%; 1 mg/kg) while seed oil
treated (14–15 and 6–10%) at two doses 1 and 2 mg/kg respectively (Figure 4). Seed
oil treated mice were significantly (p <0.001) reduced the formation of micronuclei
in PCEs and NCEs comparable with tumor control and doxorubicin treated group.
The micronucleus assay has been used in cytogenetic studies to detect chromosomal
changes such as acentric chromosome, chromatid fragments, and chromosome lag-
ging at anaphase. Formation of MN in the interphase is dependent on factors such as
cell cycle stage and types of mutagens. Micronuclei were considered an indication
of a mutation effect (Auerbach, 1962). Results of this study indicate that the seed oil
reduced the frequency of micronuclei per PCEs and NCEs. The decrease in PCEs and
NCEs in tumor alone mice reflects the early effects on cell cycle leading to mitotic
inhibition. The observed antimutagenic efficacy showed the similar trends of chromo-
somal aberration assay.
Figure 4. Effect of the Eruca sativa seed oil on frequency of micronuclei formation per PCEs and NCEs
against standard doxorubicin.
HISTOPATHOLOGY STUDY
It is evidenced that tumor growth and lethality are dependent on angiogenesis. The
decrease in tumor growth by test samples in mice may be attributed to decreased host
angiogenesis. Representative photographs of melanoma after excision and photomi-
crographs of stained tumor micro sections are illustrated in Slide 2. A marked and
dense microvasculature was observed in the control tumors. Tumors treated with SO
(31.23 ± 6.3%) and doxorubicin (27.6 ± 6.7%) had significantly fewer micro-vessels
compared with the control (62.6 ± 8.7%). The findings are in the harmony of earlier
observation (Barnhill et al., 1998) that the suppression of melanoma is based on the
triggering of apoptosis and angiogenesis. The improved angiogenesis inhibition ob-
served with seed oil treatment is the indicative of high test sample accumulation in
the tumor. The fact also finds support from the decreased tumor micro-vessel density
resulting from seed oil treatment, suppressing the expression of angiogenic vascular-
ization, tumor cell proliferation, and increased tumor cell apoptosis in melanoma.
Slide 2. Histological observation of micro vessels among the tumor cells with solid tumor: (A) Control
tumor (B) Doxorubicin treated (C) Seed oil treated are tissue sections stained by HE (200×). Arrows
indicate micro vessels. Histological study demonstrated that numerous micro vessels with larger
cavity and better integrity could be seen among the tumors of the mice injected with B16F10 cells.
In contrast, micro vessels were few in the tumor of the mice treated with seed oil and doxorubicin.
CHEMISTRY PROFILE OF SEED OIL

Head Space/Solid Phase Micro Extraction analysis of the crude oil resulted in the
identification of isothiocyanates by GC-MS. Identification of ITCs was accomplished
by comparison with NIST 05 MS-library (f-fit > 700; r-fit > 650) and was confirmed
using authentic standards in all cases (Figure 5). Seed oil revealed the presence of sig-
nificant amount of allyl-ITC (40.30 µg/gm), 3-butenyl-ITC (259.60 µg/gm), 2-phenyl-
ethyl-ITC (158.50 µg/gm), 4-methyl sulfinyl butyl isothiocyanate (743.10 µg/gm) and
bis(4-isothiocyanatobutyl)disulphide (~5000 µg/gm) and traces of erucin (Table 2)
(Khoobchandani et al., 2010).
Table 2. Chemical structure of isothiocyanates identified in taramira seed oil.

Figure 5. HS-SPME-GC-MS chromatograms obtained from analysis of E. sativa seed oil in Total ion
Count (A) and in Selected Ion Monitoring of m/z 99 (B), m/z 72 (C), m/z91 (D), m/z 160 (E), m/z 86 (F).
Like other cruciferous vegetables, taramira plant is rich sources of sulfur-contain-

ing compounds known as glucosinolates, have recently garnered great interest for their
potential role in the maintenance of human health. Chopping or chewing crucifer-
ous vegetables results in the formation of bioactive glucosinolate hydrolysis products,
such as isothiocyanates (Chen and Andreasson, 2001; Fahey et al., 1997; Zhang et al.,
1992).
Formation of an isothiocyanate by hydrolysis of a glucosinolate

(Source: Chen & Andreasson, 2001)
Epidemiological studies suggest that high intake of cruciferous vegetables has

been associated with lower risk of cancer (Jeffery and Jarrell, 2001; Poppel et al.,
1999). Many organizations, including the National Cancer Institute, recommend the
consumption of ﬁve to nine servings (2.5–4.5 cups) of fruits and vegetables daily.
The isothiocyanates rich seed oil triggering of cell cycle arrest, enzymatic free radical
scavenging, and blockage of DNA damage in the suppression of melanoma has been
proposed in the schematic pattern (Figure 6).
Figure 6. Tentative mechanism depicting isothiocyanates inhibition of cancer.
CONCLUSION
ITCs-rich seed oil was, notably, the only preparation of E. sativa capable of significant
reduction of melanoma in vivo at 21st day, at doses only twice as large as those effec-
tive for the reference drug doxorubicin. Overall, the results nicely complement other
investigations depicting the safe and health promoting value of dietary consumption of
E. sativa, highlighting its potential for clinical applications and lend support to its use
in traditional medicine without any toxicity and loss of body weight.
KEYWORDS
•• Cancer
•• Eruca sativa
•• Melanoma
•• Micronucleated polychromatic erythrocytes
•• Mutations
ACKNOWLEDGMENT
The authors gratefully acknowledge Prof. V. G Dass, Director, Dayalbagh Educational
Institute, Dayalbagh, Agra, for providing necessary research facilities. M. Khoobchan-
dani acknowledges Department of Science and Technology (New Delhi) for Financial
Assistant.
Chapter 12
Optical and Mechanical Investigations of
Nanostructures for Biomolecular Detection
G. Malegori, D. Nardi, F. Banfi, C. Giannetti, and G. Ferrini
INTRODUCTION
Since Luigi Galvani (1737–1798) began his studies, physics and biology have inter-
acted and many tools from physics have been used in the biological sciences. Today
the availability of new microscopic techniques has pushed the boundaries from the μm
to the (sub-) nm level. These possibilities stimulate to find ever more creative ways of
using physics, material science, and biology combined together.
In particular, the development of techniques capable of measuring the chemical
and mechanical state of biological samples, in vivo and with attention to molecular dy-
namics localized at surfaces is of great interest. The way organic thin films properties
are affected by molecular interactions at surfaces makes such films a model system for
biological research and applications ranging from light-emitting diodes and solar cells
to chemical sensors and nanomedicine. Moreover, the possibility of studying surface
chemical reactions in biological samples without treatments and in vivo is an impor-
tant issue for the understanding of the complex chemical machinery of living cells.
Many cell functions depend on surface ligand-receptor complexes or surface chemi-
cal reactions and many kind of tumors start from the first cellular layer inside hollow
organs. Therefore, non-invasive techniques that allow an in vivo study of chemical
processes located at surfaces constitute important tools to develop and test biological
models and target diseases.
An approach trying to combine these aspects will be reviewed here, based on the
following techniques: (a) Optical detection based on evanescent wave spectroscopy,
(b) femtosecond laser pulses used to excite thermal and mechanical transients in na-
noengineered materials, (c) Non-Contact Atomic Force Microscopy (NC-AFM) and
force spectroscopy.
The complementarity of these seemingly not related techniques aims to foster
an approach beneficial for the problems at hand in nanomedicine and drug delivery.
While chemical information is provided by optical spectroscopy, mechanical, and
structural parameter could be retrieved by NC-AFM and optically induced mechani-
cal transients. The measured parameters could in principle be related to a single
theoretical model, thus characterizing the response of the system with a multimodal
approach.
Optical and Mechanical Investigations of Nanostructures 171
MOLECULAR DETECTION AT SURFACES USING EVANESCENT WAVE

SPECTROSCOPY
One interesting possibility to develop surface sensitive spectroscopic techniques is the
use of evanescent waves (Knoll, 1998). The term evanescent wave optics refers to a
number of optical phenomena and techniques associated with the total internal reflec-
tion of light at the boundary between two media of different optical properties, usually
described by their refraction indexes, as can be observed at the boundary between a
glass prism and water (see Figure 1). In this case, glass is the incidence medium, with
refraction index ni, and water is the transmitting medium, with refraction index nt.
A laser light beam (wavelength λ) impinging upon that interface from the glass
side, that is from the side of the material with the higher refractive index, will be
totally (internally) reflected if the angle of incidence exceeds a critical value θc = sin–
1
(ni/nt). However in the transmitting medium the optical field does not fall abruptly to
zero. The optical E-field in the rarer medium along the propagation direction, Ex, has
the usual oscillatory character of an electromagnetic wave. Instead, the component
perpendicular to the interface, Ez, is bounded and decays exponentially into the opti-
cally rarer medium with a decay length l which is a function of the angle of incidence,
l = λ/(2π ((nsinθ)2‑1)1/2), for θ > θc.
Such inhomogeneous electromagnetic wave in the rarer medium is called an ev-
anescent wave. The advantage using this kind of waves resides in the selective il-
lumination of the near-interface range, resulting in a surface selectivity for optical
experiments due to this surface-bounded light. Moreover, its intensity is enhanced
compared to the incoming wave, which results in a sensitivity enhancement for opti-
cal experiments. When an absorbing layer of molecules is present at the interface, the
evanescent field interacts with molecular resonances, giving an absorption spectrum
comparable to that observed in a transmission experiment. When films are much thin-
ner than the penetration depth and the electric field amplitude can be considered con-
stant over the film thickness, it is possible to associate an “effective thickness” to the
film equivalent to that of a transmission experiment (Harrick and and du Pré, 1966). It
results that the electric field amplitude in the thin film is determined by the refractive
indexes of the incidence and transmission media.
Using a broadband spectral source it is possible to retrieve absorbance spectra of
the molecular species absorbed on the surface and thus obtain a selective spectral fin-
gerprint of the molecules at the surface. A particularly promising broadband spectral
source is constituted by the white light continuum produced in nonlinear fibers seeded
by a femtosecond laser oscillator. In this way, broadband continuum, extending from
450 to 1,600 nm with a nearly flat spectral intensity, can be obtained from few nano-
joule pulses produced by a standard 120 fs-800 nm Ti:sapphire oscillator (Cilento et
al., 2010). The key elements in continuum generation by high repetition rate and low
energy per pulse sources are the newly developed micro structured photonic-crystal
fibers (PCF), engineered to be nearly dispersion-free at particular frequencies in the
near-infrared/visible range (see Figure 1). Nonlinear interactions between an infrared
laser pulse propagating into the fiber and the silica core generate a broadband pulse
output.
To demonstrate that this kind of light source could be effectively used in evanes-
cent wave spectroscopy of biomolecules, the formation of thin films of Methylene
Blue (MB) in aqueous solution at a fused quartz surface was investigated with eva-
nescent wave absorption spectroscopy (Ferrini et al., 2009). The MB has a variety of
aggregation states (monomers, H-dimers, J-dimers, trimers) that depend on the con-
centration and surface proximity. The spectra of the various aggregates are known and
can thus be used to test new optical techniques.
Figure 1. Evanescent wave optics.
The continuum spectrum is produced by delivering from 790 nm to 100 fs laser

pulses with a maximum energy of 20 nJ from a Ti:Sapphire laser oscillator into a
nonlinear fiber. The nonlinear fiber output is recollimated and split in two beams by a
broadband beam splitter. One beam is sent into a fused quartz prism in a Kretchmann
configuration, as shown schematically in Figure 1, the other is used as a reference.
To directly probe MB in water solution, a drop of liquid is prepared on the surface of
the prism. Both beams are collimated and successively spectrally filtered by the same
monocromator, adjusting the angle with respect to the input slit to spatially separate
the beams. The continuum is modulated by a chopper constituted by a photo elastic
modulator at 50 kHz and the photodiode signal amplitude at the same frequency is
detected by means of a lock-in amplifier. By taking the ratio between the probe beam
and the reference beam, the attenuation produced by the MB solution can be measured
at every wavelength.
Figure 2. Molar absorbance spectra of Methylene Blue at the prism fused quartz surface.
The molar absorbance spectra of MB at the prism fused quartz surface are reported
in Figure 2 (Ferrini et al., 2009). The thick gray line represent the retrieved spectrum
for MB dimers at a fused silica surface after spin coating, complete solvent (ethanol)
evaporation and reduction (Ohline et al., 2001). The square points represent the molar
absorbance of the MB water solution retrieved from the evanescent white-light con-
tinuum absorption, with a surface molecular density of 1015 cm–2 (Ferrini et al., 2009)
The continuous line is representative of the bulk molar absorption spectrum.
From the experimental data two main conclusions can be drawn. The first regards
the aggregation state of MB near the fused silica surface. At the highest concentration
available in this experiment, the MB at the surface is almost entirely organized in the
form of dimers, as it is apparent from the prominence of the dimers peak in the absor-
bance data. The second regards the fact that the molar absorbance spectrum measured
directly from a liquid water solution agree quantitatively with the dimer absorbance
spectrum retrieved from an experiment using spin coating and complete solvent (etha-
nol) evaporation (Ohline et al., 2001), confirming that, at comparable concentrations,
the dimers spectral features are the same in different experimental conditions.
From the spectra the aggregation state of MB near the fused silica surface was
determined to be due almost entirely to dimers even if the molar concentration to
obtain dimers aggregation in bulk water solution were much higher than that used in
the experiment. This imply that MB dimer aggregation is favored by the vicinity of a
quartz surface, a conclusion identical to those obtained by (Fujita, 2005) using optical
waveguide spectroscopy with a broadband CW xenon lamp. While the combination
of attenuated total internal reflection with a broadband light source allows to recover
absorbance rapidly at all wavelengths, using short laser pulses opens the possibility to
study the molecular dynamics at surfaces by means of pump and probe techniques. In
fact, by adding a delayed pump pulse that excites the MB solution at the surface (either
from the solution side or from the prism) it would be possible to study the temporal
behavior of absorbance and/or orientation dynamics after an optical excitation, with a
temporal resolution in the sub-ps time range. Moreover, the use of the asynchronous
optical sampling technique (Bartels et al., 2007) to perform pump and probe experi-
ments without mechanical delay line, with a time resolution below 100 fs in a 10 ns
time measurement window and high speed scanning, opens the possibility to use eva-
nescent wave absorption spectroscopy to follow in real time the modifications of the
molecules electronic or vibrational dynamics in evolving chemical reactions.
Since, evanescent wave spectroscopy is a surface sensitive technique, its applica-
tion in fluorescence resonance energy transfer (FRET) should give interesting insights,
especially in situations where donor and acceptor chromophores labels molecules resi-
dent on surfaces and experiments in bulk solution are not possible.
MECHANICAL STUDY OF SURFACE NANOSTRUCTURES USING LASER

GENERATED THERMAL TRANSIENTS
In recent years, the use of femtosecond laser pulses to excite thermal and mechanical
transients in matter (Maris, 1998) led, in recent years, to the development of applied
acoustics in the domains of material science and biology. Recently, this approach has
been applied to nanoengineered materials to optically generate and detect acoustic
waves in the gigahertz–terahertz frequency range. A review of the latest advances on
ultrafast generation and detection of thermal gradients and pseudo-surface acoustic
waves in lattices of metallic nanostructures, that is, elastic meta-materials, is of inter-
est both to physicists and life scientists due to the development of new molecular sen-
sors and manipulation techniques based on acoustic waves.
Nanostructured meta-materials emerged as model systems to investigate both the
mechanical (Giannetti et al., 2009; Nardi et al., 2009) and thermal (Banfi et al., 2010)
energy transfer at the nanoscale. The sensitivity of the all-optical time-resolved tech-
nique to deposited mass and thermal fluxes, coupled with the phononic crystal proper-
ties induced by the nanopatterned periodic lattices, opens the way to a variety of ap-
plications ranging from nanocalorimetry to mass sensors. The approach reported here
is based on optical time-resolved experiments with femtosecond resolution over a 5
decades time window (100 fs to 10 ns) and does not require piezoelectric substrates.
This approach extends the range of exploitable materials with respect to standard in-
terdigital transducer-based mass sensor technology and paves the way to an increased
miniaturization. The basic idea is to use a subpicosecond light pulse (pump pulse)
focused to a small spot (the laser wavelength diffraction limit constituting the smaller
achievable spot’s diameter) to induce a nonequilibrium local heating of both electrons
and lattice of the sample surface on the picosecond timescale. The local temperature
increase triggers a sudden lattice expansion via the thermal expansion coefficient. The
photoinduced thermoelastic stress launches strain pulses propagating away from the
pump-excited spot, propagating at the sound velocity. Since the refractive index of the
material depends on its local strain, through the photoelastic constant, it is possible to
follow the propagation of the strain pulses monitoring the reflectivity variation of a
second delayed pulse (probe pulse) focused at the same or different locations on the
sample. An energy per pulse of the order of 1 nJ, which is easily available by means of
Ti:sapphire oscillators producing 100 fs light pulses at 100 MHz repetition rate, can be
exploited to impulsively heat semiconductor or metal samples, leading to temperature
raises of the order of 0.1‑10 K, implying thermoelastic stresses ranging from 0.1 to 1
Mbar.
Among the mechanical modes excited by short laser pulses, Surface Acoustic
Waves (SAWs) have the greatest practical relevance. The SAWs are solutions of the
elastic eigenvalue equation in which the displacement field is confined to the surface
within a depth of the order of the wavelength (Landau and Lifschitz, 1986), very much
like the evanescent electromagnetic waves addressed in the previous section. In par-
ticular, when the pump pulse is focused on a small area of 1–10 μm2 of a surface, the
large Fourier spectrum of the excited acoustical waves allows launching of SAWs at
different k-wave vectors. Time-resolved imaging techniques have been employed to
follow the picosecond-timescale The SAW propagation on free surfaces (Sugawara et
al., 2002), through grain boundaries (Hurley et al., 2006), in phononic crystals (Profunser
et al., 2006), and in resonators (Maznev, 2009). In addition, SAWs in the hypersonic
frequency range (> 1 GHz) are currently used to manipulate electrons in semiconduc-
tor devices (Cecchini et al., 2006) and photons in microcavities (de Lima et al., 2005).
Notably, the same pump-probe technique can be applied to study heat transport in
matter (Stoner and Maris, 1993). The pump-induced temperature variation triggers a
heat flow on the subnanosecond timescale from the heated volume to the rest of the
sample. The dependence of the refractive index on the temperature enables following
the propagation of heat pulses by means of the optical probe pulse. This technique,
named time-domain thermo reflectance, has been employed to investigate the ther-
mal conductance at metal–metal (Gundrum et al., 2005) and metal–dielectric (Lyeo
and Cahill, 2006; Stoner and Maris, 1993) interfaces and to disentangle the energy
transport related to electron diffusion (Gundrum et al., 2005), anharmonic phonon
decay (Lyeo and Cahill, 2006), and ballistic phonon transport (Highland et al., 2007;
Siemens et al., 2010). The signature of ballistic heat transport (von Gutfeld and
Nethercot, 1966), has been recently reported at cryogenic temperatures in a GaAs
crystal covered by a metallic thin film transducer (Perrin et al., 2006). The extension of
this technique to the study of thermal transport between a single metallic nanoparticle
and the environment is a more difficult task, due to the difficulties in controlling the
properties of the nanoparticle-environment interface (Juvé et al., 2009; Voisin et al.,
2000).
The frontier, in this intriguing research field, is the investigation of the thermo
mechanical transients occurring between in lattices of metallic nanostructures and the
underlying substrate (Giannetti et al., 2007; Hurley et al., 2006, 2008; Lin et al., 1993;
Robillard et al., 2007; Siemens et al., 2009; Tobey et al., 2004). State-of-the-art nano-
lithography and patterning techniques allow obtaining metallic nanostructures, whose
shapes, dimensions, periodicities, and interface properties can be carefully tuned. The
interest in these systems is inherent to the following features: (i) the periodicity, poten-
tially scalable down to the 10 nm range (Chao et al., 2005), can be exploited to launch
quasi-monochromatic SAWs in the substrate beyond the 10 GHz range (Siemens et al.,
2009); (ii) the opening of a band gap in the acoustic modes (Nardi et al., 2009); and
(iii) the fine control over the nanostructures/substrate interface, as required to investi-
gate heat transport at the nanoscale.
The above-mentioned approach is here shown in a paradigmatic experiment. A
scheme of the pump and probe experiment is shown in Figure 3.
Figure 3. Schematic representation of the pump and probe experiment.
The pump beam selectively heats the permalloy (iron-nickel alloy, Fe20Ni80 )
nanodisks patterned in a square periodic array, leaving the temperature of the silicon
substrate substantially unaltered. The sudden (ps time scale) thermal expansion of the
nanodisk triggers two concurrent dynamics: (a) a SAW is launched in the substrate,
finally transferring mechanical energy, dW, to the Si bulk (ns time scale), and (b) the
disks thermalize with the silicon substrate transferring heat, dQ, on a ns timescale. The
time-delayed probe pulse investigates these dynamics.
As the delay from the pump pulse increases, the disks temperature decreases and
the transient average radius of the disks shrinks accordingly. The intensity of the dif-
fracted probe pulse decreases because of a shrinking reflecting surface (nanosisks
area). In the meanwhile the disks oscillate at the SAW frequency, inducing the oscilla-
tions in the diffracted probe signal intensity. The oscillation is exponentially dumped
due to mechanical energy radiation to the bulk (Giannetti et al., 2007; Nardi et al.,
2009).
In Figure 4 (Giannetti et al., 2009), we report the time-resolved measurements on

two-dimensional square lattices of permalloy nanodisks, with a thickness of 30‑50 nm
and a diameter of 400‑600 nm. The laser source is a Ti:sapphire oscillator, delivering
light pulses with 120 fs time duration, 30 nJ energy/pulse, and 790 nm wavelength, at
a repetition rate of 76 MHz. The output is split into an intense component (pump, 10
nJ) and a weak component (probe, less than 1 nJ). The delay between the pump and
probe pulses is changed by a mechanical delay-line. A feedback system that drives two
piezo-nanomotors mounted on optical mirrors is used to keep the alignment between
pump and probe. To optimize the optical signal-to-noise ratio, the pump and probe
beams are focused on the same point of the sample with a diameter of 60 and 40 μm,
respectively. This size allows exciting and probing a large number of nanostructures
(about 3,000), while keeping the laser fluence high enough to significantly excite the
system. A Peltier device is used to keep the sample back side temperature constant
during the experiment. The pump beam intensity is modulated at 100 kHz by a photo-
elastic modulator (PEM), placed between two crossed polarizers (chopper). In order to
improve the signal to noise ratio the probe is detected recording the relative intensity
variation of the first diffraction order spot, the nanostructured surface lattice (surface
phononic crystal) serving as a diffraction grating. This technique avoids all nonperi-
odic contributions in the probe signal, such as Brillouin scattering from the acoustic
pulses propagating into the substrate. The first-order diffracted beam is detected by a
photodiode and filtered by a lock-in amplifier referenced at the PEM frequency (Gi-
annetti et al., 2007). Relative intensity variations of the order of 10–6 can be measured
with a time resolution of 120 fs in a time window of 3 ns.
Figure 4. The time-resolved measurements on two-dimensional square lattices.

In the experimental trace (see Figure 4), at zero delay, it is possible to see a fast
increase of the transient signal while a nanosecond decay superimposed to an oscil-
lation with much shorter period is detected for positive delays. Considering the laser
energy density absorbed by the nanostructures and by the silicon substrate, we can es-
timate that, within 5 ps, the temperature of the nanodisks is homogeneously increased
by about 10 K. In contrast, the substrate temperature is essentially unvaried due to the
different penetration depth of the 800 nm radiation (Giannetti et al., 2007, 2009).
The light penetration depth is comparable with the nanostructure height, hence
a uniform heating of the nanodisks is obtained and the impulsive temperature mis-
match triggers a nonequilibrium expansion of the nanostructures dimensions (of the
order of 10–5 considering the effective thermal expansion coefficient of the Py/Si sys-
tem). The expanded periodic nanostructures induce a spatially modulated stress on the
silicon surface. Such stress launches a pseudo-SAW with a wavelength matching the
nanodisks two-dimensional lattice periodicity. The variation of the diffracted light in-
tensity as a function of the delay evidences that the nanostructures, due to the thermal
expansion, oscillates around a larger diameter with respect to the unperturbed value,
which is proportional to the average temperature. The transient average diameter de-
cays following the disk cool down due to the energy exchange with the substrate.
The oscillation dynamic is well represented by the fit function in Figure 4(a) (black
line), obtained by the sum of a damped oscillating function (continuous gray line,
representing the SAW) and a simple exponential decay (dashed gray line, due to disk
cool down). The oscillating and the exponentially damped curves have been scaled for
graphical reasons.
The period of the oscillations (T), superposed on the exponential decay, are related
to the lattice spatial periodicity (λ), and are connected by the sound velocity (v): λ/T
= v. The frequency υ = 1/T is the oscillation Eigen frequency of the surface waves
associated to the 2D nanostructured lattice. Finally, the decaying amplitude of the
oscillations is due to the elastic energy dissipation in the bulk, much like by a spring
affected by viscous damping.
It is important to note that the disks thermalization with the silicon substrate on
a nanosecond timescale can be followed by this technique, providing also important
thermo dynamical information at the nanoscale. The reader is referred to (Banfi et al.,
2010; Giannetti et al., 2007; Siemens et al., 2010) for a fuller account.
The possibility to optically control the excitation of SAW and thermal gradients in
arrays of metallic nanostructures on substrates opens the way to fundamental applica-
tions in the field of hypersonic phononics, nanocalorimetry, and biology. The sensi-
tivity of the time-resolved techniques can be exploited to develop mass sensors with
picosecond time resolution. Considering that a difference of 10 ps in the oscillation pe-
riod has been measured for the samples reported in Figure 4, we can easily estimate the
sensitivity of these devices. The nanostructures volume difference in the two samples
is ΔV = 5·10–17 cm3, corresponding to a mass difference/disk of Δm = 5·10–16 g/disk. In
the probe area, the number of nanodisks is about 1,250, giving an absolute mass varia-
tion of Δm = 625 fg. Shorter SAW wavelengths imply higher surface confinement and,
hence, higher surface sensitivity (Auld, 1990). The proposed device periodicity can be
scaled to tens of nanometers, thus enhancing the sensitivity.
The experimental scheme reported in this work proves useful to access the specific
heat, that is nanocalorimetry (Banfi et al., 2010), or thermal conductivity of mesoscale/
nanoscale samples (Wilson, 2007). Without entering in details, the quantities control-
ling the thermal dynamics can be extracted from the exponentially decaying contribu-
tion to the diffracted probe intensity, see Figure 4. Such informations could be of inter-
est for the thermodynamics at the nanoscale of biomolecules or receptor aggregates.
A NEW APPROACH TO ATOMIC FORCE SPECTROSCOPY USING WAVELET

TRANSFORMS
Atomic force microscopy (AFM) (Garcia and Perez, 2002; Giessibl, 2003; Morita et
al., 2002) has developed into a powerful technique, delivering not only topographical
images with sub-molecular resolution (Gross et al., 2009) but also providing sensitive
force measurements on the atomic scale (Lantz et al., 2001; Sugimoto et al., 2007).
Such force measurements are commonly referred to as force spectroscopy. The sim-
plest technique used for quantitative force measurements exploit the static deflection
of the cantilever, from which the force is determined using Hooke’s law (Butt et al.,
2005).
The investigation of mechanical properties of biological samples with AFM has
received considerable attention in the past years (Cross et al., 2007; Radmacher et
al., 1996; Rosenbluth et al., 2006). In addition to fundamental research interests,
monitoring local mechanical properties of cells allows a better understanding of
the mechanism of some diseases causing cell stiffness changes (Costa, 2003). The
elastic properties of bacteria or cell walls can be determined by taking force curves
using AFM. The stiffness of the exterior cell surfaces is measured by indenting
them with a cantilever tip, achieving a lateral resolution of few tens of nm (Butt
et al., 2005).
When the surface of a biological object is deformed by the pressure exerted by the
tip of an AFM cantilever, the amount of displacement with respect to the under-formed
surface (the indentation) carries information on its elastic properties. The deflection
of the cantilever as its tip indents an object is usually described with models of linear
elasticity theory (Landau et al., 1959).
In the literature there are examples of force spectroscopy applied to cell walls or
soft samples in the absence of strong interaction between the tip and the surface. In this
case, soft cantilevers can be used for indentation studies without taking into account
instabilities caused by jump-to-contact or force modulations due to the interactions
between tip and surface. However, in the natural environment, the presence of strong
adhesion of the tip to the cell surfaces is often found. Moreover, there is frequently
the need to immobilize mobile samples (cells, bacteria, spores). As an example, we
mention that the Young modulus of the external surface of Clostridium tyrobutyricum
spores, with an atomic force microscope and in air, can be reliably measured despite
the strong tip-spore adhesion forces and the need to immobilize the spores due to their
slipping on most substrates (Andreeva et al., 2009).
More refined techniques with respect to static force spectroscopy rely on measur-
ing of the cantilever’s dynamical parameters while it is excited at or near its resonant
frequency and interacts with the force field provided by a sample surface (Albrecht et
al., 1991). A recent development of this dynamic technique is known as three-dimen-
sional (3D) force spectroscopy (Albers et al., 2009). It emerges that the so called “non-
contact” atomic force microscopy (NC-AFM) is a powerful tool to study not only
the surface topography, but also the mechanical and chemical characteristics of the
sample at the nanoscale. The tip of an excited cantilever is sensitive to both forces and
force gradients, when approaching the sample surface. The response of the interacting
cantilever may show a modification of the oscillation amplitude, frequency, phase, or
damping. The measurement of these cantilever parameters provides information on
the physical properties of the sample with (sub) molecular resolution. In dynamical
force spectroscopy, the influence of the local environment on the cantilever oscilla-
tions around the equilibrium position is usually detected by an optical beam deflection
method, and the cantilever dynamics is analyzed by the Fourier transform (FT), that
represents the temporal oscillations of the cantilever in the frequency domain. By do-
ing so, the Eigen modes of the cantilever oscillations are displayed in the spectrum as
resonance peaks. However, FT analysis is correctly interpreted only in the case of sta-
tionary systems that is, the frequency spectrum must be correlated with a temporally
invariant physical system. In many cases, even if the interacting sample–cantilever
system changes its spectral response during the acquisition, as in the tapping mode
technique, the origin of the spectral features can be traced to the interaction dynam-
ics from reasonable assumptions. However, in these cases, the FT of the signal only
displays an average spectrum over the collection time and prevents a direct correla-
tion of the frequency features with the signal modifications in time and their temporal
evolution.
To go beyond these limitations, a mathematical tool that combines time domain
and frequency domain analysis is useful for non-stationary signals. There are several
mathematical approaches providing a time–frequency representation of a signal, us-
ing basis functions that do not extend indefinitely in time as the Fourier basis (e.g.,
windowed Fourier transform). One of the most refined approaches is the wavelet trans-
form (WT) analysis (Mallat, 1999). The WTs are computed by correlating the signal
f(t) with families of time–frequency atoms, called wavelets. The wavelets are smooth
functions Ψ(t) with a limited support in time (unlike the Fourier basis) whose oscilla-
tion behavior sets the frequency resolution. Being limited in time, the wavelet func-
tions are subjected to translation in time (d) and dilation by a positive scale factor (s).
Time translation (d) is connected to time (t) and dilations (s) to frequency (f). By cor-
relating the translated and dilated wavelets (Ψs,d(t)) with the signal for every delay (d)
and every scale (s) in a range, a two dimensional map of “resemblance coefficients”
Wf(s,d) is obtained, giving at every delay (d) the resemblance of the signal with the
wavelet scaled by the coefficients.
Figure 5. Comparision of Fourier transform (FT) and wavelet transform (WT). (a) Shows the time
signal, a cosine function for negative times and a cosine with quadratic chirp. (b) WT of the signal,
coded in gray scale describes the evolution of its spectral content.
In Figure 5 (Malegori and Ferrini, 2010(b)), the FT and the WT analysis are
compared. Figure 5 (a) shows the time signal, a cosine function for negative times
and a cosine with quadratic chirp (i.e., a frequency proportional to the square of
time) for positive times. Two wavelet functions with different dilations and delays
are superposed to the signal to show the local resemblance between signal and wave-
let. The WT of the signal, coded in gray scale (see Figure 5(b)), correctly describes
the evolution of its spectral content. The white line superposed on the WT is the
calculated instantaneous frequency. In Figure 5(c) the FT (power spectral density)
of the signal is displayed. Only an average of the signal instantaneous frequencies
is observed.
From the previous example, it is clear that WT converts a one-dimensional time
signal into a two-dimensional time–frequency image, which displays the time and
frequency information of the signal in a time–frequency plane. The square modulus
of the wavelet coefficients |Wf(s,d)|2 is proportional to the local energy density of the
signal at the given delay and scale. Thus, WT represents the temporal evolution of the
spectral energy content of the signal (Malegori and Ferrini, 2010b, 2011a).
The dynamics of a free cantilever in air can be reasonably modeled as a harmonic

oscillator with viscous dissipation. In NC-AFM, the frequency shift of the cantilever is
proportional to the gradient of the interaction force for small frequency shifts (Morita
et al., 2002). The frequency shift can be followed in real time by the WT, allowing to
capture transient force interactions that could not be measured otherwise.
An example of this technique is presented in Figure 6 (Malegori and Ferrini,
2010b). In panel (a) is shown the power spectral density of the Brownian motion of
the first flexural mode of the cantilever (Malegori and Ferrini, 2010a) while the tip is
moved into interaction with a graphite surface.
Figure 6. (a) Power spectral density of the Brownian motion of the first flexural mode of the cantilever.
(b) WT of the same signal that is the cantilever thermal fluctuations around its instantaneous
equilibrium position.
The Brownian motion is enhanced in correspondence of the first resonance fre-

quency of the cantilever at about 11 kHz. However the power spectral density gives no
information on the frequency shift due to the cantilever interaction. Figure 6(b) shows
the WT of the same signal that is the cantilever thermal fluctuations around its instan-
taneous equilibrium position, as the tip approaches the surface at constant velocity of
about 225 nm/s. The wavelet coefficients |Wf(f,t)| are coded in grayscale. In this case,
a clear frequency shift as a function of time is detected. The origin of the time axis cor-
responds to the jump-to-contact onset, a phenomenon due to strong surface forces that
induce the cantilever tip to land on the surface almost at once. The wavelet frequency
resolution ∆Ω and time resolution ∆t are limited by a time-frequency uncertainty prin-
ciple, much like the Heisenberg principle in quantum mechanics, which states that ∆t
∆Ω ≥ 1/2. The black box at the left side of Figure 6(b) represents the so called Heisen-
berg box, the minimum uncertainty area for the analyzing wavelet. It is important to
note that the entire measurement takes only a few tens of ms, a time compatible with
force imaging acquisition rates.
Figure 7. Quantitative analysis based on the WT of the Brownian motion.
Figure 7 (Malegori and Ferrini, 2010b) presented a quantitative analysis based on

the WT of the Brownian motion shown in Figure 6. In this case the time axis of Figure
6 is converted into tip-sample distance taking into account tip velocity and cantilever
static deflection. Then, the WT “ridges” are calculated to provide the instantaneous
frequencies evolution within the limits of the wavelet resolution. The wavelet ridges
are the crest of the wavelet coefficients, that is, the local maxima of the normalized
WT above a specified threshold, as schematically shown in the inset. The threshold
is represented by a horizontal line and the maximum point is indicated by an arrow
for a vertical cut of the data at constant tip-sample distance. The frequency shift is
converted into the interaction force gradient (Morita et al., 2002) allowing to map
the force gradient versus tip-sample distance curve. The continuous black line is a
Hamaker-like force-gradient function fitted to the wavelet ridges by considering a Van
der Waals type interaction potential. The dashed line is the interaction force calculated
by integration.
The technique used to retrieve the measurements contained in Figure 7 could be
of interest not only to study the force fields near solid surfaces, but also to character-
ize force fields in the proximity of surfaces covered with receptors or other kinds
of biomolecules, thus characterizing the chemical specificity via mechanical interac-
tions. Moreover, the same analysis can be used to follow the frequency evolution of
the cantilever torsional modes (Malegori and Ferrini, 2011b), which give important
information on the friction between tip and sample and allow the nanotribology study
of surfaces (Schirmeisen, 2010). Another important evolution of the wavelet analysis
is towards force spectroscopy in liquid environments that are of great interest for bio-
logical studies (Fukuma et al., 2007).
CONCLUSIONS
Mechanical interactions and forces are fundamental to biology. Those of chemical
origin govern transport on the molecular scale and determine motility and adhesion
on the cellular scale. Measuring mechanical interactions at the nanoscale provides
unique opportunities to measure forces, displacements, and mass changes from cel-
lular and subcellular processes. Optical spectroscopy is a powerful technique that
allows detecting and identifying various samples through the “fingerprint” of their
specific molecular vibrations. The progress in the studies of biological sample has led
to the possibility of realizing optical biodiagnostics. Femtosecond laser technology is
mature enough to be part of complex optical systems to be used in potential applica-
tions not only in biochemistry but also as biodiagnostics of viruses, bacteria, or even
cancer cells at the molecular level. The merging of mechanical interactions, probed
with AFM force spectroscopy, with surface specific femtosecond optical spectroscopy
techniques (evanescent wave optical spectroscopy) provides new opportunities to fol-
low the dynamics of a single molecule in time. Moreover, the merging of femtosecond
light excitation with mechanical surface waves prospects the possibility of manipulat-
ing the mechanical degrees of freedom of single molecules (e.g., vibrational modes)
and having ultrasensitive and ultrafast mass diagnostics down to the femtogram level.
The ultimate goal of these efforts is to create a multiprobe laboratory on a common
platform to apply the techniques described in previous sections to the same (biologi-
cal) system.
KEYWORDS
•• Atomic force microscopy
•• Fluorescence resonance energy transfer
•• Methylene Blue
•• Photonic-crystal fibers
•• Surface Acoustic Waves
ACKNOWLEDGMENT
This work was partially supported by MIUR under contract PRIN 2008JWKYXB and
by Università Cattolica through D.2.2 grants.
Chapter 13
Suffering and Comfort in Portuguese Cancer
Patients
João Luís Alves Apóstolo, Rita Susana Soares Capela,
and Inês Barata Sá Castro
INTRODUCTION
Someone who is diagnosed with a severe illness experiences feelings of threat, loss,
uncertainty, finitude, anxiety, and of deprivation of basic needs, which cause discom-
fort and suffering. Suffering is part of the personal experience of cancer patients,
particularly terminal patients who not only have to face physical symptoms, but are
also confronted with the idea of death being near and, therefore, feel their integrity is
threatened. In addition, cancer patients experience discomfort resulting from the treat-
ment itself, which can add to this sense of threat to physical integrity.
Nevertheless, individuals may have health projects encompassing a vital capacity
and resilience to fight for life, in order to overcome the ontological condition of suf-
fering and try to achieve levels of comfort that are necessary for existence, for life to
go on.
ANALYSING THE CONCEPT OF SUFFERING

Suffering is a fundamental and universal experience of the human condition. It has
been described as an inevitable human experience (Frankl, 2004), as the state of severe
distress associated with events that threaten the integrity or continued existence of the
person as a whole (Cassell, 1991). Suffering involves the construction of personal
meanings that carry a strong affective load. Therefore, the way in which people deal
with suffering is highly subjective and individual, and in order to understand it, one
must access the individual meanings attributed and ways of responding to suffering,
that is the subjective experiences of suffering (Gameiro, 2000; Mcintyre, 2004).
Most people find it hard to distinguish pain from suffering; however, these are very
different entities that can be inter-related. Suffering is not limited to physical pain. It
is always experienced by the whole-person and not merely on the body level (Cassell,
1991; Cassel, 1999; Fleming, 2003; Neto, 2004).
OPERATIONALIZING THE CONCEPT OF SUFFERING

Suffering can be understood as a complex, personal, and multidimensional phenom-
enon comprising five dimensions: psychological, physical, existential, sociorelational,
and positive experiences related to suffering (Gameiro, 2000).
Physical suffering covers pain, discomfort, and loss of physical strength. This type
of suffering focuses on the dimensions of pain and management of symptoms resulting
from the disease or treatments, and also on energy loss and functional limitations. To
put it in a simple way, one can say that pain, physical symptoms, or other disabilities
(such as loss of energy or strength) limit the patients’ access to the world, thus causing
suffering (Barbosa, 2006; Gameiro, 2000).
Psychological suffering covers cognitive (disturbing and pessimistic ideas, etc.)
and emotional disorders (such as depressed mood, anxiety, irritability, and psychologi-
cal tension). Mental suffering and emotional suffering are both dimensions of psycho-
logical suffering. Mental suffering is mainly due to memory loss and concentration
problems, lack of cognitive control due to preoccupations and difficulty in solving
problems. Emotional suffering includes mood disorders, insomnia, ideas of abandon-
ment, and desire to die (suicidal ideation/intent) (Barbosa, 2006; Gameiro, 2000).
Existential suffering is related to changes in personal identity (low self-esteem;
changes in body image; sense of loss of function etc.), changes in the sense of control
(loss of self-control; loss of perceived control over the situation; fatalism; lack of con-
fidence in one’s own skills; perceived loss of freedom, of control over one’s life etc.),
existential limitations (loss of purpose in life; sense of futility and personal insignifi-
cance; disappointment etc.), limitations in life projects (perceived threat or inability
to develop one’s own project etc.), and lack of harmony with oneself. In other words,
existential suffering expresses the loss of life’s meaning and the sense of uselessness
and despair that can be experienced (Barbosa, 2006; Gameiro, 2000).
Socio-relational suffering refers to affective relational changes (due to separation
from loved ones, empathic suffering, inability to perform family role etc.) and socio-
professional changes (due to changes in socio-professional status and roles, loss of
income etc.). This type of suffering is divided into two main components: family and
social. The social component includes problems with health professionals, economic
and professional problems, and lack of social and community support. The family
component is related to patient-family communication problems (conspiracy of si-
lence), self-blame for being dependent, deep concerns about the future, and sexual
problems (Barbosa, 2006; Gameiro, 2000).
The dimension of positive experiences of suffering during illness resulted from the
validation of “The Inventory of Subjective Suffering Experiences in Illness”—ISSEI
conducted on a sample of 125 hospitalized patients in central hospitals. This new di-
mension of suffering accounts for the possibility of there being positive aspects about
suffering, such as optimism and hope, which result from the illness process (Gameiro,
2000). Whereas loss is inevitable in illness, its negative consequences, particularly
in the psychosocial and existential domains, may not be inevitable (Mcintyre, 2004).
In fact, it is possible for someone to adjust to the situation of suffering. Even in
face of the most adverse conditions, it is possible to cope with the existential crisis and
find a purpose in life (Frankl, 2004). In order to do this, patients have to believe they
are fulfilling a role and have a purpose that is unique, living life to its fullest in accor-
dance with their human potential. In this way, they can achieve a sense of plenitude,
inner peace, and even transcendence (Neto, 2004).
Suffering and Comfort in Portuguese Cancer Patients 187
ANALYSING THE CONCEPT OF COMFORT

Comfort has come to acquire a significant role in health care philosophy and it is
recognized as a holistic outcome related to the responses of the whole-person. The
comfort theory has been used to explain and predict phenomena of human responses
to the health-illness process, and has contributed to a proper evaluation of care and to
the assessment of intervention outcomes (Kolcaba, 1991, 2003).
In this way, comfort as a process (of comforting) or as a product (of interventions)
is a noble concept that underlies the interventions of health professionals in the health-
illness continuum, as well as the human responses to this process (Kolcaba, 1991;
Kolcaba, 2003).
The word comfort comes from the Latin word confortare which means “to give
back strength, vigor, and energy; make strong, reinforce, invigorate” (Instituto de
Lexicologia e Lexicografia da Academia das Ciências de Lisboa, 2001). In view of
these meanings, the concept of comfort could be misunderstood and considered too
vague to be studied and assessed as a health and well-being component. In addition,
comfort has a much more complex and diverse meaning than the one inferred from its
etymology. Comfort is a dimension composed of dynamic processes, experiences, and
concepts, such as quality of life, hope, control, and decision-making. Thus, control and
absence of pain are often considered a synonym for comfort, while the presence and
feeling of pain very often describe the meaning of the word discomfort. At the same
time, discomfort is usually referred to as unmet needs, and when needs are satisfied
they result in the experience of comfort. However, the state of comfort does not pre-
suppose complete absence of discomfort because sensitivity to discomfort is relative
and individual (Kolcaba, 1991, 2003).
One can consider four different meanings of comfort. The first meaning has to do
with comfort as a cause, either of the state of comfort itself or of relief from discom-
fort. The second meaning associates comfort with a state of ease and peaceful content-
ment. Thus, comfort as a cause (first meaning) is supposed to produce comfort as an
effect (second meaning) (Kolcaba and Kolcaba, 1991).
Many of life’s conditions, such as concerns, pain, or suffering, mean absence of
the state of comfort. The existence of these conditions is designated as discomfort,
whose outcome is contrary to the state of comfort. It should be stressed that the state
of comfort can exist without a prior state of discomfort (ease). However, when dis-
comfort cannot be avoided, it is often neutralized with additional comforts, relieving
discomfort.
The third meaning of comfort is in the sense of relief from discomfort and it can be
understood through the first two meanings. The cause of relief is specified by the first
meaning, whereas the state of comfort is specified by the second meaning. When relief
itself is called comfort, it need not to be equivalent to the state of comfort as it may
be incomplete, partial, or temporary if, for example, it is relief from only one of many
discomforts. It can be partial because only a degree of relief is attained and temporary
as it may last only until discomfort arises again.
According to the fourth meaning, comfort is whatever makes life easy or pleasur-
able. This refers to the goal of maximizing pleasure and in this aspect it is not appli-
cable to nursing care.
A fifth and a sixth meaning of comfort can also be considered: one that comes from
the Latin word confortare meaning to “strengthen greatly” (expressing the actions of
strengthening, encouragement, incitement, aid, succor, support, and countenance); and
another one that indicates physical refreshment or sustenance. These meanings are
related to causes of renewal, amplifications of power, positive mindsets, and readiness
for action. Thus, comfort is based on things that strengthen and encourage, support
and/or physically refresh or invigorate a person (Kolcaba and Kolcaba, 1991).
OPERATIONALIZING THE CONCEPT OF COMFORT

In order to operationalize the concept of comfort, Kolcaba described it as the immedi-
ate experience of feeling strengthened when the basic human needs of relief, ease, and
transcendence are addressed in four contexts of experience (physical, psychospiritual,
sociocultural, and environmental) (Kolcaba, 1991, 2003).
Relief is the state in which a need has been met, essential for the person to re-es-
tablish her/his normal functioning; ease is a state of calmness or contentment and it is
necessary for an effective performance; transcendence is the state in which people feel
they have skills or potential to plan, control their destiny, and resolve their problems.
These three states of comfort can be experienced in the following four contexts:
physical, psychospiritual, sociocultural, and environmental. The physical context per-
tains to bodily sensations; the psychospiritual context pertains to the internal aware-
ness of self (including self-concept, sexuality, and purpose in one’s life) and can also
encompass one’s relationship to a higher order or being; the sociocultural context
pertains to interpersonal, familiar, and societal relationships; the environmental con-
text involves items such as light, noise, equipment (furniture), color, temperature, and
natural versus synthetic elements.
These four contexts, when combined with the three senses of comfort form a taxo-
nomic structure (TS) of 12 cells, which represents the total Gestalt of patient comfort
from the perspective of patients’ needs and their fulfillment (see Table 1).
Table 1. Kolcaba’s conceptual framework of comfort (Kolcaba, 1991).

States
Relief Ease Transcendence
Contexts
Physical Physical Physical
Physical
Psychospiritual Psychospiritual Psychospiritual
Psychospiritual
Sociocultural Sociocultural Sociocultural
Sociocultural
Environmental Environmental Environmental
Environmental
COMPARING THE FINDINGS OF STUDIES ON COMFORT AND SUFFERING

OF PORTUGUESE CANCER PATIENTS AND RELATIVES
Instruments used in the different studies:
(1) Sociodemographic and clinical questions;
(2) Comfort Chemotherapy Scale (CCS) (Apóstolo et al., 2006): five point Likert-
type scale with 33 items, based on the operational model of “comfort” to as-
sess the three states of comfort (relief, ease, and transcendence) in the four
contexts described;
(3) The Inventory of Subjective Suffering Experiences in Illness—ISSEI—
(Gameiro, 2000): five point Likert-type scale with 44 items to assess the five
dimensions of suffering (psychological, existential, sociorelational, and posi-
tive experiences of suffering).
Study 1
Descriptive-analytic study aiming to describe the characteristics of suffering and com-
fort experienced by female patients undergoing chemotherapy, to analyze the relation-
ship between the comfort and suffering experienced by these patients and to determine
whether these states would be related to the number of chemotherapy cycles and to the
patients’ age (Apóstolo et al., 2006).
The CCS and the ISSEI were administered to a consecutive sample of 50 female
patients diagnosed with cancer (breast and gynecological) undergoing chemotherapy
in outpatient care, in a hospital of the Centre Region of Portugal. Mean age 51.94; SD
12.41, ranging from 30 to 74 years. Regarding education (years), 46% 4, 26% between
5 and 9, 8% between 10 and 12, and 20% higher education. Data was collected be-
tween 16 August and 30 October, 2004.
Main results: Number of chemotherapy cycles: 64% less than 5; 14% between 6
and 10; 4% between 11 and 15; 6% between 16 and 20; 12% more than 20 cycles.
The analysis of the dimensions showed that female patients undergoing chemo-
therapy reported higher discomfort and suffering in the state of relief and in the physi-
cal context, as well as higher comfort in the sociocultural context.
The analysis of the scales’ items that revealed higher levels of suffering (assessed
by the ISSEI) showed that these were related to the sociorelational level, particularly
to the items that addressed the concern with the fact that being ill would bring suffer-
ing to relatives, that is the aspects of empathic suffering1 (see Figure 1). Nevertheless,
the aspects that registered a higher level of perceived comfort (assessed by the CCS)
were related to family support2.
1
“I wish my family would not suffer so much because I am ill; My disease makes me worry about the future
of people close to me; The idea that I cannot help my family as I did before makes me worried; I dread the
fact that I may have to leave the people I care about”.
2
“My family/friends help me face the disease; To know that I am loved gives me strength to go on; People’s
affection around me brings me comfort; Visits from friends make me happy; The state of mind of the people
around me gives me strength; If I need help, I have people who take care of me”.
Figure 1. Profile of the suffering of patients undergoing chemotherapy.
These patients suffer because they understand how much suffering is brought to
their loved ones due to the process of illness. On the other hand, being close to loved
ones is understood as something that provides comfort, showing how important it is to
have the presence and support of the family.
The high mean levels of suffering associated with inability to perform activities
they used to assume before becoming ill and with the severity and worsening of health
conditions are noteworthy. From a physical point of view, although there were few
reports on high levels of pain, patients suffered with the anticipation of future pain and
due to weariness and lack of physical vigor and energy.
Comfort and suffering were not correlated with the number of chemotherapy cy-
cles or with the patients’ age. Comfort correlated negatively with suffering and posi-
tively with the positive experiences of suffering. These two concepts are closely re-
lated and can be seen as part of a continuum in which one of the extremities‑‑suffering/
discomfort‑‑corresponds to the existence of unmet human needs.
Study 2
Descriptive study aiming to characterize the patients’ suffering in the context of oncol-
ogy palliative care (Capela, 2010).
The ISSEI was administered to a consecutive sample of 50 inpatients or outpatients
attending a palliative care unit in the North Region of Portugal. Mean age (years) 61,0;
SD 14.0, ranging from 27‑88 years; 48% male and 52% female; 66% married, 18 %
widowed, 8% divorced, and 8% single. Regarding education (years), 58% 4, 20% 6,
2% 9, 4% 12, 6% undergraduate degree, 10% no education. Data was collected be-
tween 7 May and 15 October, 2009.
Main results: Patients showed high levels of sociorelational suffering, particularly

affective-emotional suffering, that is they suffered from the negative impact of the
disease on their closest relatives. The loss of physical vigor was also a great source
of suffering because of its impact on the performance of activities of daily living, as
well as psychological suffering, depressive mood, despair, existential limitations, loss
of autonomy, and meaningfulness. The socio-professional changes are not a primary
source of suffering. In addition, pain seems to be the symptom that brings less suffer-
ing to terminal patients.
Study 3
Descriptive-analytic study aiming to analyze the suffering of women undergoing che-
motherapy after mastectomy and to identify the extent to which social support, physi-
cal and psychological morbidity, and some sociodemographic and clinical variables
are related to such suffering (Ferreira, 2009).
The ISSEI was administered to a consecutive sample of 84 women undergoing
chemotherapy after mastectomy in outpatient consultations in a day hospital. Mean
age 52.99; SD 10.66, ranging from 35 to 76 years; 76.2% married, 11.8% widowed
and 12% single, divorced or separated. Regarding education (years), 64.3% between
4 and 9, 25.0% between 13 and 19, 10.7% between 10 and 12. Data was collected
between 24 July and 5 September, 2003.
Main results: number of chemotherapy cycles: 54.8% less than 8, 34.5% between
8 and 15, 10.7% between 16 and 25.
These findings revealed that patients experienced high psychological and sociore-
lational suffering and few positive experiences of suffering.
The patients who received more social support experienced less suffering, whereas
those with more physical and psychological morbidity showed higher levels of suffer-
ing, namely older and single patients.
Patients with higher academic qualifications experienced less suffering and, as the
number of chemotherapy cycles increased, patients experienced more and more suf-
fering in all dimensions (except in the sociorelational dimension in the case of number
of chemotherapy cycles).
Patients who were given information about breast reconstruction and those who
planned to take this step showed less suffering when compared to those who were not
informed and those who did not consider having a reconstruction.
Suffering was positively correlated with age.
Study 4
Qualitative phenomenological study aiming to describe the experience lived by rela-
tives of inpatients with oncological illness in terminal condition, in a palliative care
unit, with a sample of five close relatives (three male and two female participants)
(Apóstolo et al., 2004).
Results revealed that families of cancer patients experienced a process that was
focused on the patient and on themselves and that was surrounded by circumstantial
elements typical of hospital environments.
The feelings and emotions shared by these relatives were essentially related to
pain (because of the anticipation of an inevitable loss), impairment, sadness, anguish,
anger, emptiness, and uncertainty. They suffered from witnessing the suffering of their
ill relatives, their physical and psychological deterioration, and subsequent loss of role
in the family system.
Daily life experienced a deep change when family members decided to be close
to their ill relatives, that is, in the hospital setting where everything could be done to
provide the best possible quality of life. Family needs became secondary in face of the
patient’s needs and they felt that they were losing their social identity.
However, there were some experiences that can be considered as positive, such as
a feeling of personal growth since this experience can make the family reflect on the
purpose of life and the importance of the patient in their lives.
CONCLUSION
The relation between suffering, number of chemotherapy cycles and patients’ age
is not unanimous in all studies. Results from study 3, unlike the ones from study
1, showed that suffering is positively correlated with the number of chemotherapy
cycles and with age. Both studies included women undergoing chemotherapy after
mastectomy with similar mean ages. However, study 1 comprised a smaller sample
that included also women with gynecological cancer hampering the possibility of
comparing both samples. In addition, 64% of patients in study 1 had undergone less
than 5 cycles, whereas in study 3 45.2% of patients had undergone between 8 and
25 cycles.
Nevertheless, the main results from all studies point to the great importance
of the family in the comfort and suffering of cancer patients. Family members are
a source of comfort, making these patients feel loved. They help them cope with
the disease and provide support, encouragement and comfort in the most difficult
times, easing their sense of loss, and of impending threat to their integrity. However,
family is also a source of concern and suffering for patients because of their fear of
becoming a burden. Because the patients’ close relatives are seen both as a source
of comfort and as a potential focus of suffering, health care interventions should
encompass not only physical relief, but also an understanding of the patients’ so-
ciorelational contexts, in order to help them finding a purpose in their life, suffering
and existence.
Therefore, it seems that in order for interventions intending to relief suffering to
be effective, they will have to include some support of family relationships because
of the major importance assigned by these patients to the negative impact of the
disease on their families. Health professionals can intervene to alleviate patients’
suffering, particularly by establishing an open patient-family communication and
adopting attitudes of sincerity, respect and trust that facilitate the sharing of vulner-
abilities, experiences, thoughts, emotions, and feelings. In this way, a conspiracy of
silence can be avoided while mutual personal development and relief from suffering
are promoted.
KEYWORDS
•• Comfort
•• Comfort Chemotherapy Scale
•• Existential suffering
•• Psychological suffering
•• Relief
•• Taxonomic structure
References
1 Koirala, S., Corfas, G., D’Amato, R. J., and

Folkman, J. (2008). An orally delivered small-
Abdollahi, A., Hahnfeldt, P., Maercker, C., molecule formulation with anti-angiogenic and
Grone, H. J., Debus, J., Ansorge, W., Folkman, anticancer activity. Nat. Biotechnol. 26, 799–
J., Hlatky, L., and Huber, P. E. (2004). End- 807.
ostatin’s anti-angiogenic signaling network. Mol.
Blind, M., Kolanus, W., and Famulok, M. (1999).
Cell. 13, 649–663.
Cytoplasmic RNA modulators of an inside-out
Akao, Y., Nakagawa, Y., and Naoe, T. (2006). signal-transduction cascade. Proc. Natl. Acad.
Let-7 microRNA functions as a potential growth Sci. USA 96, 3606–3610.
suppressor in human colon cancer cells. Biologi-
Brooks, P. C. (1996). Role of integrins in angio-
cal and Pharmaceutical Bulletin 29, 903–906.
genesis. Eur. J. Cancer 32A, 2423–2429.
Ambros, V. (2001). microRNAs: Tiny regulators
Calin, G. A., Dumitru, C. D., Shimizu, M., Bi-
with great potential. Cell 107, 823–826.
chi, R., Zupo, S., Noch, E., Aldler, H., Rattan, S.,
Ambros, V. (2004). The functions of animal mi- Keating, M., Rai, K., Rassenti, L., Kipps, T., Ne-
croRNAs. Nature 431, 350–355. grini, M., Bullrich, F., and Croce, C. M. (2002).
Antonelli-Orlidge, A., Saunders, K. B., Smith, Frequent deletions and down-regulation of mi-
S. R., and D’Amore, P. A. (1989). An activated cro- RNA genes miR-15 and miR-16 at 13q14 in
form of transforming growth factor beta is pro- chronic lymphocytic leukemia. Proceedings of
duced by cocultures of endothelial cells and peri- the National Academy of Sciences of the United
cytes. Proc. Natl. Acad. Sci. USA 86, 4544–4548. States of America 99, 15524–15529.
Aznavoorian, S., Murphy, A. N., Stetler-Steven- Caplen, N. J., Parrish, S., Imani, F., Fire, A.,
son, W. G., and Liotta, L. A. (1993). Molecular and Morgan, R. A. (2001). Specific inhibition of
aspects of tumor cell invasion and metastasis. gene expression by small double-stranded RNAs
Cancer 71, 1368‑1383. in invertebrate and vertebrate systems. Proc.
Natl. Acad. Sci. USA 98, 9742–9747.
Baratchi, S., Kanwar, R. K., and Kanwar, J. R.
(2010). Survivin: A target from brain cancer to Cavallaro, U. and Christofori, G. (2000). Mo-
neurodegenerative disease. Critical Reviews in lecular Mechanisms of Tumor Angiogenesis and
Biochemistry and Molecular Biology 45, 535– Tumor Progression. Journal of Neuro-Oncology
554. 50, 63–70.
Baratchi, S., Kanwar, R. K., Khoshmanesh, K., Cevc, G. (2004). Lipid vesicles and other col-
Vasu, P., Ashok, C., Hittu, M., Parratt, A., Krish- loids as drug carriers on the skin. Adv. Drug. De-
nakumar, S., Sun, X., Sahoo, S. K., and Kanwar, liv. Rev. 56, 675–711.
J. R. (2009). Promises of nanotechnology for Chan, J. A., Krichevsky, A. M., and Kosik, K. S.
drug delivery to brain in neurodegenerative dis- (2005). MicroRNA-21 is an antiapoptotic factor
eases. Current Nanoscience 5, 15–25. in human glioblastoma cells. Cancer Research
Bartlett, D. W. and Davis, M. E. (2006). Insights 65, 6029–6033.
into the kinetics of siRNA-mediated gene silenc- Chen, Y. and Gorski, D. H. (2008). Regulation of
ing from live-cell and live-animal biolumines- angiogenesis through a microRNA (miR-130a)
cent imaging. Nucleic Acids Res. 34, 322–333. that down-regulates anti-angiogenic homeobox
Bawa, R. (2009). NanoBiotech 2008: Exploring genes GAX and HOXA5. Blood 111, 1217–1226.
global advances in nanomedicine. Nanomedicine Cherian, A. K., Rana A. C., and Jain, S. K.
5, 5–7. (2000). Self-assembled carbohydrate-stabilized
Benny, O., Fainaru, O., Adini, A., Cassiola, F., ceramic nanoparticles for the parenteral delivery
Bazinet, L., Adini, I., Pravda, E., Nahmias, Y., of insulin. Drug Dev. Ind. Pharm. 26, 459–463.
References 195
Cheung, C. G. A., Kanwar, J. R., and Krissansen, ligand ephrin-A3. Journal of Biological Chemis-
G. W. (2006). A cell-permeable dominant-nega- try 283, 15878–15883.
tive survivin protein as a tool to understand how Ferrara, N., Gerber, H. P., and LeCouter, J.
Survivin maintains tumour cell survival. Euro- (2003). The biology of VEGF and its receptors.
pean Journal of Cancer 4, 149. Nat. Med. 9, 669–676.
Clark, R. A., Tonnesen, M. G., Gailit, J., and Fire, A., Xu, S., Montgomery, M. K., Kostas, S.
Cheresh, D. A. (1996). Transient functional ex- A., Driver, S. E., and Mello, C. C. (1998). Po-
pression of alphaVbeta 3 on vascular cells during tent and specific genetic interference by double-
wound repair. Am. J. Pathol. 148, 1407–1421. stranded RNA in Caenorhabditis elegans. Na-
Cleland, J. L. (1997). Protein delivery from bio- ture 391, 806–811.
degradable microspheres. Pharm. Biotechnol. Fish, J. E., Santoro, M. M., Morton, S. U., Yu,
10, 1–43. S., Yeh, R. F., Wythe, J. D., Ivey, K. N., Bru-
Conradi, R. A., Hilgers, A. R., Ho, N. F., and neau, B. G., Stainier, D. Y. R., and Srivastava, D.
Burton, P. S. (1992). The influence of peptide (2008). miR-126 Regulates Angiogenic Signal-
structure on transport across Caco-2 cells. II. ing and Vascular Integrity. Developmental Cell
Peptide bond modification which results in im- 15, 272–284.
proved permeability. Pharm. Res. 9, 435–439. Foley, S., Crowley, C., Smaihi, M., Bonfils, C.,
Das, M. and Sahoo, S. K. (2010). Epithelial cell Erlanger, B. F., Seta, P., and Larroque, C. (2002).
adhesion molecule targeted nutlin-3a loaded im- Cellular localisation of a water-soluble fullerene
munonanoparticles for cancer therapy. Acta Bio- derivative. Biochem. Biophys. Res. Commun.
mater 7, 355–369. 294, 116–119.
Devalapally, H., Duan, Z., Seiden, M. V., and Folkman, J. (1990). How the field of controlled-
Amiji, M. M. (2008). Modulation of drug resis- release technology began, and its central role in
tance in ovarian adenocarcinoma by enhancing the development of angiogenesis research. Bio-
intracellular ceramide using tamoxifen-loaded materials 11, 615–618.
biodegradable polymeric nanoparticles. Clin. Folkman, J. (1995). Angiogenesis in cancer, vas-
Cancer Res. 14, 3193–3203. cular, rheumatoid and other disease. Nat. Med.
Devi, G. R. (2006). SiRNA-based approaches in 1, 27–31.
cancer therapy. Cancer Gene Ther. 13, 819–829. Folkman, J. (1996). New perspectives in clini-
Dews, M., Homayouni, A., Yu, D., Murphy, D., cal oncology from angiogenesis research. Eur. J.
Sevignani, C., Wentzel, E., Furth, E. E., Lee, W. Cancer 32A, 2534–2539.
M., Enders, G. H., Mendell, J. T., and Thomas- Folkman, J. and Kalluri, R. (2004). Cancer with-
Tikhonenko, A. (2006). Augmentation of tumor out disease. Nature 427, 787.
angiogenesis by a Myc-activated microRNA
cluster. Nat. Genet. 38, 1060–1065. de Fougerolles, A., Vornlocher, H. P., Maragan-
ore, J., and Lieberman, J. (2007). Interfering with
Duncan, R. (2006). Polymer conjugates as an- disease: A a progress report on siRNA-based
ticancer nanomedicines. Nat. Rev. Cancer 6, therapeutics. Nat. Rev. Drug Discov. 6, 443–453.
688–701.
Gregory, R. I. and Shiekhattar, R. (2005). Mi-
Elbashir, S. M., Harborth, J., Lendeckel, W., croRNA biogenesis and cancer. Cancer Res. 65,
Yalcin, A., Weber, K., and Tuschl, T. (2001). 3509–3512.
Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells. Nature Griffin, L. C., Tidmarsh, G. F., Bock, L. C.,
411, 494–498. Toole, J. J., and Leung, L. L. (1993). In vivo anti-
coagulant properties of a novel nucleotide-based
Fasanaro, P., D’Alessandra, Y., Di Stefano, V., thrombin inhibitor and demonstration of regional
Melchionna, R., Romani, S., Pompilio, G., Ca- anticoagulation in extracorporeal circuits. Blood
pogrossi, M. C., and Martelli, F. (2008). MicroR- 81, 3271–3276.
NA-210 modulates endothelial cell response to
hypoxia and inhibits the receptor tyrosine kinase Haas, T. L., Milkiewicz, M., Davis, S. J., Zhou,
A. L., Egginton, S., Brown, M. D., Madri, J. A.,
and Hudlicka, O. (2000). Matrix metalloprotein- Kanwar, J. R., Mahidhara, G., and Kanwar, R. K.
ase activity is required for activity-induced an- (2009). Recent advances in nanoneurology for
giogenesis in rat skeletal muscle. Am. J. Physiol. drug delivery to the brain. Current Nanoscience
Heart Circ. Physiol. 279, H1540–1547. 5, 441–448.
Harris, S. L. and Levine, A. J. (2005). The p53 Kanwar, J. R., Mahidhara, G., and Kanwar, R. K.
pathway: Positive and negative feedback loops. (2010). MicroRNA in human cancer and chronic
Oncogene 24, 2899–2908. inflammatory diseases. Front. Biosci. (Schol
Hockenbery, D., Nunez, G., Milliman, C., Sch- Ed.) 2, 1113–1326.
reiber, R. D., and Korsmeyer, S. J. (1990). Bcl- Kanwar, J. R., Mohan, R. M., Kanwar, R. K.,
2 is an inner mitochondrial membrane protein Roy, K., and Bawa, R. (2010). Application of
that blocks programmed cell death. Nature 348, aptamers in nano-delivery systems in cancer,
334–336. eye and inflammatory diseases. Nanomedicine
Isik, F. F., Rand, R. P., Gruss, J. S., Benjamin, 5, 1435–1445.
D., and Alpers, C. E. (1996). Monocyte chemoat- Kanwar, J. R., Palmano, K. P., Sun, X., Kan-
tractant protein-1 mRNA expression in hemangi- war, R. K., Gupta, R., Haggarty, N., Rowan, A.,
omas and vascular malformations. J. Surg. Res. Ram, S., and Krissansen, G. W. (2008). “Iron-
61, 71–76. saturated” lactoferrin is a potent natural adjuvant
Jiang, G., Li, J., Zeng, Z., and Xian, L. (2006). for augmenting cancer chemotherapy. Immunol.
Lentivirus-mediated gene therapy by suppress- Cell Biol. 86, 277–288.
ing survivin in BALB/c nude mice bearing oral Kanwar, J. R., Shen, W. P., Kanwar, R. K., Berg,
squamous cell carcinoma. Cancer Biology and R. W., and Krissansen, G. W. (2001a). Effects
Therapy 5, 435–440. of survivin antagonists on growth of established
Johnson, S. M., Grosshans, H., Shingara, J., tumors and B7-1 immunogene therapy. J. Natl.
Byrom, M., Jarvis, R., Cheng, A., Labourier, Cancer Inst. 93, 1541–1552.
E., Reinert, K. L., Brown, D., and Slack, F. J. Kerr, J. F., Wyllie, A. H., and Currie, A. R.
(2005). RAS is regulated by the Let-7 microR- (1972). Apoptosis: A basic biological phenom-
NA family. Cell 120, 635–647. enon with wide-ranging implications in tissue
Josephson, L., Tung, C. H., Moore, A., and kinetics. British Journal of Cancer 26, 239–257.
Weissleder R. (1999). High-efficiency intracel- Khalil, I. A., Kogure, K., Futaki, S., and Ha-
lular magnetic labeling with novel superpara- rashima, H. (2006). High density of octa arginine
magnetic Tat-peptide conjugates. Bioconjug. stimulates macropinocytosis leading to efficient
Chem. 10, 186–191. intracellular trafficking for gene expression. J.
Kanwar, J., Berg, R., Lehnert, K., and Krissan- Biol. Chem. 281, 3544–3551.
sen, G. (1999). Taking lessons from dendritic Knudson, A. G., Jr. (1971). Mutation and cancer:
cells: Mmultiple xenogeneic ligands for leu- Statistical study of retinoblastoma. Proc. Natl.
kocyte integrins have the potential to stimulate Acad. Sci. USA 68, 820–823.
anti-tumor immunity. Gene Ther. 6, 1835–1844. Kobayashi, N., Matsui, Y., Kawase, A., Hirata,
Kanwar, J. R., Berg, R. W., Yang, Y., Kanwar, K., Miyagishi, M., Taira, K., Nishikawa, M., and
R. K., Ching, L. M., Sun, X., and Krissansen, G. Takakura, Y. (2004). Vector-based in vivo RNA
W. (2003). Requirements for ICAM-1 immuno- interference: dose- and time-dependent suppres-
gene therapy of lymphoma. Cancer Gene Ther. sion of transgene expression. J. Pharmacol. Exp.
10, 468–476. Ther. 308, 688–693.
Kanwar, J. R., Kanwar, R. K., Pandey, S., Ching, Kondo, K. and Kaelin, W. G., Jr. (2001). The von
L. M., and Krissansen, G. W. (2001b). Vascular Hippel-Lindau tumor suppressor gene. Exp. Cell
attack by 5, 6-dimethylxanthenone-4-acetic acid Res. 264, 117–125.
combined with B7.1 (CD80)-mediated immuno- Krammer, P. H. (2000). CD95’s deadly mission
therapy overcomes immune resistance and leads in the immune system. Nature 407, 789–95.
to the eradication of large tumors and multiple
tumor foci. Cancer Res. 61, 1948–1956. Krissansen, G. W., Singh, J., Kanwar, R. K.,
Chan, Y. C., Leung, E., Lehnert, K. B., Kanwar,
References 197
J. R., and Yang, Y. (2006). A pseudosymmetric nanoparticles allow in vivo tracking and recov-
cell adhesion regulatory domain in the beta7 tail ery of progenitor cells. Nat. Biotechnol. 18,
of the integrin alpha4beta7 that interacts with fo- 410–414.
cal adhesion kinase and src. Eur. J. Immunol. 36, Li, F. (2003). Survivin study: What is the next
2203–2214. wave? J. Cell Physiol. 197, 8–29.
Kuehbacher, A., Urbich, C., Zeiher, A. M., and Li, F. and Ling, X. (2006). Survivin study: An
Dimmeler, S. (2007). Role of Dicer and Drosha update of “what is the next wave”? J. Cell Physi-
for endothelial microRNA expression and angio- ol. 208, 476–486.
genesis. Circulation Research 101, 59–68.
Liang, J. F. and Yang, V. C. (2005). Synthesis of
Kulshreshtha, R., Ferracin, M., Wojcik, S. E., doxorubicin-peptide conjugate with multidrug
Garzon, R., Alder, H., Agosto-Perez, F. J., Da- resistant tumor cell killing activity. Bioorg. Med.
vuluri, R., Liu, C. G., Croce, C. M., Negrini, M., Chem. Lett. 15, 5071–5075.
Calin, G. A., and Ivan, M. (2007). A microRNA
signature of hypoxia. Molecular and Cellular Bi- Luo, L., Qiao, H., Meng, F., Dong, X., Zhou, B.,
ology 27, 1859–1867. Jiang, H., Kanwar, J. R., Krissansen, G. W., and
Sun, X. (2006). Arsenic trioxide synergizes with
Kuwabara, K., Ogawa, S., Matsumoto, M., B7H3-mediated immunotherapy to eradicate
Koga, S., Clauss, M., Pinsky, D. J., Lyn, P., hepatocellular carcinomas. Int. J. Cancer 118,
Leavy, J., Witte, L., Joseph-Silverstein, J. et al. 1823–1830.
(1995). Hypoxia-mediated induction of acidic/
basic fibroblast growth factor and platelet-de- Mahidhara, G., Kanwar, R. K., and Kanwar, J. R.
rived growth factor in mononuclear phagocytes (2011 ). A novel nanoplatform for oral delivery
stimulates growth of hypoxic endothelial cells. of anti-cancer biomacromolecules. International
Proc. Natl. Acad. Sci. USA 92, 4606–4610. Journal of Nanotechnology (Accepted).
Lameiro, M. H., Lopes, A., Martins, L. O., Maisonpierre, P. C., Suri, C., Jones, P. F., Bar-
Alves, P. M., and Melo, E. (2006). Incorporation tunkova, S., Wiegand, S. J., Radziejewski, C.,
of a model protein into chitosan-bile salt mic- Compton, D., McClain, J., Aldrich, T. H., Papa-
roparticles. Int. J. Pharm. 312, 119–130. dopoulos, N., Daly, T. J., Davis, S., Sato, T. N.,
and Yancopoulos, G. D. (1997). Angiopoietin-2,
Lee, D. Y., Deng, Z., Wang, C. H., and Yang, B. a natural antagonist for Tie2 that disrupts in vivo
B. (2007). MicroRNA-378 promotes cell sur- angiogenesis. Science 277, 55–60.
vival, tumor growth, and angiogenesis by target-
ing SuFu and Fus-1 expression. Proceedings of Maliyekkel, A., Davis, B. M., and Roninson, I.
the National Academy of Sciences of the United B. (2006). Cell cycle arrest drastically extends
States of America 104, 20350–20355. the duration of gene silencing after transient
expression of short hairpin RNA. Cell Cycle 5,
Lee, R. C., Feinbaum, R. L., and Ambros, V. 2390–2395.
(1993). The C. elegans heterochronic gene lin-4
encodes small RNAs with antisense complemen- Martin, C. R. and Kohli, P. (2003). The emerging
tarity to lin-14. Cell 75, 843–854. field of nanotube biotechnology. Nat. Rev. Drug
Discov. 2, 29–37.
Lee, Y., Ahn, C., Han, J., Choi, H., Kim, J., Yim,
J., Lee, J., Provost, P., Radmark, O., Kim, S., and Matsumura, Y. and Maeda, H. (1986). A new
Kim, V. N. (2003). The nuclear RNase III Dro- concept for macromolecular therapeutics in can-
sha initiates microRNA processing. Nature 425, cer chemotherapy: Mechanism of tumor itropic
415–419. accumulation of proteins and the antitumor agent
smancs. Cancer Res. 46, 6387–6392.
Lee, Y., Jeon, K., Lee, J. T., Kim, S., and Kim,
V. N. (2002). MicroRNA maturation: Stepwise Maxwell, P. H., Dachs, G. U., Gleadle, J. M.,
processing and subcellular localization. EMBO Nicholls, L. G., Harris, A. L., Stratford, I. J.,
J. 21, 4663–4670. Hankinson, O., Pugh, C. W., and Ratcliffe, P. J.
(1997). Hypoxia-inducible factor-1 modulates
Lewin, M., Carlesso, N., Tung, C. H., Tang, X. gene expression in solid tumors and influences
W., Cory, D., Scadden, D. T., and Weissleder, both angiogenesis and tumor growth. Proceed-
R. (2000). Tat-peptide-derivatized magnetic
ings of the National Academy of Sciences of the cancer cells. Molecular Cancer Therapeutics 5,
United States of America 94, 8104–8109. 179–186.
Meade, B. R. and Dowdy, S. F. (2008). Enhanc- Pasquinelli, A. E., Reinhart, B. J., Slack, F., Mar-
ing the cellular uptake of siRNA duplexes fol- tindale, M. Q., Kuroda, M. I., Maller, B., Hay-
lowing noncovalent packaging with protein ward, D. C., Ball, E. E., Degnan, B., Muller, P.,
transduction domain peptides. Adv. Drug Deliv. Spring, J., Srinivasan, A., Fishman, M., Finnerty,
Rev. 60, 530–536. J., Corbo, J., Levine, M., Leahy, P., Davidson,
Mi, J., Zhang, X., Rabbani, Z. N., Liu, Y., Red- E., and Ruvkun, G. (2000). Conservation of the
dy, S. K., Su, Z., Salahuddin, F. K., Viles, K., sequence and temporal expression of Let-7 het-
Giangrande, P. H., Dewhirst, M. W., Sullenger, erochronic regulatory RNA. Nature 408, 86–99.
B. A., Kontos, C. D., and Clary, B. M. (2008). Peer, D., Karp, J. M., Hong, S., Farokhzad, O. C.,
RNA aptamer-targeted inhibition of NF-kappa B Margalit, R., and Langer, R. (2007). Nanocarri-
suppresses non-small cell lung cancer resistance ers as an emerging platform for cancer therapy.
to doxorubicin. Mol. Ther. 16, 66–73. Nat. Nanotechnol. 2, 751–760.
Michael, M. Z., O’Connor, S. M., Van Holst Plank, M. J., Sleeman, B. D., and Jones, P. F.
Pellekaan, N. G., Young, G. P., and James, R. J. (2004). The role of the angiopoietins in tumour
(2003). Reduced Accumulation of Specific Mi- angiogenesis. Growth Factors 22, 1–11.
croRNAs in Colorectal Neoplasia. Molecular Plate, K. H., Breier, G., Weich, H. A., and Risau,
Cancer Research 1, 882–891. W. (1992). Vascular endothelial growth factor is
Morimoto, K., Yamaguchi, H., Iwakura, Y., Mi- a potential tumour angiogenesis factor in human
yazaki, M., Nakatani, E., Iwamoto, T., Ohashi, gliomas in vivo. Nature 359, 845–848.
Y., and Nakai, Y. (1991). Effects of proteolytic Poliseno, L., Tuccoli, A., Mariani, L., Evangelis-
enzyme inhibitors on the nasal absorption of ta, M., Citti, L., Woods, K., Mercatanti, A., Ham-
vasopressin and an analogue. Pharm. Res. 8, mond, S., and Rainaldi, G. (2006). MicroRNAs
1175–1179. modulate the angiogenic properties of HUVECs.
Naumov, G. N., Akslen, L. A., and Folkman, J. Blood 108, 3068–3071.
(2006). Role of angiogenesis in human tumor Prokop, A., Kozlov, E., Newman, G. W., and
dormancy: Animal models of the angiogenic Newman, M. J. (2002). Water-based nanopar-
switch. Cell Cycle 5, 1779–1787. ticulate polymeric system for protein delivery:
O’Rourke, G. E. and Ellem, A. O. (2000). John Permeability control and vaccine application.
Kerr and apoptosis. Medical Journal of Australia Biotechnol. Bioeng. 78, 459–466.
173, 616–617. Rawat, M., Singh, D., and Saraf, S. (2006).
Ota, A., Tagawa, H., Karnan, S., Tsuzuki, S., Nanocarriers: Promising vehicle for bioactive
Karpas, A., Kira, S., Yoshida, Y., and Seto, M. drugs. Biol. Pharm. Bull. 29, 1790–1798.
(2004). Identification and Characterization of a Rawat, M., Singh, D., and Saraf, S. (2008). De-
Novel Gene, C13orf25, as a Target for 13q31- velopment and in vitro evaluation of alginate
q32 Amplification in Malignant Lymphoma. gel-encapsulated, chitosan-coated ceramic nano-
Cancer Research 64, 3087–3095. cores for oral delivery of enzyme. Drug Dev. Ind.
Paddison, P. J., Caudy, A. A., Bernstein, E., Han- Pharm. 34, 181–188.
non, G. J., and Conklin, D. S. (2002). Short hair- Reinhart, B. J., Slack, F. J., Basson, M., Pas-
pin RNAs (shRNAs) induce sequence-specific quinelli, A. E., Bettinger, J. C., Rougvie, A. E.,
silencing in mammalian cells. Genes Dev. 16, Horvitz, H. R., and Ruvkun, G. (2000). The
948–958. 21-nucleotide let-7 RNA regulates developmen-
Paduano, F., Villa, R., Pennati, M., Folini, M., tal timing in Caenorhabditis elegans. Nature
Binda, M., Grazia, M., and Zaffaroni, N. (2006). 403, 901–906.
Silencing of suvivin gene by small interfering Rothbard, J. B., Garlington, S., Lin, Q., Kirsch-
RNAs produces supra-additive growth sup- berg, T., Kreider, E., McGrane, P. L., Wender, P.
pression in combination with 17-allylamino- A., and Khavari, P. A. (2000). Conjugation of
17-demethoxygeldanamycin in human prostate arginine oligomers to cyclosporin A facilitates
References 199
topical delivery and inhibition of inflammation. Stamatovic, S. M., Keep, R. F., Mostarica-
Nat. Med. 6, 1253–1257. Stojkovic, M., and Andjelkovic, A. V. (2006).
Salcedo, R., Ponce, M. L., Young, H. A., Was- CCL2 regulates angiogenesis via activation
serman, K., Ward, J. M., Kleinman, H. K., Op- of Ets-1 transcription factor. J. Immunol. 177,
penheim, J. J., and Murphy, W. J. (2000). Human 2651–2661.
endothelial cells express CCR2 and respond to Suárez, Y., Fernández-Hernando, C., Pober, J.
MCP-1: Direct role of MCP-1 in angiogenesis S., and Sessa, W. C. (2007). Dicer dependent mi-
and tumor progression. Blood 96, 34–40. croRNAs regulate gene expression and functions
Sargiannidou, I., Zhou, J., and Tuszynski, G. P. in human endothelial cells. Circulation Research
(2001). The role of thrombospondin-1 in tumor 100, 1164–1173.
progression. Exp. Biol. Med. (Maywood) 226, Sudhakar, A., Sugimoto, H., Yang, C., Lively,
726–733. J., Zeisberg, M., and Kalluri, R. (2003). Human
Sarraf-Yazdi, S., Mi, J., Moeller, B. J., Niu, X., tumstatin and human endostatin exhibit distinct
White, R. R., Kontos, C. D., Sullenger, B. A., anti-angiogenic activities mediated by alpha v
Dewhirst, M. W., and Clary, B. M. (2008). Inhi- beta 3 and alpha 5 beta 1 integrins. Proc. Natl.
bition of in vivo tumor angiogenesis and growth Acad. Sci. USA 100, 4766–4771.
via systemic delivery of an angiopoietin 2-spe- Sun, X., Kanwar, J. R., Leung, E., Lehnert, K.,
cific RNA aptamer. J. Surg. Res. 146, 16–23. Wang, D., and Krissansen, G. W. (2001). Angio-
Scaffidi, C., Fulda, S., Srinivasan, A., Friesen, statin enhances B7.1-mediated cancer immuno-
C., Li, F., Tomaselli, K. J., Debatin, K. M., therapy independently of effects on vascular en-
Krammer, P. H., and Peter, M. E. (1998). Two dothelial growth factor expression. Cancer Gene
CD95 (APO-1/Fas) signaling pathways. EMBO Ther. 8, 719–727.
J. 17, 1675–1687. Sun, X., Kanwar, J. R., Leung, E., Vale, M., and
Schaffert, D. and Wagner, E. (2008). Gene ther- Krissansen, G. W. (2003a). Regression of solid
apy progress and prospects: Synthetic polymer- tumors by engineered overexpression of von
based systems. Gene Ther. 15, 1131–1138. Hippel-Lindau tumor suppressor protein and
antisense hypoxia-inducible factor-1alpha. Gene
Semenza, G. L. (1996). Transcriptional Regula- Ther. 10, 2081–2089.
tion by Hypoxia-Inducible Factor 1 Molecular
Mechanisms of Oxygen Homeostasis. Trends in Sun, X., Vale, M., Leung, E., Kanwar, J. R., Gup-
Cardiovascular Medicine 6, 151–157. ta, R., and Krissansen G., W. (2003b). Mouse
B7-H3 induces antitumor immunity. Gene Ther.
Sengupta, S., Eavarone, D., Capila, I., Zhao, G., 10, 1728–1734.
Watson, N., Kiziltepe, T., and Sasisekharan, R.
(2005). Temporal targeting of tumor cells and Sun, X., Qiao, H., Jiang, H., Zhi, X., Liu, F.,
neovasculature with a nanoscale delivery sys- Wang, J., Liu, M., Dong, D., Kanwar, J. R., Xu,
tem. Nature 436, 568–572. R., and Krissansen, G. W. (2005). Intramuscu-
lar delivery of anti-angiogenic genes suppresses
Shah, M. H. and Paradkar, A. (2005). Cubic secondary metastases after removal of primary
liquid crystalline glyceryl monooleate matrices tumors. Cancer Gene Ther. 12, 35–45.
for oral delivery of enzyme. Int. J. Pharm. 294,
161–171. Szebenyi, G. and Fallon, J. F. (1999). Fibroblast
growth factors as multifunctional signaling fac-
Shichiri, M. and Hirata, Y. (2001). Antiangio- tors. Int, Rev, Cytol. 185, 45–106.
genesis signals by endostatin. FASEB J. 15,
1044–1053. Tam, W., Hughes, S. H., Hayward, W. S., and
Besmer, P. (2002). Avian bic, a gene isolated
Shweiki, D., Itin, A., Soffer, D., and Keshet, E. from a common retroviral site in avian leukosis
(1992). Vascular endothelial growth factor in- virus-induced lymphomas that encodes a non-
duced by hypoxia may mediate hypoxia-initiated coding RNA, cooperates with c-myc in lympho-
angiogenesis. Nature 359, 843–845. magenesis and erythroleukemogenesis. Journal
Smyth, S. S. and Patterson, C. (2002). Tiny danc- of Virology 76, 4275–4286.
ers: The integrin-growth factor nexus in angio-
genic signaling. J. Cell Biol. 158, 17–21.
Thai, T. H., Calado, D. P., Casola, S., Ansel, K. As Oncogenes in Testicular Germ Cell Tumors.
M., Xiao, C., Xue, Y., Murphy, A., Frendewey, Cell 124, 1169–1181.
D., Valenzuela, D., Kutok, J. L., Schmidt-Sup- Wajant, H. (2002). The Fas signaling pathway:
prian, M., Rajewsky, N., Yancopoulos, G., Rao, More than a paradigm. Science 296, 1635–1636.
A., and Rajewsky, K. (2007). Regulation of the
germinal center response by microRNA-155. Wang, S., Aurora, A. B., Johnson, B. A., Qi,
Science 316, 604–608. X., McAnally, J., Hill, J. A., Richardson, J. A.,
Bassel-Duby, R., and Olson, E. N. (2008). The
Thompson, C. B. (1995). Apoptosis in the patho- Endothelial-Specific MicroRNA miR-126 Gov-
genesis and treatment of disease. Science 267, erns Vascular Integrity and Angiogenesis. Devel-
1456–1462. opmental Cell 15, 261–271.
Tili, E., Michaille, J.-J., Cimino, A., Costinean, Weber, K. S., Nelson, P. J., Grone, H. J., and
S., Dumitru, C. D., Adair, B., Fabbri, M., Alder, Weber C. (1999). Expression of CCR2 by endo-
H., Liu, C. G., Calin, G. A., and Croce, C. M. thelial cells: Implications for MCP-1 mediated
(2007). Modulation of miR-155 and miR-125b wound injury repair and in vivo inflammatory
Levels following Lipopolysaccharide/TNF- activation of endothelium. Arterioscler Thromb.
{alpha} Stimulation and Their Possible Roles in Vasc. Biol. 19, 2085–2093.
Regulating the Response to Endotoxin Shock. J.
Immunol. 179, 5082–5089. Wooddell, C. I., Van Hout, C. V., Reppen, T.,
Lewis, D. L., and Herweijer, H. (2005). Long-
Tonini, T., Rossi, F., and Claudio, P. P. (2003). term RNA interference from optimized siRNA
Molecular basis of angiogenesis and cancer. On- expression constructs in adult mice. Biochem.
cogene 22, 6549–6556. Biophys. Res. Commun. 334, 117–127.
Torchilin, V. P., Rammohan, R., Weissig, V., and Xin Hua, Z. (1994). Overcoming enzymatic and
Levchenko, T. S. (2001). TAT peptide on the absorption barriers to non-parenterally adminis-
surface of liposomes affords their efficient intra- tered protein and peptide drugs. Journal of Con-
cellular delivery even at low temperature and in trolled Release 29, 239–252.
the presence of metabolic inhibitors. Proc. Natl.
Acad. Sci. USA 98, 8786–8791. Yagihashi, A., Ohmura, T., Asanuma, K., Ko-
bayashi, D., Tsuji, N., Torigoe, T., Sato, N., Hi-
Torchilin, V. P., Tischenko, E. G., Smirnov, V. rata, K., and Watanabe, N. (2005). Detection of
N., and Chazov, E. I. (1977). Immobilization of autoantibodies to survivin and livin in sera from
enzymes on slowly soluble carriers. J. Biomed. patients with breast cancer. Clin. Chim. Acta.
Mater. Res. 11, 223–235. 362, 125–130.
Tuerk, C. and Gold, L. (1990). Systematic evolu- Yang, W. J., Yang, D. D., Na, S., Sandusky, G.
tion of ligands by exponential enrichment: RNA E., Zhang, Q., and Zhao, G. (2005). Dicer is re-
ligands to bacteriophage T4 DNA polymerase. quired for embryonic angiogenesis during mouse
Science 249, 505–510. development. Journal of Biological Chemistry
Volinia, S., Calin, G. A., Liu, C. G., Ambs, S., 280, 9330–9335.
Cimmino, A., Petrocca, F., Visone, R., Iorio, M., Yi, R., Qin, Y., Macara, I. G., and Cullen, B. R.
Roldo, C., Ferracin, M., Prueitt, R. L., Yanaihara, (2003). Exportin-5 mediates the nuclear export
N., Lanza, G., Scarpa, A., Vecchione, A., Negri- of pre-microRNAs and short hairpin RNAs.
ni, M., Harris, C. C., and Croce, C. M. (2006). A Genes Dev. 17, 3011–3016.
microRNA expression signature of human solid
tumors defines cancer gene targets. Proceedings Zapata, J. M., Pawlowski, K., Haas, E., Ware, C.
of the National Academy of Sciences of the Unit- F., Godzik, A., and Reed, J. C. (2001). A diverse
ed States of America 103, 2257–2261. family of proteins containing tumor necrosis fac-
tor receptor-associated factor domains. J. Biol.
Voorhoeve, P. M., le Sage, C., Schrier, M., Gil- Chem. 276, 24242–24252.
lis, A. J. M., Stoop, H., Nagel, R., Liu, Y. P., van
Duijse, J., Drost, J., Griekspoor, A., Zlotorynski, Zhang, L., Gao, L., Li, Y., Lin, G., Shao, Y., Ji,
E., Yabuta, N., De Vita, G., Nojima, H., Looi- K., Yu, H., Hu, J., Kalvakolanu, D. V., Kopecko,
jenga, L. H. J., and Agami, R. (2006). A Genetic D. J., Zhao, X., and Xu, D. Q. (2008). Effects of
Screen Implicates miRNA-372 and miRNA-373 plasmid-based Stat3-specific short hairpin RNA
References 201
and GRIM-19 on PC-3M tumor cell growth. Frappaz, D., Chinot, O., Bataillard, A., Ben Has-
Clin. Cancer Res. 14, 559–568. sel, M., Capelle, L., Chanalet, S., Chatel, M.,
Zhang, S., Zhao, B., Jiang, H., Wang, B., and Figarella-Branger, D., Guegan, Y., Guyotat, J.,
Ma, B. (2007). Cationic lipids and polymers me- Hoang-Xuan, K., Jouanneau, E., Keime-Guibert,
diated vectors for delivery of siRNA. J. Control F., Laforet, C., Linassier, C., Loiseau, H., Maire,
Release 123, 1–10. J. P., Menei, P., Rousmans, S., Sanson, M., and
Sunyach, M. P. (2003). Summary version of the
Zhang, Y., Boado, R. J., and Pardridge, W. M. Standards, Options and Recommendations for
(2003). In vivo knockdown of gene expression in the management of adult patients with intracra-
brain cancer with intravenous RNAi in adult rats. nial glioma (2002). Br. J. Cancer 89(S1), S73‑83.
J. Gene Med. 5, 1039–1045.
Haberland, C. (2007). Clinical Neuropathology.
Zhou, X. H. and Li Wan, Po A. (1991). Com- Text and color atlas. Demos Medical Publishing,
parison of enzyme activities of tissues lining LLC, New York.
portals of absorption of drugs: Species differ-
ences. International Journal of Pharmaceutics Hochberg, F. H. and Pruitt, A. (1980). Assump-
70, 271–283. tions in the radiotherapy of glioblastoma. Neu-
rology 30, 907–911.
Zuhorn, I. S., Engberts, J. B., and Hoekstra, D.
(2007). Gene delivery by cationic lipid vectors: International Agency for Research on Cancer
Overcoming cellular barriers. Eur. Biophys. J. (2008). World Cancer Report. http://www.iarc.
36, 349–362. fr/en/publications/pdfs-online/wcr/2008/index.
php (accessed 17/08/10).
Katz, A. and Alfano, R. R. (1996). Optical Bi-
2 opsy: Detecting Cancer with Light. In Biomedi-
cal Optical Spectroscopy and Diagnostics. E.
Ball, G. F. M. (1998). Bioavilability and Analy-
Sevick-Muraca and D. Benaron (Eds.). OSA
sis of Vitamins in Food. In Methods of Biochemi-
Trends in Optics and Photonics Series (Optical
cal Analysis. Chapman and Hall, London, vol.
Society of America), 3, paper FT1.
10, pp. 293‑313.
Kuri Morales, P., Vargas Cortés, M., López
Burger, P. C. and Green, S. B. (1987). Patient
Sibaja, Z., and Rizo Ríos, P. (2010). Registro
age, histologic features, and length of survival in
Histopatológico de Neoplasias Malignas (Histo-
patients with glioblastoma multiforme. Cancer
pathological Register of Malignant Neoplasia).
59(9), 1617‑1625.
http://www.dgepi.salud.gob.mx/diveent/RHNM.
Butte, P. V., Pikul, B. K., Hever, A., Young, W. htm (accessed 17/08/10).
H., Black, K. L., and Marcu, L. (2005). Diagno-
Lin, W. C., Toms, S. A., Johnson, M., Jansen, E.
sis of meningioma by time resolved fluorescence
D., and Mahadevan-Jansen, A. (2001). In vivo
spectroscopy. J. Biomed. Opt. 10(6), 064026.
Brain Tumor Demarcation Using Optical Spec-
Chung, Y. G., Schwartz, J. A., Gardner, C. M., troscopy. Photochem. Photobiol. 73(4), 396‑402.
Sawaya, R. E., and Jacques, S. L. (1997). Diag-
López, T., Figueras, F., Manjarrez, J., Bustos,
nostic Potential of Laser-Induced Autofluores-
J., Alvarez, M., Silvestre-Albero, J., Rodríguez-
cence Emission in Brain Tissue. J. Korean Med.
Reinoso, F., Martínez-Ferre, A., and Martínez,
Sci. 12(2), 135‑142.
E. (2010). Catalytic nanomedicine: A new field
Croce, A. C., Fiorani, S., Locatelli, D., Nano, R., in antitumor treatment using supported platinum
Ceroni, M., Tancioni, F., Giombelli, E., Beneri- nanoparticles. In vitro DNA degradation and in
cetti, E., and Bottiroli, G. (2003). Diagnostic Po- vivo tests with C6 animal model on Wistar rats.
tential of Autofluorescence for an Assisted Intra- Eur. J. Med. Chem. 45(5), 1982‑1990.
operative Delineation of Gioblastoma Resection
López, T., Recillas, S., Guevara, P., Sotelo, J.,
Margins. Photochem. Photobiol. 77(3), 309‑318.
Alvarez, M., and Odriozola, J. A. (2008). Pt/
DaCosta, R. S., Andersson, H., and Wilson, B. TiO2 brain biocompatible nanoparticles: GBM
C. (2003). Molecular Fluorescence Excitation treatment using C6 model in Wistar rats. Acta
Emission Matrices Relevant to Tissue Spectros- Biomaterialia 4(6), 2037‑2044.
copy. Photochem. Photobiol. 78(4), 384‑392.
Marcu, L., Jo, J. A., Butte, P. V., Yong, W. H., sive glioblastoma multiforme: A prospective
Pikul, B. K., Black, K. L., and Thompson, R. C. trial. Surg. Neurol. 68(3), 250‑254.
(2004). Fluorescence Lifetime Spectroscopy of Wagnieres, G., MacWilliams, A., and Lam, S.
Gioblastoma Multiforme. Photochem. Photo- (2003). Lung Cancer Imaging with Fluorescence
biol. 80, 98‑103. Endoscopy. In Handbook of Biomedical Fluo-
Policard, A. (1924). Etude sur les aspects of- rescence. M. A. Mycek and B. W.Pogue (Eds.).
ferts par des tumeurs experimentales examinees Marcel Dekker, Inc., New York, p. 365.
a la lumiitre de Wood. CR. Soc. Biol. Paris 91, Wallner, K. E., Galicich, J. H., Krol, G., Arbit
1423‑1424. E., and Malkin, M.G. (1989). Patterns of failure
Ricchelli, F., Gobbo, S., Jori, G., Salet, C., and following treatment for glioblastoma multiforme
Moreno, G. (1995). Temoperature-induced and anaplastic astrocytoma. Int. J. Radiat. On-
changes in fluorescence properties as a probe of col. Biol. Phys 16(6), 1405–1409.
porphyrin microenviroment in lipid membranes.
2. The partition of hematoporphyrin and pro-
toporphyrin in mitochondria. Eur. J. Biochem. 3
233(1), 165‑170.
Allan, I., Newman, H., and Wilson, M. (2001).
Rimington, C. (1960). Spectral-absorption Coef- Antibacterial activity of particulate bioglass
ficients of some Porphyrins in the Soret-Band against supra- and subgingival bacteria. Bioma-
Region. Biochem. J. 75(3), 620‑623. terials 22, 1683‑1687.
Roggan, A., Albrecht, H. J., Doerschel, K., Mi- Allen, M., Bulte, J. W., Liepold, L., Basu, G., Zy-
net, O., and Mueller, G. J. (1995). Experimental wicke, H. A., Frank, J. A., Young, M., and Doug-
set-up and Monte-Carlo model for the determi- las, T. (2005). Paramagnetic viral nanoparticles
nation of optical tissue properties in the wave- as potential high-relaxivity magnetic resonance
length range 330‑1100 nm. Proc. SPIE. 2323, contrast agents. Magn. Reson. Med. 54, 807‑812.
21‑36.
Balakrishnan, B., Mohanty, M., Umashankar,
Saraswathy, A., Jayasree, R. S., Baiju, K. V., P. R., and Jayakrishnan, A. (2005). Evaluation
Gupta, A. K., and Pillai, V. P. (2009). Optimum of an in situ forming hydrogel wound dressing
wavelenght for the differentiation of brain tumor based on oxidized alginate and gelatin. Biomate-
tissue using autofluorescence spectroscopy. Pho- rials 26, 6335‑6342.
tomed. Laser Surg. 27(3), 425‑433.
Batrakova, E. V. and Kabanov, A. V. (2008). Plu-
Scott, T. G., Spencer, R. D., Leonard, N. G., ronic block copolymers: Evoltion of drug deliv-
and Weber, G. (1970). Emission properties of ery concept from inert nanocarriers to biological
NADH: Studies of fluorescence lifetimes and response modifiers. Journal of controlled release
quantum efficiencies of NADH, AcPyADH, and 130, 98‑106.
simplified synthetic models. J. Am. Chem. Soc.
Brand, G. D., Leite, J. R., de Sá Mandel, S. M.,
92, 687‑695.
Mesquita, D. A., Silva, L. P., Prates, M. V., Bar-
Stupp, R., Mason, W. P., Van den Beuf, M. J., bosa, E. A., Vinecky, F., Martins, G. R., Galasso,
Weller, M., Fisher, B., Taphoorn, M. J., Be- J. H., Kuckelhaus, S. A., Sampaio, R. N., Furta-
langer, K., Brandes, A. A., Marosi, C., Bogdahn, do, J. R. Jr., Andrade, A. C., and Bloch, C. Jr.
U., Curschmann, J., Janzer, R. C., Ludwin, S. K., (2006). Novel dermaseptins from Phyllomedusa
Gorlia, T., Allgeier, A., Lacombe, D., Cairncross, hypochondrialis (Amphibia). Biochem. Biophys.
J. G., Eisenhauer, E., and Mirimanoff, R.O. Res. Commun. 347, 739‑746.
(2005). Radiotherapy plus concomitant and ad-
Bruno, J. G. and Kiel, J. L. (1999). In vitro se-
juvant temozolomide for newly diagnosed glio-
lection of DNA aptamers to anthrax spores with
blastoma. N. Engl. J. Med. 352(10), 987–996.
electrochemiluminescence detection. Biosensors
Terasaki, M., Ogo, E., Fukushima, S., Sakata, K., and Bioelectronics 14(5), 457‑464.
Miyagi, N., Abe, T., and Shigemori, M. (2007).
Costerton, J. W., Stewart, P. S., and Greenberg,
Impact of combination therapy with repeat sur-
E. P. (1999). Bacterial biofilms: A common cause
gery and temozolomide for recurrent or progres-
of persistent infections. Science 284, 1318‑1322.
References 203
Croy, S. R. and Kwon, G. S. (2004). The effects nanofibre mats with antibacterial properties from
of Pluronic block copolymers on the aggregation quaternised chitosan and poly(vinyl alcohol).
state of nystatin. J. Control. Release 95, 161‑171. Carbohydr. Res. 341(12), 2098‑2107.
Croy, S. R. and Kwon, G. S. (2005). Polysorbate Jia, H., Hou, W., Wei, L., Xu, B., and Liu,
80 and Cremophor EL micelles deaggregate and X. (2008). The structures and antibacterial
solubilize nystatin at the core-corona interface. properties of nano-SiO2 supported silver/zinc
J. Pharm. Sci. 94, 2345‑2354. silver materials. Dent. Mater. 24(2), 244‑249.
Dong, H., Huang, J., Koepsel, R. R., Ye, P., Jones, N., Ray, B., Ranjit, K. T., and Manna,
Russell, A. J., and Matyjaszewski, K. (2011). A. C. (2008). Antibacterial activity of ZnO
Recyclable antibacterial magnetic nanoparticles nanoparticle suspensions on a broad spectrum of
grafted with quaternized poly (2-(dimethyl microorganisms FEMS. Microbiol. Lett. 279(1),
amino) ethyl methacrylate) brushes. 71‑76.
Biomacromolecules 12(4), 1305‑1311. Kabanov, A., Nazarova, I. R., Astafieva, I. V.,
Edgar, R., McKinstry, M., Hwang, J., Oppen- Batrakova, E. V., Alakhov, V. Y., Yaroslavov, A.
heim, A. B., Fekete, R. A., Giulian, G., Mer- A., and Kabanov, V. A. (1995). Micelle forma-
ril, C., Nagashima, K., and Adhya, S. (2006). tion and solubilization of fluorescent probes in
High-sensitivity bacterial detection using biotin- poly(oxyethylene-boxypropilene-b-oxyethyl-
tagged phage and quantum-dot nanocomplexes. ene) solutions. Macromolecules 28, 2303‑2314.
Proc. Natl. Acad. Sci. USA 103(13), 4841‑4845. Kang, S., Pinault, M., Pfefferle, L. D., and
Faulk, W. P. and Taylor, G. M. (1971). An Elimelech, M. (2007) Single-walled carbon
immunocolloid method for the electron nanotubes exhibit strong antimicrobial activity.
microscope. Immunochemistry 8(11), 1081‑1083. Langmuir 23, 8670‑8673.
Flenniken, M. L., Liepold, L. O., Crowley, B. Kanwar, J. R., Mohan, R. R., Kanwar, R. K.,
E., Willits, D. A., Young, M. J., and Douglas, Roy, K., and Bawa, R. (2010). Applications of
T. (2005). Selective attachment and release of aptamers in nanodelivery systems in cancer, eye,
a chemotherapeutic agent from the interior of and inflammatory diseases. Nanomedicine 5(9),
a protein cage architecture. Chem. Commun. 1435‑1445.
447–449. Kostarelos, K. (2006). The emergence of nano-
Flenniken, M. L., Willits, D. A., Harmsen, A. L., medicine: A field in the making. Nanomedicine
Liepold, L. O., Harmsen A. G., Young, M. J., and 1(1), 1‑3.
Douglas, T. (2006). Melanoma and lymphocyte Matthews, L. Kanwar, R. K., Zhou, S., Punj, V.,
cell-specific targeting incorporated into a heat and Kanwar, J. R. (2010). Application of nano-
shock protein cage architecture. Chem. Biol. 13, medicine in antibacterial medicine therapeutics
161‑170. and diagnostics. Open Tropical Medicine Jour-
Hermerén, G., Marczewski, K., and Nielsen, L. nal 3(1), 1‑9.
(2007). Ethicasl aspects of nanomedicine: A con- Merli, D., Ugonino, M., Profumo, A., Fagnoni,
densed version of the EGE opinion 21. M., Quartarone, E., Mustarelli, P., Visai, L.,
Huang, L., Li, D. Q., Lin, Y. J., Wei, M., Ev- Grandi, M. S., Galinetto, P., and Canton, P.
ans, D. G., and Duan, X. (2005). Controllable (2011). Increasing the antibacterial effect of
preparation of Nano-MgO and investigation of lysozyme by immobilization on multi-walled
its bactericidal properties. J. Inorg. Biochem. carbon nanotubes. Journal of Nanoscience and
99(5), 986‑993. Nanotechnology 11(4), 3100‑3106(7).
Ignatova, M., Starbova, K., Markova, N., Mano- Muzzarelli, R. A., Guerrieri, M., Goteri, G.,
lova, N. and, Rashkov, I. (2006a). Electrospun Muzzarelli, C., Anneni, T., Ghiselli, R., and Cor-
nanofibre mats with antibacterial properties from nelissen, M. (2005). The biocompatibility of di-
quaternised chitosan and poly(vinyl alcohol). butyryl chitin in the context of wound dressings.
Carbohydr. Res. 341, 2098‑2107. Biomaterials 26, 5844‑5854.
Ignatova, M., Starbova, K., Markova, N., Mano- Leite, J. R., Brand, G. D., Silva, L. P., Kückel-
lova, N., and Rashkova, I. (2006b). Electrospun haus, S. A., Bento, W. R., Araújo, A. L., Martins,
G. R., Lazzari, A. M., and Bloch, C., Jr. (2008). Hoyt, K. O., Codd, S., Stewart, P. S., Young,
Dermaseptins from Phyllomedusa oreades and M., and Douglas, T. (2007). High-density target-
Phyllomedusa distincta: Secondary structure, ing of a viral multifunctional nanoplatform to a
antimicrobial activity, and mammalian cell tox- pathogenic, biofilm-forming bacterium. Chemis-
icity. Comp. Biochem. Physiol. A Mol. Integr. try & Biology 14, 387‑398.
Physiol. 151, 336‑343. Tam, P. J., Lu, Y. A., and Yang, J. L. (2002). Anti-
LiPuma, J. J., Rathinavelu, S., Foster, B. K., Ke- microbial dendrimeric peptides. Eur. J. Biochem.
oleian, J. C., Makidon, P. E., Kalikin, L. M., and 269, 923‑932.
Baker, J. R., Jr. (2009). In vitro activites of a nov- Waltimo, T., Brunner, T. J., Vollenweider, M.,
el nanoemulsion against burkholderia and other Stark, W. J., and Zehnder, M. (2007). Antimicro-
multi-drug-resistant cystic fibrosis-associated bial effect of nanometric bioactive glass 45S5. J.
bacterial species. Antimicob. Agents Chemother. Dent. Res. 86, 754‑757.
53, 249‑255.
Wang, Y., Zhang, C. L., Zhang, Q., and Li, P.
Lyon, D. Y., Fortner, J. D., Sayes, C. M., Colvin, (2011). Composite electrospun nanomembranes
V. L., and Hughes, J. B. (2005). Bacterial cell of fish scale collagen peptides/chito-oligosac-
association and antimicrobial activity of a C60 charide antibacterial properties and potential for
water suspension. Environ. Toxicol. Chem. 24, wound dressing. International Journal of Nano-
2757‑2762. medicine, 6, 667‑676.
Pini, A., Giuliani, A., Falciani, C., Runci, Y., Yacoby, I. (2006). Targeting antibacterial agents
Ricci, C., Lelli, B., Malossi, M., Neri, P., Rosso- by using drug-carrying filamentous bacterio-
lini, G. M., and Bracci, L. (2005). Antimicrobial phages. Antimicrob. Agents Chemother. 50,
activity of novel dendrimeric peptides obtained 2087‑2097.
by phage display selection and rational modi-
fication. Antimicrob. Agents Chemother. 49, Yacoby, I., Bar, H., and Benhar, I. (2007). Target-
2665‑2672. ed drug-carrying bacteriophages as anti bacterial
nanomedicines. Antimicrob. Agents Chemother.
Salmaso, S., Elvassore, N., Bertucco, A., Lante, 51(6), 2156‑2163.
A., and Caliceti, P. (2004). Nisin-loaded poly-
l-lactide nano-particles produced by CO2 anti- Yacoby, I. and Benhar, I. (2008). Antibacterial
solvent precipitation for sustained antimicrobial nanomedicine. Nanomedicine 3(3), 329‑341.
activity. Int. J. Pharma. 287(1/2), 163‑173. Zampa, M. F., Araújo, I. M., Costa, V., Nery
Saski, W. and Shah, S. G. (1965a). Availability Costa, C. H., Santos, J. R. Jr., Zucolotto, V., Ei-
of drugs in the presence of surface-active agents ras, C., and Leite, J. R. (2009). Leishmanicidal
II. Effects of some oxyethylene oxypropylene activity and immobilization of dermaseptin
polymers on the biological activity of hexetidine. 01 antimicrobial peptides in ultrathin films for
J. Pharm. Sci. 54, 277‑280. nanomedicine applications. Nanomedicine:
Nanotechnology, biology, and medicine 5(3),
Saski, W. and Shah, S. G. (1965b). Availability 352‑358.
of drugs in the presence of surface-active agents
I. Critical micelle concentrations of some oxy- Zhang, L. and Falla, T. J. (2006). Antimicro-
ethylene polymers. J. Pharm. Sci. 54, 71‑74. bial peptides: Therapeutic potential. Exp. Opin.
Pharmacother. 7, 653‑663.
Sepulveda, P., Jones, J. R., and Hench, L. L.
(2002). In vitro dissolution of melt-derived 45S5
and sol-gel derived 58S bioactive glasses. J.
Biomed. Mater. Res. 61, 301‑311.
4
Agnihotri, S. A., Mallikarjuna, N. N., and Amin-
Sondi, I. and Salopek-Sondi, B. (2004). Silver
abhavi, T. M. (2004). Recent advances on chito-
nanoparticles as antimicrobial agent: A case
san-based micro- and nanoparticles in drug de-
study on E coli as a model for Gram-negative
livery. Journal of Controlled Release 100, 5–28.
bacteria. J. Coll. Inter. Sci., 275(1), 177‑182.
Agrawal, P., Strijkers, G. J., and Nicolay, K.
Suci, P. A., Berglund, D. L., Liepold, L., Brum-
(2010). Chitosan-based systems for molecular
field, S., Pitts, B., Davisonn, W., Oltrogge, L.,
References 205
imaging. Advanced Drug Delivery Reviews, 62 De Campos, A. M., Sanchez, A., and Alonso, M.
42–58. J. (2001). Chitosan nanoparticles: a new vehicle
Arhewoh, I. M., Ahonkhai, E. I., and Okhamafe, for the improvement of the delivery of drugs to
A. O. (2005). Optimising oral systems for the de- the ocular surface‑Application to cyclosporin
livery of therapeutic proteins and peptides. Afri- A. International Journal of Pharmaceutics 224,
can Journal of Biotechnology 4(13), 1591‑1597. 159–168.
Badawi, A. A., Laithy, H. M. E., Qidra, R. K. E., Chakravarthi, S. S, Robinson, D. H., and De, S.
Mofty, H. E., and Dally, M. E. (2008). Chitosan (2007). Nanoparticles prepared using natural and
Based Nanocarriers for Indomethacin Ocular synthetic polymers. In Nanoparticulate Drug
Delivery. Arch. Pharm. Res. 31(8), 1040‑1049. Delivery Systems. D. Thassu,, M. Deleers, and
Y. Pathak (Eds.). Informa Healthcare, New York,
Bae, K. H., Moon, C. W., Lee, Y., and Park, T. G. USA, pp. 51‑60.
(2009). Intracellular Delivery of Heparin Com-
plexed with Chitosan-g-Poly(Ethylene Glycol) Chang, Y. C., Shieh, D. B., Chang, C. H. and
for Inducing Apoptosis. Pharmaceutical Re- Chen, D. H. (2005). Conjugation of Monodis-
search 26(1), 93‑100. perse Chitosan-bound Magnetic Nanocarrier
with Epirubicin for Targeted Cancer Therapy.
Banerjee, T., Mitra, S., Singh, A. K., Sharma, R. Journal of Biomedical Nanotechnology 1, 196–
K., and Maitra, A. (2002). Preparation, charac- 201.
terization and biodistribution of ultrafine chi-
tosan nanoparticles. International Journal of Chen, F., Zhang, Z. R., Yuan, F., Qin, X., Wang,
Pharmaceutics 243, 93–105. M., and Huang, Y. (2008). In vitro and in vivo
study of N-trimethyl chitosan nanoparticles for
Bao, H., Li, L., and Zhang, H. (2008). Influ- oral protein delivery. International Journal of
ence of cetyltrimethylammonium bromide on Pharmaceutics 349, 226–233.
physicochemical properties and microstructures
of chitosan–TPP nanoparticles in aqueous solu- Chen, Y., Mohanraj, V. J., and Parkin, J. E.
tions. Journal of Colloid and Interface Science (2003). Chitosan-dextran sulfate nanoparticles
328, 270–277. for delivery of an anti-angiogenesis Peptide. Let-
ters in Peptide Science 10, 621–629.
Bhumkar, D. R. and Pokharkar, V. B. (2006).
Studies on Effect of pH on Cross-linking of Chi- Chen, Y., Mohanraj, V. J., Wang, F., and Benson,
tosan with Sodium Tripolyphosphate: A Techni- H. A. E. (2007). Designing Chitosan–Dextran
cal Note. AAPS PharmSciTech. 7(2) Article 50 Sulfate Nanoparticles Using Charge Ratios.
(http://www.aapspharmscitech.org) AAPS PharmSciTech. 8(4), Article 98 (http://
www.aapspharmscitech.org).
Borges, O., Silva, A. C. da., Romeijn, S. G.,
Amidi, M., Sousa, A. de, Borchard, G., and Chun, W., Xiong, F., and Sheng, Y. L. (2007).
Junginger, H. E. (2006). Uptake studies in rat Water-soluble chitosan nanoparticles as a novel
Peyer’s patches, cytotoxicity and release studies carrier system for protein delivery. Chinese Sci-
of alginate coated chitosan nanoparticles for mu- ence Bulletin 52(7), 883‑889.
cosal vaccination. Journal of Controlled Release Coppi, G. and Iannuccelli, V. (2009). Alginate/
114, 348–358. chitosan microparticles for tamoxifen delivery to
Calvo, P., Lopez, C. R., Jato, J. L. V., and Alonso, the lymphatic system. International Journal of
M. (1997). Chitosan and chitosan/ethylene ox- Pharmaceutics 367, 127–132.
ide- propylene oxide block copolymer nanopar- Coppi, G., Sala, N., Bondi, M., Sergi, S., and
ticles as Novel carriers for proteins and vaccines. Iannuccelli, V. (2006). Ex-vivo evaluation of al-
Pharmaceutical research 14(10), 1431‑1436 ginate microparticles for Polymyxin B oral Ad-
De Campos, A. M., Diebold, Y., Carvalho, E. L. ministration. Journal of Drug Targeting 14(9),
S., Sanchez, A., and Alonso, M. J. (2004). Chito- 599–606.
san Nanoparticles as New Ocular Drug Delivery Csaba, N., Fuentes, M. G., and Alonso, M. J.
Systems: In Vitro Stability, In Vivo Fate, and Cel- (2008). Nanoparticles for nasal vaccination.
lular Toxicity. Pharmaceutical Research 21(5), Advance drug delivery reviews. Doi: 10.1016/j.
803‑810. addr.2008.09.005.
Dudhani, A. R. and Kosaraju, S. L. (2008). Bio- with Chitosan Nanoparticles. Journal of phar-
adhesive Chitosan Nanoparticles: Preparation maceutical Sciences 91(6), 1396‑1404.
and Characterisation. Carbohydrate Polymers, Mao, S., Sun, W., and Kissel, T. (2010). Chito-
doi:10.1016/j.carbpol.2010.02.026. san-based formulations for delivery of DNA and
Hamidi, M., Azadi, A., and Rafiei, P. (2008). Hy- siRNA. Advanced Drug Delivery Reviews 62,
drogel nanoparticles in drug delivery. Advanced 12–27.
Drug Delivery Reviews 60, 1638–1649. Mladenovska, K., Raicki, R. S., Janevik, E.
Huang, H. N., Li, T. L., Chan, Y. L, Chen, C. L., I., Ristoski, T., Pavlova, M. J., Kavrakovski,
and Wu, C. J. (2009). Transdermal immunization Z., Dodov, M. G., and Goracinova, K. (2007).
with low-pressure-gene-gun mediated chitosan- Colon-specific delivery of 5-aminosalicylic
based DNA vaccines against Japanese encepha- acid from chitosan-Ca-alginate microparticles.
litis virus. Biomaterials 30, 6017–6025. International Journal of Pharmaceutics 342,
Illum, L., Gill, I. J., Hinchcliffe, M., Fisher, A. 124–136.
N., and Davis, S. S. (2001). Chitosan as a novel Motwani, S. K., Chopra, S., Talegaonkar, S.,
nasal delivery system for vaccines. Advanced Kohli, K., Ahmad, F. J., and Khar, R. K. (2008).
Drug Delivery Reviews 51, 81–96. Chitosan–sodium alginate nanoparticles as sub-
Issa, M. M., Hoggard, M. K., and Artursson, P. microscopic reservoirs for ocular delivery: For-
(2005). Chitosan and the mucosal delivery of mulation, optimization and in vitro characteriza-
biotechnology drugs. Drug Discovery Today: tion. European Journal of Pharmaceutics and
Technologies 2(1), 1‑6. Biopharmaceutics 68, 513–525.
Janes, K. A., Calvo, P., and Alonso, M. J. (2001). Nagamoto, T., Hattori, Y., Takayama, K., and
Polysaccharide colloidal particles as delivery Maitani, Y. (2004). Novel Chitosan Particles and
systems for Macromolecules. Advanced Drug Chitosan-Coated Emulsions Inducing Immune
Delivery Reviews 47, 83–97. Response via Intranasal Vaccine Delivery. Phar-
maceutical Research 21(4), 671‑674.
Janes, K. A., Fresneau, M. P., Marazuela, A.,
Fabra, A., and Alonso, M. J. (2001). Chitosan Patel, J. K. and Jivani, N. P. (2009). Chitosan
nanoparticles as delivery systems for doxorubi- Based Nanoparticles in Drug Delivery. Interna-
cin. Journal of Controlled Release 73, 255–267. tional Journal of Pharmaceutical Sciences and
Nanotechnology 2(2), 517‑522.
Kadiyala, I., Loo, Y., Roy, K., Rice, J., and Le-
ong, K. W. (2010). Transport of chitosan–DNA Prego, C., Paolicelli, P., Diaz, B., Vicente, S.,
nanoparticles in human intestinal M-cell model Sanchez, A., Fernandezb, A. G., and Alonso, M.
versus normal intestinal enterocytes. European J. (2010). Chitosan-based nanoparticles for im-
Journal of Pharmaceutical Sciences 39, 103– proving immunization against hepatitis B infec-
109. tion. Vaccine 28, 2607–2614.
Kao, H. J., Lin, H. R., Lo, Y. L., and Yu, S. P. Qi, L., Xu, Z., Jiang, X., Hu, C., and Zou, X.
(2006). Characterization of pilocarpine-loaded (2004). Preparation and antibacterial activity of
chitosan/Carbopol nanoparticles. Journal of chitosan nanoparticles. Carbohydrate Research
pharmacy and pharmacology 58, 179‑186. 339, 2693–2700.
Kumar, M. N. V. R. (2000). Nano and Micropar- Rieux, A. des., Fievez, V., Garinot, M., Schnei-
ticles as Controlled Drug Delivery Devices. J. der, Y. J., and Preat, V. (2006). Nanoparticles as
Pharm. Pharmaceut. Sci. 3(2), 234‑258. potential oral delivery systems of proteins and
vaccines: A mechanistic approach. Journal of
Li, G., Liu, Z., Liao, B., and Zhong, N. (2009). Controlled Release 116, 1–27.
Induction of Th1-Type Immune Response by
Chitosan Nanoparticles Containing Plasmid Sandri, G., Poggi, P., Bonferoni, M. C., Rossi, S.,
DNA Encoding House Dust Mite Allergen Der p Ferrari, F., and Caramella, C. (2006). Histologi-
2 for Oral Vaccination in Mice. Cellular & Mo- cal evaluation of buccal penetration enhance-
lecular Immunology 6(1), 45‑50. ment properties of chitosan and trimethyl chito-
san. Journal of pharmacy and pharmacology 58,
Ma, Z., Yoeh, H. H., and Lim, L. Y. (2002). For- 1327‑1336.
mulation pH Modulates the Interaction of Insulin
References 207
Sarmento, B., Ribeiro, A., Veiga, F., Sampaio, Weecharangsan, W., Opanasopit, P., Ngawhirun-
P., Neufeld, R., and Ferreira, D. (2007). Algi- pat, T., Rojanarata, T., and Apirakaramwong, A.
nate/Chitosan Nanoparticles are Effective for (2006). Chitosan Lactate as a Nonviral Gene De-
Oral Insulin Delivery. Pharmaceutical Research livery Vector in COS-1 Cells. AAPS PharmSci-
24(12), 2198‑2206. Tech. 7(3), Article 66 (http://www.aapspharmsci-
Schmidt, C. and Lamprecht, A. (2009). Nano- tech.org)
carriers in drug delivery-design, manufacture Wilson, B., Samanta, M. K., Santhi, K., Kumar,
and physicochemical properties. In Nanothera- K. P. S., Ramasamy, M., and Suresh, B. (2010).
peutics- drug delivery concepts in nanoscience. Chitosan nanoparticles as a new delivery system
A. Lamprecht (Ed.). Pan Standford Publishing for the anti-Alzheimer drug tacrine. Nanomedi-
Pte Ltd, Mainland Press Pvt Ltd, Singapore, pp. cine: Nanotechnology, Biology, and Medicine 6,
1‑38. 144–152.
Schmitt, F. et al. (2010). Chitosan-based nano- Wu, Y., Yang, W., Wang, C., Hu, J., and Fu, S.
gels for selective delivery of photosensitizers (2005). Chitosan nanoparticles as a novel de-
to macrophages and improved retention in and livery system for ammonium glycyrrhizinate.
therapy of articular joints. Journal of Control Re- International Journal of Pharmaceutics 295,
lease, doi:10.1016/j.jconrel.2010.02.008. 235–245.
Singh, D. (2010). Development and character- Yan, C., Chen, D., Gu, J., and Qin, J. (2006).
ization of chitosan nanoparticles loaded with Nanoparticles of 5-fluorouracil (5-FU) loaded
amoxicillin. [Online] available from Pharmain- N-succinyl-chitosan (Suc-Chi) for cancer che-
fo.net (accessed 5th May, 2010) motherapy: Preparation, characterization-in-vi-
Slutter, B., Plapied, L., Fievez, V., Sande, M. tro drug release and anti-tumour activity. Journal
A., Rieux, A. des., Schneider, Y. J., Riet, E. V., of pharmacy and pharmacology 58, 1177–1181.
Jiskoot, W., and Preat, V. (2009). Mechanistic Yang, S. J., Shieh, M. J., Lin, F. H., Lou, P. J.,
study of the adjuvant effect of biodegradable Peng, C. L., Wei, M. F., Yao, C. J., Lai, P. S.,
nanoparticles in mucosal vaccination. Journal of and Young, T. H. (2009a). Colorectal cancer
Controlled Release 38, 113–121. cell detection by 5-aminolaevulinic acid-loaded
Soppimath, K. S., Aminabhavi, T. M., Kulkarni, chitosan nano-particles. Cancer Letters 273,
A. R., and Rudzinski, W. E. (2001). Biodegrad- 210–220.
able polymeric nanoparticles as drug delivery Yang, S. J., Shieh, M. J., Lin, F. H., Lou, P. J.,
devices. Journal of Controlled Release 70, 1–20. Peng, C. L., Wei, M. F. et al. (2009b). Colorectal
Tiyaboonchai, W. and Limpeanchob, N. (2007). cancer cell detection by 5-aminolaevulinic acid-
Formulation and characterization of amphoteri- loaded chitosan nano-particles. Cancer Letters
cin B–chitosan–dextran sulfate nanoparticles. 273, 210–220.
International Journal of Pharmaceutics 329, Yu, S., Zhao, Y., Wu, F., Zhang, X., Lu, W.,
142–149. Zhang, H., and Zhang, Q. (2004). Nasal insulin
Tokumitsu, H., Ichikawa, H., and Fukumori, Y. delivery in the chitosan solution: In vitro and in
(1999). Chitosan‑Gadopentetic acid complex vivo studies. International Journal of Pharma-
nanoparticles for gadolinium neutron capture ceutics 281, 11–23.
therapy of cancer: Preperation by novel emul-
sion droplet coalescence technique and char-
acterization. Pharmaceutical research 16(12), 5
1830‑1835. Abid, J. P., Wark, A. W., Brevet, P. F., and Girault,
Urrusuno, R. F., Calvo, P., Lopez, C. R., Jato, J. H. H. (2002). Preparation of silver nanoparticles
L. V., and Alonso, M. J. (1999). Enhancement in solution from a silver salt by laser irradiation.
of nasal absorption of insulin using chitosan Chemical communications (Cambridge, Eng-
nanoparticles. Pharmaceutical research 16(10), land) 7, 792‑793.
1576‑1581. Ahmad A., Mukherjee P., Senapati S., Mandal
D., Khan M. I., Kumar R. and M. (2003). Ex-
tracellular biosynthesis of silver nanoparticles
using the fungus Fusarium oxysporum. Colloids Bae, C. H., Nam, S. H., and Park, S. M. (2002).
Surf. B. Biointerf. 28, 313–318. Formation of silver nanoparticles by laser abla-
Ahmad, A., Senapati, S., Khan, M. I., Kumar, R., tion of a silver target in NaCl solution. Applied
and Sastry, M. (2003). Extracellular biosynthesis Surface Science 197, 628–634.
of monodisperse gold nanoparticles by a novel Baker, R. A. and Tatum, J. H. (1998). Novel
extremophilic actinomycete Thermomonospora anthraquinones from stationary cultures of Fu-
sp. Langmuir 19, 3550. sarium oxysporum. Journal of Fermentation and
Ahmad, N., Sharma, S., Singh, V. N., Shamsi, S. Bioengineering 85, 359‑361.
F., Anjum F., and Mehta, B. R. (2011) Biosyn- Balaji, D. S., Basavaraja, S., Deshpande, R.,
thesis of Silver Nanoparticles from Desmodium Bedre Mahesh, D., Prabhakar, B. K., and Ven-
triflorum: A Novel Approach towards Weed Uti- kataraman, A. (2009) Extracellular biosynthesis
lization. Biotechnology Research International of functionalized silver nanoparticles by strains
2011, 8. of Cladosporium cladosporioides fungus. Col-
Ahmad, R. S., Ali, F., Hamid, R. S., and Sara, M. loids Surf. B Biointerfaces 68, 88‑92.
(2007). Synthesis and effect of silver nanopar- Basavaraja, S., Balaji, S. D., Arun kumar, L., Ra-
ticles on the antibacterial activity of different jasab, S., and Venkataraman, A. (2007). Extra-
antibiotics against Staphylococcus aureus and cellular biosynthesis of silver nanoparticles us-
Escherichia coli. Nanomedicine: Nanotechnol- ing the fungus Fusarium semitectum. Materials
ogy, Biology and Medicine 3(2), 168‑171. Research Bulletin 43, 1164‑1170.
Alfredo, R., Vilchis-Nestor, Victor Sánchez- Bell, A. A., Wheeler, M. H., Liu, J., Stipanovic,
Mendieta, Marco Camacho-López, A., Rosa, R. D., Puckhaber, L. S., and Orta, H. (2003).
M.,Gómez-Espinosa, Miguel, A., Camacho- United States Department of Agriculture‑‑Agri-
López, and Jesús Arenas-Alatorre, A. (2008). cultural Research Service studies on polyketide
Solventless synthesis and optical properties of toxins of Fusarium oxysporum f sp vasinfectum:
Au and Ag nanoparticles using Camellia sinensis Potential targets for disease control. Pest Man-
extract. Materials Letters 62, 3103‑3105. agement Science 59, 736–747.
Amanulla, M. F., Balaji, K., Girilal, M., Yadav, Beveridge, T. J., Hughes, M. N., Lee, H., Leung,
R., Pudupalayam Thangavelu Kalaichelvan, K. T., Poole, R. K., Savvaidis, I., Silver, S., and
and Ramasamy Venketesan. (2010). Biogenic Trevors, J. T. (1997). Metal–microbe interac-
synthesis of silver nanoparticles and their syn- tions: Contemporary approaches. Advances in
ergistic effect with antibiotics: A study against Microbial Physiology 38, 177‑243.
gram-positive and gram-negative bacteria. Beveridge, T. Y. and Murray, R. G. E (1980).
Nanomedicine: Nanotechnology, Biology and Sites of metal deposition in the cell wall of
Medicine 6, 103‑109. Bacillus subtilis. Journal of Bacteriology 141,
Ankamwar, B, Damle, C, Ahmad, A, and Sas- 876‑887.
try M. (2005a). Biosynthesis of gold and silver Bhainsa, K. C. and D’Souza, S. F. (2006). Extra-
nanoparticles using Emblica officinalis fruit ex- cellular Biosynthesis of Silver Nanoparticles us-
tract, their phase transfer and transmetallation in ing the Fungus Aspergillus fumigatus. Colloids
an organic solution. Journal of Nanoscience and Surf. B Biointerfaces 47(2) 160‑164.
Nanotechnology 5, 1665‑1671.
Birla, S. S., Tiwari, V. V., Gade, A. K., Ingle, A.
Ankamwar, B., Chaudhary, M., and Mu- P., Yadav, A. P., and Rai, M. K. (2009). Fabrica-
ral, S. (2005b). Gold nanotriangles biologi- tion of silver nanoparticles by Phoma glomerata
cally synthesized using Tamarind leaf extract and its combined effect against Escherichia coli,
and potential application in vapor sensing. Pseudomonas aeruginosa and Staphylococcus
Synthesis and Reactivity in Inorganic and Metal- aureus. Letters in Applied Microbiology 48,
Organic Chemistry 35, 19. 173‑179.
Atiyeh, B. S., Costagliola, M., Hayek, S. N., Blakemore, R. P. (1982). Magnetotactic bacteria.
and Dibo, S. A. (2007). Effect of silver on burn Annual Review of Microbiology 36, 217–238.
wound infection control and healing: Review of
the literature. Burn 33(2), 139‑148.
References 209
Bonneman, H., Brijoux, W., Brinkmann, R., Till- Chaki, N. K., Sudrik, S. G., Sonawane, H. R.,
ing, A. S., Schilling, T., Tesche, B., Seevogel, K., and Vijayamohanan, K. (2002). Single phase
Franke, R., Hormes, J. G., Pollmann, J. R., and preparation of monodispersed silver nanoclus-
Jvogel, W. (1998). Selective oxidation of glucose ters using a unique electron transfer and cluster
on bismuth-promoted Pd-Pt/C catalysts prepared stabilising agent. Triethylamine, Chemical Com-
from NOct4Cl-stabilized Pd-Pt colloids. Inor- munications 8(1), 76‑77.
ganica. Chimica. Acta 270, 95–110. Chan, W. C. W. and Nie, S. M. (1998). Quantum
Brierley, J. A. (1990). Production and applica- dot bioconjugates for ultrasensitive nonisotopic
tion of a Bacillus–based product for use in met- detection. Science 281, 2016−2018.
als biosorption. In Biosorption of heavy metals. Chandran, S. P., Chaudhary, M., Pasricha, R.,
B. Volesky (Ed.) Boca Raton, FL CRC Press, pp. Ahmad, A., and Sastry, M. (2006). Synthesis of
305‑312. gold nanotriangles and silver nanoparticles using
Brown, T. and Smith, D. (1976). The effects of Aloe vera plant extract. Biotechnology Progress
silver nitrate on the growth and the ultrastructure 22, 577–583.
of the yeast Cryptococuss albidus. Microbios let- Chen, D., Qiao, X., Qui, X., and Chen, J. (2009).
ters 3, 155‑162. Synthesis and electrical properties of uniform
Bruins, R. M., Kapil, S., and Oehme, S. W. silver nanoparticles for electronic applications.
(2000). Microbial resistance to metal in the en- Journal of Materials Science 44, 1076‑1081.
vironment. Ecotoxicology and Environmental Chen, J, Wang, K, Xin, J., and Jin, Y. (2008). Ma-
Safety 45, 198–207. ter. Chem. Phys. 108, 421.
Brust, M. and Kiely, C. J. (2002). Some recent Chen, J. C., Lin, Z. H., and Ma, X. X. (2003).
advances in nanostructure preparation from gold Evidence of the production of silver nanopar-
and silver particles: A short topical review. Col- ticles via pretreatment of Phoma sp 32883 with
loids and Surfaces A: Physicochem. Eng. As- silver nitrate. Letters in Applied Microbiology
pects 202, 175–186. 37, 105‑108.
Burda, C. X., Chen, R., Narayanan, M. A., and Chen, X. and Schluesener, H. J. (2008). Nanosil-
El-Sayed (2005). Chemistry and properties of ver: A nanoproduct in medical application. Toxi-
nanocrystals of different shapes. Chem. Rev. cology Letters 176(1), 1‑12.
105, 1025‑1102.
Chen, Y. Y., Wang, C., Liu, L. H., Qiu, J. S., and
Callegari, A., Tonti, D., and Chergui, M. (2003). Bao, X. H. (2005). Chemical Communications,
Photo chemically grown silver nanoparticles Cambridge 42, 5298.
with wave length–controlled size and shape.
Nano Letters 3, 1565‑1568. Cho, K. H., Park, J. E., Osaka, T., and Park, S.
G. (2005).The study of antimicrobial activity
Canizal, G., Ascencio, J. A., Gardea-Torresday, and preservative effects of nanosilver ingredient,
J., and Jose-Yacaman, M. (2001). J. Nanopart. Electrochimica Acta 51(5), 956‑960.
Res. 3, 475–481.
Chu, C. S., McManus, A. T., Pruitt, B. A., and
Cao, G. (Ed.). (2004). Nanostructures and Nano- Mason, A. D. (1988). Therapeutic effects of sil-
materials: Synthesis, properties and applica- ver nylon with weak direct current Pseudomonas
tions. Imperial college press, London. aeruginosa infected burn wounds. The Journal
Cao, Y. C., Jin, R., and Mirkin, C. A. (2002). of Trauma 28(10), 1488‑1492.
Nanoparticles with Raman Spectroscopic Fin- Corinne, P., Dewilde, A., Pierlot, C., and Aubry,
gerprints for DNA and RNA Detection. Science J. M. (2000). Bactericidal and virucidal activi-
297, 1536‑1540. ties of singlet oxygen generated by thermolysis
Castro-Longoria, E., Vilchis-Nestor, A. R., and of naphthalene endoperoxides. Methods in Enzy-
Avalos-Borja, M. (2011). Biosynthesis of sil- mology 319, 197‑207.
ver, gold, and bimetallic nanoparticles using the Cushing, B. L., Kolesnichenko, V. L., and Con-
filamentous fungus Neurospora crassa. Colloids nor, C. J. O. (2004). Recent Advances in the Liq-
and Surfaces B: Biointerfaces 83(1), 42‑48. uid-Phase Syntheses of Inorganic Nanoparticles.
Chemical Reviews 104, 3893–3946.
Daizy, P. (2009). Spectrochim. Biosynthesis of Elechiguerra, J. L., Justin L Burt, Jose R. Mo-
Au, Ag and Au-Ag nanoparticles using edible rones, Alejandra Camacho-Bragado, Xiaoxia
mushroom extract. Acta A 73, 374‑381. Gao, Humberto H Lara, and Miguel Jose Yaca-
Darnall, D. W., Greene, B., Henzl, M. J., Hosea, man (2005). Interaction of silver nanoparticles
M., Mc Pherson, R. A., Sneddon, J., and Alexan- with HIV-1. Journal of Nanobiotechnology 3, 6.
der, M. D. (1986). Selective recovery of gold and Elumalai, E. K., Prasad, T. N. V. K. V., Ven-
other metal ions from an algal biomass. Environ- kata Kambala, Nagajyothi, P. C., and David, E.
ment and Science and Technology 20, 206‑208. (2010). Green synthesis of silver nanoparticle
Darroudi, M., Ahmad, M. B., Shameli, K., using Euphorbia hirta L and their anti fungal
Abdullah, A. H., and Ibrahim, N. A. (2009). Syn- activities. Archives of Applied Science Research
thesis and characterization of UV-irradiated sil- 2(6), 76‑81.
ver/montmorillonite nanocomposites. Solid State Ershov, B. G., Janata, E., Henglein, A., and Fo-
Sciences 11, 1621–1624. jtlk A. (2007) unplublished report.
Darroudi, M., Ahmad, M. B., Zamiri, R., Zak, A. Esau, S. R., Roberto, S. B., Ocotlan-Flores, J.,
K., Abdullah, A. H., and Ibrahim, N. A. (2011). and Saniger, J. M. (2010). Synthesis of AgNPs
Time dependent effect in green synthesis of sil- by sonochemical induced reduction applications
ver nanoparticles. International journal of nano- in SERS. Journal of Nanoparticle Research 9,
medicine 6, 677–681. 77.
David S. G. (2004). Bionanotechnology: Les- Eutis, S., Krylova, G., Eremenko, A., Smirno-
sons from Nature. John Wiley and sons, Inc., va, N., Schill, A. W., and El-Sayed, M. (2005).
Publication. Growth and fragmentation of silver nanopar-
Deendayal, M., Bolander, E. M., Mukhopadhy- ticles in their synthesis with a fs laser and CW
ay, D., Sarkar, G., and Mukherjee, P. (2006). The light by photo-sensitization with benzophenone.
use of microorganisms for the formation of metal Photochemical and Photobiological Sciences 4,
nanoparticles and their application. Applied Mi- 154‑159.
crobiology and Biotechnology 69, 485‑492. Falletta, E., Massimo Bonini, Emiliano Fra-
Deitch, E. A., Marino, A. A., Malaleonok, V., and tini, Antonella Lo Nostro, Giovanna Pesavento,
Albricht, J. A. (1987). Silver nylon cloth: In vitro Alessio Becheri, Pierandrea Lo Nostro, Patrizia
and in vivo evaluation of antimicrobial activity. Canton., and Piero Baglioni. (2008). Clusters of
The Journal of Trauma 27, 301. Poly(acrylates) and Silver Nanoparticles: Struc-
ture and Applications for Antimicrobial Fabrics.
Devendra, J., Hemant, K. D., Sumita, K., and The Journal of Physical Chemistry C 112(31),
Kotharia, S. L. (2009). Digest Journal of Nano- 11758–11766.
materials and Biostructures 4(4), 723.
Fortin, D. and Beveridge, T. J. (2000). In Biomin-
Dunn, K. and Edwards-Jones, V. (2004). The eralisation. E. Baeuerlein (Ed.). Wiley-VCH,
role of Acticoat with nanocrystalline silver in the Verlag, Germany,p.294.
management of burns. Burns 30(1), S1‑9.
Frilis, N. and Myers-Keith, P. (1986). Biosorp-
Duran, N., Teixeria, M. P. S., De Conti, R., and tionof uranium and lead by Streptomyces long-
Esposito (2002). Ecological-friendly pigments woodensis. Biotechnology and Bioengineering
from fungi. Critical Reviews in Food Science 28, 21‑28.
and Nutrition 42, 53‑66.
Furno, F., Morley, K. S., Wong, B., Sharp, B.
Duran, N., Marcato, P. L., Alves, O. L., and De L., Arnold, P. L., Howdle, S. M., Bayston, R.,
Souza, G. I. (2005). Mechanistic aspects of bio- Brown, P. D.,Winship, P. D., and Reid, H.
synthesis of silver nanoparticles by several Fu- J.(2004). Journal of Antimicrobial Chemothera-
sarium oxysporum strains. Journal of Nanobio- py 54(6), 1019‑1024.
technology 3(8), 1‑7.
Furr, J. R. and Russell, A. D. (1994). Antibac-
Egorova, E. M. and Revina, A. A. (2000). Syn- terial activity of Actisorb Plus, Actisorb and
thesis of metallic nanoparticles in reverse mi- silver nitrate. Journal of Hospital Infection 27,
celles in the presence of quercetin. Colloids and 201‑208.
Surfaces, ser.A 168, 87.
References 211
Gad, F., Zahra, T., Francis, K. P., Hasan, T., and ization of silver nanoparticles using Escherichia
Hamblin, M. R. (2004). Targeted photodynamic coli. Colloids and Surfaces B: Biointerfaces 74,
therapy of established soft-tissue infections in 328‑335.
mice. Photochemical and Photobiological Sci- Haes, A. J. and Van Duyne, R. P. (2002). A
ences 3, 451‑458. Nanoscale Optical Biosensor: Sensitivity and
Gao, F., Lu, Q.,and Komarneni, S. (2005). In- Selectivity of an Approach Based on the Lo-
terface reaction for the self-assembly of silver calized Surface Plasmon Resonance Spectros-
nanocrystals under microwave-assisted solvo- copy of Triangular Silver Nanoparticles. Jour-
thermal conditions. Chemistry of Materials 17, nal of the American Chemical Society 124(35),
856‑860. 10596‑10604.
Gardea-Torresdey, J. L., Parsons, J. G., Gomez, Haoran, Z., Qingbiao, L., Huixuan, W., and Dao-
E., Peralta-Videa, J., Troiani, H., Santiago, P., hua, S. (2005). Journal of Chemical Technology
and Jose-Yacaman, M. (2002). Formation and & Biotechnology 80, 285‑290.
Growth of Au Nanoparticles Inside Live Alfalfa Harekrishna, B., Bhui, D. K., Gobinda, P. S.,
Plants. Nano letters 2, 397‑401. Priyanka, S., Sankar, P. De., and Ajay Misra.
Gericke, M and Pinches, A. (2006). Biological (2009a). Green synthesis of silver nanoparticles
synthesis of metal nano particles. Hydrometal- using latex of Jatropha curcas. Colloids and
lurgy 83, 132–140. Surfaces A-physicochemical and Engineering
Ghosh, S. K., Kundu, S., Mandal, M., Nath, S., Aspects 339, 134‑139.
and Pal, T. (2003). Studies on the evolution of Harekrishna, B., Bhui, D. K., Gobinda, P. S., Pri-
silver nanoparticles in micelle by UV-photoac- yanka, S., Santanu Pyne, and Misra, A. (2009b).
tivation. Journal of Nanoparticle Research 5, Green synthesis of silver nanoparticles using
577‑587. seed extract of Jatropha curcas. Colloids and
Gogoi, S. K., Chattopadhyay, A., and Ghosh, Surfaces A: Physicochemical and Engineering
S. S. (2006). Activities of silver nanoparticles. Aspects 348, 212‑216.
Langmuir, 22(22), 9322‑9328. Harfenist, St A., Wang, Z. L., Alvarez, M. M.,
Goia, E. and Matijevic, N. (1998). Preparation Vezmar, I., and Whetten, R. L. (1996). Highly
of monodispersed metal particles. Journal of Oriented Molecular Ag Nanocrystal Arrays. The
Chemical Education 22, 1203‑1215. Journal of Physical Chemistry 100, 13904.
Golab, Z. (1981). Bioaccumulation of heavy Hayat, M. A. (1989). Colloidal Gold: Principles,

metals by the bacterium Bacillus mycoides. Pro- methods and applications. Acdemic Press, Cali-
bl. Mineralurgii 13, 217‑224. fornia.
Gopidas K. R., Whitesell, J. K., and Fox, M. A. He, S. T., Yao, J. N., Jiang, P., Shi, D. X., Zhang,
(2003). Synthesis, Characterization, and Cata- H. X., Xie, S. S., Pang, S. J., and Gao, H. J.
lytic Applications of a Palladium-Nanoparticle- (2001). Formation of Silver Nanoparticles and
Cored Dendrimer. Nano Letters 3, 1757‑1760. Self-Assembled Two-Dimensional Ordered Su-
perlattice. Langmuir 17, 1571‑1575.
Govindaraju, K., Tamilselvan, S., Kiruthiga,
V., and Singaravelu, G. (2010). Biogenic Silver He, Y., Wu, Y., Lu, G., and Shi, G. (2006). A fac-
anopartilces by Solanum torvum and their prom- ile route to silver nanosheet. Materials Chemis-
ising antimicrobial activity. Journal of Biopesti- try and Physics 98(1),178‑182.
cides 3(1), 394–399. Henglein, A. (2001), Article ChemPort. Lang-
Gulbranson, S. H., Hud, J. A., and Hansen, R. muir 17, 2329–2333.
C. (2000). Argyria following the use of dietary Henglein, A. and Giersig, M. (1999). Formation
supplements containing colloidal silver protein. of Colloidal Silver Nanoparticles: Capping Ac-
Cutis 66, 373–374. tion of Citrate. The Journal of Physical Chemis-
Gurunathan, S., Kalishwaralal, K., Vaidyana- try 103(44), 9533‑9539.
than, R., Venkataraman, D., Pandian, S. R. K., Huang, C. P., Juang, C. P., Morehart, K., and Al-
Muniyandi, J., Hariharan, N., and Eom, S. H. len, L. (1990). The removal of copper (II) from
(2009). Biosynthesis, purification and character-
dilute aqueous solutions by Saccharomyces cere- Ratio. The Journal of Chemical Communica-
visiae. Water Research 24, 433‑439. tions 7, 617‑618.
Huang, H. H., Ni, X. P., Loy, G. H., Chew, C. Jana, N. R., Gearheart, L., and Murphy, C. J.
H., Tan, K. L., Loh, F. C., Deng, J. F., and Xu, (2001b). Seeding Growth for Size Control of
G. Q. (1996). Photo chemaical formation of sil- 5−40 nm Diameter Gold Nanoparticles. Lang-
ver nanoparticles in poly(N–vinyl pyrrolidone). muir 17, 6782.
Langmuir 12, 909‑912. Joerger, R., Klaus, T., and Granqvist, C. G.
Huang, J., Li Q., Sun, D., Lu, Y., Su, Y., Yang, (2000). Biologically Produced Silver–Carbon
X., Wang, H., Wang, Y., Shao, W., He, N., Hong, Composite Materials for Optically Functional
J., and Chen C. (2007). Biosynthesis of silver Thin-Film Coatings. Advanced Materials 12,
and gold nanoparticles by novel sundried Cin- 407‑409.
namomum camphora leaf. Nanotechnology 18, Jortner, J. and Rao, C. N. R. (2002). Nanostruc-
105104‑105114. tured Advanced Materials: Perspectives and Di-
Huang, J., Lin, L., Li, Q., Sun, D., Wang, Y., Lu, rections. Pure and Applied Chemistry 74, 1491.
Y., He, N., Yang, K., Yang, X., Wang, H., Wang, Kalimuthu, K., Suresh Babu, R., Venkatara-
W., and Lin, W. (2008). Continuous-Flow Bio- man, D., Bilal, M., and Gurunathan, S. (2008).
synthesis of Silver Nanoparticles by Lixivium of Biosynthesis of silver nanocrystals by Bacillus
Sundried Cinnamomum camphora Leaf in Tu- licheniformis. Colloids Surf. B Biointerfaces
bular Microreactors. Industrial & Engineering 65(1)150‑153.
Chemistry Research 47, 6081‑6090.
Kalishwaralal, K., Deepak, V., and Guruna-
Hussain, Brust, M., Papworth, A. J., and Cooper, than, S. (2010). Biosynthesis of silver and gold
A. I. (2003). Preparation of acrylate-stabilized nanoparticles using Brevibacterium casei. Col-
gold and silver hydrosols and gold-polymer loids and Surfaces B: Biointerfaces 77, 257–262.
composite films. Langmuir 19, 4831–4835.
Kamala Nalini, S. P. (2008). Synthesis of silver
Ingle, A. P., Gade, A. K., Pierrat, S., Sonnichsen, nanoparticles using flower extract of Hibiscus
C., and Rai, M. K. (2008a). Mycosynthesis of sabdariffa. Nati. level symposium biotech vel
silver nanoparticles using the fungus Fusarium Tech, India, 34‑38.
acuminatum and its activity against some hu-
man pathogenic bacteria. Current Nanoscience Kamat, P. V. (2002). Photochemical and pho-
4, 141‑144. tocatalytic aspects of metal nanoparticles. The
Journal of Physical Chemistry B 106, 7729–
Ingle, A., Rai, M., Gade, A. and Bawaskar, M. 7744.
(2008b). Fusarium solani: A novel biological
agent for the extracellular synthesis of silver Karbasian, M., Atyabi, S. M., Siadat, S. D., Mo-
nanoparticles. Journal of nanoparticle research men, S. B., and Norouzian, D. (2008). Optimiz-
11(8), 2079‑2085. ing Nano-silver Formation by Fusarium oxyspo-
rum PTCC 5115 Employing Response Surface
Jae, Y. S. and Beom, S. K. (2009). Rapid Bio- Methodology. American Journal of Agricultural
logical Synthesis of Silver Nanoparticles Using and Biological Sciences 3(1), 433‑437.
Plant Leaf Extracts. Bioprocess and Biosystems
Engineering 32, 79‑84. Kauffman, C. A. and Carver, P. L. (1997). An-
tifungal agents in the 1990s: Current status and
Jain, D., Daima, H. K., Kachhwaha, S., and Ko- future developments. Drugs 53, 539‑549.
thari, S. L. (2009). Synthesis of plant mediated
silver nanoparticles using papaya fruit extract Kearns, G. J., Foster, E. W., and Hutchison, J. E.
and evaluation of of their anti microbial activi- (2006). Substrates for direct imaging of chemi-
ties. Digest Journal of Nanomaterials and Bio- cal functionalized SiO2 surfaces by transmission
structures 4(3), 557. electron microscopy. Analytical Chemistry 78,
298‑303.
Jana, N. R., Gearheart, L., and Murphy, C.
(2001a). Wet Chemical Synthesis of Silver Khomutov, G. B. and Gubin, S. P (2002). Inter-
Nanorods and Nanowires of Controllable Aspect facial synthesis of noble metal nanoparticles.
Materials Science and Engineering: C 22, 141–
146.
References 213
Kim, J. S., Kuk, E., Yu, K. N., Kim, J. H., Park, Kuo, C. H. and Huang, M. H. (2005).Synthesis
S. J., and Lee, H. J. (2007). Antimicrobial effects of Branched Gold Nanocrystals by a Seeding
of silvernanoparticles. Nanomedicine: Nano- Growth Approach. Langmuir 21, 2012‑2016.
technology, Biology and Medicine 3, 95‑101. Kvitek Libor, Prucek Robert, Panacek Ales,
Kim, K. J., Sung, W. S., Moon, S. K., Choi, J. Novotny Radko, Hrbác Jan, Zbořil, and Radek
S., Kim, J. G., and Lee, D. G. (2008). Antifungal (2005). Journal of Materials Chemistry 15,
effect of silver nanoparticles on dermatophytes. 1099.
Journal of Microbiology and Biotechnology Lara, H. H., Ayala-Nunez, N. V., Turrent, L. d. C.
18(8), 1482‑1484. I., and Padilla, C. R. (2010). Bacterial effect of
Kim, Y. H., Lee, D. K., and Kang, Y. S. (2005). silver nanoparticles against multidrug-resistant
Colloids Surf. A, Physio. Chemical and Engi- bacteria. World Journal of Microbiology. Bio-
neering Aspects 273, 257‑258. technology 26, 615‑621.
Kishimoto, N., Takeda, Y., and Okubo, S. (2004). Law, N., Saadia Ansari,Francis R. Livens, Jo-
Jap. Patent JP2004091817. anna C. Renshaw, and Jonathan R. Lloyd (2008).
Klaus, T., Jeorger, R., Olsson, E., and Granqvist, Formation of Nanoscale Elemental Silver Par-
C., (2004). Bacteria as workers in the living ticles via Enzymatic Reduction by Geobacter
factory: Metal accumulating bacteria and their sulfurreducens. Applied and Environmental Mi-
potential for material science. Trends in Biotech- crobiology 74(22). 7090‑7093.
nology 19, 15‑20. Lee, G. J., Shin, S. I., Kim, Y. C., and Oh, S. G.
Klaus, T., Joerger, R., Olsson, E., and Granqvist, (2004). Preparation of silver nanorods through
C. G. (1999). Silver-based crystalline nanopar- the control of temperature and pH of reaction
ticles, microbially fabricated. Proceedings medium. Materials Chemistry and Physics 84,
of the National Academy of Sciences 96(24), 197–204.
13611‑13614. Lee, H. J., Yeo, S. Y., and Jeong, S. H. (2003).
Kloepfer, J. A., Mielke, R. E., and Nadeau, J. L. Antibacterial effect of nanosized silver colloidal
(2005). Uptake of CdSe and CdSe/ZnS Quantum solution on textile fabrics. Journal of Materials
Dots into Bacteria via Purine-Dependent Mecha- Science 38, 2199.
nisms. Applied and Environmental Microbiology Leela, A. and Vivekanandan, M. (2008a). Tap-
71(5), 2548‑2557. ping the unexploited plant resources for the syn-
Kowshik, M., Ashtaputre, S., Kharrazi, S., Vo- thesis of silver nanoparticles. African Journal of
gel, W., Urban, J., Kulkarni, S. K., and Paknikar, Biotechnology 7, 3162‑3165.
K. M. (2003). Extracellular synthesis of silver Leela, A. and Vivekanandan, M. (2008b). Tap-
nanoparticles by a silver-tolerant yeast strain. ping the unexploted plant resources for the syn-
MKY3. Nanotechnology 14, 95–100. thesis of silver nanoparticles. African Journal of
Krishna, B. and Dan, G. V. (2009). Silver Biotechnology 7(17), 3162‑3165.
nanoparticles for printable electronics and bio- Lengke, M F., Michael E Fleet, and Gordon
logical applications. Journal of Materials Re- Southam (2007). Biosynthesis of silver nanopar-
search 24(9), 2828‑2836. ticles by filamentous cyanobacteria from a silver
Kroger, N., Deutzmann, R., and Sumper, M. (I) nitrate complex. Lagmuir 23, 2694.
(1999). Polycationic peptides from diatom bio- Li, Q., Mahendra, S., Lyon, D. Y., Brunet, L.,
silica that direct silica nanosphere formation. Liga, M. V., Li D., and Alvarez, P. J. J. (2008).
Science 286, 1129–1132. Antimicrobial Nanomaterials for Water Disin-
Kuber, C. B. and D’Souza, S. F. (2006). Colloids fection and Microbial Control: Potential Ap-
Surf. B 47, 160–164. plications and Implications. Water Research,
42(18), 4591‑4602.
Kumar, S. A., Abyaneh, M. K., Gosavi, S. W.,
Kulkarni, S. K., Pasricha, R., Ahmad, A., and Li, S., Yuhua Shen, Anjian Xie, Xuerong Yu,
Khan, M. I. (2007). Nitrate reductase-mediated Lingguang Qiu, Li Zhang, and Qingfeng Zhang.
synthesis of silver nanoparticles from AgNO3. (2007). Green synthesis of silver nanoparticles
Biotechnology Letters 29, 439‑445.
using Capsicum annuum L. extract. Green Manish, B., Seema, B., and Kushwah, B. S.
Chemistry 9, 852‑858. (2009). Green synthesis of nanosilver particles
Li, Z., Lee, D., Sheng, X. X., Cohen, R. R., and from extract of Eucalyptus hybrida (Safeda) leaf.
Rubner, M. F.(2006). Two-Level Antibacterial Digest Journal of Nano materials and Biostruc-
Coating with Both Release-Killing and Contact- tures 4(3), 537‑543.
Killing Capabilities. Langmuir 22, 9820‑9823. Mann, S. (1993). Molecular tectonics in biomin-
Linga Rao, M. and Savithramma, N. (2011). Bi- eralization and biomimetic materials chemistry.
ological synthesis of silver nanopartilces using Nature 365, 499.
Svensonia hyderbadensis Leaf extract and evalu- Mann, S, (Ed.) (1996). BIomimetric Materials
ation of their antimicrobial efficacy. Journal of Chemistry. VCH, Weinheim.
Pharmaceutical Sciences 3(3), 1117‑1121. Matsumura, Y., Kuniaki Yoshikata, Shin-ichi
Link, S., Wang, Z. L., and El-Sayed, M. A. (1999). Kunisaki., and Tetsuaki Tsuchido. (2003). Mode
Alloy Formation of Gold-Silver Nanoparticles of Bactericidal Action of Silver Zeolite and Its
and the Dependence of the Plasmon Absorption Comparison with That of Silver Nitrate. Ap-
on their Composition. The Journal of Physical plied and Environmental Microbiology 69(7),
Chemistry B 103, 3529. 4278‑4281.
Liu, S., Weiping Huang, Siguang Chen, Sigalit Maxwell, D. J., Taylor, J. R., and Nie, S. (2002).
Avivi, and Aharon Gedanken (2001). Synthesis Self-Assembled nanoparticle probes for recogni-
of X-ray amorphous silver nanoparticles by the tion and detection of biomolecules. Journal of
pulse sonoelectrochemical method. Journal of the American Chemical Society 124, 9606.
Non-Crystalline Solids 283(1‑3), 231‑236. Mazur, M. (2004). Electrochemically prepared
Liz-Marzán, L. M. and Philipse, A. P. (1995). silver nanoﬂakes and nanowires. Electrochemis-
Stable hydrosols of metallic and bimetallic try Communications 6, 400‑403.
nanoparticles immobilized on imogolite fibers. Mc Farland, A. D. and Richard P. Van Duyne
The Journal of Physical Chemistry 99(41), (2003). Single Silver Nanoparticles as Real-
15120‑15128. Time Optical Sensors with Zeptomole Sensitiv-
Long, D., Wu, G., and Chen, S. (2007). Prepara- ity. Nano Letters 3(8), 1057–1062.
tion of oligochitosan stabilized silver nanopar- Medentsev, A. G and Akimenko, V. K. (1998).
ticles by gamma irradiation. Radiation Physics Naphthoquinone metabolites of the fungi. Phy-
and Chemistry, 76, 1126‑1131. tochemistry 47, 935‑959.
Luoma, S. N. (2008). Silver nanotechnologies Melaiye, A. Sun, Z., Hindi, K., Milsted, A., Ely,
and the environment: Old problems or new chal- D., Reneker, D., Tessier, C., Youngs, W. (2005).
lenges? Project on Emerging Nanotechnologies, Silver (I)-imidazole Cyclophane Gem-diol Com-
the Pew Charitable Trusts. plexes Encapsulated by Electrospun Tecophilic
Maillard, M., Giorgio, S., and Pileni, M. P. Nanofibers: Formation of Nanosilver Particles
(2002). Silver nanodisks. Advanced Materials and Antimicrobial Activity. Journal of the Amer-
14(15), 1084–1086. ican Chemical Society 127, 2285‑2291.
Maliszewska, I. and Sadowski, Z. (2009). Miller, L. P. and McCallan, S. E. A. (1957).
Synthesis and antibacterial activity of silver Toxic action of metal ions to fugus spores.
nanoparticles. J.of Phyc., Conference Series Journal of Agricultural and Food Chemistry 5,
146,1‑7. 116‑122.
Mallin, M. P. and Murphy, C. J. (2002). Solution- Mohanpuria, P., Rana, N. K., and Yadav, S. K.
Phase Synthesis of Sub-10 nm Au−Ag Alloy (2008). Biosynthesis of nanoparticles : Techno-
Nanoparticles. Nano Letters 2, 1235‑1237. logical concepts and future applications. Journal
Mandal, S., Arumugam, S. K., Pasricha, R., and of Nanoparticle Research 10, 507‑517.
Sastry, M. (2005). Silver nanoparticles of vari- Morones, J. R., Elechiguerra, J. L., Camacho,
able morphology synthesized in aqueous foams A., Holt, K., Kouri, J. B., Ramírez, J. T., and
as novel templates. Bulletin of Material Science Yacaman, M. J. (2005). The bactericidal effect
28, 503‑510.
References 215
of silver nanoparticles. Nanotechnology 16, Nidhi, N., Santosh, K., Ghosh, T., and Dutta, P.
2346‑2353. K. (2009). Preparation of Chitosan-based silver
Moyer, C. A, (1965). A treatment of burns. Trans. nanocomposites by a facile method. Internation-
Stud. Coll. Physicians Philadelphia 33, 53‑103. al. Conference on Optics and Photonics. Chan-
digiah: CSIO.
Mukherjee, P., Ahmad, A., Mandal, D., Senapati,
S., Sainkar, S. R., Khan, M. I., Ramani, R., Pari- Nikhil, S. S., Mahesh, B., Rahul, B., Rekha, S.
scha, R., Kumar, P. A. V., Alam, M., Sastry, M., S., George Szakacs, and Ashok Pandey (2009).
and Kumar, R. (2001a). Bioreduction of AuCl(4) Biosynthesis of silver nanoparticles using aque-
(‑) Ions by the Fungus, Verticillium sp. and Sur- ous extract from the compactin producing fungal
face Trapping of the Gold Nanoparticles Formed strain. Process Biochemistry 44, 939‑943.
D.M. and S.S. thank the Council of Scientific Niu, H., Xu, X. S., and Wang, J. H. (1993). Re-
and Industrial Research (CSIR), Government of moval of lead from Aqueous solutions of Pencil-
India, for financial assistance. Angew Chem. Int. lium biomass. Biotechnology and Bioengineer-
Ed. 40, 3585–3588. ing 42, 785‑787.
Mukherjee, P., Ahmad, A., Mandal, D., Senapa- Oksanen, T., Pere, J., Paavilainen, L., Buchert,
ti, S., Sainkar, S. R., Khan, M. I., Parischa, R., J., and Viikari, L. (2000). Treatment of recycled
Ajayakumar, P. V., Alam, M., Kumar, R., and kraft pulps with Trichoderma reesei hemicellu-
Sastry, M. (2001b). Fungus-Mediated Synthesis lases and cellulases. Journal of Biotechnology
of Silver Nanoparticles and Their Immobiliza- 78(1), 39–44.
tion in the Mycelial Matrix: A Novel Biological Oliveira, M. M., Ugarte, D., Zanchet, D., and
Approach to Nanoparticle Synthesis. Nano Let- Zarbin, A. J. G. (2005). Influence of synthetic
ters 1, 515–519. parameters on the size, structure, and stability
Mukherjee, P., Roy, M., Mandal, B., Dey, G., of dodecanethiol-stabilized silver nanoparticles.
Mukherjee, P., and Ghatak. (2008). J. Green syn- Journal of Colloid and Interface Science 292(2),
thesis of highly stabilized nanocrystalline silver 429‑435.
particles by a non-pathogenic and agriculturally Pal, A., Shah, S., and Devi, S. (2007). Synthe-
important fungus T. asperellum. Nanotechnology sis of Au, Ag and Au‑Ag Alloy Nanoparticles
19,7510. in Aqueous Polymer Solution. Colloids Surface
Mukherjee, P., Senapati, S., Mandal, D., Ah- A: Physicochemical and Engineering Aspects
mad, A., Khan, M. I., Kumar, R., and Sastry, M. 302(1‑3), 51‑57.
(2002). Extracellular synthesis of gold nanopar- Pal, T. and Maity, D. S. (1986). Silver(I)-gelatin
ticles by the fungus Fusarium oxysporum. interaction: Spectrophotometric determination
Chemistry and Biochemistry 3, 461‑463. of trace amounts of silver in water. Analyst 111,
Nair, B. and Pradeep, T. (2002). Coalescence of 1413.
nanoclusters and formation of submicron crys- Panacek, A., Kolar, M., Vecerova, R. et al.
tallites assisted by Lactobacillus strains. Crystal (2009). Antifungal activity of silver nanopar-
Growth & Design 2, 293‑298. ticles against Candida spp. Biomaterials 30,
Nanda, A. and Saravanan, M. (2009). Biosyn- 6333‑6340.
thesis of Silver-nanoparticles from Staphylococ- Panacek, A., Kvitek, L., Prucek, R., Kolar,
cus aureus and its antimicrobial activity against M., Veerova, R., Pizurova, N., Sharma, V. K.,
MRSA and MRSE. Nanomedicine NBM 5, Nevena, T., and Zboril, R. (2006). Silver colloid
453‑457. nanoparticles: Synthesis, characterization and
Navaladian, S., Viswanathan, B., Viswanath, their anti bacterial activity.The Journal of Physi-
R. P., and. Varadarajan, T. K. (2007). Thermal cal Chemistry B 110, 16248‑16253.
decomposition as route for silver nanoparticles. Papp, S., Patakfalvi, R., and Dekany, I.(2008).
Nanoscale Research Letters 2, 44‑48. Metal nanoparticle formation on layer silicate
Newman, D. K. and Kolter, R. (2000). A role for lamellae. Colloid & Polymer Science 286, 3‑14.
excreted quinones in extracellular electron trans- Parashar, V., Parashar, R., Sharma, B., and Pan-
fer. Nature 405, 94‑97. dey, A. C. (2009). Parthenium leaf extract me-
diated synthesis of silver nanoparticles: A novel silver nanoparticles and their antibacterial effi-
approach towards weed utilization. Digest Jour- cacy. Journal of Nanobiotechnology 5, 185‑189.
nal of Nanomaterials and Biostructures 4(1), Pugazhenthiran, N., Anandan, S., Kathiravan,
45‑50. G., and Udaya-Prakash, N. K. (2009). Micro-
Parikh, R. Y., Singh, S., Prasad, B. L. V., Patole, bial synthesis of silver nanoparticles by Bacil-
M. S., Sastry, M., and Schouche, Y. S. (2008). lus sp. Journal of Nanoparticle Research 11,
Extracellular synthesis of crystalline silver 1811‑1815.
nanoparticles and molecular evidence of silver Purwar, V. and Pokharkar, V. (2011). Green syn-
resistance from Morganella sp.: Towards un- thesis of silver nanoparticles using marine poly-
derstanding biochemical synthesis mechanism. saccharide: Study of in vitro antibacterial activ-
Chemistry and Biochemistry 9, 1415–1422. ity. Materials Letters 65, 999–1002.
Pastoriza-Santos and Liz-Marzán, L. M. (2002). Qourzal, S., Tamimi, M., Assabbane, A.,
Formation of PVP-Protected Metal Nanopar- Bouamrane, A., Nounah, A., Laanab, L., and Ait-
ticles in DMF. Langmuir 18, 2888‑2894. Ichou, Y. (2006). Preparation of TiO2 Photocata-
Patel, K., Kapoor, S., Dave, D. P., and Murher- lyst Using TiCl4 as a Precursor and its Photocata-
jee, T. (2005). Journal of Chemical Sciences lytic Performance. Journal of Applied Sciences
117(1), 53‑60. 6(7), 1553‑1559.
Patel, K., Kapoor, S., Dave, D. P., and Murher- Rai, M., Yadav, A., and Gade, A. (2009). Silver
jee, T. (2007). Journal of Chemical Sciences nanoparticles: A novel antimicrobial agent. Bio-
117(4), 311‑315. technology Advances 27, 76‑83.
Patterson, T. F. (2007). Treatment and preven- Rajesh, R., Stringer, Sarah, J., Agarwal Gunjan,
tion of fungal infections. Focus on candidemia. Jones Sharon, E., and Stone Morley, O. (2002).
Newyork: Applied Clinical Education 100, p Biomimetris synthesis and patterning os silver
VII‑VIII. nanoparticles. Letters 169‑172.
Petit, C., Lixon, P., and Pileni, M. P. (1993). In Raveendran, P., Fu, J., and Wallen, S. L. (2003).
situ synthesis of silver nanocluster in AOT re- Completely “green” synthesis and stabilization
verse micelles. The Journal of Physical Chemis- of metal nanoparticles. Journal of the American
try 97, 12974‑12983. Chemical Society 125, 13940‑13941.
Phong, N T P., Ngo Hoang Minh, Ngo Vo Ke Rayman, M. K., Lo, T. C., and Sanwal, B. D.
Thanh, and Dang Mau Chien (2009). Green (Octuber 10, 1972). Transport of succinate in
synthesis of silver nanoparticles and silver col- Escherichia coli. II. Characteristics of uptake
loidal solutions. Journal of Physics: Confer- and energy coupling with transport in membrane
ence Series 187, 012078 doi:10.1088/1742- preparations. The Journal of Biological Chemis-
6596/187/1/012078. try 247(19), 6332‑6339.
Pileni, M. P. (2001). Self-Assemblies of nano- Revathi, N. and Prabhu, N. (2009). Fungal silver
crystals: Fabrication and Collective Properties. nanoparticles: Preparation and its pH character-
Applied Surface Sciences 171, 1‑14. ization. Trends in Biotechnology 8, 27‑29.
Pillai, Z. S. and Kamat, P. V. (2004). What Rocio Balaguera-Gelves, M. (2006). Detection
Factors Control the Size and Shape of Silver of nitroexplosives by surface enhanced Raman
Nanoparticles in the Citrate Ion Reduction Meth- spectroscopy on colloidal metal nanoparticles.
od? Journal of Physical Chemistry B 108, 945. Master thesis. University of Puerto Rico, Maya-
Porter, A. E., Muller, K., Skepper, J., Midgley, P. guez Campus.
and Welland, M. (2006) Uptake of C “6”0 by hu- Rodriguez-Sanchez, L., Blanco, M. C., and
man monocyte macrophages, its localization and Lopez-Quintela, M. A. (2000). Electrochemi-
implications for toxicity: Studied by high resolu- cal synthesis of silver nanoparticles. Journal of
tion electron microscopy and electron tomogra- Physical Chemistry B 104, 9683‑9688.
phy. Acta Biomaterialia 2, 409‑419. Roe, D., Balu Karandikar, Nathan Bonn-Sav-
Prabhu, N., Divya, T. R., Gowri, Y. K., Siddiqua, age1, Bruce Gibbins, and Jean-Baptiste Roullet.
A. S., and Innocent, J. P. D. (2010). Synthesis of (2008). Antimicrobial surface functionaliza-
References 217
tion of plastic catheters by silver nanoparticles. Saravanan, M. and Nanda, A. (2010) Extracel-
Journal of Antimicrobial Chemotherapy 61(4), lular synthesis of silver bionanoparticles from
869‑876. Aspergillus clavatus and its antimicrobial activ-
Rosei, F. (2004). Nanostructured surfaces: ity against MRSA and MRSE. Colloids and Sur-
Challenges and frontiers in nanotechnology. faces B: Biointerfaces 77, 214‑218.
Journal of Physics: Condensed Matter 16, Sastry, M., Ahmad, A., Khan, M. I., and Kumar,
S1373‑1436. R. (2003). Biosynthesis of metal nanoparticles
Rosemary, M. J. and Pradeep, T. (2003). Solvo- using fungi and actinomycete. Current Science
thermal synthesis of silver nanoparticles from 85, 162‑170.
thiolates. Colloids and Surfaces A 268, 81‑84. Sato, Y., Wang, J. J., Batchelder, D. N., and
Rosi, N. L. and Mirkin, C. A. (2005). Nanostruc- Smith, D. A. (2003). Langmuir 19, 6857.
tures in biodiagnostics. Chemical Reviews 105, Sau, T. K. and Murphy, C. J. (2004). Room
1547‑1562. Temperature, High-Yield Synthesis of Multiple
Rouch, D. A., Lee, B. T. O., and Morby, A. P. Shapes of Gold Nanoparticles in Aqueous Solu-
(1995). Understanding cellular responses to tox- tion. Journal of the American Chemical Society
ic agents: A mechanism-choice in bacterial metal 126, 8648‑8649.
resistance. Journal of Industrial Microbiology Schreurs, W. J. and Rosenberg, H. (1982). Ef-
and Biotechnology 14, 132–141. fect of silver ions on transport and retention of
Roy, N. and Barik, A. (2010). Green synthesis of phosphate by Escherichia coli. The Journal of
silver nanoparticles from the unexploited weed Bacteriology 152, 7‑13.
resources. International journal of nanotechnol- Senapati, S., Ahmad, A., Khan, M. I., Sastry, M.,
ogy and applications 4, 95‑101. and Kumar, R. (2005). Extracellular Biosynthe-
Roy, R., Hoover, M. R., Bhalla, A. S., Slaweekl, sis of Bimetallic Au‑Ag Alloy Nanoparticles.
T., Dey, S., Cao, W., Li J., and Bhaskar, S. (2008). Small 1, 517‑520.
Ultradilute Ag-auasols with extraordinary bacte- Senapati, S., Mandal, D., Ahmad, A., Khan, M.
ricidal properties: Role of the system Ag-O-H2O. I., Sastry, M., and Kumar, R. (2004). Fungus me-
Materials Research Innovations 11, 3‑18. diated synthesis of silver nanoparticles: A novel
Russell, A. D. and Hugo, W. B. (1994). Antibac- biological approach. Indian Journal of Physics
terial activity and action of silver. Progress in 78A, 101–105.
Medicinal Chemistry 31, 351–370. Setua, P., Chakraborty, A., Seth, D., Bhatta, U.
Saifuddin, N., Wong, C. W., and Nur Yasum- M., Satyam, P. V., and Sarkar, N. (2007). Syn-
ira, A. A. (2009). Rapid Biosynthesis of Silver thesis, optical properties, and surface enhanced
Nanoparticles Using Culture Supernatant of Raman scattering of silver nanoparticles in non-
Bacteria with Microwave Irradiation. E-Journal aqueous methanol reverse micelles. The Journal
of Chemistry 6(1), 61‑70. of Physical Chemistry C 111(10), 3901.
Sakaguchi, T., Tsuji, T., Nakajima, A., and Hor- Shahverdi, A. R., Fakhimi, A., Shahverdi, H. R.,
ikoshi, T. (1979). Accumulation of cadmium by and Sara, M. (2007). Synthesis and effect of sil-
green micro algae. European Journal of Applied ver nanoparticles on the antibacterial activity of
Microbiology 8, 207‑215. different antibiotics against Staphylococcus au-
reus and Escherichia coli. Nanomedicine: Nano-
Salata, O.V. (2004). Application of nanoparticles technology, Biology and Medicine 3, 168‑171.
in biology and medicine. Journal of Nanobio-
technology 2:3, 3‑6. Shankar, S. S., Rai, A., Ahmad, A., and Sastry,
M. (2005). Chemistry of Materials 17, 566.
Salkar, R. A., Jeevanandam, P., Aruna, S. T.,
Yuri Koltypin, and Gedanken, A. (1999).The Shankar, S. S., Rai, A., Ahmad, A., and Sastry.
sonochemical preparation of amorphous silver (2004). Rapid synthesis of Au, Ag, and bimetal-
nanoparticles. Journal of Materials Chemistry 9, lic Au core-Ag shell nanoparticles using Neem
1333‑1335. (Azadirachta indica) leaf broth. Journal of Col-
loid and Interface Science 275, 496–502.
Shiv Shankar, S., Ahmad, A. M., and Sastry, M. tional Meeting of Environmental Microbiology
(2003). Geranium Leaf Assisted Biosynthesis of Curtiba. pp. 25.
Silver Nanoparticles. Biotechnology Progress Sreeram, K. J., Nidhin, M., and Nair, B. U.
19, 1627‑1631. (2008). Microwave assisted template synthesis
Shon, Y. S., Choo, H., and Chim, C. R. (2003). of silver nanoparticles. Bulletin of Materials Sci-
Organic Reactions of Monolayer-Protected ence 31(7), 937‑942.
Metal Nanoparticles. In Dendrimers and Nano- Starowicz, M., Stypula, B., and Banaœ, J. (2006).
sciences. D. Astruc (Ed.). C. R. Chime,Paris, 6, Electrochemistry Communications 8, 227‑230.
1009.
Stepanov, A. L., Khaibullin, I. B., Townsend, P.
Shrivastava, S., Bera, T., Roy, A., Singh, G, D., Hole, and Bukharaev, A. A., (2000). RF Pat-
Ramachandrarao, P., and Dash, D. (2007). Char- ent, 2156490.
acterization of enhanced antibacterial effects of
novel silver nanoparticles. Nanotechnology 18, Sudeep, P. K. and Kamat, P. V. (2005). Photo-
225103. sensitized Growth of Silver Nanoparticles under
Visible Light Irradiation: A Mechanistic Inves-
Silver, S. (2003). Bacterial silver resistance: tigation. Chemistry of Materials 17, 5404‑5410.
Molecular biology and uses and misuses of sil-
ver compounds. FEMS Microbiology Reviews Summers, A. O. and Silver, S. (1978). Microbial
27(2‑3), 341–353. transformations of metals. Annual Review of Mi-
crobiology. 32, 637−672.
Simi, C. K. and Abraham, T. E. (2007). Hydro-
phobic grafted and cross-linked starch nanopar- Sun, Y. G. and Xia, Y. N. (2002). Shape-Con-
ticles for drug delivery. Bioprocess and Biosys- trolled Synthesis of Gold and Silver Nanopar-
tems Engineering 30, 173‑180. ticles. Science 298, 2176‑2179.
Singh, A., Jain, D., Upadhyay, M. K., Khandel- Sun, Y. G., Mayers, B., Herricks, T., and Xia,
wal, N., and Verma, H. N. (2010). Green syn- Y. N. (2003). Polyol Synthesis of Uniform Sil-
thesis of silver nanoparticles using Argemone ver Nanowires: A Plausible Growth Mechanism
Mexicana leaf extract and evaluation of their and the Supporting Evidence. Nano Letters 3,
antimicrobial activities. Digest Journal of Nano- 955‑960.
materials and Biostructures 5, 483‑489. Sun, Y. G., Yin, Y. D., Mayers, B. T., Herricks,
Smetana, A. B., Klabunde, K. J., and Sorensen, T., and Xia, Y. N. (2002). Uniform silver nanow-
C. M. (2005). Synthesis of spherical silver ires can be synthesized by reducing AgNO3 with
nanoparticles by digestive ripening, stabiliza- ethylene glycol in the presence of seeds and poly
tion with various agents, and their 3-D and 2-D (vinyl pyrrolidone). Chemistry of Materials 14,
superlattice formation. Journal of Colloid and 4736–4745.
Interface Science 284, 521–526. Szłyk, E., Piszczek, P., Grodzicki, A., Chaberski,
Smith, A. M., Duan, H. W., Rhyner, M. N., M., Goliński, A., Szatkowski, J., and Błaszczyk,
Ruan, G., and Nie, S. (2006). A systematic ex- T. (2001). CVD of AgI complexes with tertiary
amination of surface coatings on the optical and phosphines and perfluorinated carboxylates‑A
chemical properties of semi conductor quantum new class of silver precursors. Advanced Mate-
dots. Physical Chemistry Chemical. Physics 8, rials, Chemical Vapour Deposition 7, 111‑116.
3895‑3903. Taleb, A., Petit, C., and Pileni, M. P. (1997). Syn-
Soroushian, B., Lampre, I., Belloni, J., and Mo- thesis of Highly Monodisperse Silver Nanopar-
stafavi, M. (2005). Radiolysis of silver ion solu- ticles from AOT Reverse Micelles: A Way to 2D
tions in ethylene glycol. Solvated electron and and 3D Self-Organization. Chemistry of Materi-
radical scavenging yields. Radiation, Physics als 9, 950–959.
and Chemistry 72, 111‑118. Tan, Y., Wang, Y., Jiang, L., and Daoben Zhu
Souza, G. I. H., Marcato, P. D., Duran, N., and (2002).Thiosalicylic Acid-Functionalized Silver
Esposito, E. (2004). Utilization of Fusarium Nanoparticles Synthesized in One-Phase Sys-
oxysporum in the biosynthesis of silver nanopar- tem. Journal of Colloid and Interface Science
ticles and its antibacterial activities. In IX Na- 249, 336–345.
References 219
Tang, Z., Liu, S., Dong, S., and Wang, E. (2001). white rot fungus, Phaenerochaete chrysospo-
Electrochemical synthesis of Ag nanoparticles rium. Colloids and Surfaces B: Biointerfaces 53,
on functional carbon surfaces. Journal of Elec- 55–59.
troanalytical Chemistry 502, 146‑151. Vigneshwaran, N., Nachane, R. P., Balasubra-
Tessier, P. M., Velev, O. D., Kalambur, A. T., manya, R. H., and Varadarajan, P. V. (2006). A
Rabolt, J. F., Lenhoff, A. M., and Kalar, E. W. novel one-pot “green” synthesis of stable silver
(2000). Assembly of gold nanostructured films nanoparticles using soluble starch. Carbohy-
templated by colloidal crystals and used in sur- drate Research 341, 2012‑2018.
face enhanced Raman spectroscopy. Journal of Virender, S. K., Yngard Ria, A., and Lin Yekat-
American chemical society 122(39), 9554‑9555. erina. (2009). Silver nanoparticles: Green syn-
Toshima, N., Yonezawa, T., and Kushihashi, K. thesis and their antimicrobial activities. Colloid
(1993). 2543Polymer-protected palladium–plati- and Interface Science 145, 83‑96.
num bimetallic clusters: Preparation, catalytic Volesky, B. (Ed.) (1990). Biosorption of Heavt
properties and structural considerations. Journal metals. pp. 139‑172. Boca Raton, FL, CRC
of the Chemical Society, Faraday Transactions press.
89, 2537‑2543.
Vorobyova, S. A., Lesnikovich, A. I., and Sobal,
Townsend, P. T., Chandler, P. J., and Zhang, L. N. S. (1999). Preparation of silver nanoparticles
(1994). Optical effects of ion implantation. Cam- by interphase reduction. Colloids Surf. A 152,
bridge Univ. Press, Cambridge. 375–379.
Tripathi, A., Ashok, M. R., Chandrasekaran, N., Vyom parashar, Rashmi parashara, Bechan
Prathna, T. C. N., Prathna, T. C., and Mukherjee, Sharma, and Avinash Pandeyc. (2009). parthe-
A. (2010). Process variables in biomimetic syn- nium leaf extract mediated synthesis of silver
thesis of silver nanoparticles by aqueous extract nanoparticles: A novel approach towards weed
of Azadirachta indica (Neem) leaves. Journal of utilization. Digest Journal of Nanomaterials and
Nanoparticle Research 12, 237‑246. Biostructures 4(1), 45‑50.
Tripathi, R. M., Antariksh Saxena, Nidhi Gupta, Walter, E. C., Ng, K., Zach, M. P., Penner, R. M.,
Harsh Kapoor, and Singh, R. P. (2010). High and Favier, F. (2002). Electronic devices from
antibacterial activity of silver nanoballs against electrodeposited metal nanowires. Microelec-
E.coli mtcc 1302, S typhimurium MTCC 1254, tronic Engineering 61‑62, 555‑561.
B. subtilis MTCC 1133 and P. aeruginosa
MTCC 2295. Digest journal of nanomaterials Wei, D. and Qian, W. (2008). Facile Synthesis of
and Biostrucutres 5(2), 323‑330. Ag and Au nanoparticles utilizing chitosan as a
mediator agent. Colloid Surface B 62, 136‑142.
Troupis, A., Hiskia, A., and Papaconstantinou, E.
(2002). Angewandte Chemie International Edi- Weinstock, I. A. (1998). Chemical Reviews
tion 41, 1911. 98,113.
Vahabi, K., Ali Mansoori, G., and Sedighe Kari- Whitesides, G. M. (2003). The “right” size in
mi (2011). Biosynthesis of Silver Nanoparticles nanobiotechnology. Nature Biotechnology 21,
by Fungus Trichoderma reesei (A Route for 1161‑1165.
Large-Scale Production of AgNPs). Insciences Willems, and van den Wildenberg. (2005). Road-
Journal 1(1), 65‑79. map report on nanoparticles. Barcelona, Spain:
Vigneshwaran, N., Ashtaputrea, N. M., Varada- W&W. Espana sl.
rajana, P. V., Nachanea, R. P., Paralikara, K. M., Windt, W. D. (2009), United States Patent Appli-
and Balasubramanyaa, R. H. (2007). Biological cation Publication. Methods for producing metal
Synthesis of Silver Nanoparticles using the Fun- nanoparticles (2009) US2009/0239280 A1.
gus Aspergillus flavus. Materials Letters 61(6),
Xie, Y., Ye, R., and Liu, H. (2006). Synthesis of
1413‑1418.
silver nanoparticles in reverse micelles stabilized
Vigneshwaran, N., Kathe, A. A., Varadarajan, P. by natural biosurfactant. Colloids and Surfaces A
V., Nachane, R. P., and Balasubramanya, R. P. 279, 175‑178.
(2006). Biomimetics of silver nanoparticles by
Xiong, Y. J., Xie, Y., Du, G. O., Liu, X. M., and Zhang, J., Chen, P., Sun, C., and Hu, X. (2004).
Tian, X. (2002). B.Ultra sound assisted self-reg- Sonochemical synthesis of colloidal silver cata-
ulated route to Ag nano rods. Chemistry Letters lysts for reduction of complexing silver in DTR
1, 98–99. system. Applied Catalysis A 266, 49.
Yanagihara, N., Tanaka, Y., and Okamoto, H. Zhang, L., Yu, J. C., Yip, H. Y., Li, Q., Kwong, K.
(2001). Formation of silver nano particles in W., Xu, A.W. et al. (2003). Langmuir 19, 10372
poly(methyl methacrylate) by UV irradiation. Zhou, Y., Yu, S. H., Cui, X. P., Wang, C. Y., and
Chemistry Letters 8, 796‑797. Chen, Z. Y. (1999). Formation of silver nanow-
Yin Yadong, Li Zhi-Yuan, Zhong Ziyi, Gates By- ires by a naovel solid-liquid phase arc discharge
ron, and Venkateswaran Sagar (2002). Synthesis method. Chemistry of materials 11, 545–546.
and characterization of stable aqueous disper- Zhu Jian, Zhu Xiang, and Wang Yongchang
sions of silver nanoparticles through the Tollens (2005). Mictoelectronic Engineering 77, 58.
process. Chemistry of Materials 12, 522‑527.
Zhu, J. J., Liao, X. H., Zhao, X. N., and Chen,
Young-Ki Jo, Byung H. Kim, and Geunhwa Jung H. Y. (2001). Preparation of silver nanorods
(2009). Antifungal activity of silver ions and by electrochemical methods. Materials Letters
nanoparticles on phytopathogenic fungi. Plant 49(2), 91‑95.
Disease 93, 1037‑1043.
Zhu, J. J., Liu, S. W., Palchik, O., Koltypin, Y.,
Yu, D. and Yam, V. W. W. (2004). Controlled and Gedanken, A. (2000). Langmuir 16, 6396.
Synthesis of Monodisperse Silver Nanocubes in
Water. Journal of the American Chemical Soci-
ety 126, 13200. 6
Yu, D. and Yam, V. W. W. (2005). Hydrothermal-
Aggarwal, B., Takata, Y., and Oommen, O. V.
Induced Assembly of Colloidal Silver Spheres
(2004). From chemoprevention to chemother-
into Various Nanoparticles on the Basis of
apy: Common targets and common goals. Ex-
HTAB-Modified Silver Mirror Reaction. Journal
pert Opinion on Investigational Drugs 13(10),
of Physical Chemistry B, 109(12), 5497‑5503.
1327‑1338.
Yu, D. G. (2007). Formation of colloidal sil-
Arivazhagan, S., Velmurugan, B., Bhuvaneswari,
ver nanoparticles stabilized by Na+–poly(γ-
V., and Nagini, S. (2004). Effects of aqueous
glutamic acid)–silver nitrate complex via
extracts of garlic (Allium sativum) and neem
chemical reduction process. Colloids Surf. B 59,
(Azadirachta indica) leaf on hepatic and blood
171‑178.
oxidant-antioxidant status during experimental
Yu, Y. Y., Chang, S. S, Lee, C. L, and Wang, gastric carcinogenesis. Journal of Medicinal
C. R. C. (1997). Gold nanorods: Electrochemi- Food 7(3), 334‑339.
cal synthesis and optical properties. Journal of
Ashidi, J. S., Houghton, P. J., Hylands, P. J., and
Physical Chemistry B 101, 6661‑6664.
Efferth, T. (2010). Ethnobotanical survey and
Zeng, F., Chao Hou, Shuizhu Wu, Xinxing Liu, cytotoxicity testing of plants of South-western
Zhen Tong, and Shuning Yu. (2007). Silver Nigeria used to treat cancer, with isolation of cy-
nanoparticles directly formed on natural macro- totoxic constituents from Cajanus cajan Millsp.
porous matrix and their anti-microbial activities. leaves. Journal of Ethnopharmacology 128(2),
Nanotechnology 18, 1‑8. 501‑512.
Zhang, G., Keita, B., Dolbecq, A., Mialane, Bairwa, N. K., Sethiya, N. K., and Mishra, S. H.
P., Secheresse, F., Miserque, F., and Nadjo, L. (2010). Protective effect of stem bark of Ceiba
(2007). Green Chemistry-type One-Step Syn- pentandra linn. against paracetamol-induced
thesis of Silver Nanostructures Based on MoV- hepatotoxicity in rats. Pharmacognosy Research
MoVI Mixed Valence Polyoxometalate. Journal 2, 26‑30.
of Materials Chemistry 19, 5821‑5823.
Bingfen, X., Zongchao, L., Qichao, P., Yongju,
Zhang, J. Z. (2009). Optical properties and spec- L., Xiurong, S., Likai, W., Runmei, Z., and Hon-
troscopy of nanomaterials. World Scientific, gda, Z. (1994). The anticancer effect and anti-DNA
Singapore, Vol. xvi, pp. 359‑383. topoisomerase II effect of extracts of Camellia
References 221
ptilophylla chang and camellia sinesis Chinese cal Pouch Carcinogenesis. Ibnosina. Journal of
Journal of Cancer Research 6(3), 184‑190. Medicine and BS 2(6), 269‑277.
Bonham, M., Posakony, J., Coleman, I., Mont- Fei, Y., Xui, L., Yi, J., Zhang, W., and Zhang,
gomery, B., Simon, J., and Nelson, P. S. (2005). D. Y. (2002). Anticancer activity of Scutellaria
Characterization of chemical constituents in baicalensis and its potential mechanism. Journal
Scutellaria baicalensis with antiandrogenic of Alternative and Complementary Medicine 8,
and growth-inhibitory activities toward pros- 567‑572.
tate carcinoma. Clinical Cancer Research 11, Gálvez, M., Martín-Cordero, C., López-Lázaro,
3905‑3914. M., Cortés, F., and Ayuso, M. J. (2003). Cyto-
Chao, A., Thun, M. J., and Connell, C. J. (2005). toxic effect of Plantago spp. on cancer cell
Meat consumption and risk of colorectal cancer. lines. Journal of Ethnopharmacology 88(2‑3),
The Journal of American Medical Association 125‑130.
293(2), 172–182. Heber, D. (2004). Vegetables, fruits and phytoes-
Choi, S. U., Ryu, S. Y., Yoon, S. K., Jung, N. P., trogens in the prevention of diseases. Journal of
Park, S. H., Kim, K. H., Choi, E. J., and Lee, C. Postgraduate Medicine 50(2), 145‑149.
O. (1999). Effects of flavonoids on the growth Hirano, T., Abe, K., Gotoh, M., and Oka, K.
and cell cycle of cancer cells. Anticancer Re- (1995). Citrus flavone tangeretin inhibits leu-
search 19, 5229‑5233. kaemic HL-60 cell growth partially through in-
Chung, C. P., Park, J. B., and Bae, K. H. (1995). duction of apoptosis with less cytotoxicity on
Pharmacological effects of methanolic extract normal lymphocytes. British Journal of Cancer
from the root of Scutellaria baicalensis and its 72(6), 1380‑1388.
flavonoids on human gingival fibroblast. Planta Hsu, S. D., Singh, B. B., Lewis, J. B., Borke, J.
Medica 61, 150‑153. L., Dickinson, D. P., Drake, L., Caughman, G.
Costa-Lotufo L. V., Khan, M. T., Ather, A., Wil- B., and Schuster, G. S. (2002). Chemoprevention
ke, D. V., Jimenez, P. C., Pessoa, C., de Moraes, of oral cancer by green tea. General Dentistry
M. E., de Moraes, M. O. (2005). Studies of the 50(2), 140‑146.
anticancer potential of plants used in Bangla- Jainu, M. and Shyamala Devi, C. S. (2003).
deshi folk medicine. Journal of Ethnopharma- Potent antiulcerogenic activity of Cissus quad-
cology 99(1), 21–30. rangularis on aspirin induced gastric ulcer by
Cragg, G. M., and Newman, D. J. (2004). A tale its antioxidative mechanism. Journal of Clinical
of two tumour targets: Topoisomerase I and tu- Biochemistry and Nutrition 34, 43‑47.
bulin. The Wall and Wani contribution to cancer Janin, D., Pathak, N., Khan, S., Raghuram, G.
chemotherapy. Journal of Natural Products 67, V., Bhargava, A., Samarth, R., and Mishra, P.
232–244. K. (2011). Evaluation of Cytotoxicity and An-
Dai, H. F., Zeng., Y. B.; Xiao, Q., Han, Z., Zhao, ticarcinogenic Potential of Mentha Leaf Ex-
Y. X., and Mei, W. L. (2010). Caloxanthones O tracts. International Journal of Toxicology 30(2),
and P: Two New Prenylated Xanthones from Ca- 225‑236.
lophyllum inophyllum. Molecules 15, 606‑612. Joon Surh, Y. (2003). Cancer chemoprevention
Das, U. N. (2002). A radical approach to cancer. with dietary phytochemicals. Nature 3, 768‑780.
Medicinal Science Monitor 8, 79–92. Kathiresan, K., Sithranga Boopathy, N., and
Dhanamani, M., Devi, L., and Kannan, S. Kavitha, S. (2005). Coastal vegetation An un-
(2011). Ethnomedicinal plants for cancer ther- derexplored source of anticancer drugs. Natural
apy‑‑A Review. Hygeia. Journal of Drug and Product Radiance 5(2), 115‑119.
Medicine.3(1), 1‑10. Konczak, I, Okuno, S, Yoshimoto, M., and
Dhanarasu, S., Masoud Al-hazimi, A., Sethura- Yamakawa, O. (2004). Caffeoylquinic Acids
man, P., and Selvam, M. (2010). Chemopre- Generated In Vitro in a High-Anthocyanin-Ac-
ventive and Antilipidperoxidative Potential of cumulating Sweet potato Cell Line. Journal of
Thespesia populnea (L.) on Experimental Buc- Biomedicine and Biotechnology 5, 287‑292.
Konczak-Islam, I., Yoshimoto, M., Hou, D. X., pal, S., Kumar, R. A., Deevi, D. S., Rajagopalan,
Terahara, N., and Yamakawa, O. (2003). Poten- R., Venkateswarlu, A. (2003). Biological investi-
tial chemopreventive properties of anthocyanin- gation and structure-activity relationship studies
rich aqueous extracts from in vitro produced on azadirone from Azadirachta indica A. Juss.
tissue of sweetpotato (Ipomoea batatas L.). Bioorganic and Medicinal Chemistry Letters
Journal of Agricultural Food Chemistry 51(20), 13(22), 4111‑4115.
5916‑5922. Natesan, S., Badami, S., Dongre, S. H., and Go-
Kouidhi, B., Zmantar, T., and Bakhrouf, A. davarthi, A. (2007). Antitumor activity and anti-
(2010). Anticariogenic and cytotoxic activity oxidant status of the methanol extract of Careya
of clove essential oil (Eugenia caryophyllata) arborea bark against Dalton’s lymphoma ascites
against a large number of oral pathogens. Annals induced ascetic and solid tumor in mice. Journal
of Microbiology 60, 599–604. of Pharmacological Sciences 103(1), 12‑23.
Kumar, R. A., Sridevi, K., Kumar, N. V., Nan- Neto, C. C. (2007). Cranberry and its phyto-
duri, S., and Rajagopal, S. (2004). Anticancer chemicals: A review of in vitro anticancer stud-
and immunostimulatory compounds from An- ies. Journal of Nutrition 137, 186‑193S.
drographis paniculata. Journal of Ethnophar- Park, K. K., Chun, K. S., Lee, J. M., and Surh,
macology 92(2‑3), 291‑295. Y. J. (1998). Inhibitory effects of [6]-gingerol,
Kyung-A. H., Yu-Jin, H., Dong-Sik, P., Jaehyun, a major pungent principle of ginger, on phorbol
K., and Ae-Son, O. (2011). In vitro investigation ester-induced inflammation, epidermal ornithine
of antioxidant and anti-apoptotic activities of decarboxylase activity and skin tumors promo-
Korean wild edible vegetable extracts and their tion in ICR mice. Cancer Letter 129, 139‑144.
correlation with apoptotic gene expression in Pfisterer, P. H., Rollinger, J. M., Schyschka,
HepG2 cells. Food Chemistry 125, 483–487. L., Rudy, A., Vollmar, A. M., and Stuppner, H.
Lee, C. K., Park, K. K., Lim, S. S., Park, J. H., (2010). Neoandrographolide from Andrographis
and Chung, W. Y. (2007). Effects of the lico- paniculata as a potential natural chemosensitiz-
rice extract against tumor growth and cisplatin- er. Planta Medica 76(15), 1698‑1700.
induced toxicity in a mouse xenograft model of Romero-Jimenez, M., Campos-Sanchez, J.,
colon cancer. Biological and Pharmaceutical Analla, M., Munoz-Serrano, A., and Alonso-
Bulletin 30(11), 2191‑2195. Moraga, A. (2005). Genotoxicity and anti-geno-
Liu, J. J., Huang, T. S., Cheng, W. F., and Lu, F. J. toxicity of some traditional medicinal herbs. Mu-
(2003). Baicalein and baicalin are potent inhibi- tation Research 585, 147–155.
tors of angiogenesis: Inhibition of endothelial Sadava, D. and Winesburg, J. (2005). Contami-
cell proliferation, migration and differentiation. nants of PC-SPES are not responsible for cy-
International Journal of Cancer 106, 559‑565. totoxicity in human small-cell lung carcinoma
Lu, X., Yu, H., Ma, Q., Shen, S., and Das, U. N. cells. Cancer Letter 220, 171‑175.
(2010). Linoleic acid suppresses colorectal can- Sakarkar, D. M. and Deshmukh, V. N. (2011).
cer cell growth by inducing oxidant stress and Ethnopharmacological Review of Traditional
mitochondrial dysfunction. Lipids in Health and Medicinal Plants for Anticancer Activity. Inter-
Disease 9, 106. national Journal of PharmTech Research 3(1),
Maloney, D. (1998). The American Association 298‑308.
of Oriental Medicine’s Complete Guide to Chi- Sakpakdeejaroen, I. and Itharat, A. (2009). Cyto-
nese Herbal Medicine. The Berkley Publishing toxic compounds against breast adenocarcinoma
Group, New York. cells (MCF-7) from Pikutbenjakul. Journal of
Markowitz, S. D. and Bertagnolli, M. M. (2009). Health Research 23(2), 71‑76.
Molecular basis of colorectal cancer. The New Seeram, N. P., Adams, L. S., Hardy, M. L., and
England Journal of Medicine 361(25), 2449– Heber, D. (2004). Total cranberry extract versus
2460. its phytochemical constituents: Antiproliferative
Nanduri, S., Thunuguntla, S. S., Nyavanandi, V. and synergistic effects against human tumor cell
K., Kasu, S., Kumar, P. M., Ram, P. S., Rajago-
References 223
lines. Journal of Agricultural and Food Chemis-

try 52(9), 2512‑2517.
7
Abu-Surrah, A. S., Al-Sa’doni, H. H., and Ab-
Sharma, V. A. (2011). Polyphenolic compound
dalla, M. Y. (2008). Palladium-based chemother-
rottlerin demonstrates significant in vitro cy-
apeutic agents: Routes toward complexes with
totoxicity against human cancer cell lines: Iso-
good antitumor activity. Cancer Therapy 6, 1‑10.
lation and characterization from the fruits of
Mallotus philippinensis. Journal of Plant Bio- Ackrell, B. A. C., Armstrong, F. A., Cochran, B.,
chemistry and Biotechnology 20(2), 190‑195. Sucheta, A., and Yu, T. (1993). Classification of
fumarate reductases and succinate dehydroge-
Singh, R. P., Banerjee, S., and Rao, A. R. (2001).
nases based upon their contrasting behavior in
Modulatory influence of Andrographis panicu-
the reduced benzylviologen/fumarate. Federa-
lata on mouse hepatic and extrahepatic carcino-
tion of European Biochemical Societies 326(1,
gen metabolizing enzymes and antioxidant sta-
2, 3), 92‑94.
tus. Phytotherapy Research 15(5), 382‑390.
Aggarwal, B. B. and Shishodia, S. (2006). Mo-
Subapriya, R., Bhuvaneswari, V., and Nagini,
lecular targets of dietary agents for prevention
S. (2005). Ethanolic neem (Azadirachta indica)
and therapy of cancer. Biochemical Pharmacol-
leaf extract induces apoptosis in the hamster
ogy 71, 1397‑1421.
buccal pouch carcinogenesis model by modu-
lation of Bcl-2, Bim, caspase 8 and caspase 3. Alberts, B., Bray, D., Lewis, J., Raff, M., Rob-
Asian Pacific Journal of Cancer Prevention 6(4), erts, K., and Watson, J. D. (1989). Molecular Bi-
515‑520. ology of the Cell. Garland Publishing Inc., New
York.
Su, L. J. and Arab, L. (2002). Tea consumption
and the reduced risk of colon cancer‑‑results Antonawich, F. J., Fiore, S. M., and Welicky, L.
from a national prospective cohort study. Public M. (2004). Regulation of ischemic cell death by
Health Nutrition 5(3), 419‑425. the lipoic acid-palladium complex, Poly MVA,
in gerbils. Experimental Neurology 189, 10‑15.
Sun, J. and Hai Liu, R. (2006). Cranberry phy-
tochemical extracts induce cell cycle arrest and Bass, J and Takahashi, J. S. (2010). Circadian In-
apoptosis in human MCF-7 breast cancer cells. tegration of Metabolism and Energetics. Science
Cancer Letter 241(1), 124‑134. 330, 1349‑1354.
Tepsuwan, A., Kupradinun, P., and Kusam- Baumgartner, M. R., Schmalle, H., and Dubler,
ran, W. R. (2002). Chemopreventive Potential E. (1996). The Interaction of transition met-
of Neem Flowers on Carcinogen-Induced Rat als with the coenzyme α-lipoic acid: Synthesis,
Mammary and Liver Carcinogenesis. Asian Pa- structure and characterization of copper and zinc
cific Journal Cancer Prevention 3(3), 231‑238. complexes. Inorg. Chim. Acta 252, 319‑331.
Tyagi, S., Singh, G., Sharma, A., and Aggar- Beattie, D. S. (2002). Bioenergetics and Oxida-
wal, G. (2010). Phytochemical candidate thera- tive Metabolism. In Textbook of Biochemistry
peutics: An overview. International Journal of with Clinical Correlations. T. M. Devlin (Ed.),
Pharmaceutical Sciences 53‑55. 5th ed. Wiley-Liss, John Wiley & Sons, Inc.,
Publication, New York.
Uma Devi, P., Selvi, S., Devipriya, D., Murugan,
S., and Suja, S. (2009). Antitumor and antimi- Berliner, L. J., Eaton, S. S., and Eaton, G. R.
crobial activities and inhibition of in-vitro lipid (Eds.) (2000). Distance Measurements in Bio-
peroxidation by Dendrobium nobile. African logical Systems by EPR, Kluwer. In Biological
Journal of Biotechnology 18, 2289‑2293. Magnetic Resonance, vol 19. Academic Press/
Plenum Publishers, New York.
Zhang, M., Binns, C. W., and Lee, A. H. (2002).
Tea consumption and ovarian cancer risk: A Berrisford, J. M. and Sazanov, L., A. (2009).
case-control study in China. Cancer Epidemiol- Structural Basis for the Mechanism of Respira-
ogy, Biomarkers, and Prevention 11(8), 713‑718. tory Complex. J. Biol. Che, 284, 29773‑29783.
Biasutto, L., Dong, L. F., and Zoratti, M., and Corduneanu, O., Paquim, A. M. C., Garnett,
Neuzil, J. (2010). Mitochondrially targeted anti- M., and Oliveira Brett, A. M. (2009). Lipoic
cancer agents. Mitochondrion 10(6), 670‑681. acid-palladium complex interaction with DNA,
Bubber, P., Haroutunian, V., Fisch, G., Blass, voltammetric and AFM characterization. Talanta
J. P., and Gibson, G. E. (2005). Mitochondrial 77, 1843‑1853.
Abnormalities in Alzheimer Brain: Mechanistic Cotton, F. A. and Wilkinson, G. (1972). Ad-
Implications. Ann. Neurol. 57, 695‑703. vanced Inorganic Chemistry, 3rd ed. Interscience
Buettner, G. R. (1993). The Pecking Order of Publishers, New York, p. 415.
Free Radicals and Antioxidants: Lipid Peroxida- Dabrowiak, J. C. (2009). Metals in Medicine.
tion, α-Tocopherol, and Ascorbate. Arch. Bio- John Wiley & Sons Ltd., UK.
chem. Biophys 300, 535‑543. Dennery, P. A. (2010). Oxidative Stress in Devel-
Caires, A. C. F. (2007). Recent Advances In- opment: Nature or nurture?. Free radical Biol-
volving Palladium(II) Complexes for the Can- ogy & Medicine 49, 1147‑1151.
cer Therapy. Anti-Cancer Agents in Medicinal Dröge, W. (2006). Oxidative Stress in HIV Infec-
Chemistry 7, 484‑491. tion. In: Oxidative Stress, Disease and Cancer.
Carew, J. S. and Huang, P. (2002). Mitochondrial K. K. Singh (Ed.). Imperial College Press, Lon-
defects in cancer. Molecular Cancer 1, 9. http:// don, WC2H 9HE, pp. 885‑895.
www.molecular-cancer.com/content/1/1/9. Elliott, S. J., Léger, C., Pershad, H. R., Hirst, J.,
Carlson, D. A., Young, K. L., Fischer, S. J., and Heffron, K., Ginet, N., Blasco, F., Rothery, R.
Ulrich, H. (2008). An Evaluation of the Stabil- A., Weine,r J. H., and Armstrong, F. A. (2002).
ity and Pharmacokinetics of R-Lipoic Acid and Detection and interpretation of redox potential
R-Dihydrolipoic Acid Dosage Forms in Human optima in the catalytic activity of enzymes. Bio-
Plasma from Healthy Subjects. In Lipoic Acid: chimica. Et. Biophysica. Acta (BBA) Bioenerget-
Energy Production, Antioxidant Activity and ics 1555, 54‑59.
Health Effects. M. S. Patel and L. Packer (Eds.). Everhard, M. E., Gross, Jr. P. M., and Turner, J.
CRC press, Taylor & Francis Group, Florida, p. W. (1962). Properties of Electrolytes in Hydro-
255. gen Peroxide-Water Solutions. 1. Solvation of
Castellani, R., Hirai, K., Aliev, G., Drew, K. L., Alkali Nitrates. J. Phys. Chem. 66(5), 923‑926.
Nunomura, A., Takeda, A., Cash, A. D., Ob- Field, J. and Gyorgyi, L. (Eds.), (1993). In Chaos
renovich, M. E., Perry, G., and Smith, M. A. in Chemistry and Biochemistry. World Scientific
(2002). Role of mitochondrial dysfunction in Publishing Co., NJ 07661, USA.
Alzheimer’s disease. Journal of Neuroscience
Research, 70(3), 357‑360. Fink D. G. (Ed.). (1975). Electronic Engineers
Handbook. McGraw Hill, New York.
Chan, S. W., Chan, P. C., and Bielski, B. H. J.
(1974). Studies on the lipoic acid free radical. Frezza, C. and Gottlieb, E. (2009). Mitochondria
Biochim. Biophys. Acta, General Subjects 338, in cancer: Not just innocent bystanders. Semi-
213‑223. nars in Cancer Biology 19, 4‑11.
Chatterjee, A., Mambo, E., and Sidransky, A. Fricker, S. P. (2007). Metal based drugs: From
(2006). Mitochondrial DNA mutations in human serendipity to design. Dalton Trans 4903‑4917.
cancer. Oncogene 25, 4663‑4674. Fukushima, S., Nakanishi, S., Fukami, K., Sakai,
Chen, G., Wang, F., Trachootham, D., and S., Nagai, T., Tada, T., and Nakato, Y. (2005).
Huang, P. (2010). Preferential killing of cancer Observation of synchronized spatiotemporal re-
cells with mitochondrial dysfunction by natural action waves in coupled electrochemical oscil-
compounds. Mitochondrion 10(6), 614‑625. lations of an NDR type. Electrochemistry Com-
munications 7, 411‑415.
Corduneanu, O., Garnett, M., and Oliveira Brett,
A. M. (2007). Anodic oxidation of α-lipoic acid Gao, E., Liu, C., Zhu, M., Lin, H., Wu, Q., and
at a glassy carbon electrode and its determina- Liu, L. (2009). Current Development of Pd(II)
tion in dietary supplements. Anal. Letters 40, Complexes as Potential Antitumor Agents. Anti-
1763‑1778.
References 225
cancer Agents in Medicinal Chemistry 9(3), Oxidoreductase. J. Am. Chem. Soc. 127(43),
356‑368. 14964‑14965.
Galkin, A. and U. Brandt U. (2005). Super- Hanahan, D. and Weinberg, R. A. (2000). The
oxide Radical Formation by Pure Complex I Hallmarks of Cancer. Cell 100, 57‑70.
(NADH:Ubiquinone Oxidoreductase) from Yar- Heiden, M. G. V., Cantley, L. C., and Thompson,
rowia lipolytica. J. Biol. Chem. 280, 3019‑10135. C. B. (2009). Understanding the Warburg Effect:
Galluzzi, L., Larochette, N., Zamzami, N., and The Metabolic Requirements of Cell Prolifera-
Kroemer, G. (2006). Mitochondria as therapeutic tion. Science 324, 1029‑1033.
targets for cancer chemotherapy. Oncogene 25, Higuchi, M. (2007). Regulation of mitochon-
4812‑4830. drial DNA content and cancer. Mitochondrion 7,
Garnett, M. (1995a). Synthetic DNA Reductase, 53‑57.
Contents of 7th EBIC Conference Abstracts Is- Hoffman, M. Z. and Hayon, E. (1972). One-
sue, C 48. J. Inorg. Biochem 59, 231. Electron Reduction of the Disulfide Linakge in
Garnett, M. (October 31, 1995b). Palladium Aqueous Solution. Formation, Protonation, and
Complexes and Methods for Using Same in the Decay Kinetics of RSSR- Radical. J. Am. Chem.
Treatment of Tumors or Psoriasis, U.S.Patent, Soc. 94, 795‑797.
No. 5,463,093. Hsu, P. P. and Sabatini, D. M. (2008). Cancer
Garnett, M. (October 21, 1997). Palladium Com- Cell Metabolism: Warburg and Beyond. Cell
plexes and Methods for Using same in the Treat- 134, 703‑707.
ment of Tumors, U.S.Patent, No. 5,679,697. Hurst, J., Sucheta, A, Ackrell, B. A., and Arm-
Garnett M. (July 7, 1998). Palladium Complexes strong, F. A. (1996). Electrocatalytic Voltamme-
and Methods for using same in the Treatment of try of Succinate Dehydrogenase: Direct Quanti-
Psoriasis, U.S.Patent, No. 5,776,973. fication of the Catalytic Properties of a Complex
Genereux, J. C. and Barton, J. K (2010). Mecha- Electron Transport Enzyme. J. Am. Chem. Soc.
nisms for DNA Charge Transport. Chem. Rev. 118(21), 5031‑5038.
110, 1642‑1662. Jaruga, P., Jaruga, B., Gackowski, D., Olczak,
Gibson, G. E., Haroutunian, V., Zhang, H., Park, A., Halota, W., Pawlowska, M., and Olinski, R.
L. C. H., Shi, Q, Lesser, M., Mohs, R. C., Sheu, (2002). Supplementation with antioxidant vita-
R. K. F., and Blass, J. P. (2000). Mitochondrial mins prevents oxidative modification of DNA
Damage in Alzheimer’s Disease Varies with in lymphocytes of HIV-infected patients. Free
Apolipoprotein E Genotype. Ann. Neurol. 48, Radical Biology & Medicine 32(5), 414‑420.
297‑303. Jones, S., Zhang, X., Parsons, D. W., Lin, J.
Gogvadze, V., Orrenius, S., and Zhivotovsky, B. C. H., Leary, R. J., Angenendt, P., Mankoo, P.,
(2008). Mitochondria in cancer cells: What is Carter, H., Kamiyama, H., Jimeno, A., Hong,
so special about them?. Trends in Cell Biology S. M., Fu, B., Lin, M. T., Calhoun, E. S., Ka-
18(4), 165‑173. miyama, M., Walter, K., Nikolskaya, T., Nikol-
sky, Y., Hartigan, J., Smith, D. R., Hidalgo, M.,
Gorin, M. H. (1935). The “Salting-in” of Hydro- Leach, S. D., Klein, A. P., Jaffee, E. M., Gog-
gen Peroxide by Electrolytes. J. Am. Chem. Soc. gins, M, Maitra, A., Donahue, C. L., Eshleman,
57, 1975‑1978. J. R., Kern, S. E., Hruban, R. H., Karchin, R.,
Greenamyre, J. T., MacKenzie, G., Peng T., and Papadopoulos, N., Parmigiani, G., Vogelstein,
Stephans, S. E. (1999). Mitochondrial dysfunc- B., Velculescu, V. E., and Kinzler, K. W. (2008).
tion in Parkinson’s disease. Biochem. Soc. Symp. Core Signaling Pathways in Human Pancreatic
66, 85‑97. Cancers Revealed by Global Genomic Analyses.
Science 321, 1801‑1806.
Gwyer, J. D., Richardson, D. J., and Butt, J. N.
(2005). Diode or Tunnel-Diode Characteristics? Keasling, J. D. (2010) Manufacturing Molecules
Resolving the Catalytic Consequences of Proton through Metabolic Engineering. Science 330,
Coupled Electron Transfer in a Multi-Centered 1355‑1358.
Koper, M. T. M. (1996). Oscillations and Com- tion in Aqueous Lidocaine Hydrochloride. Int. J.
plex Dynamical Bifurcations in Electrochemical Electrochem. Soc. 4, 1085‑1099.
systems. In Advances in Chemical Physics. I. Krishnan, C. V., Garnett, M., and Chu, B.
Prigogine and S. A. Rice, (Eds.). John Wiley & (2009b). Admittance as a useful tool for inves-
Sons, New York,vol 92, pp. 161‑297. tigation of solute-solvent interactions at the
Korkina, L. G., Afanas’ef, I. B., and Diplock, A. double layer. Electrochem. Communications 11,
T. (1993). Antioxidant therapy in children affect- 2229‑2232.
ed by irradiation from the Chernobyl nuclear ac- Kroemer, G. (2006). Mitochondria in cancer.
cident. Biochemical Society Transactions 314S, Oncogene 25, 4630‑4632.
21.
Kroemer, G., Galluzzi, L., and Brenner, C.
Krishnan, C. V. and Garnett, M. (2006). Liquid (2007). Mitochondrial Membrane Permeabiliza-
crystal behavior in solutions, electrode passiv- tion in Cell Death. Physiol. Rev. 87, 99‑163.
ation, and impedance loci in four quadrants. In
Passivation of Metals and Semiconductors, and Kroemer, G. and Pouyssegur, J. (2008). Tumor
Properties of Thin Oxide Layers. P. Marcus and Cell Metabolism: Cancer’s Achilles Heel. Can-
V. Maurice, (Eds.). Elsevier, Amsterdam, pp. cer Cell 13, 472‑482.
389‑394. Kussmaul, L. and Hirst, J. (2006). The mecha-
Krishnan, C. V., Garnett, M., and B., Chu nism of superoxide production by NADH: Ubi-
(2007a). Spatiotemporal Oscillation in Peroxo- quinone oxidoreductase (complex I) from bovine
Molybdate Complexes in Acidic Solutions: heart mitochondria. Proc. Natl. Acad. Sci., USA
1. Impedance Measurements of H2Mo2O3(O- 103, 7607‑7612.
O)4(H2O)2. Int. J. Electrochem Sci. 2, 444‑461. Lasia, A. (1999). Electrochemical Impedance
Krishnan, C. V., Garnett, M., and Chu, B. Spectroscopy and its Applications. In Modern
(2007b). Solute-Solvent Interactions from Ad- Aspects of Electrochemistry. No 32, B. E. Con-
mittance Measurements: Potential Induced and way, J. O’M. Bockris, and R. E. White (Eds.).
Water Structure-Enforced Ion-Pair Formation. Kluwer Academic/Plenum Publishers, New
Int. J. Electrochem. Sci. 2, 958‑972. York.
Krishnan, C. V., Garnett, M., and Chu, B. Lázaro, M. L. (2007). Dual role of hydrogen
(2008a). Solute-solvent interactions in biologi- peroxide in cancer: Possible relevance to cancer
cal molecules: L-Cysteine. Int. J. Electrochem. chemoprevention and therapy. Cancer Letters
Sci. 3, 854‑872. 252, 1‑8.
Krishnan, C. V., Garnett, M., and Chu, B. Léger, C. and Bertrand, P. (2008). Direct Electro-
(2008b). Spatiotemporal oscillations in biologi- chemistry of Redox Enzymes as a Tool for Mech-
cal molecules: Molybdate-L-Cysteine. Int. J. anistic Studies. Chem. Rev. 108, 2379‑2438.
Electrochem. Sci. 3, 873‑890. Levine, H and Jacob, E. B. (2004). Physical
Krishnan, C. V., Garnett, M., and Chu, B. schemata underlying biological pattern forma-
(2008c). Oxidative Stress and Parkinson’s Dis- tion-examples, issues and strategies. Physical
ease: Electrochemical Behavior of Hydrogen Biology 1(1‑2), P14‑22.
Peroxide in Aqueous Sodium Chloride. Int. J. Levine, A. J. and Puzio-Kuter, A. M. (2010).
Electrochem. Sci. 3, 1348‑1363. The Control of the Metabolic switch in Cancers
Krishnan, C. V., Garnett, M., and Chu, B. by Oncogenes and Tumor Suppressor Genes.
(2008d). Spatiotemporal oscillations in bio- Sceince 330, 1340‑1344.
logical molecules: Hydrogen peroxide and Par- Livingston, R. (1928). The Activity of Hydrogen
kinson’s disease. Int. J. Electrochem. Sci. 3, Peroxide in Sodium Chloride and Sodium Sul-
1364‑1385. fate Solutions. J. Am. Chem. Soc. 50, 3206.
Krishnan, C. V., Garnett, M., Hsiao, B., and Chu, Macdonald, J. R. and Johnson, W. B. (2005).
B. (2009a). Solute-Solvent Interactions from Fundamentals of Impedance Spectroscopy. In
Impedance Measurements: ‘π–way’ Conduction Impedance Spectroscopy: Theory, Experiment,
and Water Structure-Enforced Ion Pair Forma- and Applications, 2nd ed. E. Barsoukov, J. R.
References 227
Macdonald (Eds.). Wiley Interscience, New Jer- Neustadt, J. and Pieczenik, S. R. (2008). Medica-
sey. tion-induced mitochondrial damage and disease.
Maloň, M., Trávníček, Z., Maryško, M., Zbořil, Mol. Nutr. Food Res. 52, 780‑788.
R., Mašláň, M., Marek, J., Doležal, K., Rolčík, Niki, E. and Noguchi, N. (2004). Dynamics of
J., Kryštof, V., and Strnad, M. (2001). Metal Antioxidant Action of Vitamin E. Acc. Chem.
complexes as anticancer agents 2. Iron(III) and Res. 37, 45‑51.
copper(II) bio-active complexes with N6-ben- Noodleman, L., Peng, C. Y., Case, D. A., and
zylaminopurine derivatives. Inorg. Chim. Acta Mouesca, J.–M. (1995). Orbital interactions,
323, 119‑129. electron delocalization and spin coupling in iron-
Massey, V., Gibson, Q. H., and Veeger, C. sulfur clusters. Coord. Chem. Rev. 144, 199‑244.
(1960). Intermediates in the Catalytic Action of Nualart, F., Rivas, C., Montecinos, V., Godoy,
Lipoyl Dehydrogenase (Diaphorase). Biochem. A., Guaiquil, V., Golde, D., and Vera, J. (2003).
J. 77, 341‑351. Recycling of vitamin C by a bystander effect. J.
Matesanz, A. I., Perez, J. M., Navarro, P., Mo- Bio. Chem. 278(12), 10128‑10133.
reno, J. M., Colacio, E., and Souza, P. (1999). Ogilvie, G. K. and Moore, A. S. (2000). Tumors
Synthesis and characterization of novel palla- of Bone. In Managing the Canine Cancer Pa-
dium (II) complexes of bis (thiosemicarbazone). tient: A practical Guide to Compassionate Care.
Structure, cytotoxic activity and DNA biding of R. A. Henry and P. A Yardley, (Eds.) Veterinary
Pd (II)-benzyl bis (thiosemicarbazonate). J. In- Learning Systems, pp. 565‑589.
org. Biochem. 76, 29‑37.
Ohnishi, T. (1998). Iron-sulfur clusters/semi-
Mayevsky, A. (2009). Mitochondrial function quinones in Complex I. Biochim. Biophys. Acta
and energy metabolism in cancer cells: Past 1364, 186‑206.
Overview and future perspectives. Mitochon-
drion 9, 165‑179. Olanow, C. W and Lieberman, A. N. (Eds.)
(1992). In The Scientific Basis for the Treatment
McKnight, S. L. (2010). On getting there from of Parkinson’s Disease. The Parthenon Publish-
here. Science 330, 1338‑1339. ing Group, New Jersey, 07656, USA.
Mecocci, P., MacGarvey, U., and Beal, M. F. Packer, L., Witt, E. H., and Tritschler, H. J.
(1994). Oxidative Damage to mitochondrial (1995). Alpha Lipoic Acid as a Biological An-
DNA is increased in Alzheimer’s disease. Annals tioxidant. Free Radical Biology and Medicine
of Neurology 36(5), 747‑751. 19(2), 227‑250.
Menon, A., Krishnan, C. V., and Nair, C. K. K. Parker, Jr. W. D., Boyson, S. J., and Parks, J. K.
(2009). Protection from gamma radiation insult (1989). Abnormalities of the Electron Transport
to antioxidant defense and cellular DNA by Chain in Idiopathic Parkinson’s Disease. Ann.
POLY-MVA, a dietary supplement containing Neurol 26, 719‑723.
palladium lipoic acid formulation. Int. J. Low
Radiation 6, 248‑262. Parsons, D. W., Jones, S., Zhang, X., Lin, J. C.
H., Leary, R. J., Angenendt, P., Mankoo, P., Cart-
Mukouyama, Y., Nakanishi, S., Chiba, T., Mu- er, H., Siu, I. M., Gallia, G. L., Olivi, A., McLen-
rakoshi, K., and Nakato, Y. (2001). Mechanisms don, R., Rasheed, B. A., Keir, S., Nikolskaya, T.,
of Two Electrochemical Oscillations of Differ- Nikolsky, Y., Busam, D. A., Tekleab, H., Diaz,
ent Types, Observed for H2O2 Reduction on a Pt Jr. L. A., Hartigan, J., Smith, D. R., Strausberg,
Electrode in the Presence of Small Amount of R. L., Marie, S. K. N., Shinjo, S. M. O., Yan,
Halide Ions. J. Phys. Chem. B 105, 7246‑7253. H., Riggins, G. J., Bigner, D. D., Karchin, R.,
Mukouyama, Y., Hommura, H., Nakanishi, Papadopoulos, N., Parmigiani, G., Vogelstein,
S., Nishimura, T., Konishi, H., and Nakato, Y. B., Velculescu, V. E., and K. W. Kinzler, K. W.
(1999). Mechanism and Simulation of Electro- (2008). An Integrated Genomic Analysis of Hu-
chemical Current Oscillations Observed in the man Glioblastoma Multiforme. Science 321,
H2O2-Reduction Reaction on Platinum Elec- 1807‑1812.
trodes in Acidic Solutions. Bull. Chem. Soc. Jpn. Patel, M. S. and Packer, L. (Eds.), (2008). Lipoic
72, 1247‑1254. Acid: Energy Production, Antioxidant Activity
and Health Effects. CRC Press, Taylor & Francis way and J. O’M. Bockris (Eds.). Plenum Press,
Group, New York. New York, vol 29, pp. 319‑399.
Pershad, H. R., Hirst, J., Cochran, B., Ackrell, Rouach, N., Calvo, C., Duquennoy, H., Glowin-
B. A. C., and Armstrong, F. A. (1999). Voltam- ski, J., and Giaume, C. (2004). Hydrogen Per-
metric studies of bidirectional catalytic electron oxide Increases Gap Junctional Communication
transport in Escherichia coli succinate dehydro- and Induces Astrocyte Toxicity: Regulation by
genase: comparison with the enzyme from beef Brain Macrophages. Glia 45, 28‑38.
heart mitochondria. Biochimica et Biophysica Saad, E. I., Gowilly, S. M. E., Sherhaa, M. O.,
Acta (BBA)–Bioenergetics 1412(3), 262‑272. and Bistawroos, A. E. (2010). Role of oxidative
Portakal, O., Özkaya, O., Inal, M. E., Bozan, B., stress and nitric oxide in the protective effects
Koşan, M., and Sayek, I. (2000). Coenzyme Q10 of α-lipoic acid and aminoguanidine against iso-
concentrations and antioxidant status in tissues niazid-rifampicin-induced hepatotoxicity in rats.
of breast cancer patients. Clinical Biochemistry Food and Chemical Toxicology 48, 1869‑1875.
33(4), 279‑284. Sandhya, B., Manoharan, S., Lavanya, G. S., and
Principal, S. G., Quiles, J. L., Tortosa, C. L. R., Manmohan, Ch. R. (2010). Lipid peroxidation
Rovira, P. S., and Tortosa, M. R. (2010). New and antioxidant status in prostate cancer patients.
advances in molecular mechanisms and the pre- Indian Journal of Science and Technology 3(1)
vention of Adriamycin toxicity by antioxidant 83‑86.
nutrients. Food and Chemical Toxicology 48, Sarkar, F. H., Li, Y., Wang, Z., and Kong, D.
1425‑1438. (2009). Cellular signaling perturbation by natu-
Punnonen, K., Ahotupa, M., Asaishi, K., Hyöty, ral products. Cellular Signaling 21, 1541‑1547.
M., Kudo, R., and Punnonen, R. (1994). Anti- Schapira, A. H. V. (2004). Mitochondrial dys-
oxidant enzyme activities and oxidative stress in function in Parkinson’s disease. Cell Death and
human breast cancer. Journal of cancer Research Differentiation 14, 1261‑1266.
and Clinical Oncology 120(6), 374‑377.
Senthil, K., Aranganathan, S., and Nalini, N.
Rabinowitz, J. D and White, E. (2010). Autoph- (2004). Evidence of oxidative stress in the cir-
agy and Metabolism. Science 330, 1344‑1348. culation of ovarian cancer patients. Clinica Chi-
Ramachandran, L., Krishnan, C. V., and Nair, C. mica Acta 339, 27‑32.
K. K. (2010). Radioprotection by α-Lipoic Acid Sherman, S. E. and Lippard, S. J. (1987). Struc-
Palladium Complex Formulation (POLY-MVA) tural Aspects of Platinum Anticancer Drug Inter-
in Mice. Cancer Biotherapy and Radiopharma- actions with DNA. Chem. Rev. 87, 1153‑1181.
ceuticals 25(4), 395‑399.
Simonnet, H., Alazard, N., Pfeiffer, K., Gallour,
Ramakrishnan, N., Wolfe, W. W., and Catravas, C., Béroud, C., Demont, J., Bouvier, R., Schäg-
G. N. (1992). Radioprotection of Hematopoietic ger, H., and Godinot, C. (2002). Low mitochon-
Tissues in Mice by Lipoic Acid. Radiation Re- drial respiratory chain content correlates with
search 130, 360‑365. tumor aggressiveness in renal cell carcinoma.
Rajneesh, C. P., Manimaran, A., Sasikala, K. Carcinogenesis 23, 759‑768.
R., and Adaikappan, P. (2008). Lipid peroxida- Singh, K. K. (Ed.) (2006). In Oxidative Stress,
tion and antioxidant status in patients with breast Disease and Cancer. Imperial College Press,
cancer. Singapore Med. J. 49(8), 640‑643. London, WC2H 9HE.
Rau, T., Alsfasser, R., Zahl, A., and van Eldik, Sinha, R. J., Singh, R., Mehrotra, S., and Singh,
R. (1998). Structural and Kinetic Studies on the R. K. (2009). Implications of free radicals and
Formation of Platinum(II) and Palladium(II) antioxidant levels in carcinoma of the breast: A
Complexes with L-Cysteine-Derived Ligands. never-ending battle for survival. Indian Journal
Inorg. Chem. 37, 4223‑4230. of Cancer 46(2), 146‑150.
Roscoe, S. G. (1996). Electrochemical Investiga- Strasdeit, H., von Dollen, A., and Duhme, A. K.
tions of the Interfacial Behavior of Proteins. In (1995). Metal Complexes of the Drug and Coen-
Modern Aspects of Electrochemistry. B. E. Con- zyme α-Lipoic acid and Related Ligands. Con-
References 229
tents of 7th EBIC Conference Abstracts Issue, C Warburg, O. (1956). On the Origin of Cancer
45. J. Inorg. Biochem 59, 228. Cells. Science 123, 309‑314.
Sudheesh, N. P., Ajith, T. A., Janardhanan, K. K., Weiner, W. J., Shulman, L. M., and Lang, A. E.
and Krishnan, C. V. (2009). Palladium α-lipoic (2007). Parkinson’s Disease, 2nd ed. The John
acid complex formulation enhances activities Hopkins University, Maryland, USA.
of Krebs cycle dehydrogenases and respiratory Winklhofer, K. F and Haass, C. (2010). Mito-
complexes I-IV in the heart of aged rats. Food chondrial dysfunction in Parkinson’s disease.
and Chemical Toxicology 47, 2124‑2128. Biochim Bipophys Acta (BBA) Molecular Basis
Sudheesh, N. P., Ajith, T. A., Janardhanan, K. of Disease 1802, 29‑44.
K., and Krishnan, C. V. (2010). Effect of POLY- Wood, Z. A., Poole, L. B., and Karplus, P. A.
MVA, a palladium α-lipoic acid complex formu- (2003). Peroxoredoxin Evolution and the Reg-
lation against declined mitochondrial antioxidant ulation of Hydrogen Peroxide. Science 300,
status in the myocardium of aged rats. Food and 650‑653.
Chemical Toxicology 48, 1858‑1862.
Sucheta, A., Ackrell, B. A. C., Cochran, B., and
Armstrong, F. A. (1992). Diode-like behavior of 8
a mitochondrial electron transport enzyme. Na-
Adamekova, E., Markova, M., Kubatka, P., Bo-
ture 356, 361‑362.
jkova, B., Ahlers, I., and Ahlersova, E. (2003).
Tabassum, H., Parvez, S., Pasha, S. T., Banerjee, NMU-induced mammary carcinogenesis in fe-
B. D., and Raisuddin, S. (2010). Protective ef- male rats is influenced by repeated psychoemo-
fect of lipoic acid against methotrexate-induced tional stress. Neoplasma 50, 428‑432
oxidative stress in liver mitochondria. Food and
Anokhin, P. K. (1970). The theory of a func-
Chemical Toxicology 48, 1973‑1979.
tional system. Uspekhi Matematicheskikh Nauk
Tanriverdi, T., Hanimoglu, H., Kacira, T., Sanus, 1, 19‑54.
G. Z., Kemerdere, R., Atukeren, P., Gumustas,
Bekhterev, V. M. (1998). Suggestion and its
K., Canbaz, B., and Kaynar, M. Y. (2007). Glu-
role in social life. Transaction Publishers, New
tathione peroxidase, glutathione reductase and
Brunswick, New Jersy.
protein oxidation in patients with glioblastoma
multiforme and transitional meningioma. J. Can- Bernstein, N. A. (1967). The coordination and
cer Res Clin Oncol. 133, 627‑633. regulation of movements. Pergamon Press, Ox-
ford.
Thomas, D. K. and Maass, O. (1958). Electro-
lytic Conductance in Hydrogen Peroxide-Water Dimsdale, J. E. (2008). Psychological stress and
Mixtures. Can. J. Chem. 36, 449‑455. cardiovascular disease. Journal of the American
College of Cardiology 51, 1237‑1246.
Van Hellemond, J. J., Klockiewicz, M., Gaas-
enbeek, C. P. H., Roos, M. H., and Tielens, A. Gidron, Y., Russ, K., Tissarchondou, H., and
G. M. (1995). Rhodoquinone and Complex II of Warner, J. (2006). The relation between psycho-
the Electron Transport Chain in Anaerobically logical factors and DNA-damage: A critical re-
Functioning Eukaryotes. Journal of Biological view. Biological Psychology 72, 291‑304.
Chemistry 270, 31065‑31070. Grossardt, B. R., Bower, J. H., Geda, Y. E., Colli-
Vazquez, A, Liu, J, Zhou, Y., and Oltvai, Z. N. gan, R. C., and Rocca, W. A. (2009). Pessimistic,
(2010) Catabolic efficiency of aerobic glycoly- anxious, and depressive personality traits predict
sis: The Warburg effect revisited. BMC Systems all-cause mortality: The mayo clinic cohort study
Biology 4, 58, doi 1752-0509/4/58. of personality and aging. Psychosomatic Medici-
ne 71, 491‑500.
Voet, D. and Voet, J. G. (1995). Biochemistry,
2nd ed. John Wiley & Sons, Inc., New York. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J.,
Murray, T., and Thun, M. J. (2008). Cancer Sta-
Wallace, D. C. and Fan W. (2010). Energetics,
tistics. Cancer Journal for Clinicians 58, 71‑96.
epigenetics, mitochondrial genetics. Mitochon-
drion 10, 12‑31.
Knox, S. S. (2001). Psychosocial stress and the opment and progression of cancer. International
physiology of atherosclerosis. Advances in Psy- Review of Psychiatry 17, 515‑527.
chosomatic Medicine 22, 139‑151. Rodin, G., Lo, C., Mikulincer, M., Donner, A.,
Leont’ev, A. N. (1978). Activity, consciousness, Gagliese, L., and Zimmermann, C. (2009). Path-
and personality. Englewood Cliffs, New Jersy, ways to distress: The multiple determinants of
Prentice-Hall. depression, hopelessness, and the desire for has-
Lester, D. (2009). Voodoo death. Omega (West- tened death in metastatic cancer patients. Social
port) 59, 1‑18. Science and Medicine 68, 562‑569.
Levav, I., Kohn, R., Iscovich, J., Abramson, J. Rotenberg, V. S. and Arshavsky, V. V. (1984).
H., Tsai, W. Y., and Vigdorovich, D. (2000). Search activity and adaptation. Nauka, Moscow.
Cancer incidence and survival following be- Rotenberg, V. S. (2009). Search activity concept:
reavement. American Journal of Public Health Relationship between behavior, health and brain
90, 1601‑1607. functions. Activitas Nervosa Superior 51, 12‑44.
Li, J., Johansen, C., Brønnum–Hansen, H., Roy-Byrne, P. P., Davidson, K. W., Kessler, R.
Stenager, E., Koch-Henriksen, N., and Olsen, J. C., Asmundson, G. J. G., Goodwin, R. D., Kub-
(2004). The risk of multiple sclerosis in bereaved zansky, L., Lydiard, R. B., Massie, M. J., Katon,
parents: A nationwide cohort study in Denmark. W., Laden, S. K., and Stein, M. B. (2008). Anxi-
Neurology 62, 726‑729. ety disorders and comorbid medical illness. Fo-
Lloyd-Williams, M., Shiels, C., Taylor, F., and cus 6, 467‑485.
Dennis, M. (2009). Depression–an independent Seymour, J. and Benning, T. B. (2009). Depres-
predictor of early death in patients with ad- sion, cardiac mortality and all-cause mortality.
vanced cancer. Journal of Affective Disorders Advances in Psychiatric Treatment 15, 107‑113.
113, 127‑132. Shpagina, L. A., Ermakova, M. A., Volkova, E.
Luria, A. R. (1970). The functional organization A., and Iakovleva, S. A. (2008). Clinical, func-
of the brain. Scientific American 222, 66‑78. tional and biochemical characteristics of arte-
Maslow, A. H. (1943). A theory of human moti- rial hypertension in military men under chronic
vation. Psychological Review 50, 370‑396. stress. Meditsina Truda i Promyshlennaia Ekolo-
giia 7, 24‑29.
McEwen, B. S. (2007). Physiology and neurobi-
ology of stress and adaptation: Central role of the Simon, N. M., Smoller, J. W., McNamara, K.
brain. Physiological Reviews 87, 873‑904. L., Maser, R. S., Zalta, A. K., Pollack, M. H.,
Nierenberg, A. A., Fava, M., and Wong, K. K.
Mravec, B., Gidron, Y., and Hulin, I. (2008). (2006). Telomere shortening and mood disor-
Neurobiology of cancer: Interactions between ders: Preliminary support for a chronic stress
nervous, endocrine and immune systems as a model of accelerated aging. Biological Psychia-
base for monitoring and modulating the tumoro- try 60, 432‑435.
genesis by the brain. Seminars in Cancer Biol-
ogy 18, 150‑163. Spinelli, S., Chefer, S., Suomi, S. J., Higley, J.
Dee., Barr, C. S., and Stein, E. (2009). Early-life
Nemeroff, C. B. (2008). Recent findings in the stress induces long-term morphologic changes
pathophysiology of depression. Focus 6, 3‑14. in primate brain. Archives of General Psychiatry
Ostrander, M. M., Ulrich-Lai, Y. M., Choi, D. 66, 658‑665.
C., Richtand, N. M., and Herman, J. P. (2006). Surtees, P. G., Wainwright, N. W. J., Luben, R.
Hypoactivity of the hypothalamo-pituitary-ad- N., Wareham, N. J., Bingham, S. A., and Khaw,
renocortical axis during recovery from chronic K. T. (2008). Depression and ischemic heart
variable stress. Endocrinology 147, 2008‑2017. disease mortality: Evidence from the EPIC-Nor-
Perelman, Ya. (2008). Physics for entertainment, folk United Kingdom prospective cohort study.
Book two. Hyperion, New York. American Journal of Psychiatry 165, 515‑523.
Reiche, E. M., Morimoto, H. K., and Nunes, S. Ukhtomsky, A. A. (1927). The dominant as a
M. (2005). Stress and depression-induced im- working principle of nervous centers. Russkii
mune dysfunction: Implications for the devel- Fiziologicheskii Zhurnal 6.
References 231
Ukhtomsky, A. A. (1966). The Dominant. Nau- causing Phaeoyphomycosis in humans. Nat. J.

ka, Moscou. Homeopath. 4, 1‑3.
Uznadze D. N. (1997). The theory of installation. Halliwell, B., Gutteridge, J. M. C., and Cross, C.
Metsniereba, Tbilisi. E. (1992). Free radicals, antioxidants and human
Yousef, N. (2008). From the wild side. History disease: Where are we now? J. Lab. Clin. Med.
Workshop 65, 213‑220. 119(6), 598–620.
In vivo cancer model (1976‑1982). Developmen-
tal therapeutic programme, Division of Cancer
9 Treatment, National Cancer Institute, NIH Pub-
lication No. 84–2695, Pg no. 25, Protocol No.
Anbuselvam, C., Vijayavel, K., and Balasubra-
3MBH2, 1984.
maniam, M. P. (2007). Protective effect of Oper-
culina turpethum against 7,12-dimethylbenz(a) Kanekar, S. A. (1962). Biological study of breast
anthracene induced oxidative stress with refer- cancer in the albino strain of mouse inbred at the
ence to breast cancer in experimental rats. Chem. ICRC. M.Sc. thesis. University of Bombay.
Biol. Interact. 168, 229‑236. Khoobchandani, M, Ojeswi, B. K., Hazra, D.
Barros, A. C. S. D., Muranaka, E. N. K., and K., and Srivastava, M. M. (2009). Brassica ol-
Mori, L. J. (2004). Induction of experimen- eracea extracts: Potential for antioxidative and
tal mammary carcinogenesis in rats with 7, anticancer activities for B16F10 & B16F1 mice
12-dimethylbenz(a)anthracene. Cancer Pharma. melanoma cell lines. Nat. Acad. Sci. Let. 32(9,
21, 257‑261. 10), 297‑302.
Bryle, P., Leon, M. E., and Maisonneuve, P. Khoobchandani, M, Ojeswi, B. K., Sharma, B,
(2003). Cancer control in women. Int. J. Gyn- and Srivastava, M. M. (2009). Chenopodium al-
acecol. Obes. 83, 179‑202 bum preventive progression of cell growth and
enhancement in cell toxicity in human breast
Comporti, M. (1989). Three models of free-radi-
cancer cell lines. Oxi. Med. Cellular Longivity
cals induced cell injury. Chem. Biol. Intract. 72,
2(3), 160‑165.
1–56.
Kun-Young, K., Jae, H., Sun, Y. K., and Chi, H.
Deb, L., Dubey, S. K., Jain, A., Pandian, G. S.,
C. (2003). Cadmiun induced acute lung injury
and Rout, S. P. (2007). Antidiarrhoel activity of
and tunel expression of apoptosis in respiratory
Thuja occidentalis Linn ethanol extract on ex-
cells. J. Korean Med. Sci. 18, 655‑662.
perimental animal. Ind. Drug 44, 319‑321.
Lee, S. E., Ju, E. M., and Kim, J. H. (2002). An-
DeSantis, C, Center, M. M, Sighel, R., and Je-
tioxidant activity of extracts from Euryale ferox
mal, A. (2009‑2010). Breast cancer facts and
seed. Exp. Mol. Med. 34(2), 100‑106.
figures. American Cancer Society, Department
of Surveillance and Health Policy Research, At- Millspaugh, C. F. (1974). American Medicinal
lanta, Georgia 1‑40. Plant-Thuja occidentalis. Dover Publications,
New York.
Dubey, S. K and Batra, A. (2008). Hepatoprotec-
tive activity from ethanol fraction of Thuja occi- Nam, S. H and Kang, M. Y. (2008). Antioxidant
dentalis Linn. Asian J. Chem. Res. 1, 32‑35. activity of medicinal plants. Pharma Biotech 42,
409‑415.
Frieauff, W., Hartmann, A., and Suter, W. (2001).
Automatic analysis of slides processed in the Naser, B, Bodinet, C, Tegtmeier, M., and Lind-
Comet assay. Mutagenesis 16(2), 133‑137. equest, V. (2009). Thuja occidentalis (Arbor-
vitae): A review of its pharmaceutical, pharma-
Ghumare, S. S. and Cunningham, J. E. (2007).
cological and clinical properties. Adv. Access.
Breast cancer trends in Indian residents and emi-
Publ. 2, 69‑78.
grants portend an emerging epidemic for India.
Asian Pac. J. Cancer Prev. 8, 507‑512. Ojeswi, B. K, Khoobchandani, M., and Srivas-
tava, M. M. (2009). Green Chemicals: Prospects
Gupta, G. (2002). In-vitro Antimycotic potential
and Future in designing new drugs for cancer. Int.
of Thuja occidentalis against Curvularia-lunata
J. Waste Wat. Treat. Green Chem. 1(1), 17‑21.
Ojeswi, B. K., Khoobchandani, M., Hazra, D. creases oxidative damage in human lympho-
K., and Srivastava, M. M. (2010). Protective cytes. Cancer Research 56, 12911295.
effect of Thuja occidentalis against DMBA in- Hogan, F. S., Krishnegowda, N. K., Mikhailova,
duced breast cancer with reference to oxidative M., and Kahlenberg, M. S. (2007). Flavonoid,
stress. Human Exp. Toxi. 29(5), 369‑375. Silibinin, Inhibits Proliferation and Promotes
Ojeswi, B. K., Khoobchandani, M., Hazra, D. K., Cell-Cycle Arrest of Human Colon Cancer. Jour-
and Srivastava, M. M. (2009). In vitro antibreast nal of Surgical Research 143, 58–65.
cancer efficacy of two indigenous plants on hu- Kaur, M. and Agarwal, R. (2007). Silymarin and
man cancer cell line MCF-7. National Academy epithelial cancer chemoprevention: How close
Science Letter 32 (3, 4), 105‑109. we are to bedside? Toxicology and Applied Phar-
Savage, J. R. K. (1993). Update on target theory macology 224, 350–359.
as applied to chromosomal aberrations. Env. Kedar, N., Prasad, K. N., Kumar, A., Kochupil-
Mol. Mutagen. 22, 198‑202. lai, V., and Cole, W. C. (1999). High Doses of
Shimada, K., Fujikawa, K., Yahara, K., and Na- Multiple Antioxidant Vitamins: Essential Ingre-
kamura, T. (1992). Antioxidative properties of dients in Improving the Efficacy of Standard
xanthin on autoxidation of soybean oil in cy- Cancer Therapy. Journal of American College of
clodextrin emulsion. J. Agric. Food Chem. 40, Nutrition 18(1), 1325.
945–948. Labriola, D. and Livingston, R. (1999). Possible
Sunde, R. A. and Hoekstra, W. G. (1980). Struc- interactions between dietary antioxidants and
ture, synthesis and function of glutathione per- chemotherapy. Oncology 13, 10031012.
oxidase. Nutr. Rev. 6, 265–273. Lamm, D. L., Riggs, D., Shriver, J., VanGilder,
Werneke, U, Ladenheim, D., and McCarthy, T. P., Rach, J., and DeHaven, J. (1994). Megadose
(2004). Complementary alternative medicine vitamins in bladder cancer: A double-blind clini-
for cancer: A review of effectiveness and safety. cal trial. Journal of Urology 151, 2126.
Cancer Therapy 2, 475‑500. Lamson, D. W. and Brignall, M. S. (1999). Anti-
Yao, M., Song, D. H., Rana, B., and Wolfe, M. oxidants in cancer therapy: their actions and in-
M. (2002). COX-2 selective inhibition reverses teractions with oncologic therapies. Alternative
the tropic properties of gastrin in colorectal can- Medicine Review 4, 304329.
cer. Brit. J. Can. 87, 574–579. Lamson, D. W. and Brignall, M. S. (2000) Anti-
oxidants and cancer therapy II: Quick reference
guide. Alternative Medicine Review 5, 152163.
10
Lee, I. M., Cook, N. R., and Manson, J. E.
Block, K. I. (2004). Antioxidants and Cancer (1999). Beta-carotene supplementation and in-
Therapy: Furthering the Debate. Integrative cidence of cancer and cardiovascular disease:
Cancer Therapy 3, 342348. Women’s Health Study. Journal of National
Borek, C. (2004). Dietary antioxidants and hu- Cancer Institute 91, 2102–2106.
man cancer. Integrative Cancer Therapy 3, Liu, Q. Y. and Tan, B. K. H. (2002). Dietary Fish
333341. Oil and Vitamin E Enhance Doxorubicin. Ef-
Carmia, B. (2004). Dietary Antioxidants and Hu- fects in P388 Tumor-Bearing Mice. Lipids 37,
man Cancer. Integrative Cancer Therapy 3, 333. 549–556.
Chinery, R., Brockman, J. A., Peeler, M. O., Prasad, K. N. (2004). Multiple dietary antioxi-
Shyr, Y., Beauchamp, R. D., and Coffey, R. J. dants enhance the efficacy of standard and ex-
(1997). Antioxidants enhance the cytotoxicity of perimental cancer therapies and decrease their
chemotherapeutic agents in colorectal cancer: A toxicity. Integrative Cancer Therapy 3, 10322.
p53-independent induction of p21WAF1/CIP1 via C/ Sangeetha, P., Das, U. N., Koratkar, R., and
EBP β. Nature Medicine 3, 1233–1241. Suryaprabha, P. (1990). Increase in free radical
Duthie, S. J., Ma, A., Ross, M. A., and Collins, generation and lipid peroxidation following che-
A. R. (1996). Antioxidant supplementation de- motherapy in patients with cancer. Free Radical
Biology Medicine 8, 1519.
References 233
Saxena, A., Saxena, A. K., Singh, J., and (2001). Prognostic factors analysis of 17,600
Bhushan, S. (2010). Natural antioxidants syn- melanoma patients: validation of the American
ergistically enhance the anticancer potential of Joint Committee on Cancer melanoma staging
AP9-cd, a novel lignan composition from Ce- system. Journal of Clinical Oncology 19(16),
drus deodara in human leukemia HL-60 cells. 3622–3634.
Chemico. Biological Interactions 188, 580590. Barnhill, R. L., Piepkorn, M. W., Cochran, A. J.,
Singh, S. K., Shanmugavel, M., Kampasi, H., Flynn, E., Karaoli, T., and Folkman, J. (1998).
Singh, R., Mondhe, D. M., Rao, J. M., Adwan- Tumor vascularity, proliferation, and apopto-
kar, M. K., Saxena, A. K., and Qazi, G. N. (2007) sis in human melanoma micrometastases and
Chemically standardized isolates from Cedrus macrometastases. Archive Dermatology 134(8),
deodara stem wood having anticancer activity. 991–994.
Planta Medical 73, 519526. Bianco, V. (1995). Rocket, an ancient underuti-
Weij, N. I., Hopman, G. D., Wipkink-Bakker, A., lized Vegetable crop and its potential. Rocket Ge-
Lentjes, E. G. W. M., Berger, H. M., Cleton1, F. netic Resources Network. S. Paludosi (compiler).
J., and Osanto, S. (1998). Cisplatin combination IPGRI, Rome, pp. 35–58.
chemotherapy induces a fall in plasma antioxi- Cassileth, B. (2009). Mediterranean diet. Oncol-
dants of cancer patients. Annals of Oncology 9, ogy 23(14), 1315.
13311337.
Chen, S. and Andreasson, E. (2001). Update on
Whelan, R. L., Horvath, K. D., Gleason, N. R., glucosinolate metabolism and transport. Plant
Forde, K. A., Treat, M. D., Teitelbaum, S. L., Physiology and Biochemistry 39, 743–758.
Bertram, A., and Neugut, A. I. (1999). Vitamin
and calcium supplement use is associated with Comporti, M. (1989). Three models of free-
decreased adenoma recurrence in patients with a radicals induced cell injury. Chemico Biological
previous history of neoplasia. Dis. Colon Rectum Interaction, 72, 1–56.
42, 212217. Diepgen, T. L. and Mahler, V. (2002). The epi-
demiology of skin cancer. The British Journal of
Dermatology 146(61), 1–6.
11 Fahey, J. W., Zhang, Y., and Talalay, P. (1997).
Altekruse, S. F., Kosary, C. L., Krapcho, M., Broccoli sprouts: An exceptionally rich source of
Neyman, N., Aminou, R., Waldron, W., Ruhl, inducers of enzymes that protect against chemi-
J., Howlader, N., Tatalovich, Z., Cho, H., Mari- cal carcinogens. Proceeding of Natural Academ-
otto, A., Eisner, M. P., Lewis, D. R., Cronin, K., ic Science USA 89, 10367–10372.
Chen, H. S., Feuer, E. J., Stinchcomb, D. G., and
Frieauff, W., Hartmann, A., and Suter, W. (2001).
Edwards, B. K. (2010). SEER Cancer Statistics
Automatic analysis of slides processed in the
Review, 1975–2007, National Cancer Insti-
Comet assay. Mutagenesis 16(2), 133–137.
tute. M. D. Bethesda, http://seer.cancer.gov/csr
/1975_2007/, based on November 2009 SEER Greenlee, R. T., Hill-Harmon, M. B., Murray, T.,
data submission, posted to the SEER web site. and Thun, M. (2001). Cancer statistics. CA Can-
cer Journal of Clinical 51, 15–36.
Auerbach, C. (1962). Mutation: An introduction
to research on mutagenesis Part I: Methods. Oli- Guerrero, R. F., Garcia-Parrilla, M. C., Puertas,
ver and Boyed, Edinburgh, pp. 422–428. B., and Cantos-Villar, E. (2009). Wine, resvera-
trol and health: A review. Natural Products Com-
Balch, C. M. (1992). Cutaneous melanoma:
munication 4(5), 635–658.
Prognosis and treatment results worldwide, Se-
min. Surgical Oncology 8, 400–414. Halliwell, B., Gutteridge, J. M. C., and Cross, C.
E. (1992). Free radicals, antioxidants and human
Balch, C. M., Soong, S. J., Gershenwald, J. E.,
disease: Where are we now? Journal of Labora-
Thompson, J. F., Reintgen, D. S., Cascinelli
tory Clinical Medicine 119(6), 598–620.
N., Urist, M., McMasters, K. M., Ross, M. I.,
Kirkwood, J. M., Atkins, M. B., Thompson, J.
A., Coit, D. G., Byrd, D., Desmond, R., Zhang,
Y., Liu, P. Y. Lyman, G. H., and Morabito, A.
Higdon, V., Delage, B., Williams, D. E., and Nascimento, N. C. D., Fragoso, V., Moura, D.
Dashwood, R. H. (2007). Cruciferous vegetables J., Silva, A. C. R., Fett-Neto, A. G., and Saffi,
and human cancer risk: Epidemiologic evidence J. (2007). Antioxidant and Antimutagenic ef-
and mechanistic basis. Pharmacological Re- fects of the Crude Foliar Extract and the Alka-
search 55, 224–236. loid Brachycerine of Psychotria brachyceras.
Hoey, S. H. E., Devereux, C. E. J., and Murray, Environmental and Molecular Mutagenesis 48,
L. (2007). Skin cancer trends in Northern Ireland 728–734.
and consequences for provision of dermatology Ojeswi, B. K., Khoobchandani, M., and Srivas-
services. The British Journal of Dermatology tava, M. M. (2009). Green Chemicals: Prospects
156, 1301–1307. and Future in designing new drugs for cancer.
Jeffery, E. H. and Jarrell, V. (2001). Cruciferous International Journal of Waste Water Treatment
vegetables and cancer prevention. In Handbook and Green Chemistry 1(1), 17–21.
of Nutraceuticals and Functional Foods. R. E. C. Pauwels, E. K. J. and Covas, M. I. (2009). The
Wildman, (Ed.). CRC Press LLC, Boca, Raton, Mediterranean diet, part I: The anticancer effect
FL, pp. 169–192. of olive oil. Drugs Future 34(4), 307–313.
Khoobchandani, M., Ojeswi, B. K., Ganesh, N., Poppel, V. G., Verhoeven, D. T., Terhagen, H.,
Srivastava, M. M., Gabbanini, S., Matera, R., and Goldbohm, R. A. (1999). Brassica vegeta-
Iori, R., and Valgimigli, L. (2010). Antimicrobial bles and cancer prevention. Epidemiology and
properties and analytical profile of traditional mechanisms. Advance Experimental Medicinal
Eruca sativa seed oil. Comparison with various Biology 472, 159–168.
aerial and root plant extracts. Food Chemistry Savage, J. R. K. (1993). Update on target theory
120 (1), 217–224. as applied to chromosomal aberrations. Environ-
Khoobchandani, M., Ojeswi, B. K., Hazra, D. mental and Molecular Mutagenesis 22, 198–207.
K., and Srivastava, M. M. (2009). Brassica ol- Schmid, W. (1975). The micronucleus test. Mu-
eracea extracts: Potential for antioxidative and tation Research 31, 9–12.
anticancer activities for B16F10 & B16F1 mice
melanoma cell lines. National Academy Science Shimada, K., Fujikawa, K., Yahara, K., and Na-
Letter 32 (9, 10), 297–302. kamura, T. (1992). Antioxidative properties of
xanthin on autoxidation of soybean oil in cyclo-
Kun-Young, K., Jae, H., Sun, Y. K., and Chi, H. dextrin emulsion. Journal of Agricultural and
C. (2003). Cadmiun induced acute lung injury Food Chemistry 40, 945–948.
and tunel expression of apoptosis in respiratory
cells. Journal of Korean Medical Science 18, Shureiqi, I., Reddy, P., and Brenner, D. E. (2000).
655–662. Chemoprevention: General perspective. Critical
reviews in oncology/hematology 33, 157–167.
Lee, W. R., Shen, S. C., Lin, H. Y., Hou, W. C.,
Yang, L. L., and Chen, Y. C. (2002). Wogonin Tang, F. Y., Cho, H. J., Pai, M. H., and Chen,
and fisetin induce apoptosis in human promy- Y. H. (2009). Concomitant supplementation of
eloleukemic cells, accompanied by a decrease lycopene and eicosapentaenoic acid inhibits the
of reactive oxygen species, and activation of proliferation of human colon cancer cells. Jour-
caspase 3 and Ca(2þ)-dependent endonuclease. nal of Nutrition Biochemistry 20(6), 426–434.
Biochemical Pharmacology 63, 225–236. Tseng, M., Sellers Thomas, A., Vierkant Robert,
Melchini, A., Costa, C., Traka, M., Miceli, N., A., Kushi Lawrence, H., and Vachon Celine, M.
Mithen, R., De Pasquale, R., and Trovato, A. (2008). Mediterranean diet and breast density in
(2009). Erucin, a new promising cancer chemo- the Minnesota Breast Cancer Family Study. Nu-
preventive agent from rocket salads, shows anti- trition Cancer 60(6), 703–709.
proliferative activity on human lung carcinoma Verhoeven, D. T., Goldbohm, R. A., Van Pop-
A549 cells. Food Chemical and Toxicology pel, G., Verhagen, H., and Brandt van den, P.
47(7), 1430–1436. A. (1996). Epidemiological studies on Brassica
Nam, S. H. and Kang, M. Y. (2008). Antioxidant vegetables and cancer risk. Cancer Epidemiol-
activity of medicinal plants. Pharmaceutical ogy, Biomarkers and Prevention 5, 733–748.
Biotechnology 42, 409–415.
References 235
Warwick, S. I. (1994). Guide to the wild germ- Bartels, A., Cerna, R., Kistner, C., Thoma, A.,
plasm of Brassica and allied crops. Part V. Life Hudert, F., Janke, C., and Dekorsy, T. (2007).
History and Geographical Data for wild species Ultrafast time-domain spectroscopy based on
in the tribe Brassicaceae (Cruciferae). Agricul- high-speed asynchronous optical sampling. Rev.
tural Canadian Technical Bulletin 2E, 61. Sci. Instrum. 78, 035‑107.
Zeven, A. C. and de Wet, J. M. J. (1982). Dic- Butt, H. J., Cappella, B., and Kappl, M. (2005).
tionary of cultivated plants and their regions of Force measurements with the atomic force mi-
diversity, 2nd ed. Centre for Agricultural Pub- croscope: technique, interpretation and applica-
lishing and Documentation, Wageningen, p. 107. tions. Surf. Sci. Rep. 59, 1–152.
Zhang, Y., Talalay, P., Cho, C., and Posner, G. Cecchini, M., Piazza, V., Simoni, G. D., Bel-
H. (1992). A Major Inducer of Anticarcinogenic tram, F., Beere, H. E., and Ritchie, D. A. (2006).
Protective Enzymes from Broccoli: Isolation and Acoustic charge transport in a n-i-n three termi-
Elucidation of Structure. Proceedings of the Na- nal device. Appl. Phys. Lett. 88, 212101.
tional Academy of Sciences 89, 2399–2403. Chao, W., Harteneck, B. D., Liddle, J. A., Ander-
son, E. H., and Attwood, D. T. (2005). Soft X-ray
microscopy at a spatial resolution better than 15
12 nm. Nature 435, 1210–1213.
Albers, B. J., Schwendemann, T. C., Baykara, Cilento, F., Giannetti, C., Ferrini, G., Dal Conte,
M. Z., Pilet, N., Liebmann, M., Altman, E. I., S., Sala, T., Coslovich, G., Rini, M., Cavalleri,
and Schwarz, U. D. (2009). Three-dimensional A., and Parmigiani, F. (2010). Ultrafast insula-
imaging of short-range chemical forces with pi- tor-to-metal phase transition as a switch to mea-
cometre resolution. Nat. Nanotechnol. 4, 307. sure the spectrogram of a supercontinuum light
Albrecht, T. R., Grütter, P., Horne, D., and Rugar, pulse. Appl. Phys. Lett. 96, 021‑102.
D. (1991). J. Appl. Phys. 69, 668. Cross, S. E., Jin, Y. S., Rao, J., and Gimzewski,
Andreeva, N., Ferrini, G., Bassi, D., Cappa, F., J. K. (2007). Nanomechanical analysis of cells
Cocconcelli, P. S., Prato, S., Troian, B., and Par- from cancer patients. Nat. Nanotechnol. 2, 780–
migiani, F. (2009). Preliminary results of com- 783.
bined scanning near-field optical microscopy de Lima, M. M., Hey, R., Santos, P. V., and Can-
and atomic force microscopy applied to a model tarero, A. (2005). Phonon-induced optical super-
biological system: Clostridium tyrobutyricum lattice. Phys. Rev. Lett. 94, 126‑805.
spores. Boll. Geof. Teor. Appl. 50, 396.
Ferrini, G., De Carlo, L., Giannetti, C., and Par-
Andreeva, N., Bassi, D., Cappa, F., Cocconcel- migiani, F. (2009). Evanescent wave spectros-
li, P. S., Parmigiani, F., and Ferrini, G. (2010). copy of methylene blue solutions at surfaces us-
Nanomechanical analysis of Clostridium tyrobu- ing a continuum generated by a nonlinear fiber.
tyricum spores. Micron 41, 945. Optics and Spectroscopy 107, 464; ОПТИКА И
Auld, B. (1990). Acoustic Fields and Waves in СПЕКТРОСКОПИЯ 107, 490.
Solids. Krieger, Malabar FL. Fukuma, T., Higgins, M. J., and Jarvis, S. P.
Banfi, F., Pressacco, F., Revaz, B., Giannetti, (2007). Direct Imaging of Lipid-Ion Network
C., Nardi, D., Ferrini, G., and Parmigiani, F. Formation under Physiological Conditions by
(2010). Abinitio thermodynamics calculation of Frequency Modulation Atomic Force Micros-
all-optical time-resolved calorimetry of nanosize copy. Phys. Rev. Lett. 98, 106101.
systems: Evidence of nanosecond decoupling of Garcia, R. and Perez, R. (2002). Dynamic atomic
electron and phonon temperatures. Phys. Rev. B force microscopy methods. Surf. Sci. Rep. 47,
81, 155‑426. 197.
Bartels, A., Dekorsy, T., and Kurz, H. (1999). Giannetti, C., Revaz, B., Banfi, F., Montagnese,
Coherent zone-folded longitudinal acoustic pho- M., Ferrini, G., Cilento, F., Maccalli, S., Vavas-
nons in semiconductor superlattices: Excitation sori, P., Oliviero, G., Bontempi, E., Depero, L.
and detection. Phys. Rev. Lett. 82, 1044–1047. E., Metlushko, V., and Parmigiani, F. (2007).
Thermomechanical behavior of surface acous-
tic waves in ordered arrays of nanodisks studied Landau, L. D., Pitaevskii, L. P., Kosevich, A. M.,
by near-infrared pump-probe diffraction experi- and Lifshitz, E. M. (1959). Theory of Elasticity.
ments. Phys. Rev. B 76, 125‑413. Pergamon Press Ltd., Oxford, U.K.
Giannetti, C., Banfi, F., Nardi, D., Ferrini, G., Landau, L. D. and Lifshitz, E. M. (1986 ). The-
and Parmigiani, F. (2009). Ultrafast Laser Pulses ory of Elasticity. Butterworth-Heinemann, Ox-
to Detect and Generate Fast Thermomechanical ford, U.K.
Transients in Matter. IEEE Photonics Journal 1, Lantz, M. A., Hug, H. J., Hoffmann, R., van
21. Schendel, P. J. A., Kappenberger, P., Martin, S.,
Giessibl, F. J. (2003). Advances in atomic force Baratoff, A., and Güntherodt, H. J. (2001). Quan-
microscopy. Rev. Mod. Phys. 75, 949. titative Measurement of Short-Range Chemical
Gross, L., Mohn, F., Moll, N., Liljeroth, P., and Bonding Forces. Science 291, 25‑80.
Meyer, G. (2009). The Chemical Structure of a Lin, H. Y., Maris, H. J., Freund, L. B., Lee, K.
Molecule Resolved by Atomic Force Micros- Y., Luhn, H., and Kern, D. P. (1993). Study of
copy. Science 325, 1110. vibrational modes of gold nanostructures by pi-
Gundrum, B. C., Cahill, D. G., and Averback, R. cosecond ultrasonics. J. Appl. Phys. 73, 37.
S. (2005). Thermal conductance of metal–metal Lyeo, H. K. and Cahill, D. G. (2006). Thermal
interfaces. Phys. Rev. B 72, 245‑426. conductance of interfaces between highly dis-
von Gutfeld, R. J. and Nethercot, A. H. (1966). similar materials. Phys. Rev. B 73, 144‑301.
Temperature dependence of heat-pulse propaga- Mallat, S. G. (1999). A Wavelet Tour of Signal
tion in sapphire. Phys. Rev. Lett. 17, 868–871. Processing. Academic Press, New York.
Harrick, N. J. and du Pré, F. K. (1966). Effective Malegori, G. and Ferrini, G. (2010a). Tip-sample
Thickness of Bulk Materials and of Thin Films interactions on graphite studied in the thermal
for Internal Reflection Spectroscopy. Appl. Op- oscillation regime. J. Vac. Sci. Technol. B 28,
tics 11, 17‑39. C4B18.
Highland, M., Gundrum, B. C., Koh, Y. K., Malegori, G. and Ferrini, G. (2010b). Tip-sample
Averback, R. S., Cahill, D. G., Elarde, V. C., interactions on graphite studied using the wave-
Coleman, J. J., Walko, D. A., and Landahl, E. C. let transform. Beilstein J. Nanotechnol 1, 172.
(2007). Ballistic-phonon heat conduction at the Malegori, G. and Ferrini, G. (2011a). Wavelet
nanoscale as revealed by time-resolved X-ray transforms to probe long- and short-range forces
diffraction and time-domain thermoreflectance. by thermally excited dynamic force spectrosco-
Phys. Rev. B 76, 075‑337. py. Nanotechnology 22, 195‑702.
Hurley, D. H., Wright, O. B., Matsuda, O., Su- Malegori, G. and Ferrini, G. (2011b). Wavelet
zuki, T., Tamura, S., and Sugawara, Y. (2006). Transforms to Probe the Torsional Modes of a
Time-resolved surface acoustic wave propaga- Thermally Excited Cantilever across the Jump-
tion across a single grain boundary. Phys. Rev. to-Contact Transition: Preliminary Results. J.
B 73, 125‑403. Surf. Sci. Nanotech 9, 228.
Hurley, D. H., Lewis, R., Wright, O. B., and Mat- Maris, H. (1998). Picosecond ultrasonics. Sci.
suda, O. (2008). Coherent control of gigahertz Amer 278, 86.
surface acoustic and bulk phonons using ultra-
fast optical pulses. Appl. Phys. Lett. 93, 113101. Maznev, A. A. (2009). Laser-generated surface
acoustic waves in a ring-shaped waveguide reso-
Juvé, V., Scardamaglia, M, Maioli, P., Crut, A., nator. Ultrasonics 49, 1–3.
Merabia, S., Joly, L., Del Fatti, N., and Vallée, F.
(2009). Cooling dynamics and thermal interface Morita, S., Wiesendanger, R., and Meyer, E.
resistance of glass-embedded metal nanopar- (Eds.) (2002). Noncontact Atomic Force Micros-
ticles. Phys. Rev. B 80, 195‑406. copy. Springer, Berlin.
Knoll, W. (1998). Interfaces and thin films as Nardi, D., Banfi, F., Giannetti, C., Revaz, B.,
seen by bound electromagnetic waves. Annu. Ferrini, G., and Parmigiani, F. (2009). Pseudo-
Rev. Phys. Chem. 49, 569–638. surface acoustic waves in hypersonic surface
phononic crystals. Phys. Rev. B 80, 104‑119.
References 237
Ohline, S. M., Lee, S., Williams, S., and Chang, Tobey, R. I., Gershgoren, E. H., Siemens, M. E.,
C. (2001).Chem. Phys. Lett. 346, 9. Murnane, M. M., Kapteyn, H. C., Feurer T., and
Perrin, B., Péronne, E., and Belliard, L. (2006). Nelson K. A. (2004). Appl. Phys. Lett. 85, 564.
Generation and detection of incoherent pho- Voisin, C., Del Fatti, N., Christofilos, D., and
nons in picosecond ultrasonics. Ultrasonics 44, Vallée, F. (2000). Time resolved investigation of
e1277–e1281. the vibrational dynamics of metal nanoparticles.
Profunser, D. M., Wright, O. B., and Matsuda, O. Appl. Surf. Sci. 164, 131–139.
(2006). Imaging ripples on phononic crystals re- Wilson, M. (2007). Ultrafast laser spectroscopy
veals acoustic band structure and Bloch harmon- measures heat flow through molecular chains.
ics. Phys. Rev. Lett. 97, 055‑502. Physics Today 60, 20‑23.
Radmacher, M., Fritz, M., Kacher, C. M., Cleve-
land, J. P., and Hansma, P. K. (1996). Measur-
ing the viscoelastic properties of human platelets 13
with the atomic force microscope. Biophys. J. Apóstolo, J. L. A., Batista ,A. C., Macedo, C. M.
70, 556–567. R., and Pereira, E. M. R. (2006). Suffering and
Robillard, J. F., Devos, A., and Roch-Jeune, I. comfort in patients who undergo chemotherapy.
(2007). Time-resolved vibrations of two-dimen- Referência 2ª Série (3), 55–64.
sional hypersonic phononic crystals. Phys. Rev. Apóstolo, J. L. A., Cunha, S. R. P., Cristo, J. M.
B 76, 092‑301. F., and Lacerda, R. P. P. (2004). Experience of
Rosenbluth, M. J., Lam, W. A., and Fletcher, D. the patients’ relatives with oncological illness
A. (2006). Force microscopy of nonadherent terminal condition in a center of palliative care.
cells: a comparison of leukemia cell deformabil- Revista Investigação Enfermagem 10, 28–37.
ity. Biophys. J. 90, 2994–3003. Barbosa, A. (2006). Sofrimento (Suffering).
Schirmeisen, A. (2010). Surfing on graphite In Manual de cuidados paliativos (Manual of
waves. Nat. Mat. 9, 615. palliative care), A. Barbosa and I. Neto (Eds.),
Fundação Calouste Gulbenkian, Lisboa, Portu-
Siemens, M. E., Li, Q., Murnane, M. M., gal, pp. 397–417.
Kapteyn, H. C., Yang, R., Anderson, E. H., and
Nelson, K. A. (2009). High-frequency surface Capela, R. (2010). O sofrimento de doentes on-
acoustic wave propagation in nanostructures cológicos em cuidados paliativos (Suffering of
characterized by coherent extreme ultraviolet oncological patients in palliative care). Master
beams. Appl. Phys. Lett. 94, 093‑103. Dissertation, Universidade Católica Portuguesa,
Porto, Portugal.
Siemens, M. E., Li, Q., Yang, R., Nelson, K. A.,
Anderson, E. H., Murnane, M. M., and Kapteyn, Cassell, E. J. (1991). Recognizing suffering.
H. C. (2010). Nature Materials 9, 26. Hastings Center Report 21(3), 24–31.
Stoner, R. J. and Maris, H. (1993). Kapitza con- Cassell, E. J. (1999). Diagnosing suffering: A
ductance and heat flow between solids at tem- perspective. Annals of Internal Medicine 131(7),
peratures from 50 to 300 K. Phys. Rev. B 48, 531–534.
16373–16387. Fleming, M. (2003). Dor sem nome: Pensar o so-
Sugawara, Y., Wright, O. B., Matsuda, O., frimento (Pain without a name: The thinking of
Takigahira, M., Tanaka, Y., Tamura, S., and Gu- suffering). Afrontamento, Porto, Portugal.
sev, V. E. (2002). Watching ripples on crystals. Ferreira, F. L. (2009). Suffering of women hav-
Phys. Rev. Lett. 88, 185‑504. ing chemotherapy after mastectomy. Referência
Sugimoto, Y., Pou, P., Abe, M., Jelinek, P., Pérez, 2ª Série(10), 65–76.
R., Morita, S., and Custance O. (2007). Chemi- Frankl, V. (2004). El hombre en busca de sentido
cal identification of individual surface atoms by (Man’s search for meaning). Herder, Barcelona.
atomic force microscopy. Nature 446, 64.
Gameiro, M. G. H. (2000). IESSD: Um instru- Kolcaba, K. Y. and Kolcaba, R. J. (1991). An

mento para a abordagem do sofrimento na doen- analysis of the concept of comfort. Journal of
ça (ISSEI: An instrument to approach suffering Advanced Nursing 16(11), 1301–1310.
in illness), Referência 4, 57–66. Mcintyre, T. M. (2004). Perda e sofrimento na
Instituto de Lexicologia e Lexicografia da doença: Contributo da psicologia da saúde (Loss
Academia das Ciências de Lisboa (Institute of and suffering during illness: Contributions from
Lexicology and Lexicography of the Lisbon health psychology), Psychologica 35, 167–179.
Academy of Sciences) (2001). Dicionário da Neto, I. (2004). Para além dos sintomas: A dig-
língua portuguesa contemporânea (Dictionary nidade e o sentido da vida na prática dos cui-
of contemporary Portuguese language). Editorial dados paliativos. In A dignidade e o sentido da
Verbo, Lisboa, Portugal. vida: uma reflexão sobre a nossa existência
Kolcaba, K. Y. (1991). A taxonomic structure for (Beyond symptoms: dignity and purpose of life
the concept comfort. Image: Journal of Nursing in the practice of palliative care. In Dignity and
Scholarship 23(4), 237–240. purpose of life: a reflection on our existence).
Kolcaba, K. Y. (2003). Comfort theory and I. Neto, H. Aitken, and T. Paldrön (Eds.), Per-
practice. A vision for holistic health care and gaminho, Cascais, Portugal, pp. 13–40.
research. Springer Publishing Company, New
York.
Index
A clinical applications, 36–37

DS polypeptide chains, 37
Adenosine triphosphate (ATP), 74
EPR effect, 37
Angiogenesis
immobilization of, 37
breast cancer, 3
leishmanicidal activity, 37
Down’s syndrome, 3
magnetic nanoparticles, 37–38
factors
P. oreades and P. hypochondrialis,
angiopoetins, 5
37
angiostatin, 6
antibacterial activity of lysozyme, 33
CXC-chemokines, 6
Bacillus anthracis, 31
endostatin, 6–7
bacteriophage drug-carrying platforms,
fibroblast growth, 5
35
integrins, 4–5
bioactive glasses, 34
thrombospondin-1, 6
biopolymers, 34–35
transforming growth, 5
carbon nanotubes and fullerenes, 32
vascular endothelial growth, 5
mechanism of action, 32–33
malignant metastatic cancer, 2–3
micellar nanocarriers, 36
mechanism, 2–4
multifunctional nanoplatforms
modulators of, 4
biofilm infections, 36
physiological conditions, 2
mechanism of action, 36
switch, 3
MRI, 36
tumor progression
nanoemulsion
angiogenic switch, 4
Burkholderia cepacia, 35–36
EPCs, 4
cystic fibrosis, 35
vasculogenesis and lymphangiogen-
systemic toxicity, 36
esis, 4
nanomedicines, 31
VEGF expression, 4
therapeutic and diagnostic applications,
types, 4
31
VEGF, 3
Antimicrobial peptides (AMPs)
Angiogenic factors of angiogenesis
biocidal efficiency, 37
angiopoetins, 5
clinical applications, 36–37
CXC-chemokines, 6
DS polypeptide chains, 37
fibroblast growth, 5
EPR effect, 37
integrins, 4–5
immobilization of, 37
transforming growth, 5
leishmanicidal activity, 37
vascular endothelial growth, 5
magnetic nanoparticles, 37–38
Angiostatic factors of angiogenesis
P. oreades and P. hypochondrialis, 37
angiostatin, 6
Antioxidants and combinatorial therapies
endostatin, 6–7
in cancer treatment
thrombospondin-1, 6
anticancer potential
Antibacterial therapy and diagnosis
Cedrus deodar, 156
AMPs
curcumin, 156
biocidal efficiency, 37
cytotoxic and apoptotic, 156
lignan, 156 Atomic force microscopy (AFM)

anti-neoplastic leads, 155 Brownian motion, 182
chemotherapeutic agents, 155 cantilever’s dynamical parameters, 180
cisplatin-induced oxidative damage, cell stiffness, 179
156–157 chemical specificity, 183
cytotoxic potential of chemotherapeutic force measurements, 179
in colorectal cancer, 157 Fourier transform (FT), 180–181
enhancement effect of doxorubicin, 157 frequency shift, 182
heated debates Heisenberg box, 182
cancer therapeutic agents, 158 Hooke’s law, 179
chemotherapy protocols, 159 linear elasticity theory, 179
growth-inhibitory effects, 158 mechanical properties, 179
growth promoting agents, 158–159 quantitative analysis, 183
modulating effects, 155 temporal oscillations of cantilever, 180
Silymarin time–frequency representation, 180
cancer protective effects, 158 Wavelet coefficients, 182
liver treatment, 158 Wavelet transform (WT), 180–181
polyphenolic, 158 Young modulus, 179–180
silibinin’s mechanism of action, 158 ATP. See Adenosine triphosphate (ATP)
therapeutic effects, 156
Apoptosis B
cell death, 2 Brain tumours
chromalolysis, 2 anaplasia and pleomorphism, 20
combinational therapy, 2 astrocytic, 19
disastrous effects, 2 auto fluorescence spectroscopy, 21
mechanisms, 2 catalytic nanomedicine, 21
Applications of chitosan nanoparticles chemotherapeutic agents, 21
anti-fungal delivery, 46 fluorescence characteristics, 22
anti-microbial agents, 47–48 hemorrhage and necrosis, 19
articular joint therapy, 50 histological characteristics, 19
brain delivery, 51 infiltration pattern, 20
buccal and sublingual delivery system, molar absorptivity and fluorescence
50 quantum, 21
gene delivery system, 46–47 necrotic regions, 20
hepatitis treatment, 47 optical spectroscopic methods, 21
for hyperglycemia, 45 radiation and chemotherapy, 20
molecular imaging, 44 Temozolomide, 20–21
ocular delivery system, 50–51 Breast cancer
protein delivery, 44 antioxidant levels, 86
tumor targeting, 48–49 antioxidant status, 84
as vaccination, 44–45 D-loop region, 84
via nasal route, 45–46 MDA, 85
Aptamers superoxide dismutase and catalase
advantage, 10 levels, 85–86
oligonucleic acid/peptide molecules, 10 Brownian motion
riboswitches, 10 AFM, 182
therapeutic application, 10 Burkholderia cepacia, 35–36
Index 241
C non-specific uptake, 52
specific uptake, 52–53
Caenorhabditis elegans, 16
Chronic lymphoid leukemia (CLL), 8
Cancer disease in LPD
disease dominant, 139
D
iceberg psychosomatics, 139
malignant tumors diseases, 138 Diseases in LPD
psychogenic pathological condition, 133
carcinogenesis, 138 psychogenic component, 133–134
component, 138–139 subdominants of, 133
Chitosan nanoparticles Doctrine of dominant in LPD
applications of brain and spinal marrow, 130
anti-fungal delivery, 46 cortical, 131
anti-microbial agents, 47–48 features, 130
articular joint therapy, 50 spiritual anatomy, 131
brain delivery, 51 topographic center, 130
buccal and sublingual delivery vital functions, 131
system, 50 Down’s syndrome, 3
gene delivery system, 46–47
hepatitis treatment, 47 E
for hyperglycemia, 45 Endothelial progenitor cells (EPCs), 4
molecular imaging, 44 Eruca sativa inhibits melanoma growth
ocular delivery system, 50–51 antimutagenic activity
protein delivery, 44 chromosomal aberration, 163–164
tumor targeting, 48–49 histopathology study, 165–166
as vaccination, 44–45 micronuclei formation, 165
via nasal route, 45–46 micronucleus assay, 163, 165
biomedical material, 41 MPCE and MNCE, 164–165
drug delivery system, 40 mutation effect, 165
hydrophilic drug entrapment, 40 antitumor activity, 162–163
ionic gelation and complex coacerva- cruciferous family, 160–161
tion, 40–41 effect of seed oil, 163
mechanism of transport, 51 free radical scavenging, 161–162
paracellular transport, 51–52 hydroxyl and DPPH radicals, 161–162
pharmaceutical applications, 40 isothiocyanates, 161
preparation methods mediterranean diet, 160
complex coacervation, 43 seed oil, chemistry profile
covalent cross-links, 43 chemical structure of isothiocya-
desolvation, 43 nates, 166
emulsion-droplet coalescence, 44 chromatograms, 166–167
ionic cross linking/ionic gelation, epidemiological studies, 168
42–43 isothiocyanate formation, 167
reverse micellar, 44 mechanism depicting isothiocya-
schematic representation of, 42 nates, 168
solubility, 40 taramira plant, 166–167
structure of, 41
therapeutic uses, 40 F
transcellular transport Fourier transform (FT), 180–181
G NPt treatment, 29
vascularization, 29
Gene-based therapy in mitochondrial dys-
Hooke’s law, 179
function and cancer
Human concept in LPD
Herculean challenge, 78
basic integrated functional conditions,
isocitrate dehydrogenase 1 (IDH1), 78
134
pharmacogenomics, 78
central nervous system, 131
SNPs, 78
and current subdominants, 132
Glioblastoma multiformes (GBM)
dynamics of integrated condition,
biopsies, 25
135–137
brain tissue fluorescence, 26
Homo ferus, 131
brain tumours (see Brain tumours)
iceberg psychosomatics, 135–136
cavity size, 26
mental and somatic processes, 131
diffuse reflectance spectra, 27
occurrence and loss of, 133
fluorescence measurements, 22, 25–26
organs and systems, 131
Hematoxyline-Eosine (H-E), 22–23
self-destruction dominant, 136–137
histopathology
anaplasia, 29
K
bacterial-onslaught phase, 28
coagulative necrosis, 28 Kolcaba’s conceptual framework, 188
focal bacterial/fungal infections, 28 Kretchmann configuration, 172
malignant tissue, 29
necrotic tissue, 28 L
neoplasm of astrocytes, 29 Life purpose dominant (LPD)
NPt treatment, 29 in animal, 132
vascularization, 29 cancer disease
and Masson methods, 22–23 disease dominant, 139
microsurgical techniques, 24 iceberg psychosomatics, 139
morphological change, 23 malignant tumors diseases, 138
MRI studies, 24 psychogenic carcinogenesis, 138
nanostructured biocatalysts, 22 psychogenic component, 138–139
optical spectra emissions, 25 chronic disease formation, 129–130
spectrofluorometer, 23–24 concept implications, 140
tumor removal and surgical repair, 24 diseases
and type I meningioma, 84 pathological condition, 133
WHO, 19 psychogenic component, 133–134
Glutathione (GSH), 144–145 subdominants of, 133
doctrine of dominant
H brain and spinal marrow, 130
Heisenberg box, 182 cortical, 131
Histopathology for GBM features, 130
anaplasia, 29 spiritual anatomy, 131
bacterial-onslaught phase, 28 topographic center, 130
coagulative necrosis, 28 vital functions, 131
focal bacterial/fungal infections, 28 in human
malignant tissue, 29 basic integrated functional condi-
necrotic tissue, 28 tions, 134
neoplasm of astrocytes, 29 central nervous system, 131
Index 243
and current subdominants, 132 alpha lipoic acid, 95–97

dynamics of integrated condition, Alzheimer’s disease, 95
135–137 apoptosis/programmed cell death, 81
Homo ferus, 131 ascorbic acid, 91–92
iceberg psychosomatics, 135–136 ATP
mental and somatic processes, 131 turnover rates, 74
occurrence and loss, 133 breast cancer, 84–86
organs and systems, 131 catabolic processes, 80–81
self-destruction dominant, 136–137 chain reactions and free radical produc-
nervous centers and vital functions, 130 tion, 90–91
Pavlov’s doctrine, 130 defects, 75
sole correct scientific idea, 130 depletion and deletion, 83
diameter and length, 79
M drug induced toxicity, amelioration, 126
Malondialdehyde (MDA) effects, 73–74
in breast cancer, 85 electron
Medicinal plants transfer, 76
anticancer agents, 68 transport chain complexes, subunits
baikal skullcap (Scutellaria baicalen- of, 81
sis), 68 electronic properties, 81, 116
blocking agents, 71 admittance comparison of, 114
carcinogenesis, 71 current oscillations, 113
cytotoxic activity, 69 energy-converting organelles, 74
kinase enzymes, 71 enzymatic composition, 80
phytochemicals, 68–71 Faradaic impedance, 113
Plantago sp., 69 free radicals and antioxidants
potential bioactive compounds, 68 capacitation process, 88
suppressing agents, 71 chain reactions, 88
Mendelian rules enzymes, 89
for mitochondrial dysfunction and hydroxyl and superoxide, 87
cancer, 81 phagocytosis, 88
Methylene Blue (MB), 172 physiologic reducing agents, 88
molar absorbance spectra of, 173 reduction potentials, 89–90
Micro ribonucleic acids (miRNAs), 1 therapeutic applications, 90
breast carcinomas, 8 function of, 81
CLL patients, 8 GBM and type I meningioma, 84
downstream effecter molecules, 9 gene-based therapy
genetic analysis, 8 Herculean challenge, 78
hypoxia, 9 isocitrate dehydrogenase 1 (IDH1),
messenger RNAs, 7 78
potential anticancer drugs, 9 pharmacogenomics, 78
protein coding genes, 7 SNPs, 78
stem loop structures, 7 glutathione, 93
tumor necrosis factor alpha (TNF-α), 8 glycolysis, 74–75
miRNAs. See Micro ribonucleic acids hallmarks of, 78–79
(miRNAs) heteroplasmy and homoplasmy, 83
Mitochondrial dysfunction and cancer HIV infection, oxidative stress in, 126
holistic medicine, 73 Nyquist plot, 116–117, 120, 122

hydrogen peroxide, 93–95 transition metals, 116
impedance spectroscopy, 111–112 tunnel diode characteristics, 123
Krebs cycle, enzymatic reactions, 75 specific cytotoxic drugs, 73
matrix, 74 spin coupling in electron transfer
Mendelian rules, 81 cytochromes, 124–125
mutated genes in different cancers, electron donor, 125
82–83 EPR and ESR measurement, 124
mutations, 82, 84 free radical reaction mechanism, 125
natural compounds, 74 iron-sulfur clusters, 124
NDR, 114 pulse radiolysis studies, 125
neuron degeneration, 115 semiquinone radicals, 124
Nyquist plot, 113 vectorial translocation, 124
ovarian cancer, 86 tumorogenesis, 84
oxidative damage, 82 tunnel-diode behavior, 110–111
palladium α-lipoic acid complex, vitamin E, 92–93
97–110, 123–124 Warburg effect/aerobic glycolysis,
formulation, 75–76 83–84
Parkinson’s disease, 95 Multifunctional nanoplatforms
platinum(II) and palladium(II) com- of antibacterial therapy and diagnosis
plexes biofilm infections, 36
adenocarcinoma MCF7, 77–78 mechanism of action, 36
biological action, 77 MRI, 36
cisplatin, 77
gastrointestinal toxicity, 76 N
hydrolysis, 77 Nanoemulsion of antibacterial therapy and
ovarian cancer treatment, 76 diagnosis
palladium-based drugs, 78 Burkholderia cepacia, 35–36
solid tumor chemotherapy, 76 cystic fibrosis, 35
powerhouse of cell, 79 systemic toxicity, 36
production of ATP, 82 Nanostructures, optical and mechanical
prostate cancer, 86–87 investigations
pyruvate dehydrogenase, 75 AFM
Raoult’s law, 115 Brownian motion, 182
regulation of calcium homeostasis, 79 cantilever’s dynamical parameters,
ROS, 76 180
SDH and shapes, 79 cell stiffness, 179
simple molecules, electronic properties chemical specificity, 183
of force measurements, 179
electrochemical impedance tech- Fourier transform (FT), 180–181
nique, 116 frequency shift, 182
enzyme activity, 116 Heisenberg box, 182
FAD, 119, 120 Hooke’s law, 179
impedance data, 120–121 linear elasticity theory, 179
impedance spectra, 118 mechanical properties, 179
L-lysine, 119 quantitative analysis, 183
neurotransmitter histamine, 122
Index 245
temporal oscillations of cantilever, aptamers, 10

180 carcinogenesis, 1
time–frequency representation, 180 delivering anti-angiogenic/anticancer
Wavelet coefficients, 182 molecules, 11–12
Wavelet transform (WT), 180–181 gene delivery to target cells, 16
Young modulus, 179–180 lactoferrin, 11
ligand-receptor complexes, 170 micro RNAS in, 7–9
molecular detection at surfaces miRNAs, 1
aggregation states, 172 RNA interference (RNAi)
angle of incidence, 171 Caenorhabditis elegans, 16
application, 174 endocytotic pathway, 17
evanescent wave, 171 gene therapy, 16
evanescent wave optics, 172 mechanism of, 16
Kretchmann configuration, 172 non-viral carriers, 17
Methylene Blue (MB), 172–173 therapeutic applications, 16
PCF, 171 tumor
pump and probe techniques, 174 invasion and metastasis, 1
refraction indexes, 171 suppressor genes, 1
molecular interactions, 170 uses of nano particles
single theoretical model, 170 bioactive glass, 13–15
study calcium phosphate ceramics, 13
applications, 178–179 doxorubicin and taxol loaded
energy density, 178 ACNC-NPs, 15
energy radiation, 176 lodamin, 12
features, 176 paciltaxil and ceramide, 12
gigahertz–terahertz frequency range, tumor vasculature, 12
174 VHL disease, 10–11
light penetration depth, 178 Negative differential resistance (NDR),
oscillation dynamic, 178 114
PEM, 177 Nyquist plot, 113, 116–117, 120, 122
phononic crystal properties, 174
pseudo-surface acoustic waves, 174 O
pump and probe experiment, Ovarian cancer, 86
175–176
refractive index, 175 P
relative intensity, 177
Palladium α-lipoic acid complex
SAWs, 175
antioxidant activity and prophylactic
thermal conductance, 175
effects, 108–110
thermal gradients, 174, 178
clinical human studies, 103–104
thermoelastic stress, 175
clinical veterinary studies, 101–102
time-resolved measurements, 177
effect of, 99
techniques, 170
glioblastoma tumor volume, 100–101
Nanotechnological based systems for
mitochondrial studies
cancer
antitumor activity, 106
angiogenesis, 1, 2–7
effect of, 104, 106
anti-angiogenic drugs, 1
enzyme activities, 104–105
apoptosis, 2
influence of, 107
Krebs cycle, 104 comfort and suffering, 189–192

lactate dehydrogenase and pyruvate concept of suffering
dehydrogenase, 108 dimensions, 185
lipid peroxidation and GSH level, existential and Socio-relational, 186
106 human experience, 185
phosphorylation, 108 human potential, 186
succinate dehydrogenase, 107 physical and psychological, 186
therapeutic effect, 105 profile of suffering, 190
open label veterinary oncology study, Prostate cancer, 86–87
102
oxygen radical absorbance capacity, 98 R
phase microscopy pictures, 123 Raoult’s law, 115
protection from radiation, 108 Reactive oxygen species (ROS), 76
self-assembly of, 123–124 Recent advances in cancer therapy
synthesis of, 97 causes, 67
transient ischemia studies with gerbils, chemotherapeutic agents, 67
102–103 dietary supplements, 68
in vitro cell line studies, 98–100 medicinal plants
in vivo studies, 100–101 anticancer agents, 68
voltammetric studies, 98 baikal skullcap (Scutellaria ba-
Parkinson’s disease icalensis), 68
in mitochondrial dysfunction and blocking agents, 71
cancer, 95 carcinogenesis, 71
PCF. See Photonic-crystal fibers (PCF) cytotoxic activity, 69
Photoelastic modulator (PEM), 177 kinase enzymes, 71
Photonic-crystal fibers (PCF), 171 phytochemicals, 68–71
P. hypochondrialis, 37 Plantago sp., 69
Phytosynthesis in silver nanoparticles potential bioactive compounds, 68
Capsicum annum, 59 suppressing agents, 71
green chemistry methods, 58 natural products, 67
mechanisms, 59 phytochemicals, 67–68
Menthapiperita leaf extract, 59 Taxol (paclitaxel), 67
plant materials choice, 59–60 RNA interference (RNAi)
protocols and materials, 58 Caenorhabditis elegans, 16
spectrometric analysis, 59 endocytotic pathway, 17
vascular plants leaf extracts, 59 gene therapy, 16
P. oreades, 37 mechanism of, 16
Portuguese cancer patients non-viral carriers, 17
comfort therapeutic applications, 16
ease and peaceful contentment, 187 ROS. See Reactive oxygen species (ROS)
health and well-being component,
187 S
Kolcaba’s conceptual framework,
SAWs. See Surface acoustic waves
188
(SAWs)
predict phenomena, 187
SDH. See Succinate dehydrogenase (SDH)
relief, 187–188
Silver nanoparticles
strengthen greatly, 188
applications, 54
Index 247
biomedical applications reducing and capping agent, 62

antibacterial effect, 64 Tollen’s method, 62
anti-infective topical medicine, 63 top-down approach, 55
antimicrobial activity, 63 Silymarin
bactericidal effect, 63–64 cancer protective effects, 158
dimorphic transition, 65 liver treatment, 158
as disinfectant, 63 polyphenolic, 158
fungicidal effect, 65 silibinin’s mechanism of action, 158
hygienic and healing purposes, 63 Single nucleotide polymorphisms (SNPs),
inhibitory and lethal effect, 64 78
ulcer treatment, 63 Succinate dehydrogenase (SDH)
biomemetric synthesis, 61 mitochondrial dysfunction and cancer,
biosynthetic methods, 55–56 79
chemical synthesis Surface acoustic waves (SAWs), 175
chemical reductants, 61 Synthesis from saccharides in silver
reducing agent, 60 nanoparticles
stabilizing agents, 61 irradiation method, 62
surfactants, 61 reducing and capping agent, 62
wet chemical reduction, 60 Tollen’s method, 62
classical chemical method, 55
intracellular magnetite/greigite nano- T
crystals, 56 Thuja occidentalis and breast cancer che-
microbial synthesis moprevention
antimicrobial activity, 56–57 antibreast cancer activity
bacterial strains, 58 bioassay of EtOAc fractions, 153
bactericidal effects, 56 doxorubicin drug, 149
fungal strains used for biosynthesis, EtOAc and MeOH extracts effect,
57–58 149–150
Fusarium oxysporium and Verticil- growth inhibitory activity, 146–148
lium sp, 56 TBE and MTT bioassays, 146
Fusarium semitectum, 56 tumor volume and body weight,
mechanism for, 57 148–149
physical synthesis procedures, 55 antimutagenic activity, 151–152
phytosynthesis carcinogenesis, 141
Capsicum annum, 59 cases of, 141
green chemistry methods, 58 chromosomal aberration in bone mar-
mechanisms, 59 row, 151–152
Menthapiperita leaf extract, 59 coniferous pyramidal features, 141
plant materials choice, 59–60 cupressaceae, 141
protocols and materials, 58 ethyl acetate extract, chromatographic
spectrometric analysis, 59 fractionation, 152–153
vascular plants leaf extracts, 59 experiments and protocol, 142–143
polyoxometalates method, 61 folk medicine, 141
properties, 54 free radical scavenging activity
radiation chemical method, 55 antioxidant, 143
synthesis from saccharides DMBA toxicity, 145
irradiation method, 62 effect of solvent extracts, 144
EtOAc extract, effect of, 145 Tumor progression for angiogenesis

glutathione (GSH), 144–145 angiogenic switch, 4
order of potency, 143 EPCs, 4
solvent extracts, 143 vasculogenesis and lymphangiogenesis,
haematoxylin and eosin stained slides, 4
150 VEGF expression, 4
histopathological study, 150
morbidity and mortality, 141 V
phytochemical prevention, 141 Vascular endothelial growth factor
therapies, 141 (VEGF), 3
Tollen’s method VEGF. See Vascular endothelial growth
for silver nanoparticles, 62 factor (VEGF)
Transcellular transport of chitosan
nanoparticles W
non-specific uptake, 52
Wavelet transform (WT), 180–181
specific uptake, 52–53
Tumor necrosis factor alpha (TNF-α)
in micro RNAS, 8

Unity of Mind and Body The Concept of Li

Uploaded by

Copyright:

Available Formats

Unity of Mind and Body The Concept of Li

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Unity of Mind and Body The Concept of Li

Uploaded by

Copyright:

Available Formats

Nanomedicine and

Apple Academic Press

Apple Academic Press Inc.

Exclusive worldwide distribution by CRC Press, a Taylor & Francis Group

International Standard Book Number: 978-1-926895-18-5 (Hardback)

Printed in the United States of America on acid-free paper

Library of Congress Control Number: 2012935668

Library and Archives Canada Cataloguing in Publication

R857.N34N36 2012 610.28 C2011-908742-1

Sabu Thomas, PhD

Dr. Yang Weimin

1. Nanotechnological Based Systems for Cancer................................................. 1

2. In vivo Spectroscopy for Detection and Treatment of GBM with

3. Nanobiotechnology for Antibacterial Therapy and Diagnosis..................... 31

5. Synthesis and Biomedical Application of Silver Nanoparticles.................... 54

6. Recent Advances in Cancer Therapy Using Phytochemicals....................... 67

7. Mitochondrial Dysfunction and Cancer: Modulation by Palladium

9. Thuja occidentalis and Breast Cancer Chemoprevention........................... 141

10. Antioxidants and Combinatorial Therapies in Cancer Treatment............ 155

11. Eruca sativa Inhibits Melanoma Growth: A Scientific Evidence................ 160

12. Optical and Mechanical Investigations of Nanostructures for

13. Suffering and Comfort in Portuguese Cancer Patients.............................. 185

João Luís Alves Apóstolo

Rita Susana Soares Capela

Hullathy Subban Ganapathy

Luis Felipe Hernández

Samarin Denis Mikhaylovich

Bukhtoyarov Oleg Viktorovich

5-ASA 5-Amino salicylic acid

EPCs Endothelial progenitor cells

MBCs Minimal bactericidal concentrations

siRNAs Small interfering ribo nucleic acid

ANGIOGENESIS AND ITS MECHANISM

Angiogenesis, depending on different physiological processes in which it is in-

ANGIOGENESIS AND TUMOR PROGRESSION

Vascular endothelial growth factor

Fibroblast Growth Factors (FGFs)

Transforming growth factor β (TGF β)

kininogen, ATIII, and chromogranin A; while down regulating downstream regulators

MICRO RNAs IN CANCER

genes by miR-130a expressed in HUVECs, in response to addition of serum (FBS)

We have previously reported the regression of solid tumors by using a combination

DELIVERING ANTI-ANGIOGENIC/ANTICANCER MOLECULES WITH

USES OF NANO PARTICLES IN CANCER TREATMENT

Calcium phosphate ceramics

Calcium phosphate cements

in any disease in vitro or in vivo in human or animal systems. It is tempting to ex-

GENE DELIVERY TO TARGET CELLS

RNAI FOR CANCER THERAPY

of CPP-mediated molecular delivery. Endosomal entrapment of RNAs delivered by

et al., 2003). The measurement of in vivo continuous and time-resolved fluorescence,

Figure 1. Experimental setup.

current supply regulated from 10 to 500 mA in order to produce a variable radiation

DISCUSSION AND RESULTS

Optical spectra emissions of non-neoplastic and neoplastic tissue were taken

Figure 6. Typical example of a well located GBM.

Table 1. Various antibacterial therapeutics and their mechanism of action.

Antibacterial Examples Developed Mechanism of Reference

Bacteriophage Drug-carrying Platforms