Digest Journal of Nanomaterials and Biostructures Vol. 3, No.3, September 2008, p.

115 - 122

NANOTECHNOLOGY IN MEDICINE AND ANTIBACTERIAL EFFECT OF

SILVER NANOPARTICLES

MRITUNJAI SINGH, SHINJINI SINGH, S. PRASADa, I. S.GAMBHIR

Department of Medicine, Institute of Medical Sciences, Banaras Hindu University,
Varanasi, INDIA-221005
a
Biochemistry & Molecular Biology Lab., Department of Zoology, Banaras Hindu
University, Varanasi, INDIA-221005

Nanotechnology is expected to open some new aspects to fight and prevent diseases using
atomic scale tailoring of materials. The ability to uncover the structure and function of
biosystems at the nanoscale, stimulates research leading to improvement in biology,
biotechnology, medicine and healthcare. The size of nanomaterials is similar to that of
most biological molecules and structures; therefore, nanomaterials can be useful for both
in vivo and in vitro biomedical research and applications. The integration of nanomaterials
with biology has led to the development of diagnostic devices, contrast agents, analytical
tools, physical therapy applications, and drug delivery vehicles. In all the nanomaterials
with antibacterial properties, metallic nanoparticles are the best. Nanoparticles increase
chemical activity due to crystallographic surface structure with their large surface to
volume ratio. The importance of bactericidal nanomaterials study is because of the
increase in new resistant strains of bacteria against most potent antibiotics. This has
promoted research in the well known activity of silver ions and silver-based compounds,
including silver nanoparticles. This effect was size and dose dependent and was more
pronounced against gram-negative bacteria than gram-positive organisms.

(Received April 29, 2008; accepted May 29, 2008)

Keywords: Nanotechnology, Silver Nanoparticles, Nanomedicine, Antibacterial effect,

Cancer, Drug delivery, Alzheimers disease.

1. Introduction

Most of the natural processes also take place in the nanometer scale regime. Therefore, a
confluence of nanotechnology and biology can address several biomedical problems, and can
revolutionize the field of health and medicine [1]. Nanotechnology is currently employed as a tool
to explore the darkest avenues of medical sciences in several ways like imaging [2], sensing [3],
targeted drug delivery [4] and gene delivery systems [5] and artificial implants [6]. The new age
drugs are nanoparticles of polymers, metals or ceramics, which can combat conditions like cancer
[7] and fight human pathogens like bacteria [8-12].
The development of new resistant strains of bacteria to current antibiotics [13] has become
a serious problem in public health; therefore, there is a strong incentive to develop new
bactericides [10]. Bacteria have different membrane structures which allow a general classification
of them as Gram-negative or Gram positive. The structural differences lie in the organization of a
key component of the membrane, peptidoglycan. Gram negative bacteria exhibit only a thin
peptidoglycan layer (~23 nm) between the cytoplasmic membrane and the outer membrane [14];
in contrast, Gram-positive bacteria lack the outer membrane but have a peptidoglycan layer of
about 30 nm thick [15]. Silver has long been known to exhibit a strong toxicity to a wide range of
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micro-organisms [16]; for this reason silver-based compounds have been used extensively in many
bactericidal applications [17, 18]. Silver compounds have also been used in the medical field to
treat burns and a variety of infections [19]. Several salts of silver and their derivatives are
commercially employed as antimicrobial agents [20]. Commendable efforts have been made to
explore this property using electron microscopy, which has revealed size dependent interaction of
silver nanoparticles with bacteria [11]. Nanoparticles of silver have thus been studied as a medium
for antibiotic delivery [21], and to synthesize composites for use as disinfecting filters [22] and
coating materials [23]. However, the bactericidal property of these nanoparticles depends on their
stability in the growth medium, since this imparts greater retention time for bacterium
nanoparticle interaction. There lies a strong challenge in preparing nanoparticles of silver stable
enough to significantly restrict bacterial growth.
Studies were carried out on both antibiotic resistant (ampicillin- resistant) and non-
resistant strains of gram-negative (Escherichia coli) and a non-resistant strain of gram-positive
bacteria (Staphylococcus aureus). A multi-drug resistant strain of gram-negative (Salmonella
typhus, resistant to chloramphenicol, amoxycilin and trimethoprim) bacteria was also subjected to
analysis to examine the antibacterial effect of the nanoparticles [24]. Efforts have been made to
understand the underlying molecular mechanism of such antimicrobial actions. The effect of the
nanoparticles was found to be significantly more pronounced on the gram-negative strains,
irrespective of whether the strains were resistant or not, than on the gram-positive organisms. We
attribute this enhanced antibacterial effect of the nanoparticles to their stability in the medium as a
colloid, which modulates the phosphotyrosine profile of the bacterial proteins and arrests bacterial
growth.
The bactericidal effect of silver ions on micro-organisms is very well known; however, the
bactericidal mechanism is only partially understood. It has been proposed that ionic silver strongly
interacts with thiol groups of vital enzymes and inactivates them [25, 26]. Experimental evidence
suggests that DNA loses its replication ability once the bacteria have been treated with silver ions
[19]. Other studies have shown evidence of structural changes in the cell membrane as well as the
formation of small electron-dense granules formed by silver and sulfur [19, 27]. Silver ions have
been demonstrated to be useful and effective in bactericidal applications, but due to the unique
properties of nanoparticles nanotechnology presents a reasonable alternative for development of
new bactericides. Metal particles in the nanometer size range exhibit physical properties that are
different from both the ion and the bulk material. This makes them exhibit remarkable properties
such as increased catalytic activity due to morphologies with highly active facets [2833]. We can
apply several electron microscopy techniques to study the mechanism by which silver
nanoparticles interact with these bacteria. We can use high angle annular dark field (HAADF)
scanning transmission electron microscopy (STEM), and developed a novel sample preparation
that avoids the use of heavy metal based compounds such as OsO4. High resolutions and more
accurate x-ray microanalysis were obtained.

2. Nanotechnology

The term "nanotechnology" was first defined by Tokyo Science University, Norio
Taniguchi in a 1974 paper [34] as follows: "'Nano-technology' mainly consists of the processing
of, separation, consolidation, and deformation of materials by one atom or one molecule.
Nanotechnology and nanoscience got a boost in the early 1980s with two major developments: the
birth of cluster science and the invention of the scanning tunneling microscope (STM). This
development led to the discovery of fullerenes in 1985.
As practiced at Rice University, there are three distinct nanotechnologies:
"Wet" nanotechnology is the study of biological systems that exist primarily in a
water environment. The functional nanometer-scale structures of interest here are genetic material,
membranes, enzymes and other cellular components. The success of this nanotechnology is amply
demonstrated by the existence of living organisms whose form, function, and evolution are
governed by the interactions of nanometer-scale structures.
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"Dry" nanotechnology derived from surface science and physical chemistry,

focuses on fabrication of structures in carbon (for example, fullerenes and nanotubes), silicon, and
other inorganic materials. Unlike the "wet" technology, "dry" techniques admit use of metals and
semiconductors. The active conduction electrons of these materials make them too reactive to
operate in a "wet" environment, but these same electrons provide the physical properties that make
"dry" nanostructures promising as electronic, magnetic, and optical devices. Another objective is
to develop "dry" structures that possess some of the same attributes of the self-assembly that the
wet ones exhibit.
Computational nanotechnology permits the modeling and simulation of complex
nanometer-scale structures. The predictive and analytical power of computation is critical to
success in nanotechnology: nature required several hundred million years to evolve a functional
"wet" nanotechnology; the insight provided by computation should allow us to reduce the
development time of a working "dry" nanotechnology to a few decades, and it will have a major
impact on the "wet" side as well.
These three nanotechnologies are highly interdependent. The major advances in each have
often come from application of techniques or adaptation of information from one or both of the
others.

3. Nanotechnology in the area of Medicine

Applying nanotechnology for treatment, diagnosis, monitoring, and control of diseases has
been referred to as nanomedicine. Although the application of nanotechnology to medicine
appears to be a relatively recent trend, the basic nanotechnology approaches for medical
application date back several decades. The first example of lipid vesicles which later became
known as liposomes were described in 1965 [35]; the first controlled release polymer system of
macromolecules was described in 1976 [36]; the first long circulating stealth polymeric
nanoparticle was described in 1994 [37]; the first quantum dot bioconjugate was described in
1998 [38, 39]; and the first nanowire nanosenser dates back to 2001 [40]. Recent Studies on new
targeted nanoparticle contrast agents for early characterization of atherosclerosis and
cardiovascular pathology at the cellular and molecular levels that might represent the next frontier
for combining imaging and rational drug delivery to facilitate personalized medicine [41].
Nanotechnology-based highly efficient markers and precise, quantitative detection devices for
early diagnosis and for therapy monitoring will have a wide influence in patient management, in
improving patients quality of life and in lowering mortality rates, in diseases like cancer and
Alzheimers disease.

Multi-functional nanoparticles for cancer therapy

Biodegradable chitosan nanoparticles encapsulating quantum dots were prepared by D. K.

Chatterjee and Y. Zhang, with suitable surface modification to immobilize both tumor targeting
agent and chemokine on their surfaces. The interactions between immune cells and tumor cells
were visualized using optical microscope. Use of Quantum dots in the treatment of cancer is a
great advancement in this area. Quantum dots glow when exposed to UV light. When injected they
seep into cancer tumour. The surgeon can see the glowing tumour. Nanotechnology could be very
helpful in regenerating the injured nerves. During the last decade, however, developments in the
areas of surface microscopy, silicon fabrication, biochemistry, physical chemistry, and
computational engineering have converged to provide remarkable capabilities for understanding,
fabricating and manipulating structures at the atomic level. The rapid evolution of this new science
and the opportunities for its application promise that nanotechnology will become one of the
dominant technologies of the 21st century.

Nanoscience enables early detection of Alzheimers Disease

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Brain represents one of the most complex systems in biomedicine. With an improved
understanding of brain functioning, better diagnosis and treatment for neurodegenerative diseases
like Alzheimers is offered by nanotechnology [42]. Presently, the prevailing problem is early
detection for effective treatment of the disease. An ideal diagnostic tool for Alzheimers disease
(AD) must have specificity & sensitivity more than 80% for its early diagnosis & excluding other
causes. Since the neurodegeneration process begins well before AD becomes symptomatic the
potential for early detection is another important characteristic of an ideal diagnostic tool.
Nanotechnology can be the basis of new tools for very early detection of AD. Nanotechnology in
the diagnosis of AD came into light after two articles were published in February 2005. The two
detection approaches proposed in those papers were the Bio-barcode assay (BCA) & Localized
surface plasmon resonance (LSPR) technology [43-44].

4. Health and safety implications from nanoparticles

Nanopollution is a generic name for all waste generated by nanodevices or during the
nanomaterials manufacturing process. This kind of waste may be very dangerous because of its
size. It can float in air the and might easily penetrate animal and plant cells causing unknown
effects. Most human-made nanoparticles do not appear in nature, so living organisms may not
have appropriate means to deal with nanowaste.
The smaller a particle, the greater its surface area to volume ratio and the higher its
chemical reactivity and biological activity. The greater chemical reactivity of nanomaterials results
in increased production of reactive oxygen species (ROS), including free radicals [45]. ROS
production has been found in a diverse range of nanomaterials including carbon fullerenes, carbon
nanotubes and nanoparticle metal oxides. ROS and free radical production is one of the primary
mechanisms of nanoparticle toxicity; it may result in oxidative stress, inflammation, and
consequent damage to proteins, membranes and DNA [45].
Nanomaterials have proved toxic to human tissue and cell cultures, resulting in increased
oxidative stress, inflammatory cytokine production and cell death [46]. Unlike larger particles,
nanomaterials may be taken up by cell mitochondria [47] and the cell nucleus [48, 49]. Studies
demonstrate the potential for nanomaterials to cause DNA mutation [49] and induce major
structural damage to mitochondria, even resulting in cell death [47, 50]. Size is therefore a key
factor in determining the potential toxicity of a particle. However it is not the only important
factor.Other properties of nanomaterials that influence toxicity include: chemical composition,
shape, surface structure, surface charge, aggregation and solubility,[45] and the presence or
absence of functional groups of other chemicals [51].
Significant environmental, health, and safety issues might arise with development in
nanotechnology since some negative effects of nanoparticles in our environment might be
overlooked. However nature itself creates all kinds of nano objects, so probable dangers are not
due to the nanoscale alone, but due to the fact that previously non-toxic materials can become
harmful when ingested or inhaled as nanoparticles. Social risks related to nanotechnology
development include the possibility of military applications of nanotechnology in biological
warfare, chemical warfare, ammunitions and armaments and even as implants for soldier
"enhancement." Enhanced surveillance capabilities through nano-sensors are also of concern to
privacy rights advocates.In discussing issues related to nanotechnology, the acronym NELSI is
used to signify nanotechnology's ethical, legal, and social implications.

5. Silver as a biocide

Silver (Ag) is a transition metal element having atomic number-47 and atomic mass-
107.87. The medicinal uses of silver have been documented since 1000 B.C. Silver is a health
additive in traditional Chinese and Indian Ayurvedic medicine [52]. Its action as an antibiotic
comes from the fact that it is a non-selective toxic "biocide." Silver based antimicrobial biocides
are used as wood preservatives. In water usage, silver and copper based disinfectants are used in
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hospital and hotel distribution systems to control infectious agents (for example, Legionella).
Silver together with copper, is commonly used to inhibit bacterial and fungal growth in chicken
farms and in post harvested cleaning of oysters. Silver used to sterilize recycled water aboard the
MIR space station and on the NASA space shuttle [53]. Microdyn (colloidal silver in gelatin) is
sold in supermarkets to disinfect salad vegetables and drinking water. Johnson Mathey Chemicals
(Nottingham, UK) developed an inorganic composite (immobilized slow-release silver product)
for use as a preservative in cosmetics, toiletries, and similar retail hygine-sensitive products [54].
In Japan, a new compound (Amenitop, silica gel microspheres containing a silver-thiosulfate
complex) is mixed into plastics for lasting antibacterial protection [18]. Silver halide is often
incorporated into prescription eye glasses for reversible photochromatic protection, as it
decreases transmitted visible light. Silver resistance is important to monitor because modern
technology has developed a wide range of products that depend on silver as a key microbial
component. In the late 1970s, Robert O. Becker discovered that silver ions promote bone growth
and kill surrounding bacteria. Silver kills some 650 different disease organisms. Silver based
topical dressing has been widely used as a treatment for infections in burns, open wounds and
chronic ulcers. The Silver nanoparticles and Ag+ carriers can be beneficial in delayed diabetic
wound healing as diabetic wounds are affected by many secondary infections. These nanoparticles
can help the diabetic patients in early wound healing with minimal scars [55]. Silver nitrate is still
a common antimicrobial used in the treatment of chronic wounds [56].

6. Colloidal Silver

Scientists have discovered that the body's most important fluids are colloidal in nature:
suspended ultra-fine particles. Blood, for example, carries nutrition and oxygen to the body cells.
This led to studies with colloidal silver (electrical silver atoms). An electro-colloidal process,
which is known to be the best method, is used for manufacturing the Colloidal silver. Colloidal
silver appears to be a powerful, natural antibiotic and preventative against infections. Acting as a
catalyst, it reportedly disables the enzyme that one-celled bacteria, viruses and fungi need for their
oxygen metabolism. They suffocate without corresponding harm occurring to human enzymes or
parts of the human body chemistry. The result is the destruction of disease-causing organisms in
the body and in the food.

7. Synthesis of silver nanoparticles

Nanometer sized silver particles were synthesized by inert gas condensation and co-
condensation techniques. Both techniques are based on the evaporation of a metal into an inert
atmosphere with the subsequent cooling for the nucleation and growth of the nanoparticles. The
size and morphology of the nanoparticles were analysed with a transmission electron microscope
(TEM). The stability of nanoparticles was examined by exposing them to ambient conditions for
one month. The antibacterial efficiency of the nanoparticles was investigated by introducing the
particles into a media containing bacteria. The antibacterial investigations were performed in
solution and on petri dishes. The silver nanoparticles were found to exhibit antibacterial effects at
low concentrations. The antibacterial properties were related to the total surface area of the
nanoparticles. Smaller particles with a larger surface to volume ratio provided a more efficient
means for antibacterial activity. The nanoparticles were found to be completely cytotoxic to E. coli
for surface concentrations as low as 8 g of Ag/cm2 [12].

8. Effect of silver nanoparticles on the phosphotyrosine profile of

bacterial proteins

By the examination of the phosphotyrosine content of proteins derived from gram positive
and negative bacteria using a specific monoclonal antibody, the possible effect of nanoparticles on
120

bacterial signal transduction affecting growth can be studied. Phosphorylation of various protein
substrates is now well established in bacterial species [57] and is found to influence bacterial
signal transduction [58]. Hardly any change in the profile of tyrosine phosphorylated proteins was
observed for S. aureus upon treatment with silver nanoparticles; however, there was noticeable
dephosphorylation of two peptides of relative masses 150 and 110 kDa in E. coli and S. typhi
exposed to nanoparticles [24]. As tyrosine phosphorylation in bacteria would lead to activation of
various protein substrates like RNA polymerase sigma factors and UDPglucose dehydrogenases
[59], decreased phosphorylation may reflect inhibition of activity of these enzymes with critical
implications on bacterial growth. A recent report has described tyrosine phosphorylation of
bacterial single-stranded DNA-binding proteins (BsSSB), ubiquitous molecules binding DNA in
various functional stages like replication and recombination [60]. A phospho-signalling pathway
has also been shown to be critical for bacterial cell cycle progression [61]. The identity of the
substrate peptides and that of the putative tyrosine phosphatases responsible for observed
dephosphorylation in gram-negative bacteria, as described by Siddhartha et al., are yet to be
established and can be the subject for future research. The present findings, along with reported
interactions of silver nanoparticles with thiol rich enzymes and bacterial genomic DNA [11], can
explain the inhibitory effect of the nanoparticles on growth of gram negative bacteria.
Interestingly, phosphorylation of protein tyrosine kinases involved in exopolysaccharide and
capsular polysaccharide biosynthesis and transport has been reported in a number of gram-
negative and gram-positive bacteria [62].

9. Conclusions

Silver nanoparticles exhibit a broad size distribution and morphologies with highly
reactive facets. The major mechanism through which silver nanoparticles manifested antibacterial
properties is by anchoring to and penetrating the bacterial cell wall, and modulating cellular
signalling by dephosphorylating putative key peptide substrates on tyrosine residues. Silver
nanoparticles act primarily in three ways against Gram-negative bacteria: (1) nanoparticles mainly
in the range of 110 nm attach to the surface of the cell membrane and drastically disturb its
proper function, like permeability and respiration; (2) they are able to penetrate inside the bacteria
and cause further damage by possibly interacting with sulfur- and phosphorus-containing
compounds such as DNA;
(3) nanoparticles release silver ions, which have an additional contribution to the
bactericidal effect of the silver nanoparticles [19]. Although bacterial cell lysis could be one of the
reasons for the observed antibacterial property, nanoparticles also modulate the phosphotyrosine
profile of putative bacterial peptides, which could thus affect bacterial signal transduction and
inhibit the growth of the organisms. The effect is dose dependent and is more pronounced against
gram negative organisms than gram-positive ones. The antibacterial effect of nanoparticles is
independent of acquisition of resistance by the bacteria against antibiotics. However, further
studies must be conducted to verify if the bacteria develop resistance towards the nanoparticles
and to examine cytotoxicity [63] of nanoparticles towards human cells before proposing their
therapeutic use.

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