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Toxicity of therapeutic nanoparticles

A total of six nanotherapeutic formulations are already approved for medical use and more are in the
approval pipeline currently. Despite the massive research effort in nanotherapeutic materials, there is
relatively little information about the toxicity of these materials or the tools needed to assess this toxicity.
Recently, the scientific community has begun to respond to the paucity of information by investing in the
field of nanoparticle toxicology. This review is intended to provide an overview of the techniques needed
to assess toxicity of these therapeutic nanoparticles and to summarize the current state of the field. We
begin with background on the toxicological assessment techniques used currently as well as considerations
in nanoparticle dosing. The toxicological research overview is divided into the most common applications
of therapeutic nanoparticles: drug delivery, photodynamic therapy and bioimaging. We end with a
perspective section discussing the current technological gaps and promising research aimed at addressing
those gaps.
keywords: contrast agent, dosing, drug delivery, nanoparticle, photodynamic Melissa A Maurer-
therapy, toxicity Jones, Kyle C Bantz*,
Sara A Love*,
Common toxicological the ethical concerns regarding the treatment Bryce J Marquis*
assessment methods of laboratory animals. Yet, in vivo studies are & Christy L Haynes†
Currently, there is a significant worldwide necessary to explore nanoparticle biodistribu- †
Author for correspondence:
research effort to fabricate novel nanoscale tion to determine appropriate cell types to be University of Minnesota,
materials and characterize their size-dependent used for in vitro studies. In vitro methods are Department of Chemistry,
chemical and physical properties. The potential relatively fast and inexpensive and minimize 207 Pleasant Street SE,
applications seem endless as researchers vary the ethical concerns regarding animals. However, Minneapolis, MN 55455, USA
chemical composition, size, shape and assembly many of these methods require more extensive Tel.: +1 612 626 1096;
of nanoparticles, in addition to the capability validation with in vivo studies to evaluate their Fax: +1 612 626 7541;
of nanoparticles to be multifunctionalized. The toxicological predictive capability. E-mail: [email protected]
proposed applications for many of these new The first step when assessing in vitro toxicity *
These authors contributed
nanomaterials are as therapeutic agents and requires choosing a model for study. Choices equally.
citation records demonstrate a nearly sevenfold that must be made when selecting a model
increase in evaluation of nanoscale materials cell include: whether to use primary culture
for therapeutic purposes over the last decade. or immortal cell lines, whether to use cells of
Toxicity assessment of these nanomaterials is human origin or animal origin (typically, rat,
necessary to facilitate integration into future mouse or hamster), whether the physiologi-
medical applications. It is likely that exploration cal function of the model cell is relevant and
of nanoparticle toxicity will yield nanoparticle whether the model cell is found in the tissue
design rules that will enable rational prediction that is likely to be exposed to the nanoparticle.
and control of toxicity. Primary culture of human cells presents the
In vivo toxicity studies provide vital informa- ideal model for studying human toxicity but
tion to assess the health and safety of nanopar- these models are generally restricted to com-
ticles [1] . Depending on the potential applica- mercially available cells capable of continuous
tion of the nanomaterial, any number of animal propagation or cell types isolated from small
models or physiological mechanisms can be samples of donor blood, such as mononuclear
studied, such as zebrafish embryo develop- cells [7] , B lymphocytes and T lymphocytes
ment [2] , rabbit ocular toxicity [3] , rat pulmo- [8] . An alternative is to use primary culture of
nary toxicity [4] and LD50 [5] , or immune cell cells from animal models, which require less
distributions in mice [6] . These types of stud- regulation [9] . Primary cell culture yields hetero
ies have several limitations, including the long geneous mixtures of cells that then typically
experimental time required, the high cost and require a density gradient or flow cytometry
10.2217/17435889.4.2.219 © 2009 Future Medicine Ltd Nanomedicine (2009) 4(2), 219–241 ISSN 1743-5889 219
Review Maurer-Jones, Bantz, Love, Marquis & Haynes
sorting to isolate the model cell of interest. colorimetrically and give a static picture of cell
These separation techniques often damage cells health. To examine the proliferation in a more
in the process of isolation. Additionally, pri- dynamic fashion, changes in the numbers of live
mary culture cells are often obtained in limited cells can be monitored over time using the same
numbers and have a limited lifetime in culture. viability assays. Another approach to assessing
For these reasons, immortal cell lines are used proliferation is through the incorporation of
commonly for in vitro toxicological testing and radiolabeled (3H) thymidine into DNA, with
a wide variety are available commercially for which increased proliferation yields increased
use as both cancer cell models and immortal- uptake of (3H) thymidine into newly synthesized
ized representations of normal cells. The major DNA [18] . An alternative to this technique is the
drawback of using immortal cell lines is that the analysis of 5‑bromo-deoxyuridine incorporation
mutations required for the immortalization of into newly synthesized DNA [19] , which is less
the lines may affect the way the cells respond to expensive and less toxic to cells.
a therapeutic nanoparticle. To improve the reli- Cell death, both through apoptosis and necro-
ability of interpretations from immortal mod- sis, is also measured during in vitro toxicological
els, studies can be compared using different cell assessment. Compromised membrane integrity
lines that have the same physiological function. is the most commonly used marker for cellular
Co-culture models can also be used to yield a death, arising from both necrosis and late-stage
better representation of in vivo conditions [10,11] , apoptosis. Trypan blue [20–22] and propidium
although the presence of two cells in culture can iodide [23,24] are charged molecules that are
complicate interpretation. excluded from cells with intact membranes; their
The most common in vitro assays for nano- presence in cells is used to quantify cell death
particle toxicity will be reviewed only briefly colorimetrically or fluorescently. Neutral red
herein because they have been reviewed in accumulates in all cells but undergoes protona-
greater depth elsewhere [12,13] . The in vitro tech- tion and, accordingly, a color change only in liv-
niques reviewed later, including those involving ing cells; thus, stained cells enable quantitation
viability, proliferation, apoptosis and necrosis, of cell death. Additionally, a combination assay
oxidative stress and the inflammatory response, can be performed using calcein acetoxymethyl
provide examples of the techniques used thus ester and ethidium homodimer, in which cal-
far in assessing therapeutic nanoparticle toxic- cein acetoxymethyl ester undergoes a hydrolysis
ity; they represent only a fraction of the tradi- reaction within living cells producing a green-
tional in vitro toxicological techniques available. fluorescent product and ethidium homodimer,
Many of the assays herein rely on the detection whereas a red-fluorescent dye accumulates only
of changes in probe molecules to determine the within dead cells [25] . The leakage of lactate dehy-
correlating toxicity biomarker concentration as drogenase (LDH) into the culture medium is
a result of probe–biomarker reactions. Owing another marker for membrane integrity, in which
to the increased reactivity of nanomaterials, it the LDH activity is assessed through a two-step
is important to determine probe–nanomaterial assay based on NADH production during the
reactivity to eliminate false-positive results conversion of lactate to pyruvate [26] . Apoptosis,
a rising from these reactions. or programmed cell death, is also assayed during
Changes in cellular viability, after exposure in vitro toxicological assessment and is often com-
to therapeutic nanoparticles, result from either bined with membrane-integrity measurements
or both changes in proliferation and/or cellular to yield a more complete picture of cell death.
death and is measured as a general assessment of Phosphatidylserine is found commonly on the
cellular health. The most common method of this outer cellular membrane during apoptosis and is
assessment is through the mitochondrial reduc- assayed using a fluorescein isothiocyanate-tagged
tion by enzymatic cleavage of (3-(4,5-dimeth- annexin V label [27] . The caspases are a series
ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide of proteases found in an activity cascade associ-
(MTT) [14] or variations of tetrazolium-based ated closely with apoptosis [28] . This activity is
dyes, such as XTT [15] , WST-1 [16] or MTS, with detected as a biomarker for apoptosis using any
which mitochondrial activity is used as a proxy of a number of commercially available caspase-
for viability. Increasingly popular is the moni- activity kits, which involve the conjugation of a
toring of the reduction of the resazurin-based fluorescent- or colorimetric-probe molecule with
Alamar Blue dye, which also correlates cellular a peptide cleaved specifically by an initiator cas-
reduction with viability [17] . All of these dyes pase (caspase 2, 8, 9 or 10) or an effector caspase
generate cleavage products that can be detected (caspase 3, 6 or 7). Enzymatic digestion of DNA
220 Nanomedicine (2009) 4(2) future science group

Toxicity of therapeutic nanoparticles Review
occurs during apoptosis and the resultant DNA may not be mutually exclusive. This variety
fragments can be assayed during in vitro toxicity enables a wealth of information to be gained but
experiments using DNA-laddering techniques [29], the lack of a standard set of toxicological assess-
the comet assay [30] or TUNEL assay [31] . ment methodology (i.e., model, dose, assay, con-
DNA fragmentation may also be the result of trols and data analysis) makes cohesive inter-
oxidative stress, a common toxicity mechanism pretation among results complicated and does
that is explored during in vitro assessment of ther- not actively lead to understanding the mode of
apeutic nanoparticles. During oxidative stress, toxicity. Rather, these tools can only rule out a
increases in reactive oxygen species (ROS) or reac- particular mode in a particular model.
tive nitrogen species (RNS) result in oxidative
damage to biomolecules, including nucleic acids, Nanotherapeutic dosing
proteins and lipids. ROS and RNS are assayed by Dosing, or the amount of therapeutic nanoma-
the oxidative production of fluorescent dyes using terial administered to cells or tissues, is critical
probes, such as 2´,7´-dichlorofluorescein diace- to assessing toxicity. Although this may seem
tate (DCFDA) [32] or dihydrorhodamine123 [33] . obvious, dosage becomes complicated to estab-
Other probes, such as C11-BODIPY, which local- lish because nanoparticle behavior differs from
izes in the plasma membrane [34] , or MitoSOX that of a molecular therapeutic. That is, physi-
Red, which localizes in the mitochondria [35,36] , cal properties of nanomaterials, such as size
can give information regarding the location of oxi- and surface chemistry, alter particle–particle
dative stress. The oxidative products can also be (e.g., aggregation) or particle–cell (e.g., uptake
used as markers for oxidative stress. For example, rates) interactions, which, in turn, affect the
malondialdehyde is the end product of lipid per- number of nanoparticles internalized by the cell
oxidation by ROS and RNS and is assayed with [43] . Therefore, unlike molecular therapeutics,
thiobarbituric acid [37] and detected by either fluo- for which cellular or tissue doses can be pre-
rescence or UV–visable absorption. Glutathione dicted based on structure–activity relationships,
is an important cellular antioxidant and can be exposure to given amounts of therapeutic nano-
a biomarker for oxidative stress. Owing to the materials may not predict the dose that impacts
presence of both disulfide-bridged glutathione the cell (Figure 1) [44] .
and monomeric glutathione, total glutathione is Potential applications of therapeutic nanopar-
quantified by reducing the disulfide with glutathi- ticles include drug delivery and photodynamic
onereductase and then assaying total glutathione therapy (PDT), in which the nanoparticles act
with a 5,5´‑dithio-bis(2‑nitrobenzoic acid) probe, as vehicles for molecular therapeutics or sensitiz-
yielding a product that can be detected colori- ers; in these cases, ideally, the nanoparticle would
metrically [38] . Superoxide dismutase (SOD) is not influence the overall toxicity. In some cases,
a cellular enzyme used to reduce the oxidative however, the impact of the nanoparticle vehicle
stress a cell undergoes and its activity is used as (e.g., without drug or sensitizer) induces uninten-
a biomarker for oxidative stress. SOD activity is tional toxicity, possibly owing to intrinsic material
determined indirectly by the inhibition of the properties, immunogenicity and/or bioaccumula-
oxidation of an absorptive substrate, nitro blue tion, and therefore, it is important to consider the
tetrazolium, by exogenously generated superox- dose of both the therapeutic and vehicle itself.
ide. Because SOD is an inducible enzyme, the Azarmi et al. and Gil‑Tomas et al. evaluated
interpretation of SOD activity is complicated the unintentional toxicity of drug-delivery and
because increases or decreases of SOD activity can photodynamic-sensitizer vehicles, respectively, by
be interpreted as indirect evidence of increases in performing careful control experiments during
oxidative stress. For this reason, it is often prefer- nanoparticle therapeutic evaluation [45,46] .
able to use the aforementioned direct detection It is important to quantify the number of
assays of ROS or RNS [39] . nanoparticles to which cells are exposed as
To predict therapeutic nanoparticle effects on well as the amount of nanoparticles the cells
the inflammatory response, the production of uptake in order to calculate commonly used
inflammatory mediators is assayed in vitro. Pro- dose metrics and determine administered dose
inflammatory mediators are assayed using ELISA accurately. Many techniques have been used to
[40] and include cytokines, such as IL‑2, IL‑8, characterize uptake. For example, inductively
IL‑6, TNF-α, IFN-γ and EMAP-II [41,42] . coupled plasma mass spectrometry (ICP-MS)
Whether using an in vivo or in vitro approach, was used to probe the Au content of HeLa and
there is a wide variety of tools for assessing the A594 cells exposed to oligonucleotide-modified
many possible toxicological mechanisms, which Au nanoparticles [47] . ICP, with either MS or
future science group www.futuremedicine.com 221

Improving measures of dose
Mass administered
Exposure
Media mass, SA or
no. concentration
Delivered dose
Deposited mass, SA or no.
Deposited mass, SA or no. per

cell or cm2
Cellular dose
Internalized mass, SA or no.
per cell or cm2
Target site mass, SA or no.

Signal transduction
Figure 1. Effective dose of nanoparticles in vitro may differ from the dose administered to the dose internalized by the cell
as the relationship between the two is difficult to predict. SA: Surface area.
Adapted with permission from [44] .
atomic emission spectroscopy detection, enables that may induce artifacts while providing only
easy sample preparation and quick results, a static picture of a small sample population.
although it gives limited information about the In another technique, fluorescence spectroscopy
cellular location of the nanoparticles, cannot has been used to determine nanoparticle uptake.
distinguish between internalized or externally For example, Moghe and coworkers used con-
adherent nanoparticles, is applicable to only a focal fluorescence microscopy to evaluate the
subset of nanomaterial elements and does not dis- location of fluorescently labeled micelles within
criminate between nanoparticles and preexisting individual cells [50] . Fluorescence spectroscopy
ions of the same element. Transmission-electron offers highly sensitive measurements with time
microscopy (TEM) enables the location of the resolution but requires confocal-fluorescence
nanoparticles to be visualized as Chithrani et al. techniques to determine nanoparticle localiza-
showed when they determined the location of Au tion. For nanoparticles that are not natively
nanoparticles in HeLa cells [48] . Monteiro-Riviere fluorescent, a tag or label is crucial but may
and coworkers also used TEM to determine not alter nanoparticle characteristics and toxicity.
only the location of quantum dots (QDs) but the Although advancements are being made with
behavior of the particles within the cell by exam- TEM and confocal-fluorescence microscopy,
ining nanoparticle aggregation as well [49] . Some new techniques are also being used to perform
of the advantages of TEM include the ability dynamic nanoparticle tracking in cells [2] . As
to determine internalized nanoparticle localiza- stated previously, understanding nanomate-
tion, concurrent characterization of nanoparticle rial internalization is essential for determining
and cell and/or tissue morphology and comple- dosage of nanotherapeutics and the limitations
mentary elemental analysis using spectroscopic of these common techniques necessitate the
methods. Conversely, TEM analysis of biologi- further development of new methods to assess
cal samples entails time-intensive preparation nanoparticle uptake.

In nanoparticle-exposure experiments, it is deliver them to a targeted cell or tissue. These
imperative to justify the dose so that the cel- vehicles for drug delivery may also be designed
lular response accurately represents a realistic to slowly release the drug for optimal therapy
exposure [51] , yet, few experiments include dose [55–57] . Beyond the great advantage of targeted
reasoning. A good example of dose justification chemotherapies, formulation of a drug into a
is by Kallinteri et al., who demonstrated the nanoparticle delivery vehicle may enable use of
toxicity of an unloaded drug-delivery vehicle drugs that were abandoned originally as thera-
in an exposure range relevant to administer the peutics because they were not bioaccessible after
correct dose of drug [52] . Dose justification is dosing or toxic doses were necessary to achieve
also important when results from in vitro experi- a therapeutic effect. A wide range of nano-
ments are extrapolated to in vivo exposures. materials has been identified as drug-delivery
However, correlation of in vivo and in vitro vehicles, including liposomes, polymeric nano-
toxicity assessments is only possible if the same particles, dendrimers and fullerenes, among oth-
nanoparticle dose can be delivered in both cases. ers. Although the concept of using hydrophilic
Equivalent dosing is difficult to achieve because materials with steric stabilization originated with
the nanoparticle experiences a vastly more com- the use of polymeric drug-delivery vehicles [58] , it
plicated delivery route and environment when has now been generalized for the design of drug-
administered in vivo. Accordingly, in vitro tox- delivery vehicles of many different materials.
icity response tends to overestimate the in vivo There are several commercially available drug-
impact. By using a good dose metric, or measure delivery nanoparticles, including Emend® [201]
of the dose, in vivo versus in vitro doses can be and TriCor ® [202] (milled nanocrystalline for-
justified and an accurate determination of the mulations of an antinausea drug and cholesterol-
therapeutic effect or toxicity can be achieved. lowering drug, respectively, US FDA approved
Donaldson et al. and Duffin et al. were success- in March 2003), Abraxane® [203] (130‑nm diam-
ful recently in this endeavor by representing the eter albumin-bound paclitaxel for breast cancer
dose in terms of nanoparticle surface area and treatment, FDA approved in January 2005) and
both groups showed correlated toxicity between Doxil® [204] (doxorubicin [DOX]-containing
in vivo and in vitro experiments [53,54] . Owing to lipid nanoparticles with a poly[ethylene glycol]
the complex nature of nanomaterial dosing and [PEG] coating for ovarian cancer treatment,
recent emergence of nanotoxicology as a disci- FDA approved in February 2005). It is impor-
pline, few standards for toxicity evaluation exist tant to assess the unintentional toxicity of the
and are an area of much needed research. drug-delivery nanoparticle to identify either
Amidst the challenges of nanotoxicology, unintended effects of the delivery vehicle itself
such as the application of a myriad of toxicology or synergistic effects in which the nanomaterial
assays in a wide range of in vivo and in vitro influences drug delivery or drug efficacy.
systems and the often ambiguous description of Liposomes, nanosized vesicles of phopholip-
nanoparticle dose, the nanomedicine commu- ids, are among the most extensively considered
nity has begun to recognize the importance of drug-delivery nanoparticles, based on the facile
exploring toxicity. The remainder of this article variation of their size, lipid composition, drug-
reviews recent consideration of toxicity in the loading capacity and surface modification [59,60] .
three most common applications of nanothera- These nanostructures have the potential to
peutics: drug delivery, PDT and bioimaging. deliver therapeutic agents to various tumor sites
The majority of the following reviewed stud- [61,62] , the lungs [63,64] , the eye [65,66] , the brain
ies performed toxicity assays to assess the effi- [67] and through the skin [68] after drug localiza-
cacy of the therapeutic nanoparticle instead tion either within the lipid bilayer (lipophilic
of assessing the unintentional toxicity of the drugs) or in the inner aqueous region (hydro-
nanoparticle vehicle. There are few studies that philic drugs). The most significant technical
examine unintentional toxicity systematically challenge in using injected liposomal drug deliv-
and purposefully. ery is avoiding liposome uptake by macrophages
or localization in the reticuloendothelial system
Drug-delivery nanoparticles (RES). The short blood half-life limits drug-
The promise of nanoparticle drug-delivery agents delivery capacity, although surface functional-
lies in the potentially biochemically stealth ization with species, such as PEG or sphingomy-
nature of particles, owing to size, hydrophilic- elin, a ubiquitous sphingolipid in the exoplasmic
ity and/or surface charge, which can be designed leaflet of the cell membrane [69] , has enhanced
to carry a wide variety or cocktail of drugs and circulation time significantly. Additional

targeting can be achieved by covalent modifica- long circulation times and a hydrophobic inte-
tion of the outer lipid leaflet with antibodies. rior so that a wide variety of therapeutic agents
Kasid and coworkers evaluated the toxicity of can be carried [73] . In addition, by complexing
467-nm diameter cationic liposomes contain- negatively charged DNA with a polymeric car-
ing the raf antisense oligonucleotide as well as rier, it is possible to achieve cellular uptake of
the blank liposome itself using multiple assays DNA. Unlike liposome drug-delivery vehicles,
in multiple species [6] . Although these loaded in which drug can fill the interior cavity, poly-
nanoparticles do show promise for anticancer mer nanoparticles are limited in their payload
treatment, the liposomes themselves exhibit tox- capacity by the space available in the polymeric
icity with significant increases in total neutro- network but offer significant advantages in
phil counts and liver enzymes in mouse studies terms of the wide variety of available monomers.
and changes in complement activity in mon- The most commonly used polymers for DNA
key studies. Hussain et al. fabricated 125-nm delivery are poly-(d,l‑co‑glycolicacid) (PLGA),
diameter cationic liposomes with a PEG coating poly(ethyleneimine) (PEI), PEI–PEG co‑poly-
that contained a cancer-therapy oligonucleotide mers, poly(l‑lysine) (PLL), PLL–PEG co-poly-
and targeted them to a cell-adhesion molecule mers, biodegradable cationic polymers, chitosan
expressed nearly exclusively on solid tumors [70] . and cyclodextrins. Core-shell polymer micelles
Exposure of cancerous cell lines to these drug- for drug delivery are made commonly using
delivery nanoparticles sensitized the cells suc- PEG–poly(amino acid), PEG–poly(d,l‑lactic
cessfully to further drug treatment. However, acid), PEG–polyester, hydrophobically modified
a MTT assay demonstrated that nearly half polysaccharides, cyanoacrylates and even PEG–
the sensitization effect was due to the liposome lipids (Figure 2) [74] . Recently, Lee et al. compared
alone; the mode of liposome toxicity was not the drug-delivery capability and toxicity of a
identified, although monitoring the caspase‑3- commercially available liposome, BioPorter ®,
like protease activity demonstrated that it could with that of a cationic and amphiphilic copo-
not be attributed to induced apoptosis. Smistad lymer micelle for delivery of lectin A‑chain,
and coworkers screened empty liposome toxicity an anticancer glycoprotein [75] . The small size
using the MTS assay in immortal buccal cells (150 nm diameter) and positive zeta potential
while varying the main phospholipid used, the of the polymer micelle promoted more efficient
lipid concentration (12–35 mM) and, finally, endocytotic uptake than that measured using
the liposome charge and size (100–2000 nm) BioPorter. However, the empty polymer lipo-
[71] . They found that cationic liposomes were somes display unintentional toxicity, assessed
significantly more toxic than anionic liposomes, using the MTT assay in four different immor-
although it was not through a simple relationship tal cell lines, which increases with increasing
to the net liposomal charge. In addition, neither polymer concentration up to a 40% reduction
lipid concentration nor liposome size had a sig- in cell viability. Azarmi et al. incubated lung
nificant influence on toxicity. In a final example, cancer cell lines with 173 ± 43 nm diameter
Lal and coworkers encapsulated cisplatin into poly(butylcyanoacrylate) nanoparticles with
200-nm diameter liposomes and incubated incorporated DOX that could be delivered
the drug-delivery nanoparticles with immortal directly into the lungs using spray freeze-dried
ovarian cancer cells [72] . Using esterase activity carrier microparticles [45] . Results of the XTT
to identify live cells and membrane integrity assay after nanoparticle exposure showed that
to identify dead cells, the group reported ‘sig- this system did amplify the desired cytotoxic-
nificant’ cell death among cells incubated with ity on delivering the chemotherapeutic DOX
cisplatin-containing liposomes and no apparent to the cells; however, the non-DOX-containing
cell death among cells incubated with empty nanoparticles also showed an unintentional tox-
liposomes. Liposome-delivery vehicles facilitate icity of up to 50%. Similarly, Lee and cowork-
improved delivery of drugs for certain diseases; ers synthesized core-shell polymer nanoparticles
however, it is clear that their advantages are ranging in size from 75 to 100 nm diameter
overshadowed by concerns about and desires to using varied percentages of two different cyano-
control unintentional toxicity. acrylates in their copolymer formulation in the
Although unmodified liposomes display a hopes of creating a low toxicity drug-delivery
hydrophilic exterior that promotes protein adsorp- nanoparticle that could cross the blood–brain
tion and opsonization, polymeric gene therapy and barrier [76] . Micelles made of only poly(2‑octyl
drug-delivery nanoparticles are usually designed cyanoacrylate) showed no unintentional toxicity
to present a hydrophilic exterior that promotes using the MTT screen in immortal fibroblasts,

Hydrophilic
Drug polymer
Linker
DNA
Protein
Figure 2. Various nanoscale polymer drug-delivery vehicles. (A) Drug covalently bound to a
simple polymer backbone, (B) a protein covalently bound to polymers, (C) DNA interacting with
polymer, (D) drug-loaded polymer micelle and (E) drug-loaded dendrimer.
Reprinted with permission from [74] .
whereas the addition of any percentage of the course of 30 days [52] . Using the MTT
n‑butyl cyanoacrylate decreased cell viability to assay, the group demonstrated, that the novel
50%. In another study, Diebold and coworkers polymer micelles are not toxic to either adher-
exploited chitosan, a mucoadhesive polymer, for ent or nonadherent immortal cells. Although
ocular drug delivery with 289 ± 13 nm diam- these particular micelles have great potential to
eter nanoparticles [65] . When assessing in vitro deliver steroids based on the specific interaction
toxicity by counting cells and performing the between steroids and acyl functional groups,
Trypan blue exclusion assay, they found that cell different polymer backbones will have to be
survival was always greater than 40% (although developed for each class of drug.
it did not follow expected concentration or Dendrimer drug-delivery vehicles are a natural
dosing-time trends) and the viability of the extension from polymeric nanoparticles because
surviving cells was not compromised. In vivo dendrimers are spherical, multigeneration and
testing in rabbits showed good distribution of regularly branched polymers with significant
the chitosan nanoparticles in the eye with little intradendrimer volume. Dendrimers maintain
or no ocular damage. In further work, Diebold the advantage of functional-group flexibility
and coworkers added a phospholipid shell onto while potentially increasing the drug capacity per
the chitosan nanoparticle in the hopes of pro- unit volume over normal polymer nanoparticles
moting biocompatibility [66] . This modification based on the branched structure. Overall nano-
did improve cell viability after exposure, based particle size is also vastly different, with polymer
on XTT assay data, and the nanoparticles were nanoparticles greater than 100 nm in diameter
still retained and tolerated during in vivo evalu- and dendrimers usually less than 10 nm in diam-
ation in rabbits. New polymer syntheses are also eter. A 2005 review article by Duncan and Izzo
being pursued to increase drug-loading capacity discussed toxicity assessment of dendrimers up
and decrease unintentional toxicity. Kallinteri to that point and aptly noted that these studies
et al. synthesized a series of acyl-substituted were often done with low dendrimer doses, short
polymers that form monodisperse micelles incubation times and immortal cell lines [77] .
with diameters between 160 and 240 nm, load Herein, a few additional examples are discussed.
steroids efficiently and release the steroid over Simanek and coworkers synthesized a series of

generation three melamine-based dendrimers, 15% hemolysis, a significant improvement

each with 48 peripheral functional groups, and over polycationic dendrimers (in all cases) and
examined how surface charge and functional even the chloroquine alone (in the case of the
group influenced hemolysis and toxicity owing galactose-coated dendrimers). Unfortunately,
to dendrimer exposure [78] . Their spectropho- the unintentional toxicity of the dendrimer
tometric hemolysis measurements demonstrate vehicles (without loaded chloroquine) was not
that anionic and PEG-modified dendrimers investigated, although these nanomaterials
induced minimal hemolysis after 1 h, whereas show great promise in both in vitro and in vivo
cationic dendrimers lyse a significant percent- studies. Overall, dendrimers show great prom-
age of red blood cells. With 24 h exposure, ise for drug delivery based on the large variety
the anionic dendrimers also induce significant of accessible chemical functionalities and the
hemolysis but the PEG-modified vehicles have highly homogeneous nanoparticle distribution;
little effect. MTT assessment of dendrimer tox- additionally, the toxicity data garnered using
icity in immortal rat epithelial cells showed sig- polymer nanoparticles directly inform material
nificant decreases in cell viability after exposure choices for these drug-delivery vehicles.
to even the lowest concentration (0.001 mg/ml) Fullerenes are the most recently discovered
of cationic dendrimer. Cell viability after expo- carbon allotropes, consisting of pentagonal
sure to anionic dendrimers was better, although and hexagonal carbon rings that form hollow
they were still cytotoxic at higher concentra- sphere or tube structures. Based on their hol-
tions (1 mg/ml). Even the PEG-modified den- low nature, fullerenes have been proposed
drimers caused a decrease in cell viability to extensively as potential drug-delivery vehicles,
70% on exposure at the 10 mg/ml level. The especially for use in treating cancer and HIV.
in vitro assay results showing the promise of Most recent studies in this area are focused on
PEG-modified dendrimers as drug-delivery fundamental materials development and studies
vehicles was validated with in vivo exposure of the nanoparticles’ interaction with biological
studies using mice; after both intraperitoneal species. For example, GeWirth and coworkers
and intravenous administration, no mortality used scanning-probe microscopy in the hopes
was observed and there was no apparent change of clarifying how fullerenes interact with cel-
in serum liver-enzyme levels. lular membranes [81] . They demonstrated that
Berna et al. synthesized a series of poten- water-soluble fullerenes adhere to zwitterionic
tial drug-delivery dendrimers using various and cationic lipid headgroups on model lipid
PEG species to define the dendrimer internal bilayers but do not penetrate into the hydro-
structure and varying the functional groups phobic inner leaflet. Mori et al. assessed in vivo
(NH 2 and COOH) on the dendrimer exterior fullerene toxicity by giving a 2000 mg/kg-dose
[79] . On exposing immortal ECV 304 cells to mixture of C60 and C70 to rats as a single oral
the dendrimers for 72 h and performing the exposure [82] . Measurement of rat bodyweight
MTT assay, the group found little evidence for for 2 weeks following exposure yielded no
dendrimer toxicity, even from the often prob- deviations from controls. This group also
lematic NH 2-terminated dendrimers. Jain and used the Ames test (bacterial reverse-mutation
coworkers synthesized peptide dendrimers in assay) to look for mutagenicity with doses of
the hopes of achieving minimal unintentional up to 5000 µg/plate and saw no evidence for
toxicity using a PEG core with grafted third- mutagenesis. In vitro cell culture with similarly
and fourth-generation lysine dendrimers [80] . high doses showed no numerical or morphologi-
Both vehicles were approximately 5 nm in cal chromosomal abnormalities. The authors
diameter and used both as synthesized and suggest that the results of this study, showing
with a galactose coating. Each was loaded no unintentional toxicity, may be in contrast
with chloroquine, a drug used to treat malaria with other studies because production of ROS
that is most effective when delivered slowly is more efficient in pristine fullerene samples,
at a low dose. Chloroquine loading was both unlike those used herein. Some groups are
dendrimer-generation and galactose-coating exploring the drug-delivery potential and toxic-
dependent, with optimal loading in fourth- ity of even more exotic fullerene structures. For
generation galactose-coated dendrimers. The example, Conyers and coworkers synthesized
vehicle that loaded the largest chloroquine dose amphiphilic C60 by adding hydroxyl terminated
was also the slowest to release the drug, having dendrimers over a portion of the cage structure
released only 40% of the payload after 24 h. All and lipophilic alkyl moieties on the remainder
four synthesized vehicles exhibited less than (F igure 3) [83] . These modified fullerenes then

OH OH
OHHO OH
O O O OH
HO
O O O OH O
HO
HO O
OH
HO O
O NH O
O N O O NH
H O
O O
O
NH
HO NH
NH OH
HO O NH
O OH
O O O O
HO O O OH
O O
O OO
O
O
O
O O
O O O
O O
O O
O O
O O O
O O
Figure 3. Amphiphilic fullerene. (A) Structure of the amphiphilic fullerene and (B) fluorescent and
bright-field images of cells exposed to fluorophore-labeled amphiphilic fullerenes.
Adapted with permission from [83] .
self-assemble into vesicle structures (diam- uptake of fullerenes is not well characterized
eters ranging from 6.5 to 400.0 nm) with [84–88] , thus complicating the choice of species,
the potential to deliver larger drug payloads tissue or cell, as well as nanoparticle dose, to
than the standard fullerenes. To assess uptake, use for in vitro toxicity analysis.
fluorophore-labeled fullerenes were incubated
with immortal kidney, epithelial and mac- Nanoparticles for PDT
rophage cells and were visualized using fluo- PDT is an alternative to conventional therapies
rescence microscopy (although internalization used to treat cancers, choroidal neovascular-
and exact location are unknown). The group ization and age-related macular degeneration
assessed cytotoxicity with in vitro MTT and [89–91] . In PDT, a photosensitizer (PS) is intro-
LDH assays and found no unintentional toxic- duced into the body by injection [92] , eye drops
ity with doses up to 2 mg/ml. Fullerenes, like [90] or topical application [91] . After a waiting
other materials that are finding new application period, the target tissue is illuminated with the
in drug delivery (e.g., perfluorocarbon and mag- appropriate wavelength of light, which causes
netic core nanoparticles), are in the early stages the PS to convert molecular oxygen to ROS
of development; ideally, assessments of unin- that have a toxic effect on the cells. An ideal
tentional toxicity will be performed with the PS should:
maturation of each new nanomaterial towards n Display no unintentional toxicity;
the drug-delivery application. However, these
studies are complicated by the fact that the n Enable facile delivery;

n Be activated by wavelengths of light that can will be released and fluoresce again. Initial studies
penetrate the tissue; demonstrated that the porpholactol was released
n Target the tumor selectively; preferentially once the PLGA nanoparticle inter-
acted with a lipid solution; this was confirmed
n Be eliminated from the body efficiently, which when increased cellular fluorescence was observed
will minimize prolonged skin sensitivity to after the porpholactol-loaded PLGA was incu-
light. bated with 9L glioblastoma, B16 melanoma
and BT breast carcinoma cells. When cells with
Therapeutic nanoparticles are now being used internalized nanoparticles were irradiated with
in conjunction with PSs and targeting agents to 42 mW/cm 650 nm laser light for 180 s, 95% cell
increase the efficacy of PDT. The small size of death was achieved (compared with only 9% cell
the nanoparticles and the ability to functionalize death for noninternalized nanoparticles).
their surface facilitate ‘Trojan horse’ delivery [93] In another study, verteporfin-loaded PLGA
of PS inside the cell where the photocytotoxic nanoparticles of two different sizes (diameter:
effect is greater. Liposomes, polymers, fullerenes, 167 and 370 nm) were evaluated for PDT in
metallic and magnetic nanoparticles are all being EMT-6 mammary mouse tumor cells [97] .
used in PDT. Visudyne® [205] (verteporfin encap- Phototoxicity was evaluated with the MTT assay
sulated in a liposome to treat age-related macular after exposure to a 70 ng/ml dose of Vertaporfin
degeneration, FDA approved in April 2000) is in the 167‑nm diameter nanoparticles irradiated
the one FDA-approved nanoparticle drug for with 6 J/cm2 of 690 nm laser light. The result
PDT. Although many toxicity studies have been was a 67% decrease in cell viability compared
performed with molecular PSs, there are only a with an 11 or 29% decrease caused by free PS
few that investigate the possible unintentional or 370-nm diameter PS-loaded nanoparticles,
toxicity of the nanoparticle vehicle. respectively. To investigate the PDT mecha-
Polymer nanoparticles used in PDT act mainly nism, PS fluorescence was monitored following
as a drug-delivery vehicle to localize the PS in exposure to fetal bovine serum, revealing that
tumor tissue. Most PSs are hydrophobic and the nanoparticle rapidly transfers the PS to the
contain a complex mixture of porphyrins that serum proteins. This suggests that, in vivo, the
are needed to absorb light and transfer energy PS would probably be dispersed within the cell
to molecular oxygen. These hydrophobic PSs membrane and able to produce cytotoxic singlet
can be encapsulated or intercalated into poly- oxygen. To evaluate nonspecific skin sensitivity,
mer micelles or nanoparticles. Ricci-Junior et al. mice were injected with the nanoparticle–PS for-
used zinc (II) phthalocyanine (ZnPc) encapsu- mulation and examined for skin abnormalities;
lated in 200-nm diameter PLGA nanoparticles for a short period (60 min) after the injection,
and evaluated the PDT effect in P388-D1 lym- the skin was photosensitive. A tumor size-control
phoma cells [94] . Cell viability after exposure to assay was also performed in vivo to evaluate the
drug-free nanoparticles and 5 µM ZnPc-loaded body’s ability to clear the nanoparticle–PS con-
nanoparticles were assessed using the MTT jugate. Based on declining therapeutic efficacy
assay. The drug-free nanoparticles did not dis- when light exposure was carried out more than
play unintentional toxicity (>97% viability) or 30 min after injection, rapid nanoparticle clear-
phototoxicity after exposure to the therapeu- ance was obvious. An alternative to nanoparticle
tic laser irradiation, whereas the ZnPc-loaded clearance is to design biodegradable PS vehicles.
PLGA nanoparticles showed 92% viability prior Toward this end, Rijcken et al. synthesized 75-nm
to irradiation with 60% cell death after exposure diameter block copolymer micelles (ω‑methoxy
to 30 J/cm2 of 675 nm light. poly(ethyleneglycol)-block-poly(N‑(2‑hydroxy-
In another study, PLGA nanoparticles were propyl)methaacrylamide-dilactate) loaded with
used to encapsulate meso-tetraphenylporpholactol 2 mg/ml PS, an axially solketal-subsituted sili-
(98-nm diameter) to make a photodynamic thera- con phthalocyanine and incubated them with
peutic with minimal nonspecific sensitization [95] . B16F10 melanoma and 14C human and neck
Porpholactol was chosen as the PS because it is squamous cancerous cell lines [92] . This novel PS
known to aggregate on encapsulation, quench- vehicle has an advantageous degradation mecha-
ing chromaphore fluorescence and, thus, reduc- nism whereby physiological conditions induce
ing skin sensitization. Phorpholactol can either an increase in the critical micelle temperature
form nanoparticles directly [96] or can be loaded that leads to the breakdown of the micelles after
into polymer vehicles. Once the loaded nano- 1 week. Cellular viability was evaluated using
particle is taken up into a cell, the porpholactol the XTT assay after exposure to the free PS and

PS-loaded nanoparticles; no unintentional tox- lung carcinoma and colon adenocarcinoma)
icity was observed in either case and the IC50 using the MTT assay [100] . BF4, a mono-substi-
values after irradiation with 3.5 mW/cm2 light tuted pyrrolidinium fullerene, killed 80% of the
for 10 min were 5 nM for the B16F10 and 3 nM cells after exposure to 150 mW/cm2 white light
for the 14C cells. Khdair et al. used a novel sur- at 1–80 J/cm2 and displayed no unintentional
factant-polymer nanoparticle (39-nm diameter) toxicity. The cell death was observed with BF4
comprising Arosol OT™ and alginate to encap- concentrations as low as 2 µM in as little as
sulate methylene blue (MB) and DOX for the 4–6 h after exposure to the light source. The
PDT treatment of NCI/ADR-RES ovary ade- great success of this fullerene PS is attributed
nocarcinoma, a drug-resistant cell line [98] . MB to its slight hydrophobicity and single cationic
both shuts down the transporter at least partially charge that enables it to localize in intracellular
responsible for multidrug resistance and acts as a membrane organelles, such as the mitochondria.
PS. Cell viability was assessed after 24 h exposure Fullerenes show great potential as PS for PDT
to 3.3 µg/ml DOX-MB-loaded nanoparticles by but bioaccumulation of these particles is also
the MTS assay. The drug-free nanoparticles did a concern to researchers. Roberts et al. used a
not display unintentional toxicity or phototoxic- pristine sample of hydroxylated fullerenes (or
ity; however, cells exposed to the combination fullerols) to assess phototoxicity and cytotoxicity
therapy nanoparticles displayed significant cell in human lens epithelial cells, as may be experi-
death both before and after irradiation with the enced when the fullerene drug-delivery vehicle
laser (665 nm, 2400 mJ/cm2). The increase in crosses the blood-ocular barrier [101] . Previous
cytotoxicity of the DOX–MB nanoparticle was work had demonstrated that photoexcitation of
contributed to by the localization of the nano- fullerene derivatives by light with wavelengths
particles within the nucleus and irradiation with longer than 295 nm produced singlet oxygen
the laser increased ROS generation. Chatterjee and superoxide, toxic species that may cause
and Yong investigated nanoparticles compris- lens damage. In this work, the group verified
ing a zinc phthalocyanine (ZnPc) PS bound to (but did not quantitate) fullerol uptake after
a poly(ethylene imine)-coated sodium yttrium cell exposure to 0–50 µM solutions by measur-
fluoride nanoparticle co-doped with ytterbium ing fullerol absorbance at 405 nm. Following
and erbium [99] . After 24 h incubation with cell incubation with fullerol, metabolic activity
HT29 human colon adenocarcinoma cells at and LDH release were monitored using com-
varying nanoparticle (50-nm diameter) doses, mercial MTS and LDH assays under condi-
viability was assessed by the MTT assay and tions of controlled light exposure. MTS assays
no unintentional toxicity was observed (>90% showed decreased metabolic activity and drastic
viability). On irradiation of cell-bound nano- increases in LDH release at fullerol concentra-
particles, based on the charged PEI, with the tions as low as 5 µM when the cells were also
980 nm laser for 5 min, cell viability decreased exposed to either UVA or visible light. Flow
for all concentrations of nanoparticles but was cytometry analysis suggests that the damage is
not concentration dependent. These multi- owing to early apoptosis. Concurrent quenching
functional nanoparticles are promising because experiments suggest that light exposure leads to
they had a small effect on cells before irradia- singlet oxygen production and cell membrane
tion, showed good phototoxicity and will not damage. Even without light exposure, the ful-
be excited by exposure to sunlight. As in poly- lerol showed cytotoxicity at concentrations of
mer drug delivery, flexibility in monomer choice 20 µM and higher. Although substituted fuller-
enables application of previously unusable PSs enes show great potential as PDT agents, there
but careful choice of polymer is still necessary are still many opportunities for further develop-
to minimize unintentional toxicity. ment, such as loading of additional PSs into the
Although polymer nanoparticles are best fullerene vehicle or cell/tissue targeting of the
exploited when exploring a variety of PSs, fuller- fullerene PDT agent.
enes can either act as a vehicle or can be used as Noble metal nanoparticles are also being
a PS themselves. Fullerene complexes produce a explored for PDT, although loading of the
long-lived excited triplet state that can transfer PS requires surface self-assembly rather than
energy to molecular oxygen to produce oxygen encapsulation, and photothermal therapy, where
radicals. Hamblin and coworkers examined localized heating of the nanoparticle causes cell
six different substituted fullerenes to evalu- death. Wieder and coworkers exposed HeLa cells
ate their therapeutic effect for PDT in three to a phthalocyanine PS attached to 2- to 4-nm
murine cell lines (reticulum sarcoma, Lewis diameter gold nanoparticle by thiol linkage and

assessed viability using the MTT assay [93] . Cell well-understood optical properties and simple
viability was concentration, exposure-time and modification chemistry. However, recent work
irradiation-time dependent with optimal expo- in our group demonstrates that noble metal
sure conditions of 4 h incubation in 0.55 µM nanoparticles do alter cell function even when
nanoparticles and a 20 min irradiation with a viability is unaffected [103] .
690 nm diode laser at 1.84 mW/cm 2, leading
to 46% cell death by apoptosis. However, they Nanoparticle contrast agents
found that the phthalocyanine–nanoparticle Nanoparticle contrast agents hold great promise
conjugate had significant unintentional toxic- in bioimaging based on their ability to provide
ity and, on further testing, determined that the high contrast and to label targeted cells and tis-
toxicity was a cumulative effect of the nanopar- sue, thus increasing sensitivity when compared
ticle, phthalocyanine PS and the phase-transfer with molecular contrast agents and enabling
agent. Although many noble metal nanoparti- imaging of smaller, specific biological features
cles use a PS to induce cell death, PS-free nano- (e.g., vasculature). Because traditional molecu-
particles may also be used, such as in the work lar contrast agents, such as gadolinium-based
by El-Sayed et al., in which the nanoparticles agents for MRI, have been linked to overdos-
have a photothermal effect [102] . El-Sayed and ing and cases of nephrogenic systemic fibrosis
coworkers looked at the photothermal efficacy [206] , nanoparticle-based contrast agents have
of 40-nm diameter gold nanoparticles function- become an increasingly attractive alternative
alized with an anti-EGF receptor antibody and for MRI and non-MRI applications. Some
PEG. They then examined the targeting of func- of the nanomaterials being developed as con-
tionalized nanoparticles and their PDT effect trast agents include QDs and magnetic-core
in three different cell lines, a benign HeCaT nanoparticles. In addition, multifunctional
(human keratinocytes) and two malignant cell nanomaterials are being developed in which
lines (human oral squamous cell carcinoma), contrast enhancement is combined with either
HOC 313 clone 8 and HCS3, both overpro- drug delivery or PDT capability. There is one
ducing EGF receptors on their surface (Figure 4) . commercially available nanoparticle-based
Cell viability was assessed by Trypan blue after MRI contrast agent called Feridex IV® [207]
exposure to approximately 2 nM nanoparticles (superparamagnetic iron oxide nanoparticles
at varying laser powers. The optimal laser pow- [SPION] associated with dextran for detec-
ers, at which complete cell death was observed, tion of liver lesions, FDA approved in August
were 25 and 19 W/cm 2 for the HSC and HOC, 1987). Additionally, there are several others in
respectively. The group observed no toxicity the pipeline, including Combidex ® (SPION
caused by the light source alone and phototox- with associated dextran for detection of can-
icity to the benign cell line only at high (>57 W/ cerous lymph nodes) and Resovist ® (SPION
cm 2 ) laser powers. No unintentional toxicity coated with carboxydextran for detection of
study was conducted in which the toxicity of the liver metastases). Various review articles have
nanoparticle, with or without functionalization, addressed both the progress in the materials
was probed. Clearly, noble metal nanoparticles aspects of bioimaging nanoparticles [104–107] as
act as a foundation for the development of mul- well as their applications [108–121] . Although con-
tifunctional nanoparticle therapeutics based on sideration of bioimaging agent unintentional
Figure 4. Light-scattering image of human keratinocyte benign cells, HSC malignant cells and HOC malignant cells treated
with anti-EGF receptor-conjugated gold nanoparticles. The figure demonstrates the ability of El-Sayed and coworkers to efficiently
target malignant cells with a functionalized gold nanoparticle.
Reprinted with permission from [102] .

toxicity is critical for medical use, most studies
neglect this aspect and, thus, it is largely absent
from the referenced reviews.
QDs, high quantum-yield semiconductor
nanoparticles, are used for in vivo and in vitro
bioimaging based on their photostability, large
fluorescence intensity and size-tunable emis-
sion (Figure 5) [108–110,112,122] . One of the limita-
tions of in vivo QD use is the potential tox-
icity of the nanoparticle, stemming from the
10 µm
possible release of heavy metal ions from the
QD core. To overcome this limitation, QDs
are functionalized with biopolymers to pre-
vent ion leeching and increase biocompatibil-
ity and bioavailability [111,112,123] . Additionally,
the inherent toxicity of heavy metals may also
be detrimental long term, including effects
resulting from bioaccumulation. The effective-
ness of the protective polymer shells should be
evaluated with unintentional toxicity studies
to ensure that QDs are safe. In one of a lim-
ited number of examples, Bhatia and cowork-
ers examined 24 h primary-culture hepatocyte 1 µm
exposure to 1 mg/ml CdSe QDs while varying
QD synthesis conditions [123] . Cell viability was
monitored using the MTT assay and changes
in cell morphology. They found that QDs were
cytotoxic when they were exposed to conditions
that would oxidize surface species, such as air
or UV light exposure. Under both of these con-
ditions, cytotoxic Cd 2+ and CdSe complexes
were released from the deteriorated QD surface,
causing cell death. Surface passivation with ZnS
or BSA-conjugated ZnS was used to ameliorate
the toxicity but could not completely eliminate
unintentional toxicity. Additionally, in vivo
hepatocyte studies revealed that cell migration
and differentiation were unaffected by PEG-
coated ZnS/CdSe QD exposure. In a second
study, Maysinger and coworkers examined the
localization and toxicity of CdTe nanoparticles,
ranging from 2 to 6 nm in diameter, which
had either anionic or cationic functionaliza-
tion [124] . Using immortal cells, they examined Figure 5. Quantum dot imaging in live animals and immortal cells.
(A) Compares the fluorescence between quantum dots (QDs; red, injected in the
morphological and metabolic changes (using right flank of the mouse) and green-fluorescent protein (injected in the left flank,
the MTT assay), nuclear entry and protec- indicated by the circle) at approximately equal numbers both in vivo and in vitro
tive measures (e.g., conjugation of BSA to the and (B) examines three QDs (emitting green, yellow and red fluorescence) for
surface or antioxidant application) with QD simultaneous multicolor imaging.
concentrations ranging from 1 to 37.5 µg/ml. Reproduced with permission from [122] .
They found that smaller cationic QDs were able
to enter the nucleus and that this entry corre- Parak and coworkers examined the in vitro toxic-
lated with decreased metabolism and changes ity of CdSe and ZnS/CdSe QDs with a variety
in nuclear composition, indicating toxicity. The of surface coatings, including silica, mercapto
application of antioxidants or BSA improved propanonic acid (MPAA) or an amphiphilic
cell viability, indicating a probable role for Cd 2+ polymer [125] . After exposure to the various QDs,
in the toxicity mechanism. In a final example, uptake was monitored with confocal microscopy

while cell adhesion and ion-channel function changes. They found that there was an approxi-
were monitored to correlate uptake with toxic- mately 20% increase in apoptosis and there were
ity. After normalizing results to reflect the num- corresponding morphological and cytoskel-
ber on surface atoms on the QDs, they found etal changes after exposure to both types of
that MPAA-coated QDs had decreased adhe- SPION. Combined with their uptake studies,
sion, indicating decreased cell viability at less they concluded that the increase in apoptosis
than 1 µM surface Cd atoms, whereas a silica was a result of SPION accumulation rather than
coating protected cells up to 30 µM surface particle- or coating-induced toxicity. In another
Cd atom exposure. They also found that there example, Arbab et al. examined the toxicity of
was no alteration in ion-channel function after Feridex IV conjugated with protamine sulfate
exposure. Overall, these studies conclude that (1–100 µg/ml) to increase uptake in nonphago-
leeching of toxic heavy metal ions is the probable cytic cells [129] . Toxicity was assessed in primary
mode of unintentional toxicity but that protec- culture with the Typan blue assay, MTT assay,
tive coatings improve the biocompatibility of annexin V and DCF, which is a fluorescent
contrast agent QDs. ROS probe. All assays supported that there was
Although QDs inherently suffer from unin- no significant decrease in viability after expo-
tentional toxicity, owing to their heavy metal sure, even though there was an increase in the
cores, SPIONs use a more biofriendly core. nonphagocytic cell uptake. In a final example,
SPIONs are used in MRI for their large mag- Gupta and Gupta examined the in vitro toxicity
netic moment that enables contrast enhance- of SPIONs with (diameter: ~45 nm) or with-
ment at lower levels of metal than traditional out (diameter: ~15 nm) a coating of pullulan (a
contrast agents. Although there has been a com- nonionic polysacharride) in an effort to decrease
mercially available SPION used in MRI since surface reactivity while increasing solubility and
1987, much work is still being done to improve bioavailability [118] . In their immortal fibroblast
the utility of these particles beyond the local- cells, they examined toxicity with 50 μg/ml and
ization and visualization of the liver to other 20 mg/ml exposure to both SPION species by
typical RES sites and to improve overall con- MTT assay and immunofluorescence to examine
trast enhancement (Figur e 6) [115,116,126,127] . In cytoskeletal-organization changes. They found
one toxicity study, Berry et al. examined the that there was no significant toxicity for the pul-
effects of 24 and 48 h exposure of underivatized lulan-coated particles, even at the highest con-
and dextran-derivatized SPIONs (diameter: centrations, whereas a 50% decrease in viability
10–15 nm) in vitro using cultured fibroblast cells was measured with uncoated SPION exposure.
[128] . They used annexin V to measure apoptosis, Although derivitization of SPIONs mediates
scanning-electron microscopy to examine cell- toxicity, future development in this area would
morphology changes and immunofluorescence benefit from a fundamental understanding of
to examine β-tublin and Rac (cytoskeletal) bare SPION unintentional toxicity.
Figure 6. MRI (T2) images before and after superparamagnetic iron oxide nanoparticles administration. This resulted in a 38%
change in contrast for the Tu compared with 13% change for the Mus, where the superparamagnetic iron oxide nanoparticles were
targeted to the tumor using folate receptor-mediated uptake.
Mus: Muscle; Tu: Tumor.
Reproduced with permission from [135] .

Although other nanomaterials, including Au
[130–132] and carbon nanotubes [133] , are being
Core matrix MRI contrast agents
developed as contrast agents for bioimaging, the
remainder of this nanoparticle contrast-agent sec-
tion will focus on the combination of imaging
with other desirable diagnostic and therapeutic
options [134] , such as drug delivery and tissue tar-
geting [135,136] and PDT [137,138] (Figure 7) . In one
drug-delivery example, Hong et al. examined the Photosensitizer
behavior of nontargeted and folate-targeted drug- PEG coating
delivery micelles containing SPIONs and DOX
[139] . They used Prussian blue staining to exam-
ine uptake in an immortal hepatic cell line and
confirmed targeting with MRI while evaluating
nanoparticle toxicity with the MTT assay. They
found that folate-targeted micelles were internal-
ized more effectively than nontargeted micelles,
resulting in decreased cellular proliferation and
increased contrast in MRI.
There are more expansive examples of the Molecular targets
synergistic use of nanoparticles to perform bio-
imaging and PDT. In one PDT example, Ross
and coworkers used PEGylated polyacrylamide
Singlet oxygen Cytoplasm
nanoparticles for both imaging and PDT treat-
ment by encapsulating both iron oxide and the PS
Photofrin within the polymer [140] . Additionally, Figure 7. Multifunctional nanoparticle that contains an MRI agent,
they used the vascular homing peptide F3 to photosensitizer and an antibody-tagging agent for photodynamic therapy.
examine tumor targeting by appending F3 to the PEG: Poly(ethylene glycol).
PEGylated surface of the nanoparticle. In vitro Reproduced with permission from [134] .
studies, using MDA-MB‑435 human breast
carcinoma cells and 1,3‑diphenylisobenzofuran toxicity. However, when the nanoparticles were
(DPIBF) in place of the iron oxide as a singlet oxy- used in combination with the therapeutic light
gen probe, found the nanoparticles to be cytotoxic source, significant damage to the cell, including
in a dose-dependant manner. Additionally, in vivo shrinkage, loss of organelles and a fractionated
studies using 9L glioma rats compared standard cytoplasm were observed.
intravenous delivery of Photofrin with delivery of Using noble metal nanoparticles as the PDT
Photofrin from targeted and nontargeted poly- vehicle enables concurrent imaging when the
mer nanoparticles. These studies showed that nanoparticles are also functionalized with a
the nanoparticles were targeted successfully to targeting moiety. Drezek and coworkers used
the tumor site, increasing the nanoparticle resi- 120 nm-diameter Au nanoshells functionalized
dence half-life threefold and doubling the imag- with anti-HER2 PEG to target SKBr3 breast
ing contrast. Another example of combined PDT cancer cells and compared them with the same
and imaging technology is the work by Chou and Au nanoshell functionalized with a nonspecific
coworkers using SPIONs coated with a silica-irid- antibody (anti-IgG)–PEG complex [138] . Imaging
ium shell [141] . SPIONs facilitate MRI while the of plasmonic scattering was performed to verify
silica-iridium shell generates oxygen radicals for effective targeting to the SKBr3 cells and fur-
PDT and emits fluorescence for confocal imaging ther evaluation was accomplished by electro-
of the location of the nanoparticle within a cell. A less Ag-plating amplification of the bound Au
total of 1 mg/ml of these multifunctional nano- nanoparticles. Cell viability was evaluated with
particles were incubated with HeLa cells for 1 h, calcein acetoxymethyl staining after exposure to
then they were exposed to a 200 mW laser light 3109 nanoshell/ml for 1 h followed by NIR laser
source for 5 min; afterwards, the phototoxicity irradiation at 0.008 W/m2 for 7 min. Without
and cytotoxicity were evaluated using the MTT PS, neither light exposure alone nor light expo-
assay (Figure 8) . The nanoparticles were local- sure with PS-free nanoparticles showed a PDT
ized on the cytoskeleton and also distributed in effect. The anti-HER2 functionalized nanopar-
the cytoplasm but do not display unintentional ticles targeted the cells effectively and facilitated

0 µg 25
Figure 8. Confocal image of HeLa cells containing Fe3O4 /SiO2 (Ir) from Lai et al. demonstrating the ability to concurrently
image and treat cancerous cells with functionalized nanoparticles. The DNA, cytoskeleton and nanoparticles are shown in blue,
green and red, respectively.
Reproduced from [141] © Wiley-VCH Verlag GmbH & Co.
a significant PDT effect. Although there have identified priority areas of research in the com-
been toxicity studies of imaging agents, further mentary ‘Safe Handling of Nanotechnology’,
toxicity studies are warranted [13,108,113,119,142] , in which five grand challenges were issued with
which go beyond viability to consider critical cell specific goal timeframes (Figure 9) [143] . These
functions, differentiation and proliferation. challenges provide a good framework for under-
standing the obstacles associated with assessing
Future perspective therapeutic nanoparticle risks, especially because
Nanotoxicology, including the study of unin- of the rapidly increasing applications of nanoma-
tentional nanotherapeutic toxicity, poses many terials. The grand challenges emphasize the need
scientific challenges. Recently, Maynard et al. for both ‘combin[ing] applicable existing testing

methods with new cutting edge technologies’ complicates interpretation of toxicity. Although
and ‘international nanotoxicology testing proto- a single unit of quantification will not be suit-
cols’ [143] . We believe that it is critical to develop able in all cases, the nanotoxicology community
a toxicological methodology with specific in vitro would greatly benefit from a chosen preferred
techniques and models that are high-throughput dosage unit relevant to the type of nanoparticle.
and reproducible (both intra- and inter-lab), as In addition to accurate representation of nano-
well as predictive of the toxic response in vivo. particle vehicle and/or cargo dose, it is critical
In the realm of model systems, complex culture to explore nanotherapeutic uptake pathways, for
systems enable more reliable results [10] and are both intentional and unintentional exposure.
worthy of further exploration. Although tradi- This relates directly to another of the grand
tional toxicological assays correlate well with LD50 challenges to ‘creat[e] new instrumentation to
studies [144] , they have struggled to accurately measure possible exposure to nanomaterials
predict more complex toxicological mechanisms through air by 2010 and water by 2012, leading
during in vivo studies [10] and have yielded mis- to ‘smart’ nanosensors with the ability to detect
leading results owing to nanoparticle reactivity to potential exposure hazards and identify potential
the probe molecules in vitro [145] . Although it is environmental and/or health reactions by 2017’
clear that we need new assessment tools to study [143] . Clearly, this challenge will only be met with
nanotherapeutic toxicity, only a few have been collaboration between scientists with expertise in
attempted to date. For example, Zhang et al. and instrument design and toxicologists. In addition,
Cuddihy et al. used high-content screening with many more studies are needed on the topics of
a multitude of traditional in vitro assays simulta- chronic exposure, biodistribution, clearance and
neously, facilitating parallel analysis of multiple possible bioaccumulation of nanotherapeutics.
samples, including controls [146,147] . In a less tra- If nanotoxicology is ever going to achieve pre-
ditional approach, toxicity is examined without dictive capability, it is necessary to have a com-
the use of a probe molecule by using single-cell plete and accurate description of the nanoparticle
amperometry to explore biophysical characteris- therapeutic within a realistic biological environ-
tics of cells both with and without nanoparticle ment when performing toxicological assessment.
exposure [103] . Prenner and coworkers measured The most common techniques used to character-
Langmuir isotherms to study the interaction of ize nanoparticles provide only a static picture of
nanoparticles with lung surfactant by monitor- the nanoparticle (e.g., TEM, X‑ray diffraction
ing changes in surfactant surface area and charge or atomic spectroscopy) without consideration
versus pressure [148] . The gaps in assessment of the relevant biological environment. For
technology for nanoparticle therapeutics must example, characterization is either performed
be addressed to effectively address other areas of where the biological environment is ignored,
nanotoxicology, such as dosing. in water or organic solvent alone or under vac-
Determining the dose of therapeutic nano- uum, or in oversimplified models systems, such
particles is complex and inconsistencies in the as adding BSA to model protein coverage. New
dosing, as is evident by the multitude of con- characterization methods are needed to study
centration units used for dose reporting, further how protein adsorption (i.e., opsonization) on
2 Assess whether fiber-shaped 3 Models for predicting engineered-

nanoparticles present a unique nanomaterial behavior in the
health risk environment 2 Validated alternatives
for in vivo nanomaterial
5 Strategic research 2 High-throughput toxicity- 1 ‘Smart sensors’ that indicate potential toxicity tests
programs testing protocols harm
2006 2008 2010 2012 2014 2016 2018 2020 2022
1 A universal aerosol sampler

for airborne nanostructure 3 Methods for engineering
materials nanomaterials that are
1 Instruments to monitor safe-by-design
waterborne-engineered
3 Models for predicting
nanomaterials
engineered-nanomaterial
4 The ability to evaluate the impact of behavior in the body
engineered nanomaterials from
cradle to grave
Figure 9. A timeline for the five ‘grand challenges’ in nanoparticle toxicology.

Reproduced with permission from [143] .

the surface of the particle or particle aggregation a broad survey of the nanomaterials literature
or agglomeration may impact the uptake and/or and/or expanded collaboration with experts in
clearance mechanisms and drug diffusion and nanoparticle fabrication to make nanotherapeu-
release from vehicles. Ideally, new methods would tics with the most desirable properties and the
provide dynamic nanoparticle characterization least toxicity.
both before and after they have been internalized The challenge of creating new nanotherapeu-
by the cells or tissue, considering surface proper- tics and making a realistic assessment of their
ties and particle integrity. Unfortunately, there are toxicity will be pursued optimally with the col-
currently few techniques available for such ana laboration of scientists from materials science,
lysis, even though characterization of nanomateri- chemistry, biomedical engineering, medicine
als both before and during exposure is important and toxicology, with each scientist performing
for understanding mechanisms of toxicity. the task at which they are expert. Although an
In addition, it is imperative to have good and important goal in nanotoxicology would be to
consistent controls for both in vivo and in vitro have predictive guidelines, there currently lacks
experiments to interpret toxicity-assessment systematic and purposeful studies, thus inhibit-
results. First, the toxicity of the nonfunction- ing their creation. Provided the field can address
alized, unloaded nanotherapeutic vehicle needs the need for nanoparticle-specific toxicity
to be measured to characterize unintentional assays, understanding and consistent expression
toxicity. Thereafter, toxicity assessments must of nanoparticle dosing, relevant controls and
be done with each added component, both with appropriate model systems, the full potential of
and without the nanotherapeutic vehicle; this nanotherapeutics can be realized safely.
systematic analysis enables understanding of
how each constituent enhances or diminishes Conclusion
overall toxicity. It is important throughout all The field of nanoparticle therapeutics is expand-
these assessments to ensure that the nanopar- ing rapidly to include new materials and new
ticles are isolated from the starting materials applications; however, evaluations of nanopar-
and trace impurities because these have been ticle toxicity are not currently keeping pace
found, in some cases, to be toxic [149–151] . This with these advancements. This article reviews
effort plays into another of the issued grand the methods being used to assess in vitro nano-
challenges to ‘Develop systems and methods particle toxicity and addresses the challenges in
that enable scientists to assess the potential choosing an appropriate nanoparticle dose for
impact of nanomaterials during their entire life- these studies. Then, relevant examples of nano-
cycle from cradle to grave’ [143] . Additionally, toxicity evaluations using a variety of nano-
although many agents used in drug delivery or particle materials are detailed for three major
PDT are intended to have a cytotoxic effect, it applications of nanotherapeutics: drug delivery,
is still important to understand what portion phototherapy and imaging. In all three nano-
of the effect is owing to the drug and what therapeutic classes, it is clear that current nano-
portion is owing to the vehicle, in order to toxicity studies are not systematic enough to
design the next generation of nanotherapeu- draw generalizable conclusions about controlling
tics. Particularly in the case of nanotherapeu- nanoparticle toxicity. The field of nanotoxicology
tics, the discovery of toxicity mechanisms may presents many opportunities for future research,
be exploited for the use in therapies, such as for including development of new instrumentation,
chemotherapy. assays and appropriate in vitro systems, as well as
As is evident in the examples of nanotherapeu- exploration of nanoparticle uptake mechanisms
tic applications presented herein, even though and dosing characterization.
multiple materials have been exploited to accom-
plish a given task (e.g., liposomes, polymers, den- Financial & competing interests disclosure
drimers and fullerenes for drug delivery), the This work was funded by the National Science Foundation
variation of material properties within each class (CHE-0645041) and NSF Graduate Research Fellowship
is narrow (e.g., only a small set of the many avail- awarded to MA Maurer-Jones. The authors have no other
able polymer monomers have been considered). relevant affiliations or financial involvement with any
Often, it seems that the chosen nanomaterial organization or entity with a financial interest in or finan-
is based only on convenience instead of a full cial conflict with the subject matter or materials discussed
consideration of the desirable and available in the manuscript apart from those disclosed.
material properties. Future work in designing No writing assistance was utilized in the production of
nanoparticle therapeutics would benefit from this manuscript.

Executive summary
Common toxicological assessment methods
In vitro assessments of toxicity measure a variety of cellular characteristics, from DNA damage to membrane integrity, to determine
cell viability.
In vivo toxicity protocols vary widely and it is difficult to correlate with in vitro protocols.
Nanotherapeutic dosing
Dosing of nanotherapeutics depends on both the physical characteristics and the ability of the cell or tissue to internalize
the nanomaterial.
Nanotherapeutic dose must consider not only the dose of pharmaceutical but also the dose of the nanoparticle vehicle because the
unloaded nanoparticle may influence toxicity.
Uptake pathways must be known to characterize the nanotherapeutic dose; owing to the limitations of common techniques, such as
transmission-electron microscopy, the development of new tools to assess uptake is therefore required urgently.
Drug-delivery nanoparticles
Nanoparticle vehicles enable the targeted, stealth delivery of previously unusable pharmacological agents.
Drug delivery can be accomplished with a range of vehicles: liposomes, polymers, dendrimers, fullerenes and others. With a few
exceptions, the drug-delivery vehicles exhibit some cytotoxicity, even without the loaded drug.
Nanoparticles for photodynamic therapy
Targeted nanoparticles can either carry a photosensitizer payload or act as the photosensitizer themselves to facilitate light-activated cell
death, often with increased efficacy owing to the long tissue dwell time that can be achieved.
Photodynamic therapy can be accomplished with a range of vehicles: polymers, fullerenes, metallic and magnetic nanoparticles, among
others. Vehicle surface functionalization strategies minimize unintentional toxicity in the studies that have been completed to date.
Nanoparticle contrast agents
Nanoparticle contrast agents yield increased sensitivity compared with their molecular counterparts, facilitating dynamic imaging of small
physiological features.
Bioimaging can be accomplished with a range of nanomaterials, including quantum dots, superparamagnetic nanoparticles and
multifunctional nanoparticles. Major contributors to unintentional toxicity are well-understood for quantum dots but less
well-characterized for the magnetic contrast agents.
Creative design of multifunctional nanotherapeutics facilitates the synergistic combination of bioimaging with drug delivery and/or
photodynamic therapy.
Future perspective
Major areas for future development include: new toxicity assessment tools specifically for consideration of nanoparticle toxicants,
new model in vitro systems, new nanoparticle-characterization methods and new nanotherapeutic materials.
The nanotoxicology community would benefit by agreeing on a nanoparticle dose metric and arranging round-robin
material exchanges.
Controls are of the utmost importance in nanotoxicology studies. Each component of the nanomaterial must be considered separately
and in combination along with positive and negative controls.
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Toxicity of therapeutic nanoparticles

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Improving measures of dose

Deposited mass, SA or no. per

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generation three melamine-based dendrimers, 15% hemolysis, a significant improvement

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2 Assess whether fiber-shaped 3 Models for predicting engineered-

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1 A universal aerosol sampler

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