Nanomaterials in the Environment: From Materials to High-Throughput Screening to Organisms

Photoactive Nanomaterials

Emerging In Vitro Models for Safety Screening of High-Volume Production Nanomaterials under Environmentally Relevant Exposure Conditions Mustafa Hussain Kathawala , Sijing Xiong , Mark Richards , Kee Woei Ng , Saji George , * and Say Chye Joachim Loo *

1© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim wileyonlinelibrary.com

The rising production of nanomaterial-based consumer products has raised safety concerns. Testing these with animal and other direct models is neither ethically nor economically viable, nor quick enough. This review aims to discuss the strength of in vitro testing, including the use of 2D and 3D cultures, stem cells, and tissue constructs, etc., which would give fast and repeatable answers of a highly specifi c nature, while remaining relevant to in vivo outcomes. These results can then be combined and the overall toxicity predicted with relative accuracy. Such in vitro models can screen potentially toxic nanomaterials which, if required, can undergo further stringent studies in animals. The cyto- and phototoxicity of some high-volume production nanomaterials, using in vitro models, is also reviewed.

Introduction .............................................. 1. 2

Paradigm Shift in Toxicity Testing ............... 2. 3

Emerging In vitro Models and Tools 3. to Enhance Predictability of Nanotoxicity .......................................... 4

In Vitro Safety Screening Under 4. Simulated Environmental Exposure Conditions ..................................9

A Class of High-Volume Production 5. Nanomaterials: Photoactive Nanomaterials .......................................... 10

Conclusion and Perspectives 6. ................... 13

From the Contents

small 2012, DOI: 10.1002/smll.201201452

M. H. Kathawala et al.reviews

DOI: 10.1002/smll.201201452

M. H. Kathawala, S. Xiong, Prof. K. W. Ng, Prof. S. C. J. LooNanyang Technological UniversitySchool of Materials Science and Engineering50 Nanyang Avenue, Singapore 639798, SingaporePhone: + 65 6790-4603; Fax: + 65 6790-9081 E-mail: [email protected]

Dr. M. Richards, Dr. S. GeorgeCentre for Sustainable NanotechnologySchool of Chemical & Life SciencesNanyang Polytechnic180 Ang Mo Kio Avenue 8, Singapore 569830, SingaporePhone: + 65 6550-1517; Fax: + 65 6552-0844E-mail: [email protected]

Figure 1 . (A) Total Products Listed by Nanotechnology consumer product inventory until 2010. (B) Product categories as of March 2011. Reproduced with permission. [ 2 ] Copyright The Project on Emerging Nanotechnologies (PEN).

1. Introduction

The quest to reduce size beyond reasonable proportions

where materials exhibit ‘novel’ properties has perhaps led to

the conquest of nanomaterials. Rapid advances in the fi eld

of nanotechnology have created a whole array of nano-sized

particles with very different chemical and physical properties

as compared to particles in their bulk matter form. [ 1 ] Gener-

ally, nanomaterials can be classifi ed as the category of mate-

rials with at least one of their dimension(s) in the size range

of 1–100 nm. In detail, materials with one out of three dimen-

sions less than 100 nm fall into the categories of nano-fi lms,

coatings, etc., materials with two out of three dimensions

below 100 nm are identifi ed as nanorods, -prisms, -wires, etc.,

while materials with all three dimensions below 100 nm are

classifi ed into the categories of nanoparticles, -fl akes, etc.

When the size of material is reduced to microscopic levels,

there is little effect on the material properties. However,

when sizes reach down to nanoscopic levels, quantum effects

kick in possibly due the exponential increase in surface area

to volume ratio. Surface properties start dominating, and

quantum mechanical and electronic effects are exhibited.

Materials that are generally regarded as ‘inert’ can become

‘active’ at nanometric size and may display unique properties.

These properties offer many attractive possibilities in terms

of cultivable high performance and value-added applications.

Understandably so, nanomaterials are fast impregnating their

footmarks into various fi elds ranging from aerospace, bio-

medical, cosmetics, electronics, food and beverages, to sports

goods, structural components, water purifi cation industries,

and many more.

Because of the vast exploitation of these novel materials,

many of these nanomaterials are commercially available and

are being produced in high volumes, whereby these synthetic

nanomaterials are often referred to as engineered nano-

materials (ENMs). The nanotechnology consumer products

inventory maintained by the Project on Emerging Nanotech-

nologies (PEN) contained 1317 nanomaterial based consumer

products as of March 2011, representing a massive increase of

521% since March 2006. [ 2 ] Figure 1 shows the rising trend of

consumer products in the market and the product categories.

As refl ected from Figure 1 , it is evident that nanomate-

rials have penetrated many fi elds. The various possibilities

2 www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA,

encompass medicine, drug delivery, regen-

erative sciences, ceramics, electronics,

micro-packaging, sports goods, cosmetics,

antibiotics, etc. [ 3–12 ] Most of the high

volume production nanomaterials belong

to the class of photoactive nanomaterials

which is a group of nanomaterials that are

activated under visible or invisible elec-

tromagnetic waves, and are beginning to

gain importance in a variety of applica-

tions. [ 13–17 ] TiO 2 and ZnO nanoparticles

fi nd themselves in the thick of this class of

materials, and are the third and fi fth most

abundant constituent in nanotechnology

consumer products according to PEN. [ 2 ]

We will discuss more on their potency and

drawbacks in the latter part of this review.

In the recent years of industrial development in exploiting

nanotechnology, ENMs may have inevitably leached and dis-

charged into the environment as pollutants and will continue

to accumulate over the years. However, many of these ENMs

have not been adequately scrutinized for potential harm. It

is therefore imperative to have not only a comprehensive

knowledge about these ENMs, but also an understanding of

their potential health effects. An important lesson that can

therefore be drawn from asbestos, for which the discovery

of its toxicity came too late and its impact on the health of

humans was detrimental. However, unlike drugs intended

for the treatment of human diseases, the majority of these

ENMs have also not been subjected to the stringent criteria

in determining toxicity by way of pre-clinical safety assess-

ment in animal models and ensuing clinical trials. Further-

more, exposure to these substances is usually unintentional,

thus parameters that typically contribute to toxicity, such as

dose, duration of exposure are diffi cult to assess.

These considerations coupled with increasing public

awareness have led to a renewed interest in predictive toxi-

cology assessment for chemical hazards in the environment

and potentially harmful effects on human health. Exposure of

high volume production (HVP) ENMs to workers and con-

sumers is growing as nanotechnology becomes increasingly

more pervasive in society. [ 18–21 ] Human health concerns for

Weinheim small 2012, DOI: 10.1002/smll.201201452

http://doi.wiley.com/10.1002/smll.201201452

In Vitro Models for Safety Screening Nanomaterials in Relevant Exposure Conditions

Joachim Loo is an Associate Professor in the

School of Materials Science and Engineering,

Nanyang Technological University (NTU). He

received his PhD from NTU as a recipient of

the A ∗ STAR Graduate Scholarship, and his

post-doctoral training in Mayo Clinic (MN,

USA). His research interests include con-

trolled drug-delivery systems and nanotoxicol-

ogy. On the nano-safety front, he is a member

in both the ISO/TC 229 Nanotechnology

National Working Group under SPRING

Singapore, and the National Environment

Agency (NEA) Expert Resource Panel, deal-

ing with nano-safety issues in Singapore.

Saji George is a lecturer at the School of

Chemical and Life Sciences at Nanyang

Polytechnic (NYP) Singapore. He obtained

his PhD from the National University of

Singapore and joined the Centre for Envi-

ronmental Implications of Nanotechnology

at University of California, Los Angeles, for

his postdoctoral studies. At the Centre for

Sustainable Nanotechnology, NYP Singapore,

Dr. George is overseeing research activities

related to nanomaterial properties that lead

to benefi cial and hazardous outcomes in dif-

ferent biological systems, and in developing

high-throughput screening platforms for the

safety screening of nanomaterials. His research interests include environmental fac-

tors that shape nano-bio interactions, and risks.

ENMs are established historically by epidemiologic and clin-

ical studies on naturally occurring fi bres and particles such

as asbestos and silica. [ 18 , 19 , 21 ] Furthermore, although nano-

toxicology research is well-established, the reported effects

of bulk-production ENMs such as nano-Ag and nano-ZnO in

vitro appears to be quite varied and dependant on a variety

of factors such as cell type, particle size, species, coating and

manufacturing process. [ 22–35 ] For these reasons, new screening

assays are urgently required to assess a plethora of chemi-

cally and physically diverse ENMs present in consumer prod-

ucts. The prohibitive cost of in vivo experiments and negative

public sentiment towards animal testing also favours the use

of novel in vitro models for preliminary testing of ENMs

to assess potential toxicity and their ability to elicit human

disease.

The aim of this review is therefore to emphasize the

applicability of various screening models that have been

developed to address the potential hazardous effects of

ENMs. The next section would discuss the paradigm shifts

in toxicity testing and the efforts that scientifi c boards have

directed towards this cause. We then delve straight into the

thick of emerging in vitro toxicity screening models, which

are gaining popularity due to the speed of output or the spe-

cifi city of the outcome. Subsequently, some attempts to simu-

late actual exposure conditions and mimic real life conditions

through in vitro models are also discussed. Finally, a group of

high volume production nanomaterials, i.e., the photoactive

nanomaterials (e.g., TiO 2 and ZnO), and their mechanisms of

action in terms of photocatalysis, and cyto- and photo toxicity

through these in vitro approaches are reviewed. In conclu-

sion, this review aims to summarize some of the key fi nd-

ings and suggests effective approaches which may expedite

solving the labyrinth, i.e., nanotoxicology, by employing novel

toxicity models, as detailed in this review.

2. Paradigm Shift in Toxicity Testing

Although modern toxicology has a history of over 80 years

as a scientifi c discipline, it is probably the only scientifi c fi eld

where core experimental protocols and methods of testing

have remained unchanged for more than 40 years. [ 36 ] Inter-

estingly enough, it is in the last 40 years that it has witnessed

an exponential growth in the science and applications of

newer materials–including nanomaterials. Furthermore, over

these recent years, the awareness and expectation about the

safety of chemicals and materials has increased among the

consumers. These developments have been the impetus for

regulatory and government agencies all around the world to

advance the safety screening of chemicals and materials, the

European Union’s regulation by legislation in 2007, known

as Registration, Evaluation, Authorisation and Restriction of

Chemicals (REACH) being one such example. One should

bear in mind that the key aspects that determine the effec-

tiveness of environmental health and safety regulations are,

(1) the capability to assess the safety of the materials and,

(2) the ability to detect chemicals and materials in complex

media. With regards to safety screening, the overwhelming

concern is that the traditional toxicology methods dominated

© 2012 Wiley-VCH Verlag Gmsmall 2012, DOI: 10.1002/smll.201201452

by animal models are too tedious and resource intensive to

cope with the infl ow of novel synthetic chemicals and mate-

rials. The ineffi ciency of traditional toxicology methods is evi-

dent when we realize that safety data for 86% of all the major

chemicals in use are lacking despite the fact that 97% of these

chemicals were produced three decades ago. [ 36 ] These exam-

ples signify the pressing need of alternate safety screening

strategies that are faster, cheaper and more reliable. [ 36 ]

Similar to the dilemma with synthetic chemicals, the

advances in nanomaterial fabrication over the past few dec-

ades have made it possible to generate thousands of nano-

materials with unique physicochemical characteristics, even

from a single element, by changing either of the nanoscale

design parameters such as size, shape, phase, aspect ratio,

surface chemistry, and surface functional groups. Here too,

the traditional toxicology approach involving animal experi-

ments may not be appropriate for the timely completions of

toxicological profi ling of the huge number of new nanomate-

rials being introduced. [ 36 , 37 ] In spite of the huge cost involved

in animal experiments, this approach suffers from the limited

mechanistic information on the toxicity of ENMs. Therefore,

the National Research Council (2007) advocated the use of

mechanistically informative in vitro assays based on human

cells or human cell constituents that measure effects on

“toxicity pathways” leading to human disease, necessitating

assaying for multiple biological endpoints. Thus, contradic-

tory to the traditional approach of using animal models in

3www.small-journal.combH & Co. KGaA, Weinheim

the front lines of toxicity screening, the new approach advo-
cates for the use of in vitro models in the initial stages of tox-

icity screening. The advantage of such a paradigm shift being

the increased throughput of toxicity screening with minimal

resource expenditure. As in the case of synthetic chemicals,

similar approach could be adopted for nanotoxicology where

the in vitro models could facilitate faster identifi cation of

potentially ‘hazardous’ nanomaterials which, at a later stage,

should undergo more stringent studies in animal models.

3. Emerging In vitro Models and Tools to Enhance Predictability of Nanotoxicity

3.1. In vitro Model 1: 2D Culture, Assays, and Towards High-Throughput Screening

As is in the case of synthetic chemicals, there is a surge in the

production and application of ENMs which hence poses the

same conundrum as synthetic chemicals on our capacity to

assess their safety. An article published in 2009 estimated that

the time taken to complete the safety testing of ENMs gener-

ated then in US alone would be 34–53 years at an estimated

cost of $1.18 billion. [ 38 ] Added with the severe market pres-

sure for newly generated nanomaterials, it is understandable

that many of them will fi nd their way to consumers without

undergoing adequate safety evaluation. Therefore, it is the

requirement of time to revitalize the safety screening methods

that are effi cient in terms of time and resource involvement.

The commission communiqué issued in Rome in the month

of August 2009 at the World Congress on Alternatives and

Animal observed that a “Faster, cheaper and more reliable

alternative methods will contribute to increased safety” while

reducing the use of animals. [ 39 ] In fact, the National Research

Council (2007) of the US National Academy of Sciences pro-

posed the use of more robust models for in vitro toxicological

testing that can be carried out for larger batches of toxicants

but still maintain relevance to in vivo outcomes. [ 40 ]

The advancements in medical science have helped in

gaining suffi cient insights into the molecular and cellular

mechanisms underpinning disease conditions in human

arising from workplace exposure to toxicants. One such

example is the ‘metal fume fever’, an acute infl ammatory

response caused by the inhalation of metal oxide (e.g., nano-

ZnO) particles generated during the welding process. Metal

fume fever is characterized by the presence of pro-infl amma-

tory cytokines such as IL-8 and TNF- α in the bronchoalve-

olar lavage fl uid of patients. [ 41 ] Noteworthy is the possibility

of demonstrating the induction of these cytokines by cellular

models of inhalation toxicology [ 42 ] that signifi es the possibility

of using in vitro models in predicting the possible infl amma-

tory response in vivo. Similarly, recent reports suggest the

potential of carbon nanotubes in causing mesothelioma, a

cancer of the lining of the lungs often caused by exposure to

asbestos. [ 43–45 ] Detailed investigation into the molecular and

cellular mechanism suggested the role of ‘frustrated phagocy-

tosis’, and subsequent induction of infl ammatory responses in

causing mesothelioma like pathological conditions in animal

4 www.small-journal.com © 2012 Wiley-VCH V

model. [ 46 ] Interestingly, many in vitro investigations that fol-

lowed could delineate the cellular markers responses of injury

mechanisms highlighting the possibility of predicting in vivo

outcomes from in vitro studies. [ 47–50 ] Another example is the

pathological condition named ‘Ardystil syndrome’ in textile

paint sprayers which is caused by the exposure to polycati-

onic components of paint, where it is possible to demonstrate

the membrainolytic action of cationic polymers using cel-

lular models with relatively high degree of in vitro to in vivo

predictability. [ 51 , 52 ]

The above-mentioned examples suggest that the knowl-

edge about the molecular and cellular injury mechanisms

underpinning pathological conditions could be fruitfully

utilized for identifying potentially harmful chemicals and

nanomaterials without relying primarily on animal models.

However, unlike animal model, molecular and cellular assays

provide a much more focused view on the effect of a poten-

tial toxicant. Accidental or therapeutic exposure of nanoma-

terials may not necessarily be restricted to a single tissue type

in human body and often it becomes necessary to test an array

of cell types for cytotoxicity evaluation. As an example, it has

been demonstrated that TiO 2 that is either injected intraperi-

toneally or ingested orally could transcytosis across epithelial

lining or across endothelial cells into the blood circulation,

respectively, could be entrapped by the reticular-endothelial

system, all the way engaging many different cell types. [ 53 , 54 ]

The knowledge on the route of exposure of the nanomaterial

and their accumulation in specifi c tissues in the body would

enable one to choose the appropriate cell lines to be selected

for the study. Often it becomes pertinent to include more

than one cell line and multiple cytotoxicity parameters assays

to improve the predictability of in vitro observations. A study

conducted by Weissleder’s group has shown that the predic-

tive power of in vitro toxicity assessment of nanomaterials

could be substantially improved by including multiple cell

lines and testing for multiple cytotoxicity parameters. [ 55 ] Dif-

ferent cell lines, representing almost entire cell types in human

body, have been successfully cultivated under defi ned growth

conditions and the versatility in the selection of cell types

and culture conditions has been of great advantage in evalu-

ating the potential health hazards due to the intentional and

unintentional exposure to nanomaterials. However, testing

for multiple parameters in multiple cell line added with the

necessity to conduct toxicity studies in physicochemical vari-

ants of a single nanomaterial is a costly and time consuming

process which again will impede the toxicity evaluation of

nanomaterials. High throughput screening approach, where

the key operations involved in the cytotoxicity screening are

automated, is therefore a viable solution to this bottleneck.

High throughput screening (HTS), has been employed

by drug industry for decades to advance the drug screening

process. Generally, a HTS platform is constituted by inter-

linked modular units that automate one or more operations

in the workfl ow of compound testing. Some of the key opera-

tions involved in the screening of molecule library are, repli-

cation of mother plate, plate stamping, preparation of working

concentrations of compounds, cell seeding to multiwell plates,

plate washing, addition of compounds and reagents to multi-

well plates, reading of the plate, data archiving and data

erlag GmbH & Co. KGaA, Weinheim small 2012, DOI: 10.1002/smll.201201452


analysis. Since all these key operations are automated in a

HTS, the time taken for entire process of lead compound

identifi cation is drastically lowered. The reagent consump-

tion in a HTS is substantially lowered since the assays are run

with microliter quantities of assay reagents. Furthermore, the

possibility of multiplexing—combining more than a single

parameter in one assay—reduces the resource requirements.

Although, HTS was conceived for drug screening, the utility

of this powerful tool for toxicity screening should not be

underestimated. In fact, HTS has also been adopted in safety

screening as an alternate tool in place of the more labour-

intensive and descriptive toxicological approaches. [ 55–58 ]

The use of HTS and libraries of well characterized nano-

materials for identifying material properties in relation

to their potential hazard has been exemplifi ed by several

reports from Andre Nel’s group at the University of Cali-

fornia Los Angeles. A multi-parametric HTS platform and

several toxicity testing methods were optimized for in vitro

toxicity screening of nanomaterials. The choice of cell lines

and cell response pathways selected has been in line with the

relevance to route of exposure of specifi c nanomaterial in

question, as discussed previously. This multi-parametric HTS

measures cellular responses encapsulated in the fi nal stage of

hierarchical oxidative stress paradigm. According to hierar-

chical oxidative stress paradigm, when a cell is subjected to

low levels of oxidative stress (Tier 1), genes for antioxidant

enzymes are activated in an attempt to restore cellular redox

homeostasis. This includes genes that encode the phase II

enzymes having Nrf-2 as the transcription factor. At higher

level of oxidative stress (Tier 2), activation of MAPK and

NF-kB cascades induces pro-infl ammatory responses, e.g.,

induced production of cytokines and chemokines. When the

oxidative stress is beyond the tolerable limit of cell (Tier 3),

cytotoxic events such as perturbation of the mitochondrial

permeability transition pore, infl ux of calcium, membrane

damage etc. takes place which ultimately lead to apoptosis or

necrosis. [ 59 , 60 ] The cellular events in cytotoxicity pathway are

common to many toxic assaults to be aptly named as ‘fi nal

common pathway of cytotoxicity’. Therefore, although, origi-

nally developed to accommodate toxicity due to high levels

of oxidative stress, the cellular events in cytotoxicity pathway

such as mitochondrial superoxide generation, mitochondrial

depolarization, and intracellular calcium fl ux are shown to be

appropriate for screening of nanomaterials. Thus their HTS

platform measures intracellular reactive oxidative species

(ROS) generation, mitochondrial depolarization, increased

intracellular calcium [Ca 2 + ] and plasma membrane damage by

using an automated epifl uoresence microscope after staining

the cells with fl uorescent dyes MitoSox Red, JC1, Fluo-4 and

Propidium iodide, respectively. [ 61 ] This platform was indeed

instrumental in delineating several nanotoxicology paradigms.

Examples include the, screening of compositional library

of nanomaterials for toxicity ranking, [ 62 ] identifi cation and

demonstration of particle dissolution and shedding of toxic

metal ions in mediating toxicities of ZnO and Qdot nanopar-

ticles, [ 61 , 62 ] demonstration of the role of band-gap energy in

determining the potential phototoxicity of TiO 2 nanoparti-

cles under visible light activation, [ 63 ] and demonstrating the

role of cationic density at the material surface of mesoporous


nanoparticles in infl uencing its toxic potential. [ 64 ] Similarly,

a HTS assay was optimized by the same group to speed up

the screening of high aspect ratio nanomaterials such as

carbon nanotubes (CNTs) for their potential profi brogenic

effects by using a combination of cell nuclear fl uorescent dye

Hoechst and a DNA synthesis fl uorescence labelled EdU

(Click-iT EdU cell proliferation assay). [ 50 ] Fibroblast prolif-

eration can be regarded as an early and important marker of

lung fi brosis, a pathological condition often associated with

chronic exposure to long aspect ratio materials. [ 65 , 66 ] This

HTS assay measurement substantiated by mice model studies

was instrumental in demonstrating the higher fi brogenesis

potential of well-dispersed multiwalled carbon nanotubes

(MWCNTs). [ 50 ] Similar strategy was also devised to demon-

strate that at lengths ≥ 200 nm and aspect ratios ≥ 22, CeO 2 nano-

rods induced progressive cytotoxicity and pro-infl ammatory

effects. [ 67 ] Recently, the same group also reported HTS plat-

form and automated image scoring algorithm for toxicity

testing in zebrafi sh embryo–an emerging in vivo model for

toxicity testing. [ 68 , 69 ]

ToxCast program initiated by Environmental Protection

Agency (EPA) in the USA practices and advocates for the

use of HTS assay platforms in the toxicity screening and iden-

tifying injury pathways induced by large number of chemi-

cals in their inventory. [ 70 ] Recently, ToxCast initiated many

cell-based HTS assays on nanomaterials, with the ultimate

goal of identifying toxicity/biological pathways affected by

nanomaterials and fi nding correlations among nanomaterial

physicochemical characteristics, testing conditions, and nano-

material toxicities/bioactivities. [ 71 ] The use of HTS in identi-

fying potentially toxic nanomaterials with potential medical

applications was shown by Shaw et al. [ 55 ] They tested fi fty

different nanomaterials (mainly of iron and quantum dots)

with potential medical application in multiple cells lines for

multiple cytotoxicity parameters. By the use of computational

tools, they identifi ed nanomaterials that behaved similarly

with regard to biologic response. [ 55 ] Their study signifi es the

utility of HTS for quick assessment of risk to benefi t ratio

of nanomaterials for medical application- exemplifying the

possibility of implementing a decision directed approach in

developing novel nanotherapeutic applications.

3.2. In vitro Model 2: Predictive Toxicity through the Use of Stem Cells

Human embryonic stem cell (hESC) cell lines fi rst derived

in 1998 from the isolation and serial sub-culture of inner

cell masses from 5-day old blastocysts are capable of dif-

ferentiating into all somatic cell types in the adult human.

hESCs have enormous potential in providing a source of tis-

sues for replacement in diseases in which native cell types

are inactivated or destroyed. [ 72–74 ] The ability of hESCs to

undergo multi-lineage spontaneous differentiation in vitro

and in vivo into all 220 cell types in the body is very well

documented. [ 75–77 ] Induced pluripotent stem cells (iPSCs)

represent another type of pluripotent stem cells artifi cially

derived in vitro from any somatic cell by inducing expression

of specifi c stem cell genes. [ 78–83 ] iPSCs are similar to hESCs


in many respects such as gene and protein expression pro-
fi les, chromatin methylation patterns, population doubling

times, embryoid body, teratoma and chimera formation, and

plasticity. [ 79–83 ] iPSCs were fi rst derived in 2006 from mouse

cells [ 78 ] and subsequently in 2007 from human cells in a series

of experiments by Yamanaka and co-workers from Kyoto

University. [ 79 ] iPSC technology is considered an important

advance in stem cell research because it allows scientists to

derive genetically matched pluripotent stem cell lines for

patients without recourse to destroying human embryos

thereby circumventing many of the ethical concerns associ-

ated with hESC research.

hESCs, iPSCs and their differentiated somatic progenies

represent a promising tool for novel toxicology paradigms

particularly if these cell types can be shown consistently to

be a reliable, renewable and unlimited source of differenti-

ated specialized human cells (e.g., neurons, hepatocytes, and

cardiac muscle cells). The potential of applying hESCs and

iPSCs in the development of new nanotoxicology paradigms

are outlined below.

First, the ability of pluripotent stem cells to consist-

ently differentiate into a variety of bona fi de specialized cell

types of wide pharmacological interest such as liver hepato-

cytes, [ 84–86 ] lung epithelia [ 87 ] and cardiomyocytes [ 77 ] and their

ability to develop even into organ systems [ 88 ] could allow

them to replace transformed cell lines and primary cells

which are currently favoured for in vitro nanotoxicity studies.

Using a well-characterized defi ned population of normal

human specialized cells in toxicity tests will greatly improve

the relevance and reproducibility of predictive assays.

Second, hESCs and iPSCs are superior to primary cells

or immortalized cell lines because they are an inexhaustible

and renewable resource. Pluripotent stem cell lines also have

normal karyotype and physiology but still retain the ability to

self-renew indefi nitely. [ 73 , 74 ] This eliminates irreproducibility

and supply limitations associated with primary cell lines as

well as issues with a tumorigenic phenotype when trans-

formed cell lines are used in toxicity screens. [ 18 , 89 ]

Third, pluripotent stem cell lines are routinely geneti-

cally modifi ed with reporter constructs, knockouts or siRNA

knockdowns. [ 90 , 91 ] The ease at which pluripotent stem cells

can be genetically manipulated suggests that stem cell lines

can be engineered with fl uorescence reporter constructs to

detect the presence of heavy metals, teratogenic compounds

or other toxic ENMs. Transgenic reporter lines can add a new

dimension and will be advantageous in developing new nano-

toxicity assays.

Fourth, the human origin of these cells would refl ect

human physiology more closely compared to live animal

models or animal cells. Testing ENMs in a human system

should increase the relevance and accuracy of predicting

toxicological outcomes and at the same time reduce reliance

on controversial and ethically contentious animal testing

procedures.

Fifth, an ability to derive iPSCs from individual human

subjects would offer unprecedented opportunities to analyze

the contribution of individual genotype and epigenetic fac-

tors that affect susceptibility to toxicity at the level of the

individual.

6 www.small-journal.com © 2012 Wiley-VCH

Sixth, pluripotent stem cells immediately offer new

models for embryotoxicity and teratogenecity testing because

of their unique ability to form embryoid bodies. [ 73 , 74 , 79 ]

Embryoid bodies are three-dimensional aggregates of differ-

entiating pluripotent stem cells that form when these cells are

grown in suspension cultures. [ 73 ] Cellular differentiation in

embryoid bodies is believed to recapitulate some of the proc-

esses during embryonic development; this special feature of

pluripotent stem cells can be easily harnessed to develop new

embryotoxicity tests for ENMs. The differentiation of mouse

embryonic stem cell embryoid bodies into cardiomyocytes

has been successfully used as a model to assess embryotox-

icity in vitro. [ 92 ] This embryonic stem cell test has been suc-

cessfully validated by the European Center for the Validation

of Alternative Methods [ 92 ] and models fundamental mecha-

nisms in embryotoxicity such as cytotoxicity and differen-

tiation. The inhibition of embryoid body differentiation into

beating cardiomyocytes, the cytotoxic effects on stem cells

and the cytotoxic effects on control fi broblasts are used col-

lectively as end-points to determine the embryotoxic poten-

tial of a test compound. This in vitro test also eliminates the

need to use embryonic cells and tissues from pregnant ani-

mals for embryotoxicity assessment and is one of the few cur-

rently adopted and accepted toxicology test protocols which

utilize stem cells.

Seventh, hESCs and iPSCs are among the best char-

acterized and studied cell types today. The scientifi c

literature is replete with reports on the characterization, deri-

vation, genomics and differentiation of pluripotent stem cell

lines. [ 73–87 , 91 , 93 , 94 ] This comprehensive background of scientifi c

literature is not only a wealth of information but also serves

as a very valuable resource on which future studies can build

on. Additionally, several research consortiums and initiatives

are already in place to oversee the development of harmo-

nized standards for pluripotent stem cell culture, differentia-

tion and characterization protocols. [ 95 , 96 ] Taken together, the

robustness of stem cell science is already clearly evident and

improvements to technical handling protocols and SOPs will

likely increase steadily over time. These considerations make

pluripotent stem cell lines an ideal foundation cell type from

which enhanced and more sophisticated nanotoxicology pro-

tocols can be developed.

Eighth, appropriate panels of several hundred hESC

or iPSC lines and their specialized derivatives can also be

used to measure ENM-induced toxicity on diverse cell types

across a milieu of different genetic backgrounds. This would

enable discovery of physiologically relevant markers, genes

and pathways specifi cally associated with nanotoxicity early

in safety studies.

The option to use a large number of cell lines with dif-

ferent genetic backgrounds in parallel for nanotoxicology

assessment also allows for the development of a “tissue-

directed in vitro pre-clinical trial”, prior to any safety trial

involving in vivo animal models. The use of a series of human

pluripotent stem cell lines with different genotypes will facili-

tate large-scale in vitro analyses, predicting nanotoxicity at

the population level against genetic backgrounds, as well

as identifying biomarkers and signalling pathways for the

development of complementary diagnostic assays to predict

Verlag GmbH & Co. KGaA, Weinheim small 2012, DOI: 10.1002/smll.201201452


nanotoxicity across different genotypes. Therefore, the idea

and proposal of developing “tissue-directed in vitro pre-

clinical trials” using stem cells represents a unique step

towards making broad-based, large-scale toxicity studies

in the domain of stratifi ed medicine a reality. Data gener-

ated from such a study would be necessarily complex but

extremely valuable in accurately predicting and pinpointing

the toxic effects of ENMs on human health.

Taken together, the use of human stem cell systems in

toxicology has the potential to dramatically increase our

ability to predict toxic responses in humans. Stem cells could

increase the predictive power of high throughput cell-based

assays by providing large quantities of reliable, high-quality

human cells for assay development and mechanistic studies.

However, it is noteworthy that the literature is also pep-

pered with studies which question the genetic stability of

pluripotent cell lines. [ 97 , 98 ] Pluripotent stem cell lines gener-

ally remain karyotypically normal but progressively tend to

acquire genetic changes in prolonged culture. These muta-

tions commonly occur in chromosomes 1, 12, 17, and 20 and

have been reported by several groups. [ 99–101 ] DNA methyla-

tion patterns have also been observed to change somewhat

haphazardly with no link to time in culture. [ 102 , 103 ] Genetic

stability issues are therefore of key concern if pluripotent

stem cell-based toxicity paradigms are to enter mainstream

toxicology. These issues must be addressed and resolved with

urgency. Proof-of-concept studies must also be performed

on new stem cell-based toxicology tests in comparison with

accepted toxicology protocols to validate their suitability for

widespread adoption.

Stem cells and their ability to differentiate into diverse cell

types have created opportunities to rethink the way in which

ENMs are evaluated for risk to human health. If certain chal-

lenges can be overcome, it might be possible to improve or

replace many conventional models of nanotoxicity screening

with more relevant human systems, resulting in improved

nanotoxicology prediction and perhaps an even better under-

standing of how genetics and environmental exposure con-

tribute to sensitivity and susceptibility to disease.

3.3. In Vitro Model 3: 3D Culture and Tissue Engineering Approaches

Conventional biomedical research has relied heavily on the

use of in vitro, 2D cell culture systems for probing cellular

behavior. [ 104 , 105 ] These offer quick, convenient and relatively

reproducible means of understanding biological processes

in relation to any form of disturbances to the cellular envi-

ronment. However, it has become clear that 2D cell culture

systems are often inadequate in recapitulating the multitude

of physiological stimuli and cellular responses that are repre-

sentative of the in vivo, 3D scenario. [ 106 ] On a 2D substrate,

cells attach to a synthetic surface via cell surface receptors

that recognize cell attachment motifs (supplemented or

secreted) that are adsorbed on the surface. In the native 3D

environment of the extracellular matrix (ECM), cells regu-

late various functions through discrete cell-matrix adhesions

that exist in confi gurations that are specifi c to each matrix

© 2012 Wiley-VCH Verlag Gmbsmall 2012, DOI: 10.1002/smll.201201452

environment. [ 107 ] Not surprisingly, the resulting phenotype

and behavior of the same cell type become highly varied and

are highly dependent on the complex interactions with the

ECM and neighbouring cells. Driven by the evolution of tissue

engineering, [ 108 ] explorations into the relevance of 3D culture

systems has permeated into the fi elds of tissue regeneration

and regenerative medicine, [ 109 ] cancer research, [ 104 ] develop-

mental biology [ 105 ] and more recently, toxicology. [ 110 , 111 ]

In 1993 Robert Langer and Joseph Vacanti from Massa-

chusetts Institute of Technology and Massachusetts General

Hospital published the landmark paper on tissue engineering

which demonstrated the realistic possibility that 3D cultures

can bring–growing transplantable tissues and organs in the

laboratory. [ 108 ] The success of tissue engineering will clearly

help to solve the acute problem of organ shortage. In addi-

tion, it has provided a platform for recreating more real-

istic tissue cultures in the laboratory for scientists to answer

a whole range of biological questions. In cancer research,

the landmark study which convincingly proved the poten-

tial biological discrepancies that 2D and 3D models can

give was reported in 1997. Mina Bissell’s group at the Law-

rence Berkeley National Laboratory showed that antibodies

against β 1-integrin resulted in breast cancer cells losing their

abnormal shapes and patterns of growth when cultured in

3D. [ 112 ] The cells appear to become non-cancerous, which was

never observed in 2D. Not surprisingly, the use of 3D culture

models has also been explored in nanotoxicology research.

Evaluating nanomaterial uptake and transport through

cells and tissues is vital in establishing fundamental under-

standing of the mechanism of nanomaterial induced toxicity.

In this respect, the availability of realistic models that imi-

tate the cellular architecture of the tissue of interest will be

important. [ 113 ] Furthermore, 3D culture systems also ensure

the relevance of the biochemical landscape within the tested

environment. For example, mammary epithelial cells only

enhance their protein secretion functions when organized in

a 3D acinus structure. [ 114 ]

3.3.1. 3D Spheroids and Cell Sheets

Cellular spheroids are perhaps the most straightforward and

easy strategy to move from 2D to 3D. [ 115 , 116 ] Early efforts in

nanotoxicology research using 3D spheroid cultures focused

on hepatocyte spheroids for testing, where efforts in liver

tissue engineering demonstrated these spheroids to remain

highly viable while preserving the relevant metabolic func-

tions. [ 117 , 118 ] Hepatocyte spheroids are often chosen because

the liver is where nanomaterials tend to accumulate. [ 119 ] Lee

et al. recently adopted a polyacrylamide based hydrogel scaf-

fold to create a 3D liver tissue spheroid model for the in vitro

testing of cadmium telluride (CdTe) and gold (Au) nano-

particles. Using a human hepatocellular carcinoma cell line

(HepG2), the group showed that the toxic effects induced by

the nanoparticles were signifi cantly reduced in the spheroid

cultures when compared to conventional 2D cultures. [ 111 ] No

mechanism for this observed difference was proposed but the

authors postulated that reduced transport of the nanoparti-

cles through the dense tissue-like spheroids and phenotypic

changes due to intensifi ed cell–cell interactions were the

reasons for the diminished toxicity. This fi nding is somewhat

7www.small-journal.comH & Co. KGaA, Weinheim

M. H. Kathawala et al.

8

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Figure 2 . Organotypic culture of human skin, recapitulating the bilayered anatomy of skin but void of all appendages.

comforting since it implies that the signifi cant toxicological

effects of nanomaterials found in 2D studies may not be

as severe as they seem in the context of real life exposure.

However, it has been rightly cautioned that the absence of

cell death does not necessarily mean that the cells are not

undergoing any undesirable changes in function or signal-

ling, which could lead to cynical outcomes. [ 120 ] Although 3D

spheroids represent an important step towards enhancing the

relevance of nanotoxicological studies, their size limitation,

typically up to a few hundreds of microns in diameter due to

diffusion limits, could restrict their widespread acceptance as

the model of choice in the fi eld.

Cell sheets are an alternative way to build up 3D constructs

that mimic native tissue architecture. These has been shown

to be feasible in producing blood vessels, [ 121 ] skin, [ 122 , 123 ] cor-

neas, [ 124 ] urothelium [ 125 ] and cardiac patches. [ 126 ] Essentially,

this strategy allows cells to be cultured to high densities to

form sheets which are then detached from culture surfaces

simply by mechanical means [ 123 ] or by changing temperature

if the cells are cultured on thermo-responsive surfaces. [ 127 ]

Using cell sheets has the advantage that an entirely natural

neo-tissue assembled by the cells can be produced. However,

there has not been any study employing the use of cell sheets

specifi cally to nanotoxicology research. This could be an

interesting approach, especially in evaluating the mass trans-

port of nanomaterials through tissues.

3.3.2. 3D Scaffold-Based Cultures

Borrowing the concept of scaffold-based tissue engineering,

toxicologists have also adopted toxicity testing in 3D cultures

achieved by growing cells in different scaffold confi gurations.

Cui et al. did this by culturing human bone marrow cells in

3D scaffolds made from combinations of collagen, Matrigel

and poly(lactic acid) fi bers. [ 110 ] These were maintained in

a multiple parallel microbioreactor platform and used for

testing of two model drugs, trimethoprim and pyrimethamine,

over a 7 day culture period. The group’s results demonstrated

that data obtained from the 3D culture system is signifi cantly

different from that of 2D cultures. However, comparisons

between the 2D and 3D systems are diffi cult given the com-

plexity of such systems where infl uence from the biomaterials

used will also need to be taken into consideration. Nonethe-

less, such systems enable the creation of a controlled and per-

fused environment to enhance reproducibility of 3D cultures.

In fact, more advanced versions of microbioreactors have

been developed to enable the co-culture of different cell

types in 3D scaffolds, for drug testing in a more complex but

realistic scenario. [ 128 ] It will be interesting to follow what 3D

scaffold systems in combination with microbioreactors will

reveal in nanotoxicology research.

3.3.3. Organotypic and Ex Vivo Cultures

An extension of 3D spheroids and scaffold-based cultures is

the strategy of using organotypic cultures to achieve physi-

ologically more realistic models for toxicology testing. These

are culture systems that take into consideration the spatial

distribution of cell type(s) to impart structural organiza-

tion that is unique to the tissue of interest. The earliest, and

www.small-journal.com © 2012 Wiley-VCH Ve

perhaps still most successful, model of that is the skin organ-

otypic culture ( Figure 2 ), which was developed with the moti-

vation of producing transplantable skin grafts. Early work by

Bell et al. showed that bovine collagen lattices seeded with

dermal fi broblasts contracted to form dermal-like tissues. [ 129 ]

Culturing keratinocytes on top of these dermal lattices in

an air-exposed condition led to their differentiation to form

the stratifi ed layers of a matured epidermis. [ 130 , 131 ] These cul-

tures retain most of the anatomical features of the epidermis

but are void of all skin appendages. Although not real skin,

they represent the next closest and most convenient model

for testing the effects of topical agents before moving onto

animal or human trials–easy to apply histological and immu-

nohistochemical techniques for analysis of cell/tissue integrity

and marker expression without having to harvest biopsies.

As a result, skin organotypic cultures have been successfully

used for the testing of carcinogens [ 132 ] and various topical

agents [ 133 ] to good effect. The enzymatic profi le in skin orga-

notypic cultures has recently been reported to be suitable for

dermatotoxicological studies. [ 134 ] Ex vivo skin cultures have

mostly been used for studying nanomaterial penetration

through the epidermal barrier. Ravichandran et al. managed

to establish the feasibility of using ex vivo human skin cul-

tures to assess the effects of tape stripping versus a depilatory

agent on quantum dot penetration through the epidermis. [ 135 ]

By optimizing fl uorescent microscopy analysis of microtomed

skin sections, the group showed that depilatory treatment

increased penetration of quantum dots but to a lesser extent

than tape stripping. In studying penetration of nanomate-

rials through skin, it was highlighted that careful considera-

tions need to be made in various factors such as time course

of treatments that may compromise skin barrier functions,

the skin model to use and the characterization methods. [ 136 ]

Ex vivo porcine skin cultures have also been used recently

to demonstrate the importance of nanomaterial biodistribu-

tion in relation to vascular physiological effects. [ 137 ] In this

study, six different nanoparticles of different size and com-

position (silica- or dextran-coated Fe 2 O 3 , silica- or citrate-

coated silver, PEG, and carboxylated quantum dots) were

rlag GmbH & Co. KGaA, Weinheim small 2012, DOI: 10.1002/smll.201201452


infused through porcine skin fl aps and were found to result

in statistically signifi cant post-infusion fl ap weight gain and

an increase in arterial perfusion pressure. Interestingly, infu-

sion with nC(60) nanoparticles did not produce these effects.

Ex vivo skin cultures are certainly useful in studying some

aspects of nanomaterial exposure and biological interaction

through dermal exposure. They are realistic models since

they are derived from real skin, can be kept viable in vitro

for a period of time and can be easily processed for different

assessments.

Besides the fi rst organ or tissue of contact, it is equally

important to understand nanotoxicology in terms of nano-

material biodistribution in the body and consequently their

excretion from the body, if at all. Different studies have

showed the potential of various nanomaterials to accumulate

in organs including the liver, lung, kidney and spleen. [ 138–141 ]

Thus, the use of organotypic cultures of these organs is an

exciting proposition that will certainly accelerate our under-

standing of nanotoxicology. As part of the group’s efforts in

developing an artifi cial kidney, Zhang et al. induced tubu-

logenesis of primary human renal proximal tubule cells on

2D surfaces by using transforming growth factor- β 1, without

any 3D matrices. The resulting renal proximal tubules were

of several millimetres in length and were easily accessible

for manipulations and imaging. [ 142 ] The group explored

using these tubules for nanotoxicology studies by investi-

gating the interactions of human and porcine renal proximal

tubules with core-shell CdSe@ZnS quantum dots. [ 143 ] Results

revealed that porcine and human proximal tubules showed

markedly different uptake behaviors, and that human prox-

imal tubule cells were most sensitive to the cytotoxic effects

of the quantum dots.

Some interesting variations of 3D organotypic cultures

for interaction studies with nanomaterials have also been

reported recently. For the purpose of testing the transfection

effi ciency of poly( β -amino ester) based nanoparticles as gene

delivery vehicles, Bhise et al. successfully suspended frag-

ments of mammary gland epithelium in Matrigel to create

“organoid” breast cancer cultures, and found the effi cacy of

transfection using the polymeric nanoparticles to be twice that

of a commercial transfection agent. [ 144 ] In another study to

develop organotypic brain cultures for nanomaterial uptake

evaluation, Meng et al. cultured medulloblastoma-derived

cell spheroids on rat cerebellum slices and maintained these

for 6 days in vitro. [ 145 ] Interestingly, poly(glycerol-adipate)

nanoparticles introduced into this system were preferentially

taken up into the medulloblastoma-derived cells rather than

the healthy brain cells in the cerebellum slices.

4. In Vitro Safety Screening Under Simulated Environmental Exposure Conditions

Nanotoxicology as a distinct discipline has its roots from

inhalation toxicology. Consequently, for historic reasons early

nanotoxicology studies were conducted with in vitro and in

vivo models of inhalation toxicology. In the fi rst generation

nanotoxicology studies, little or no attention was given to

understand the infl uence of environmental factors in shaping


the potential toxicity of ENMs. Another reason is that, over

the past decades nanotechnology was in the early phase of

research and development. The main concern over these

years of small or medium scale nanomaterial production

was the work-place-safety due to concerns over the potential

hazard arising from direct human contact during the nano-

materials synthesize. However, this decade will witness many

prototypes of nanotechnology applications being transformed

to mass production, implying large scale production and uti-

lization of nanomaterials. Although, it is diffi cult to fi gure out

the exact production volume of nanomaterials worldwide,

a recent study by Hendren et al. estimated the production

volume in the range of metric tonnes/year for fi ve of the

most popular nanomaterials. [ 146 ] Therefore, it is becoming

imperative that more contextualized environmental health

and safety studies needs to be carried out to account for the

nano-bio interactions of direct ecological relevance. More

recently, there have been some studies devoted in under-

standing possible hazardous nano-bio interactions of rele-

vance to ecotoxicology. Patricia Holden’s group, at University

of California Santa Barbara, demonstrated that the bare

CdSe quantum dots that have accumulated in Pseudomonas aeruginosa bacteria can be transferred to and biomagnifi ed in

the Tetrahymena thermophila protozoa that prey on the bac-

teria, signifying the possibility of biomagnifi cation of nano-

materials in a food chain. [ 147 ] The possibility of organ damage

in higher aquatic organism such as fi sh has been the subject

of limited studies. One of the early studies was that by Smith

et al., who reported the gill and brain injury caused in adult

rain bow trout exposed to single walled carbon nanotubes

(SWCNTs). [ 148 ] Their study suggested the possibility of oxi-

dative stress depended respiratory damage due to exposure

to SWCNTs. Recently, George et al. reported the use of cell

line derived from the gill of rain bow trout (RT-G-W1). [ 149 ]

They showed oxidative stress dependent cellular damage

caused by silver nanoplate whose toxicity was higher than

nanospheres and nanowires. Thorough examination of silver

nanoplate using high resolution transmission electron micro-

scopy (HRTEM) revealed the presence of crystal defects

the prevalence of which was more in plate morphology. [ 149 ]

While, in vivo model (zebrafi sh embryo) could also pick up

the higher toxic potential of silver nanoplates in comparison

to other shapes, the in vitro model was indispensable in

delineating the mechanism of toxicity. The excellent corre-

lation between in vitro and in vivo outcomes suggested the

utility of cellular studies in screening for toxicity and identi-

fying mechanisms of nanotoxicity without having to resort to

more expensive animal model studies. The above mentioned

studies could help in identifying the primary nanomaterial

property/ies in relation to its potential toxicity. However,

when addressing the ecotoxicity of nanomaterials, one should

realize the important role played by environmental factors in

shaping the biological outcome.

One of the environmental factors that may have more

direct and immediate infl uence on shaping the nano-bio

interactions is the sunlight. The infl uence of solar light in

determining the outcome of nanomaterial interactions with

biological systems cannot be overlooked as most, if not all,

high-volume-production nanomaterials are photoactive. TiO 2



1

reviews
nanoparticle, for example, is generally regarded safe and is
often used as a negative control nanomaterials in biological

assays. However, many studies have shown the potential

of this material to generate reactive oxygen species (ROS)

and induce deleterious biological outcomes when exposed

to near-visible [ 62 ] or simulated solar light. [ 150 ] Recently, the

potential photoxicity of TiO 2 to marine phytoplankton under

relatively low levels of ultraviolet light, consistent with those

found in nature, was reported by Miller et al. [ 151 ] While, these

studies highlight potential toxicity of TiO 2 under the infl uence

of solar light, it is worth noticing the scarcity of data on many

other high-volume-production nanomaterials. Although, ROS

generation is a mechanism for phototoxicity of nanomate-

rials, very little attention has been devoted in understanding

the infl uence of other factors such as bioavailability, light-

activated dissolution of nanomaterials, light-assisted agglom-

eration and deagglomeration etc.

5. A Class of High-Volume Production Nanomaterials: Photoactive Nanomaterials

5.1. High-Volume Production Nanomaterials

Recent years of development has seen an exponential

infl ux of nanomaterials into the market in various applica-

tions. Nanomaterials are actively making their way and even

replacing traditional materials in services like electronics,

photovoltaic, construction, biomaterials applications, drug

delivery, etc. All these applications make use of some prop-

erty of nanomaterials, which their bulk and even their micro-

sized counterparts fail to provide. The enhanced surface area

of nanomaterials not only enhances their surface properties

and makes them more reactive, it also kicks into action some

unique properties which are otherwise suppressed, unex-

pressed or non-dominating.

This section will delve exclusively into the fi rst kind of

photoactive materials, i.e., the photocatalysts, namely TiO 2

and ZnO, both of which have cemented their places in many

commercially available applications already and are making

their way into many other applications.

5.2. Cytotoxicity of Photoactive Nanomaterials

Photoactive materials are, as the name suggests, active

under exposure to light. They can be excited and/or give

rise to charged species (e.g., ROS, etc.) under photon expo-

sure. Recent research has heavily focused on these kind of

materials to extract applications which can use these excited

states, or reactive charged species for cultivating effects like

electricity in the case of solar cells, or killing bacteria in the

case of sea water purifi cation, etc. [ 14–17 , 152 , 153 ]

Generally photoactive nanomaterials can be classifi ed

in two distinct classes: photosensitizers and semiconductors.

The mechanisms for both these types of photoactive nano-

materials differ distinctly and so does the end result of their

photo activity. Photosensitizers on the other hands are organic


compounds which have electronic confi gurations that allow

for photoactivity. Fullerenes are one of the main categories

of photosensitizers. Semiconductors or photocatalysts are

mostly oxides or combinations of oxides and become active

once their band gap energy is overcome. This review will

focus on the latter class of photoactive nanomaterials.

5.2.1. Titanium Dioxide (TiO 2 )

Nano-TiO 2 is fast becoming the material of choice for appli-

cations ranging from photocatalyst in antibacterial, antifungal,

antiviral and antitumor systems to UV-fi lters and absorbers

in sunscreens and solar cells due its superior photo activity.

TiO 2 occurs as three allotropes; rutile (tetragonal), anatase

(tetragonal), and brookite (orthorhombic). [ 154 ] Rutile and

anatase are of the highest commercial importance repre-

senting 90% and 10% of the market, respectively. [ 155 ] For this

particular reason most of the research has focused on these

two crystalline forms and this review paper will be relating

extensively only to these.

The band gap energies for anatase and rutile are 3.23 eV

and 3.06 eV [ 156 ] which corresponds to light of about 385 nm

and 400 nm. Most studies conclude that the anatase phase

has greater photoactivity than the rutile phase [ 157–159 ] while

the reverse or comparable photoactivity has also been sug-

gested under certain conditions. [ 160 , 161 ] Anatase and rutile are

thermodynamically more stable for particle sizes less than

11 nm and more than 35 nm respectively. [ 154 ] Considering the

possibility that stability accounts for the lack of photoactivity,

the contradictions might be due to different crystal sizes and/

or types. However, a combination of the two (approximate

ratio = 3:1 of anatase:rutile) was found to possess superior

photoactivity than any single pure form. [ 13 , 158 ]

Tuning TiO 2 to be responsive to visible light is a poten-

tial area of research. [ 162–165 ] Doping with other elements has

been exploited e.g. using iron (1–10 wt%) to decrease Nano-

TiO 2 band gap to near-visible light. [ 166 ] When developing a

formulation with high SPF D. Nesseem showed the highest

SPF from a formulation which used ZnO as an additive to

the active TiO 2 . [ 167 ] R. Dunford et al. showed that TiO 2 (no

mention of size in study) in sunscreens can cause phenol

photodegradation which was greatest in sample containing

ZnO. [ 168 ]

Photocatalysis has been documented with regards to

these reactive oxygen species (ROS), and a detailed review

on its properties, and prospects as an effective bactericide,

anti-viral agent, anti-tumor agent and anti-fungal agent has

been published by A. Markowska. [ 13 ] Nano-TiO 2 induced

reduction in cell viability, compromised antioxidant system,

morphological alterations, intracellular ROS production and

signifi cant DNA damage in human amnion epithelial (WISH)

cells. [ 169 ] HaCaT cells also displayed drop in cell viability

when exposed to moderate concentrations (50 ppm) of two

different types of nano-TiO 2 made up of a mixture of ana-

tase and rutile of spherical shape, but not when exposed to

the same concentration of polygonal pure anatase nano-TiO 2 .

The nanoparticles were commercially available and differed

in size as well (pure anatase: 5.8 nm, mixture: 25.8 nm and

14.6 nm). [ 155 ] However, there are so many differences between

the sample in terms of shape, size and crystal structure that it



would be hard to be certain which physiochemical parameter

was the real contributing factor. Nonetheless, most studies

have shown TiO 2 nanoparticles to have low cytotoxicity until

very high doses. [ 170–173 ] Fujita et al. [ 174 ] concluded their study

on the notion that “Gene expression profi le suggests that the

ultrafi ne TiO 2 particles without illumination have no signifi -

cant impact on ROS-associated oxidative damage, but affect

the cell–matrix adhesion in keratinocytes for extracellular

matrix remodelling.” Jang et al. [ 175 ] observed an inverse rela-

tionship between nano-TiO 2 particle size and antimicrobial

effect after photoactivation suggesting directly that smaller

size increases photocatalytic abilities. Micro-particles of

TiO 2 have been generally accepted as inert [ 176 , 177 ] but their

nano-scale counterparts have cell penetrative [ 154 ] as well as

enhanced ROS production capabilities. [ 175 ]

ROS generation is widely considered as a molecular para-

digm for the toxicity of nanoparticles. [ 178 ] Excessive ROS

generation would cause oxidative stress in cells. [ 179 , 180 ] If the

oxidative stress exceeds the threshold of the cellular antioxi-

dant defenses, additional mitochondrial perturbation such as

mitochondrial depolarization would occur. [ 59 , 181 , 182 ] These dis-

turbances might further cause apoptosis or necrosis of cells

followed by an increase in cell membrane permeability.

Phototoxicity has also been widely studied, especially

in context of sunscreens and the effect is thought to be sig-

nifi cant. [ 155 , 168 , 183 ] George et al. [ 166 ] reported that iron-doped

(0–10 wt%) Nano-TiO 2 which showed a dose dependent

decrease in band gap energy increased oxidant injury and cell

death in parallel with a decrease in band gap energy. Near-

visible light exposure was used in the study. Similarly and pro-

gressively, Reeves et al. [ 184 ] observed increased damage in TiO 2

treated fi sh cells after UVA activations, whereby hydroxyl

radicals generated (extra- and intra-cellular) were the likely

suspects for damage. Nakagawa et al. [ 185 ] also pointed out the

potential photogenotoxicity of TiO 2 nanoparticles to Chinese

hamster cell line. Hee-Ok Park et al. showed using commer-

cially available TiO 2 nanoparticles that TiO 2 samples with

higher photocatalytic activity caused higher toxicity with or

without simultaneous irradiation of UV. [ 155 ] The effect of UV

was not seen on low photoactive sample which again rein-

states the potential of TiO 2 to be a phototoxic material. ROS

measured by DCF fl uorescence also followed the same trend,

but no DNA damage was seen using the comet assay.

One interesting study by Gopalan et al. [ 186 ] reported that

TiO 2 tend to introduce dose-dependent photogenotoxic effect

to human lymphocytes but not to human sperms. It has also

been reported that photoactivated TiO 2 nanoparticles could

selectively induce toxicity against cancer cells [ 187–189 ] and in

fact numerous studies on TiO 2 as an effective photocatalyst

talk about TiO 2 ’s selective anti-tumor cell action as a pros-

pect worth investing. It is thus clear that cell-line is one very

important factor when determining the cellular responses of

cell lines. Possibly, the action of TiO 2 would remain the same

i.e. its photocatalytic action and generation of ROS, but dif-

ferent cells would respond differently to the change in home-

ostasis. Oxidative stress is a residual phenomenon in cells and

antioxidant mechanisms of defense are in place. Possibly the

threshold of these defenses differ in different cells and so

does the onset of toxicity.


5.2.2. Zinc Oxide (ZnO)

Zinc oxide is another prime material candidate in the fi eld

of photoactive materials and is often discussed hand-in-hand

with TiO 2 . Like TiO 2 , it falls into the category of semicon-

ductor and has a broad spectrum adsorption with a bandgap

of 3.2 eV, [ 190 ] high refractive index of 1.95–2.10 [ 191 ] and the

ability to absorb UVA and UVB. [ 192 ] It has been studied

heavily and even employed in numerous photoactive appli-

cations ranging from solar cells, photovoltaics to the most

common UV-fi lters in sunscreens.

However, there are certain limitations to using ZnO which

restrict the practical applications of ZnO. Amongst them is

the anodic photocorrosion of ZnO which, unlike in the case

of TiO 2 , is unhindered by oxidation of water, and solubility

of ZnO especially in acidic and alkaline conditions as well as

in the presence of Zn 2 + chelators. These properties severely

limit the pH range in which ZnO can be used as a photo-

catalyst. Nonetheless ZnO displays similar photoactivity as

TiO 2 in certain conditions, [ 193 ] and has shown faster initial

degradation rate (r0) of herbicide triclopyr (3,5,6-trichloro-2-

pyridiyloxyacetic acid) compared to P25 TiO 2 . [ 194 ] There have

been various attempts to overcome the drawbacks of ZnO

and modify it to fi t photocatalytic applications. Details can be

found in these reviews. [ 190 , 195 , 196 ]

ZnO has three known crystal structures: hexagonal wur-

tzite, cubic zinc-blende structure and cubic rock-salt (NaCl-

type). The most common of them and also the most relevant

in the scope of this paper is the wurtzite form. ZnO has been

in use since a long period and was considered a safe mate-

rial in its bulk form. [ 197 ] However, the same material when

reduced to the nano-scale has raised alerts owing to its

cyto- and geno-toxic attributes. To add to that its photoac-

tivity raises further concerns over its behavior under irradia-

tive conditions especially given the superior surface area to

volume ratio of nano-ZnO. For perceptive purpose, the rate

of H 2 O 2 production is 100–1000 times faster with ZnO nano-

particles of 4 nm to 5 nm, than with larger ZnO particles of

100 nm. [ 198 ] Several theories about the toxicity of nano-ZnO

have been going around in the scientifi c community. They

mostly revolve around ROS production and Zn 2 + ions. ZnO

has even been shown to be genotoxic. [ 199 ] Here we attempt

to sum up the most relevant fi ndings, discuss the most logical

conclusions and suggest an approach for future studies.

Xia et al. published a comprehensive and detailed study

to link the dissolution of nano-ZnO to its cellular response,

albeit in the dark. Amongst other wonderful fi ndings, two of

them have notable relevance when considering the context of

phototoxicity. Firstly, it was shown that nano-ZnO is readily

soluble to upto 190 μ M and 225 μ M in BEGM and DMEM,

both of which are common cell culture mediums, and that

nano-ZnO can achieve more than 80% solubility within 3 h.

Given the rigorous sonication regimes followed by toxicity

studies to achieve homogenous suspensions that might well

be an understatement. Furthermore, it was shown in two sep-

arate experiments that shredded Fluorescein isothiocyanate

(FITC) tags from nano-ZnO particles, as well as the Zn 2 + spe-

cifi c dye, NewPort Green DCF, overlapped with the LAMP-

1 + fl uorescence (LAMP-1 + stains lysosomal membranes). [ 200 ]



Figure 3 . The toxicological pathway of ZnO-Nanoparticles. Reproduced with permission. [ 201 ] Copyright 2011, Elsevier.

Although no actual nano-ZnO particles were seen under con-

focal microscope, the group inferred that perhaps the FITC

tags were shredded off from remnants of undissolved nano-

ZnO which were taken up by the cells through endocytosis

after which they dissolved under relatively acidic conditions

of the endosomes/lysosomes giving Zn 2 + ions. This hypoth-

esis has been shared by other studies as well. [ 201 ] The second

fi nding relates to ROS production. It was shown nano-ZnO

and nano-CeO 2 , but not nano-TiO 2 , produced H 2 O 2 in abi-

otic conditions, while only nano-ZnO, but not nano-CeO 2 or

nano-TiO 2 , produced H 2 O 2 or superoxide in biotic conditions

under dark. [ 200 ]

Both of these fi ndings have serious implications in the

context of phototoxicity and also photoactivity in biotic envi-

ronments. Firstly, it is evident that solubility of nano-ZnO

is pronounced at lower concentrations probably due to the

greater surface area, and even more so inside the cellular

peripheral where lower pH further facilitates the dissolution.

If so, then the phototoxicity can be ruled out since once dis-

solved, nano-ZnO’s integrity as a semiconductor, with photo-

activation prospects, is lost. Solubility has been mentioned

as a drawback of cultivating ZnO’s photoactive properties.

However, its solubility certainly has strong implications on

the toxicity of ZnO.

The above fi nding also opens up the possibility of dif-

ferent oxidative responses of the particle in biotic and abi-

otic environments. CeO 2 , although seemingly an oxidative

agent, in cellular environments actually has anti-oxidative

effects. [ 200 ] It is thus clear, that ROS production mechanisms

are different in biotic and abiotic conditions. Studies should

be wary of such effects before prematurely extrapolating

results to and from different environments. In the case of

ZnO, this is even more imperative as the effect of Zn 2 + on

mitochondrial function is well known. Zn 2 + can cause mito-

chondria membrane potential and respiration to be disturbed

causing excess ROS to accumulate inside the mitochondria

12 www.small-journal.com © 2012 Wiley-VCH Verlag GmbH & Co. KGaA

and ultimately inducing apoptotic

death. [ 200 , 201 ] Figure 3 [ 201 ] reveals this

mechanism in detail. ROS may accentuate

ZnO’s toxicity as shown by Chin et al. [ 202 ]

Although ZnO-induced ROS spikes

have been well reported, [ 192 , 200 , 201 , 203–205 ]

well planned dissolution studies must run

parallel to cytotoxicity and ROS-measure-

ment studies in order to correctly attribute

the source of ROS production to the nan-

oparticles or the ions.

A comparison of toxicity and ROS

production due to ZnO treatment under

natural sunlight (NSL), dark, and artifi cial

ambient laboratory light (AALL) was also

studied, and revealed that for nano-ZnO

the toxicity was in the order of NSL(2h) +

AALL(22h) > NSL(2h) + dark(22h) >

AALL(24h) > AALL(2h) + dark (22h),

with [AALL(2h) + dark(22h)] being the

least toxic. [ 205 ] ROS production was also in

agreement of the trend. Considering that

NSL might be much more potent to cause

photoactivation compared to AALL based on good reason,

the study concluded that ZnO was indeed phototoxic.

However, a similar yet much older study by Dufour

et al. [ 206 ] handled the data in a much different way. They

studied the effect of two different treatments: pre-irradiation

(PI) and simultaneous irradiation (SI). They chose two sepa-

rate regimes of irradiation as well: low dose (350 mJ cm − 2 and

3 h prior to particle addition for PI) and high dose (700 mJ cm − 2

and only 1–2 h prior to particle addition for PI). Generally

SI was more cytotoxic and clastogenic than PI, however PI

should signifi cantly higher cytotoxicity and clastogenicity

when compared to dark. Even in the dark ZnO showed con-

siderable cytotoxicity and clastogenicity. Instead of concluding

that ZnO is a phototoxic and photo-clastogenic agent, several

competing factors were analyzed and it was suggested that

UV-induced DNA damage could make the cells even more

susceptible to ZnO-induced cytoxicity and clastogenicity.

This fi ts in logically as PI even should an increased in adverse

effects which were probably less enhanced than SI due to

recovery of cells during the waiting period. In the high-dose

treatment where recovery time is low, indeed there was no

observable difference between PI and SI as far as chromo-

some aberrations (clastogenicity) was concerned.

Lisa Martorano et al. [ 192 ] proved UV irradiation-induced

zinc dissolution in commercial zinc oxide sunscreens. Photo-

degradation of zinc was mentioned earlier as a drawback of

ZnO in the photocatalytic fi eld, and this may well be a con-

nected phenomenon whereby UV induces a greater avail-

ability of Zn 2 + ions which therefore cause higher toxicity.

Dadong et al. [ 207 ] when studying the effect ZnO on the uptake

of anti-cancer drug, daunorubicin, in Multi Drug Resistant

cells, noted more effi cient uptake of drug in the presence of

ZnO although no reasons were discussed. Also, in the pres-

ence of UV-irradiation, ZnO displayed greater toxicity. Com-

paring three size particles, 20, 60, and 100 nm, they were able

to show no effect of size on the action. Similarly, J. H. Yuan

, Weinheim small 2012, DOI: 10.1002/smll.201201452


et al. [ 208 ] also showed no size dependence while investigating

20, 30 and 40 nm ZnO particles. B. C. Heng et al. showed

insignifi cant difference in cytotoxicity for different shapes

(sheets and fl akes) of ZnO nanoparticles, however, there

were differences in cellular associations. [ 209 ]

6. Conclusion and Perspectives

Nanotoxicology is a relatively recent fi eld, but with the expo-

nential rate of increase in nanomaterials being developed

and commercialized, it is fast becoming a very important

fi eld nonetheless. As discussed earlier, nanomaterials are dif-

ferent from their bulk counterparts and there are numerous

contributing factors which determine their physicochemical

properties including size, shape, crystal structure, surface

area, etc., giving rise to a myriad of permutations for the pos-

sible number of nanomaterials, which can and are produced

in large volumes. This infl ux of nanomaterials has therefore

raised concerns regarding their safety to humans and the

environment. The pressing needs of nanosafety unfortunately

cannot be satisfi ed by conventional methods of toxicology

studies, which predominantly revolved around animal and

other in vivo models. One of the requirements of time for tox-

icology is the development of novel testing methods that can

keep pace with the rapid advancement of nanotechnology.

Figure 4 . Proposed tiered approach for the toxicity screening of nanomaterials: The 1st phase comprises thorough characterization of nanomaterial for its physicochemical properties and toxicity screening using biomolecular assays and 2D cell culture under in vitro conditions. The injury pathways selected should have relevance to real disease conditions and the selection of specifi c injury pathway/s should be based on the key material property identifi ed from physicochemical characterization data. The 2nd phase of the toxicity screening consists of in vitro studies in more complex biological systems such as stem cells and 3D cell culture. These assays could be aimed to answer more specifi c and contextualized nanomaterial encounters (e.g., to assay phototoxicity of nanomaterials using 3D cell culture). This phase may also include studies with small organisms of relevance to human and environmental toxicology such as zebrafi sh. Nanomaterials that show signs of hazardous properties could be tested in rodent or other relevant animal models in the 3rd phase. The toxicity and tox-ADME (absorption, distribution, metabolism and excretion) of selected nanomaterials could be studied in this phase. Human studies constitute the 4th and the fi nal phase. This is more relevant to nanomaterials to be used for therapeutic purposes. However, studies aimed at biochemical profi ling of workers exposed to nanomaterials during synthesis and application could also provide valuable information on their potential hazardous nature.

This review has discussed several

emerging in vitro models including 2D

models, 3D models, and high throughput

screening technique. Usage of stem cells as

a test subject for these in vitro screening

has also been discussed. Numerous studies

have been shown to accurately explain

and/or extrapolate in vivo effects (i.e.,

effects on organisms) through in vitro

models. This proves not only possible,

but also the practicality of using in vitro

screening as a cheaper and quicker substi-

tute to conventional methods. However, it

is also noteworthy that when dealing with

in vitro models a few things have to be

kept in strict control. First and foremost

is material characterization, which plays

a key role in explaining any results and

is imperative to know how the intrinsic

properties of nanomaterials can infl uence

biocompatibility or toxicity; and there are

numerous studies on these.

The second most important factor is

the cell line which impacts directly the

kind of effect being studied. Relevant cell

type selection is the key in getting mean-

ingful results, while multiple cell types

are important for validating the effects

observed. The environment in which the

tests are conducted must also be character-

ized well as the properties of nano materials

are dependent heavily on conditions like

pH, solvent, solute concentration, etc.


A prime example is the case of ZnO, which dissolves better

as pH diverges from neutrality and many hypothesize that

the solubility of ZnO in endosomes, where the pH is much

lower, causes Zn 2 + based toxicity. [ 200 , 201 ] From the above pool

of studies as reported earlier, it seems imperative that future

studies should consider ZnO dissolution kinetics study, as well

as under dark and irradiated conditions so that the effects that

are observed are accurately correlated to the toxicity species.

As with any technique, there are drawbacks with in vitro

models as well. The most obvious one is its very nature of

being an indirect means of testing and thus intrinsically being

slightly deviated from reality. Another limitation of in vitro

testing in contrast to in vivo is that it gives results of a highly

specifi c nature, i.e., they usually provide the effect of a certain

chemical/particle on the growth rate or metabolic activity

or specifi c protein interaction, etc., on a certain cell type. In

contrast in vivo models allow for more direct effects and out-

comes from organisms. However, extrapolating these results

using a combination of parameters is a technique growing

in stature. Furthermore, more intricate models, like the skin

constructs that mimic human skin, can closely mimic in vivo

environments for toxic studies.

It is extremely important that the scientifi c community

carefully develops strict protocols which allow comparison

of results from one lab to another. Parameters like cell-lines,

incubation time, treatment method, nanoparticle-suspension



1

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When physicochemical aspects are considered, in-depth char-

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these data to be comprehensively revealed to the scientifi c

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Nanomaterials in the Environment: From Materials to High-Throughput Screening to Organisms

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