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The innovative field of nanotechnology is most likely to benefit society and gain acceptance if environmental and human health considerations are investigated systematically, and those results are used to optimise safety as well as performance. Since nanotechnology fundamentally allows manipulation of matter at the atomic level, toxic interactions could potentially be eliminated by creative design once our knowledge of how nanomaterials interact with biological systems is sufficient. Our approach to the development of benign nanoparticles begins with the synthesis of precisely engineered, high-purity nanoparticle libraries using the principles of green chemistry. Next, evaluations for biocompatibility are performed using a rapid in vivo system (embryonic zebrafish) to assess the biological activity and toxic potential of nanomaterials at multiple levels of biological organisation (i.e., molecular, cellular, systems, organismal). Our iterative testing and redesign strategy utilises information gained from the biological studies to inform the nanomaterial design process until benign products and processes are identified. To make this information more generally available, a knowledgebase of Nanomaterial-Biological Interactions (NBI) is being developed that will offer industry, academia and regulatory agencies a mechanism to rationally inquire for unbiased interpretation of nanomaterial exposure effects in biological systems. Timely evaluation and dissemination of information on nanomaterial-biological interactions will provide much needed data, improve public trust of the nanotechnology industry, and provide nanomaterial designers in academia and industry with information to direct the development of safer nanomaterials and resulting technologies.
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124 Int. J. Nanotechnol., Vol. 5, No. 1, 2008
Copyright © 2008 Inderscience Enterprises Ltd.
Proactively designing nanomaterials to enhance
performance and minimise hazard
Stacey L. Harper, Jennifer A. Dahl,
Bettye L.S. Maddux, Robert L. Tanguay*
and James E. Hutchison*
ONAMI Safer Nanomaterials and Nanomanufacturing Initiative,
University of Oregon and Oregon State University,
Eugene, OR 97403-1253, USA
E-mail: harpers@science.oregonstate.edu
E-mail: jdahl@uoregon.edu
E-mail: bettye@greennano.org
E-mail: Robert.Tanguay@oregonstate.edu
E-mail: hutch@uoregon.edu
*Corresponding authors
Abstract: The innovative field of nanotechnology is most likely to benefit
society and gain acceptance if environmental and human health considerations
are investigated systematically, and those results are used to optimise safety as
well as performance. Since nanotechnology fundamentally allows manipulation
of matter at the atomic level, toxic interactions could potentially be eliminated
by creative design once our knowledge of how nanomaterials interact with
biological systems is sufficient. Our approach to the development of benign
nanoparticles begins with the synthesis of precisely engineered, high-purity
nanoparticle libraries using the principles of green chemistry. Next, evaluations
for biocompatibility are performed using a rapid in vivo system (embryonic
zebrafish) to assess the biological activity and toxic potential of nanomaterials
at multiple levels of biological organisation (i.e., molecular, cellular, systems,
organismal). Our iterative testing and redesign strategy utilises information
gained from the biological studies to inform the nanomaterial design process
until benign products and processes are identified. To make this information
more generally available, a knowledgebase of Nanomaterial-Biological
Interactions (NBI) is being developed that will offer industry, academia
and regulatory agencies a mechanism to rationally inquire for unbiased
interpretation of nanomaterial exposure effects in biological systems. Timely
evaluation and dissemination of information on nanomaterial-biological
interactions will provide much needed data, improve public trust of the
nanotechnology industry, and provide nanomaterial designers in academia and
industry with information to direct the development of safer nanomaterials and
resulting technologies.
Keywords: synthesis; green chemistry; knowledgebase; toxicity; zebrafish;
biocompatible; gold nanoparticles.
Reference to this paper should be made as follows: Harper, S.L.,
Dahl, J.A., Maddux, B.L.S., Tanguay, R.L. and Hutchison, J.E. (2008)
‘Proactively designing nanomaterials to enhance performance and minimise
hazard’, Int. J. Nanotechnol., Vol. 5, No. 1, pp.124–142.
Proactively designing nanomaterials to enhance performance 125
Biographical notes: Stacey L. Harper leads the Nanotoxicology Division
of the Tanguay laboratory at OSU where she employs in vivo approaches
to provide feedback on the biological activity and toxic potential of
nanomaterials. She has established a collaborative research group to
develop the knowledgebase of Nanomaterial-Biological Interactions (NBI).
She received her BS in Natural Sciences and Mathematics from Mesa State
College, Colorado in 1990; and earned her MS and PhD in Biological Sciences
from University of Nevada Las Vegas in 1998 and 2003. From 2003 to 2005,
she held a biology postdoctoral position with the Exposure and Dose Research
Branch of the EPA.
Jennifer A. Dahl was born in Wisconsin in 1976. She received her BS in
Chemistry from the University of Wisconsin-Oshkosh in 2002. She expects to
receive her PhD in Chemistry from the University of Oregon in the fall of
2007, under the supervision of Professor James E. Hutchison. Her graduate
studies have been centred on the synthesis of functionalised gold nanoparticles
designed for the fabrication of novel optical devices, with a special focus on
surface science and green chemistry. Her thesis studies were supported by an
NSF-IGERT Fellowship from 2005 to 2007.
Bettye L.S. Maddux is the Assistant Director of the Safer Nanomaterials
and Nanomanufacturing Initiative, a major research thrust of the Oregon
Nanoscience and Microtechnologies Institute and a member of the Materials
Science Institute at the University of Oregon. In 1992, she earned her PhD in
Biological Sciences with an emphasis in chemical carcinogenesis from the
University of Texas at Austin. Her postdoctoral work at the University
of California, Santa Barbara involved elucidating nature’s mechanisms
for creating environmentally benign nanomaterials. Previously, she has
published peer-reviewed research papers as ‘Bettye L. Smith’ in the fields of
nanotechnology, biophysics and chemical carcinogenesis.
Robert L. Tanguay received a BA Degree in Biology from California State
University, San Bernardino in 1988 and his PhD Degree in Biochemistry
from the University of California, Riverside in 1995. He received
postdoctoral training in molecular and developmental toxicology with
Richard E. Peterson at the University of Wisconsin between 1996 and 1999.
He is currently an Associate Professor in the Department of Environmental
and Molecular Toxicology at Oregon State University and is the Director
of the Sinnhuber Aquatic Research Laboratory. His current research interests
include developmental biology, nanotoxicology, developmental toxicology,
regenerative medicine, and chemical genetics.
James E. Hutchison is a Professor of Chemistry and Director of the Materials
Science Institute at the University of Oregon. He also directs the Safer
Nanomaterials and Nanomanufacturing Initiative of the Oregon Nanoscience
and Microtechnologies Institute and has pioneered the University’s Green
Organic Chemistry Laboratory program. A native of Oregon, he received his
BS in Chemistry from the University of Oregon in 1986 and a PhD from
Stanford University in 1991 (with James P. Collman). He then did postdoctoral
work with Royce W. Murray at University of North Carolina, Chapel Hill.
He has won numerous awards including a Postdoctoral Fellowship and
a CAREER award from the National Science Foundation, as well as awards
from the Sloan and Dreyfus Foundations. His current research interests include
the design, synthesis and study of functional organic and inorganic materials,
including functionalised surfaces and nanoparticles, green chemistry and green
nanoscience.
126 S.L. Harper et al.
1 Introduction
Nanoscience is an emerging technology that will provide a broad range of novel
applications in the electronics, healthcare, cosmetics, technologies and engineering
industries [1–6]. The National Science Foundation has estimated that by 2015
nanotechnology will be a $1 trillion market and could employ 2 million workers,
thus surpassing the industrial revolution in potential economic and societal benefits.
Thus far, nanotechnology research efforts and resources have focused on discovering
applications for new nanomaterials with minimal research aimed at evaluating health and
safety consequences of nanoparticle exposure, even though the rapid rate of discovery
and development will undoubtedly increase the potential for both human and
environmental exposures [7]. Although many applications of nanotechnology promise
benefit to human health or the environment, the potential health and environmental risks
associated with the new properties of nanoscale materials are unknown and may lead to
unintended consequences. Because the biological activity of nanomaterials will likely
depend on inherent physicochemical properties that are not routinely considered in
toxicity studies (e.g., particle size and size distribution, agglomeration status, interactions
with environmental and biological moieties); it is imperative that materials scientists and
chemists work together with biologists and toxicologists to provide critical information
on the potential biological and environmental impacts of the newly emerging
nanotechnology industry. Timely evaluation of nanomaterial-biological interactions will
provide much needed data, improve public trust of the nanotechnology industry and
provide nanomaterial designers in academia and industry with information to direct the
development of safer nanomaterials and products [8–10].
Nanomaterials are expected to interact with biological systems in unanticipated ways
because many of their attributes (e.g., magnetic, optical, tensile strength) are unique to
their size (within a transitional zone between individual atoms or molecules and
corresponding bulk materials). The principal characteristics that may be predictive of
nanomaterial interactions with biological systems have yet to be identified due to the
current lack of toxicological data. The limited data that are available reveal that
nano-sized particles can cause oxidative stress, induce pulmonary inflammation, cause
release of cytokines and induce signal transduction pathways. Additional areas of primary
concern in terms of toxicity of nanoparticles include, but are not limited to, their high
redox activity [10], ability to partition into cell membranes especially mitochondria both
in vitro [11–15] and in vivo [10,16–18], capacity to translocate from the olfactory nerve
into the olfactory bulb via a neuronal translocation pathway [14], activity as ion channel
blockers [19], and observed cytotoxicity and bioactivity [13,20]. It is anticipated that
various nanomaterials, especially those targeted for biomedical applications, will be
likely benign, or may in fact be beneficial to biological systems. Delineation of those
nanomaterials that are not biologically active and those that are biologically active
(beneficially or adversely) is essential to identifying inherent physicochemical properties
that are predictive of biological responses and consequentially, the material modifications
that can minimise hazard.
In this review, we outline a proactive approach to nanotechnology that strives to
enhance product performance and benefit to society while reducing hazards to human
health or the environment. We first summarise green nanotechnology – a strategy to
design nanoscale materials and nanomaterial production methods using the principles of
green chemistry. The balance of the review focuses on our approach to systematically
Proactively designing nanomaterials to enhance performance 127
designing safer nanomaterials. The strategy here is to utilise well-defined libraries of
structurally-analogous functionalised nanoparticles in detailed biological studies to
understand the relationships between nanoscale structure and the biological response
and then use this understanding to design safer materials. In this context we first
discuss the preparation, purification and characterisation of well-defined nanoparticle
samples. Following this we outline a tiered approach to understanding the toxicological,
biochemical and genetic influences of these nanoscale materials.
2 Toward green nanotechnology
If nanotechnology is indeed the next ‘industrial revolution’, methods for the design
of nanomaterials that minimise unintended negative consequences analogous to
those associated with previous technological advances (e.g., reduction in ozone by
chloroflurocarbons, commercial use of DDT, or lead in gasoline and paint products) must
be developed. In addition, synthesis methods and nanomanufacturing practices must also
be carefully considered because nanoparticle preparations currently require the use of
hazardous chemicals which have deleterious effects on the environment and workers
using those chemicals. Green nanoscience, pioneered at the University of Oregon (UO),
applies the principles of green chemistry to nanoscience in order to rationally design safe,
yet high performance nanoscale materials using more efficient and inexpensive
manufacturing approaches to these materials.
Green chemistry is the “utilisation of a set of principles that reduces or eliminates the
use or generation of hazardous substances in the design, manufacture and application of
chemical products”. Applied to nanomaterial synthesis and nanomanufacturing processes,
chemists and materials scientists can utilise green chemical methods of production, avoid
nanoparticles that are known to be toxic, control the physical (size, shape) or chemical
(surface groups) properties of the nanoparticles and alter nanoparticle properties to render
them nontoxic (via new reaction mechanisms or surface functionalisation). On that basis,
the Safer Nanomaterials and Nanomanufacturing Initiative (SNNI, http://greennano.org/)
of the Oregon Nanoscience and Microtechnologies Institute (ONAMI, http://www.
onami.us/) aims to proactively enhance performance while minimising adverse biological
interactions of novel nanomaterials. Our interdisciplinary group’s efforts focus on the
design of benign nanomaterials and the development of ‘greener’ nanomanufacturing
processes. Significant progress has already been made in developing greener production
methods. This progress will be briefly summarised here before we proceed to discuss the
design of benign nanoparticle materials.
To design greener nanomaterials and nanomanufacturing techniques, chemists and
materials scientists can either modify existing synthetic methods or pioneer new routes in
synthesis that take advantage of existing knowledge in order to develop greener
alternatives. Preparation of nanomaterials can be carried out within a greener context
through the consideration of toxicity profiles of starting materials, reagents and solvents
and through careful design of synthetic and purification procedures that enhance
efficiency, reduce waste and optimise overall yield.
The classic synthesis of citrate-stabilised gold nanoparticles (AuNPs) provides an
excellent example of an efficient, green nanomaterial preparation, but also illustrates how
newer methods must continuously be developed to meet the performance requirements
for new applications. The reaction begins with raw materials, trisodium citrate and a gold
128 S.L. Harper et al.
salt, that feature low toxicity and present little hazard. The reaction is carried out in
refluxing water, where citrate ions serve the dual role of reducing the gold ions to
elemental AuNPs, as well as acting as a complexing agent which forms a stabilising layer
around the newly formed particles. The reaction does not use any excess reagents, and
nearly all of the starting materials are converted to product. Furthermore, the reaction
does not generate any by-products with significant toxicity. While citrate stabilised
nanoparticles are prepared by a demonstrably green method, the synthesis of more
complex nanomaterials (e.g., those incorporating well-defined surface chemical
functionality) often relies on more sophisticated synthetic techniques that often require
the use of hazardous precursors, reagents or solvents.
Some of these more sophisticated techniques for AuNP production are now being
reexamined with the goal of improving upon established techniques to meet greener
standards and requirements for proposed applications. The Hutchison lab has successfully
applied the concepts of green nanoscience to improve reaction efficiencies and minimise
waste during the synthesis of well-defined, precisely engineered AuNPs [21–23]. One of
the traditional methods for producing AuNPs uses diborane, a highly toxic and
flammable chemical compound, as a reducing agent and benzene (another health hazard)
as solvent. This process is time-consuming, labour intensive and is difficult to scale up.
The greener method uses sodium borohydride and toluene in small quantities to achieve
a better, cleaner end-product. The process is safer and the preparation is easier than the
traditional method. With the new method, gram quantities of the AuNP building blocks
can be rapidly, and much less expensively, synthesised. The functionalisation and
purification processes for these materials have also been addressed through green
chemistry as described in the next section.
In comparison to the development of greener manufacturing methods, the rational
design of benign nanomaterials is in its infancy. Nanotechnology fundamentally allows
manipulation of matter at the molecular and atomic level so precision engineering should
permit control over nanomaterial physicochemical properties as well as their interactions
with biological systems. Thus, toxic interactions could potentially be eliminated by
creative design once our knowledge of how nanomaterials interact with biological
systems is sufficient to direct the rational development of safer, non-toxic products.
The basic protocol for the development of benign nanoparticles includes synthesis of
nanoparticle libraries, evaluations for biocompatibility and iterations of testing and
redesign until benign products and processes are identified. Our research strategy
encompasses
production of libraries of precision engineered nanoparticles with well-defined
composition, structure and purity,
usage of appropriate animal model systems to assess biological responses
to these materials and
interpretation of biological studies to inform the nanomaterial design process.
Proactively designing nanomaterials to enhance performance 129
3 Production of well-defined nanoparticle libraries for toxicological
and biological testing
In order to determine the biological effects of engineered nanoparticles, nanoparticle
libraries are needed that meet two important requirements [24].
Samples must be ‘well-defined’ wherein the size, shape, surface chemistry and purity
are known (i.e., can be measured and controlled). On this basis, many nanoparticle
samples are not ‘well-defined’. They possess known or unknown
small molecule impurities (as but one example, carbon nanotubes are routinely
mass-produced by at least four methods. Each of these methods leads to
compositionally diverse nanotubes [25,26]) and, in many cases the surface chemistry
is not known (for example, particles often oxidise presenting an oxide surface
as opposed to the core material).
Libraries must contain enough related members (depth) to perform systematic
studies of the influence of structural differences while containing enough diversity
(breadth) to investigate the broad range of functionality that may produce biological
responses. This cannot be accomplished with ‘off-the-shelf’ materials because these
are too limited in their depth and breadth. Custom libraries are needed that allow
systematic study of structural features such as core size, shape and composition;
shell thickness, composition and surface function.
During the last decade, the Hutchison group has developed an extensive library
of ligand-functionalised gold nanoparticles that contains more than 50 well-defined
nanoparticles with defined core sizes of 0.8 nm and 1.5 nm and a wide range of
surface chemical functionalities. Recent developments in nanoparticle purification
(via nanofiltration), extension of the library to include larger (up to 50 nm diameter)
metal cores, and the possibility of controlling particle shape during synthesis
(with RNA aptamer ligands) pave the way for an even larger library of precisely
engineered (and pure) nanoparticles. Thus, this library has the potential to provide the
breadth, depth and definition needed for toxicological and biological investigations.
It has recently been demonstrated that nanoparticle purity effects the reactivity and
self-assembly of functionalised nanoparticles and, one would expect this to be the
case for their biological impacts as well. A typical strategy to isolate and purify
a product involves the precipitation of a soluble nanoparticle product by the addition
of a co-solvent creating an environment where the product loses solubility and
precipitates from the bulk reaction mixture. Another approach involves extensive
washing of the solid material with a solvent selected to wash away impurities while
leaving behind the desired product. Neither of these approaches is entirely effective and
both generate large amounts of solvent waste. Despite one’s best efforts, highly pure
nanomaterials often remain elusive. However, new strategies are being developed that are
more effective and less wasteful. For example, purification of AuNPs, for instance, may
be carried out via dialysis or diafiltration (Figure 1); procedures that efficiently remove
any remaining excess salts and unreacted materials. In diafiltration, materials are placed
in the diafiltration unit and as they circulate around, the nanoparticles are retained and
any impurities are removed. Traditional methods of purification use about 15 L solvent/g
nanoparticle purified and take three days of work, whereas the greener diafiltration
method requires no organic solvent and only 15 min of work to purify the nanoparticles.
130 S.L. Harper et al.
The process of diafiltration as a purification method can effectively reduce solvent
consumption and provide cleaner, well-defined building blocks.
Figure 1 Schematic diagram of diafiltration process that employs the cross-flow of solution
through a membrane to selectively isolate those species that have apparent
cross-sections larger than the pore size in the membrane (for colours see online version)
The design of versatile, functionalisable precursor nanoparticles presents an opportunity
to generate a library of nanomaterials from a single precursor that can be functionalised
with different surface chemistries. The preparation of small AuNPs (d < 2 nm) with
a labile triphenylphosphine stabilising shell is an excellent example of such a strategy.
Nearly any thiol may be used to displace the original triphenylphosphine stabiliser
and create a thiol-stabilised nanoparticle without disrupting the integrity of the AuNP
core. The versatility of AuNP products was recently improved upon by Woehrle and
Hutchison [21], Woehrle et al. [22] and Foster et al. [27]. They developed a reliable
method of modifying the surface chemistry of nanoparticle products to facilitate a wide
range of applications on optics, sensing and nanodevice assembly. This strategy can be
extended to other nanomaterials of different size and shape. Applied to citrate-stabilised
gold nanoparticle with cores in the size range of 8–200 nm, the method allows for access
to a broader range of sizes for comparisons of the effects of particle size and surface
chemistry on nanoparticle-biological interactions. Such materials also present an
opportunity to delineate interrelated impacts of nanoparticle physicochemistry on overall
toxicity.
A strong emphasis is being placed on approaches to tailor the composition and
structure of the exterior ligand shell in order to design safer nanoparticulate materials;
given that the first contact between a nanoparticle and a biological system is the outer
Proactively designing nanomaterials to enhance performance 131
surface of the nanoparticle. The greener synthesis and purification methods described
herein provide well-defined purity profiles and compositions that are essential for
determining the importance of nanoparticle physicochemical properties on biological
interactions. Thorough characterisation of the synthesised materials is also essential to
define the physicochemical properties of a new nanomaterial because subsequent
assessments of nanoparticle-biological interactions are less robust in the absence of key
pieces of information related to the physicochemical properties of nanomaterials
(e.g., overall chemical composition and structure, reactivity and purity). To gather such
a body of data, the researcher may utilise techniques including electron microscopy
and X-ray diffraction methods to directly determine overall size and morphology.
Other techniques, including X-ray photoelectron spectrometry, electron probe
microanalysis, thermogravimetric methods, infrared spectroscopy and nuclear magnetic
resonance, are used to determine chemical composition and assess purity. It should be
noted that no single characterisation method can yield enough information to define
a nanomaterial, so we propose the use of a standard suite of complementary techniques
that allow one to describe the properties of a novel product in complete and confident
terms. We apply this same philosophy when investigating the biological impacts of
engineered nanomaterials by employing a suite of complimentary techniques to
investigate nanomaterial-biological interactions at many levels of biological organisation.
4 Assessing the biological impacts of engineered nanomaterials:
advantages of zebrafish as a whole animal model
Although it has been widely proposed that nanoparticles can be used as high-performance
diagnostic probes or as site-selective therapeutics, surprisingly little is known about how
or why these nanomaterials interact with biological systems and even less is known about
how to design them to exhibit a desired effect in vivo (in whole animals). To inform the
design of new nano-scale materials, the spectrum of interactions that these materials have
with biological systems must be investigated at multiple levels of biological organisation
(i.e., molecular, cellular, systems and whole organism). Numerous biological models can
be employed for these evaluations. In vitro techniques, such as cell culture systems,
are often preferred because of they are both cost- and time-efficient. Whilst these studies
are useful, direct translation to whole organisms and human health is often difficult to
infer. Specific to nano-biological evaluations, cell culture studies have reported
contradictory effects in the literature based on cell type, system, and primary or
secondary cells [28–31]. In vivo studies can provide improved prediction of biological
response in intact systems. Since these studies often employ rodent models; assessments
are generally expensive, time-consuming and require extensive facilities for housing
experimental animals. Cost, labour, time and infrastructure requirements can be
significantly reduced by replacing the traditional rodent model with the zebrafish model.
Just as important, assay volumes using the zebrafish model are small; thus, only limited
amounts of well-characterised nanomaterials are needed to assess nanomaterial-biological
interactions. Nanomaterial availability at sufficient quantities to perform extensive
in vivo rodent studies remains a barrier at the present time.
Inherent advantages of the zebrafish model make them an important part of an
overall integrated approach to study nanomaterial toxicity. Like many vertebrate models,
much of the anatomy and physiology of fish is highly homologous to humans [32,33].
132 S.L. Harper et al.
Zebrafish also possess all of the classical sense modalities, including vision, olfaction,
taste, touch, balance and hearing; and their sensory pathways share an overall homology
with humans. Cognitive behavioural tests suggest that anatomic substrates of cognitive
behaviour are also conserved between fish and other vertebrates. Similar to observations
of hippocampal lesions in mammals, lesions of the structural homologue of the
hippocampus in fish selectively impair spatial memory [34]. Another major advantage of
zebrafish is that the embryos develop externally and are optically transparent so it is
possible to resolve individual cells in vivo throughout the duration of an exposure using
simple microscopic techniques. Transgenic zebrafish models that express fluorescent
reporter genes in specific cell types can be employed to improve the resolution of
individual cell populations [35,36]. Several additional features of zebrafish biology
including small size, rapid embryonic development and short life cycle (reviewed in
[33,37,38]), make this model system logistically attractive for rapid assessments of
nanomaterial-biological interactions, from molecular-level responses to whole animal
effects. Many routes of exposure (i.e., ingestion, injection and dermal) can be assessed
individually or in combination. Since zebrafish are amenable to genetic manipulations,
biological targets and modes of action can be defined in a relatively short period of time.
Chronic and generational studies become logistically feasible because zebrafish attain
sexual maturity by 90 days post-fertilisation (dpf). Such features are favourable for
adapting this model system to high-throughput, rapid assessment of the ever-growing
number of novel nanomaterials.
5 Evaluation of nanomaterial-biological interactions using zebrafish
The Tanguay laboratory at Oregon State University (OSU) employs embryonic zebrafish
to investigate biological responses to nanomaterial exposure at multiple levels of
biological organisation in a single organism. Our experimental design tests for
nanomaterial effects during early vertebrate development for two important reasons.
First, fundamental processes of development are remarkably conserved across species
[39–45]. Second, vertebrates at the earliest life stages are often more responsive to
chemical insult [46]. The probable molecular explanation for increased embryonic
susceptibility is that there is no other period in an animal’s life span when the full
repertoire of molecular signalling is necessary and active. It has been postulated that
overall there are only 17 general molecular signalling pathways in vertebrates,
and that each of these is active during early development. Importantly, each of these
pathways is essential for other cells and tissues later in life (summarised in [46]).
Since development is highly coordinated, requiring cell-to-cell communications,
if nanomaterials perturb these interactions, development would be expected to be
disrupted. Perturbed development can manifest as morphological malformations,
behavioural abnormalities or death of the embryos. Collectively, zebrafish are perhaps
the most powerful in vivo model system to assess nanomaterial-biological interactions
and are an outstanding platform to detail the mechanisms by which nanomaterials elicit
adverse biological responses.
Preliminary investigations of AuNPs by the Tanguay group have employed
embryonic zebrafish as a dynamic whole animal assay to reveal the effects of exposure
on biological systems. It had largely been assumed that AuNPs would be biologically
inactive given that both the molecular and bulk forms of gold are benign; however,
Proactively designing nanomaterials to enhance performance 133
this assumption had not been formally tested. To investigate the influence of size and
shape on the biological interactions of nanomaterials, we chose two core sizes (0.8 nm
and 1.5 nm) with one of three surface groups (neutral charge = 2-(2-mercaptoethoxy)
ethanol (MEE), positive charge = N,N,N-trimethylammoniumethanethiol (TMAT),
and negative charge = 2-mercaptoethanesulfonate (MES)). Embryonic zebrafish were
waterborne exposed for five days to graded concentrations of the AuNPs and were
evaluated for mortality, morphological malformations, behavioural abnormalities and
developmental progression. Core size and surface functionalisation both had an influence
on the toxicity of AuNPs. There was a strong dependence on surface charge and purity
and a moderate influence of particle diameter. Positively charged particles had
significantly higher toxicity than negatively charged particles. Neutrally charged AuNPs
of both sizes were benign to embryonic zebrafish even at extremely high concentrations
(250 parts per million). Smaller particles were not necessarily more toxic than larger ones
dependent on surface functionalisation. These studies were a critical first step in
identifying material modifications that can minimise hazard; however, extrapolation of
these results as generalisations is entirely premature. Evaluations are continuing with
embryonic zebrafish to identify the cellular and molecular level biological responses to
nanoparticle exposure.
Unique advantages of the embryonic zebrafish model can be exploited to reveal
modes of action for nanomaterials that elicit adverse biological responses and to identify
gene expression changes that are indicative of nanomaterial exposure and effects.
In vivo cellular targets can be identified using immunohistochemical markers of cell
death or transgenic zebrafish that express fluorescent protein in specific cell populations.
Cellular death and oxidative stress measurements can be determined in a whole
mount assay to identify temporal and spatial localisation of cellular-level disruption.
Morphological effects observed during toxicological evaluations can be used to guide the
selection of reporter transgenic lines to identify cellular targets of action. For example,
if nervous system endpoints are significantly impacted (e.g., brain malformation,
necrosis, or abnormal behaviour), then specific neuronal effects can be identified using
transgenic zebrafish such as huc-GFP (green fluorescent protein, GFP produced
in all neurons), islet1-GFP (GFP produced only in 2° motor neurons), nbt1-GFP
(GFP expressed in 1° spinal motoneurons) and/or neurog1-GFP (GFP produced in
Rohon-Beard cells, dorsal root ganglion cell bodies and axons). Specific cell populations
in systems disrupted by nanomaterial exposure can be easily identified using this
approach. Finally, global gene expression profiles can be used to define the molecular
level responses to nanomaterial exposure. The relationship between physicochemical
properties of nanomaterials and the genomic responses they elicit could provide an
accurate biomarker of nanoparticle-biological interactions which could be used to predict
effects.
Once genes that respond to nanomaterials are identified, a number of approaches have
been effectively used to define the role of individual genes in toxic responses in
zebrafish. Currently, the most common approach is the use of morpholinos. Morpholinos
are chemically modified oligonucleotides that are resistant to chemical and enzymatic
degradation yet maintain base pairing properties [47]. Morpholinos can be designed to
bind to complement sequences at intron-exon spice junctions to inhibit proper mRNA
processing and have been shown to be effective in embryonic zebrafish [48–54].
Morpholinos are typically synthesised as 25mers in length, and are typically injected into
to the one to four cell stage embryos (Figure 2). The successful application of
134 S.L. Harper et al.
morpholinos in zebrafish is evident by the large number of published studies using this
approach (http://www.gene-tools.com/Publications). The ZFIN database (http://ZFIN.
org) curates and lists all the published morpholinos sequences, over 1000 morpholinos
are currently listed. With the availability of the entire zebrafish genome, conceivably any
zebrafish gene can be targeted in a matter of days using morpholinos individually,
or in combinations.
Figure 2 Morpholino injection technique. (A) Single cell stage embryos placed in channels prior
to microinjection of morpholino with finely pulled glass needles. Injection solution is
red because we include phenol red to immediately visualise injection; (B) close-up
image of a single one-cell embryo just after single injection with morpholino
and (C) typical fluorescent pattern in 1000-cell embryo following microinjection
(for colours see online version)
Genetic screens allow for the identification of gene targets essential for biological
processes with no prior functional knowledge of the genes. That is, an observed
phenotype of a selected mutant can be used to identify and to dissect complex pathways
impacted by nanomaterial exposure. To date, large-scale zebrafish mutant searches have
led to the identification of thousands of mutants with unique embryonic and larval
development defects [55,56]. A recent study using mutant zebrafish has offered an
explanation for pigment variation in human populations [57]. This example demonstrates
the rapidity by which results from zebrafish studies can be applied to the understanding
the role of human genes. More recently the Targeting Induced Local Lesions in Genomes
(TILLING) method has been adapted in zebrafish to identify mutants with specific
mutations [58,59]. TILLING can be used to identify and or select for loss-of-function
alleles (knockouts) [60]. TILLING is conceptually simple; random mutations are created
using traditional chemical mutagenesis followed by high-throughput PCR based
screening to identify point mutations in the gene of interest. Therefore TILLING is a
targeted approach and with more widespread use of this technique, each zebrafish gene
could be knocked out. Insertional mutagenesis is an alternative approach to generate
zebrafish mutants and has the benefit that the interrupted gene can be immediately
identified [61–63]. Currently thousands of insertional-derived mutants are commercially
available (Znomics, Portland Oregon), and there is a high probability that in the near
future, researcher will have the ability to obtain mutants with insertional mutations in
each zebrafish gene.
The zebrafish model is ideally suited to rapidly define the proximal gene expression
changes that are causally related to the phenotypic responses to nanomaterial exposure.
In the simple representation presented in Figure 3, it is clear that some of genes directly
influence or regulate the expression of downstream genes while other genes are affected
Proactively designing nanomaterials to enhance performance 135
in parallel and are unrelated to the phenotypic responses. The key is to predict the
relationships between these expressed genes and to have a platform to test the
predictions. We have demonstrated that the zebrafish model is ideal to functionally
determine the contribution that mis-regulated genes play in environmental-genome
interactions and toxicity. With this suite of genomic tools, individual or combinations of
genes can be functionally assessed in a matter of days, not years, as there is no need to
generate stable transgenic animals to test gene expression-derived hypotheses.
Figure 3 Schematic illustrating the relationships among genes (depicted as arrows) and how they
may be differentially expressed in response to nanomaterial exposures. Genes directly
influence or regulate the expression of downstream genes or are affected in parallel
6 Intepretation of nanomaterial effects to inform design processes
It is not clear the extent to which existing paradigms of conventional chemicals can be
applied to nanomaterials since chemical and physical forces are altered when particles are
reduced to a nanoscale size. The current paradigm for chemical analyses of toxic
potential relies on characteristics of substance composition; however, for nanomaterials,
particle size and surface chemistry are likely to be the most important features [11,12].
Again, extensive characterisation and purification of nanomaterials, like those in the UO
library of AuNPs, will be critical to identify how size, surface chemistry and exposure
concentrations relate to organismal response. Only then can dose metrics most correlated
with effects be identified. Structure-activity relationships defined from collective studies
(investigations at multiple levels of biological organisation) will produce algorithms that
provide information about the relative influence of various physicochemical parameters
and help to determine specific structural characteristics that govern nanomaterial-biology
interactions [64]. Given the distinctive properties nanomaterials possess, interactions with
biological systems may not follow biokinetic/biodynamic predictions that have been
based on the principles of chemical interactions [11]. Unique to nanotoxicology, a variety
of dose metrics need to be calculated for well-characterised nanomaterials in order to
determine which dose metrics are appropriate and predictive of biological interactions.
Dose response curves should include a plethora of identified characteristics, such as
mass, number of nanoparticles and surface area; since the importance of each of these
parameters as a dose metric has not been established.
The forecasted growth of the nanotechnology industry dictates the immediate need to
consolidate, integrate and analyse data generated on the interaction of well-defined and
characterised nanomaterials with biological systems. To meet the requirements of
industry for high performance materials with minimal hazard, a comparative integrative
database is required to consolidate and integrate data of nanomaterial effects in
136 S.L. Harper et al.
experimental animal models (including humans) and evaluate biological effects from
a variety of research platforms (i.e., in vivo and in vitro approaches). This database
should be question-directed in design such that knowledge gained will be comprehensive
and useful for industry, academia, government researchers and regulators. The level of
detail in information gained should be end-user specific and tailored to particular
applications and use of the information since features of nanoparticle-biological
interactions that are of high importance to one research group will not necessarily be
relevant to other research groups. For example, toxicologists may want to know every
detail about the biological interactions of nanomaterials that elicit an adverse biological
response, while chemists and materials scientists may only want to know about possible
material modifications that will enhance the performance and minimise toxicity without
detailed examination of the underlying causes for adverse biological interactions.
Regulatory agencies will require the knowledge of hazard (toxic potential) and exposure
in order to assess risk and prioritise testing strategies for evaluations. Yet manufacturers
of material composites may be interested in the relationship between properties
of bulk and nanoparticulate forms of their materials. Researchers of the Environmental
Health Sciences Center, Oregon State University and the Safer Nanomaterials and
Nanomanufacturing Initiative, Oregon Nanoscience and Microtechnologies Institute
(ONAMI) are developing a knowledgebase of Nanomaterial-Biological Interactions
(NBI) at Oregon State University to:
determine the inherent physicochemical properties of nanomaterials that produce
characteristic biological responses
provide this information to stakeholders to guide the rationale development
of safer and more effective nanomaterials.
The NBI knowledgebase will serve as a repository for annotated nanoparticle-biological
interaction data and will house available and relevant computational tools
(e.g., physiologically-based pharmacokinetic/pharmacodynamic models, molecular
dynamics models) that can be used to perform data integration from multiple structured
and semi-structured data sources. Scenario-based exposure-to-dose models can be used to
bring together the disparate data into a mathematical representation of whole organism
physiological processes allowing for more valid species extrapolation, dose estimation
and effects predictions. These models utilise modern concepts of pharmacokinetics,
toxicokinetics and mechanism of action to allow unbiased interpretation of results and
prediction of effects for given exposure scenarios. Numerous physiologically-based
pharmacokinetic (PBPK) and pharmacodynamic (PD) models have been developed for
use in studies of the dose-response and efficacy of pharmaceutical drugs, as well
as for risk assessments of environmental chemicals. The physiology represented by
PBPK models is well-established on the basis of known metabolic processes and
biochemical transformations that occur in animals, including humans. Chemical-specific
parameters (i.e., absorption, distribution, metabolism and elimination and biological
response) are then determined by fitting to empirical data, independent of the
experimental platform.
Computational models will provide the framework within the NBI to conduct
species, route, dose and scenario extrapolations and identify key data required to predict
effects. Because the NBI is being developed during the burgeoning years of the
nanotechnology industry; we have a timely opportunity to obtain comprehensive raw
Proactively designing nanomaterials to enhance performance 137
datasets from original researchers to populate the database, as will be the case for our
AuNP library evaluations. Subsequent, novel data will be assigned as it is generated;
thus, reducing duplicate research efforts, augmenting the dissemination of information
and increasing the power of weight-of-evidence approaches used for regulatory and
industry decision-making. Analysis of these comprehensive data sets will improve our
fundamental understanding of the relationships among currently disparate exposure,
dose and toxicity data in animal systems (including humans) and the degree to which
those relationships can accurately be extrapolated to other systems. This comparative
database information system will facilitate the identification of the experimental
platforms/methods most predictive of nanoparticle-biological interactions.
7 Conclusions
The innovative field of nanotechnology is most likely to benefit society and gain societal
acceptance if environmental and human health considerations are thoroughly investigated
and those results are used to optimise safety and performance together to produce
effective and non-toxic profitable technologies. New approaches to nanotechnology that
employ the principles of green chemistry and green nanoscience, will simultaneously
meet society’s need for high performance materials while protecting human health and
minimising harm to the environment. Green chemistry methodology has progressed to the
extent that it may be easily incorporated into novel research efforts on the first pass,
rather than serving as a tool for the modification of established procedures. Long-term
sustainability of the nanotechnology industry can be realised by creating synthetic routes
that preserve the safety of the worker, prevent environmental hazards and yield more
benign products without compromising product quality. Green chemistry principles
applied early in the formative stages of technological development prevent a perceived
dependence upon techniques which may be hazardous, but otherwise deemed acceptable
simply because it is believed to be the only way to create a desired material.
New chemical methods have been and are being developed that allow one to generate
libraries of nanomaterials from a single batch of precursor particles. In addition, one may
employ advanced filtration methods; where impurities such as molecules, salts, and
excess solvent are removed from a crude mixture. Diafiltration is an especially attractive
method as it is most suitable for solvents such as water and simple alcohols; thus,
requires the researcher to eliminate most other organic solvents from a preparation in
order to take advantage of the method. This technique does not require large amounts of
solvent and has been demonstrated as a method that provides superior levels of product
purity compared to traditional techniques such as centrifugation and washing [23].
Characterisation of nanomaterial building blocks, both in terms of their
physicochemical properties and biological interactions, ensures that we can fully
understand risks presented immediately to the pioneering researcher and minimise those
later encountered by workers in the technology sector that ultimately handle mature
technology as it goes to market. Characterisation and purification should be done
carefully so that evaluations of the biological interactions of nanomaterials can be used to
close the testing-redesign loop and optimise characteristics and safety simultaneously.
Information gained using model systems, such as the embryonic zebrafish, can be used as
rapid feedback for engineers designing novel nanomaterials, such that they can ensure the
development of materials that have the least amount of toxic potential.
138 S.L. Harper et al.
In the future, the NBI knowledgebase will be an instrumental tool to: define
physicochemical properties of nanomaterials that lead to adverse biological
consequences; define generalisations about how size, surface chemistry and exposure
concentrations relate to biological response; determine the extent to which existing
paradigms of conventional chemicals can be applied to nanomaterials; determine how
predictive one nanoparticle type is of the properties of a structurally related material;
identify correlations between gene expression changes and nanoparticle physicochemical
properties; expand traditional structure activity relationships to include novel
high-throughput data (i.e., genomics); and identify biomarkers indicative of nanomaterial
toxicity and possibly mechanism of action. Generalisations of predictive value will be
gained if relationships between exposure and effects are broadly defined using data at
many levels of biological investigation and across a variety of platforms/model systems.
The NBI will offer industry, academia and regulatory agencies a mechanism to
rationally inquire for unbiased interpretation of exposure effects in biological systems.
The opportunity to gain continual feedback on the overall safety profiles of new materials
provides an invaluable opportunity to limit the production of hazardous materials by
guiding the chemical researcher in more benign directions as technology evolves.
Acknowledgements
This research was supported by the Research Office of Oregon State University,
NIEHS Environmental Health and Sciences Center #P30 ES03850, NIEHS Toxicology
Training Grant No. T32 ES07060, NIEHS Marine and Freshwater Biomedical Sciences
Center No. ES00210, EPA STAR No. RD-833320, the Safer Nanomaterials and
Nanomanufacturing Initiative of the Oregon Nanoscience and Microtechnologies
Institute, sponsored by a grant from the Air Force Research Laboratory under agreement
number FA8650-05-1-5041, and the National Science Foundation (an IGERT Fellowship
to JAD, DGE-0549503).
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... Such examinations must be combined with specific imaging techniques such as TEM to authenticate the marker-based toxicity assays (Arora et al., 2012). p0625 Alternative animal models, such as zebrafish, have been utilized to study nanomaterials' biological interactions and determine the adverse effects of such interactions (Harper et al., 2008b). One such model is zebrafish, as it possesses various advantages such as resembling human anatomy and physiology. ...
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Since the past few decades, nanomaterials have gained much attention due to their comprehensive applications in industrial and medical fields. However, many investigations report the toxicological aspects of nanomaterial toward the cell membrane, organelles, and other cellular components. Developmental toxicity is a significant concern that impacts infertility, reproduction, and fetal development. Various aspects of formulation development and exposure are responsible for contributing to its reproductive toxicity. This chapter provides a focused discussion on the developmental toxicity of nanomaterials used in drug delivery and illustrates how to tackle them. Suggestive crosstalk on the need to improve the cell culture and animal models used to assess developmental toxicity is also presented.
... Exposure of adult and larval zebrafish to high concentrations of AgNPs is known to cause mortality, hatching delays, developmental abnormalities, neurotoxicity, genotoxicity, oxidative stress, and toxicity to gills and intestines [48,[51][52][53][54]56,59,78,79]. Furthermore, their ease of reproduction, rapid development, and transparent bodies make them a great model for high-throughput screening of materials [80][81][82][83][84][85][86]. This model is ideal to rapidly assess the effect of nanoparticle size and efficiency of uptake on toxicity without the confounding effect of Ag + ions. ...
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... For the in vivo toxicity testing, embryonic zebrafish (Danio rerio) was used as a model organism because of their rapid development and ease of use in nanotoxicology studies. [56][57][58][59][60][61] Embryonic zebrafish have very similar molecular signaling processes, cell structure, anatomy, and physiology as other higher-order vertebrates, including humans. [62][63][64][65] Hence, embryonic zebrafish allow for observation of whole organism toxicity in a model that is relevant to human health, as humans are the ultimate target of many AuNP applications. ...
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... Green nanotechnol ogy refers to the utilization of "Green Chemistry" rules to reduce or eradicate the potential hazards of nanomaterials to human health and the environment through product design and process optimization [45]. Designing greener nanomaterials and nanomanufacturing techniques may include choice of greener chemicals, exclusion of toxic nanomaterials and modification of physical and chemical properties, thereby controlling their fate in the envi ronment and rendering them nontoxic [46]. Following are a set of green chemistry princi ples that might be applied to nanoscience and technology in order to rationally design safe yet high performing nanoscale materials [47]. ...
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... Therefore, CR@Fe 3 O 4 NPs could be excreted gradually from the intestine during the feeding and digestive process. 40 ...
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