Accepted Manuscript Keeping it real: the importance of material characterization in nanotoxicology Bengt Fadeel, Andrea Fornara, Muhammet S. Toprak, Kunal Bhattacharya PII:
S0006-291X(15)30207-2
DOI:
10.1016/j.bbrc.2015.06.178
Reference:
YBBRC 34267
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 4 June 2015 Accepted Date: 23 June 2015
Please cite this article as: B. Fadeel, A. Fornara, M.S. Toprak, K. Bhattacharya, Keeping it real: the importance of material characterization in nanotoxicology, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.06.178. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Keeping it real: the importance of material characterization in nanotoxicology
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet,
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Bengt Fadeel1,*, Andrea Fornara2, Muhammet S. Toprak3, and Kunal Bhattacharya1
171 77 Stockholm, Sweden;
Unit for Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden, 114 86
Stockholm, Sweden; 3
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Functional Materials Division, Department of Materials and Nano Physics, Royal Institute of
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Technology, 164 40 Stockholm, Sweden.
*Corresponding author: Institute of Environmental Medicine, Nobels väg 13, Karolinska
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[email protected]
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Institutet, 171 77 Stockholm, Sweden; Tel. +46 8 524 877 37; Fax: +46 8 34 38 49; E-mail:
Running title: Nanomaterial characterization for safety assessment.
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Abstract Nanomaterials are small and the small size and corresponding large surface area of nanomaterials confers specific properties, making these materials desirable for various applications, not least in
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medicine. However, it is pertinent to ask whether size is the only property that matters for the desirable or detrimental effects of nanomaterials? Indeed, it is important to know not only what the material looks like, but also what it is made of, as well as how the material interacts with its
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biological surroundings. It has been suggested that guidelines should be implemented on the types of information required in terms of physicochemical characterization of nanomaterials for
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toxicological studies in order to improve the quality and relevance of the published results. This is certainly a key issue, but it is important to keep in mind that material characterization should be fit-for-purpose, that is, the information gathered should be relevant for the end-points being
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studied.
Key words: engineered nanomaterials; nanotoxicology; physicochemical characterization; bio-
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corona.
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“When things are large, they are what they are. When they are small, it’s a different game: they
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are what our measurements make them.”
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George M. Whitesides, No Small Matter. Science on the Nanoscale. [2009]
Introduction
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Engineered nanomaterials have become the focus of extensive research in many areas including biomedical applications due to the novel and unique properties arising at the nanoscale. In addition, during the past decade, there has been an exponential increase in the number of papers on the toxicological effects of nanomaterials. However, while this certainly shows that the potential risks of nanomaterials are being considered, it has been argued that numerous poorly
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controlled studies have been published, offering little insight into any ‘nanospecific’ effects (1). Indeed, Krug concluded in a recent overview of the field that while 10.000 papers have been produced on environmental and health effects of nanomaterials in the last 15 years, we are left
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with “a plethora of low-value results” due to the lack of harmonized experimental protocols, poor
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or nonexistent characterization of the nanomaterials, a lack of reference materials, the frequent reliance on unrealistically high doses both for in vitro and in vivo studies, and so on (1). Others have also complained, on the basis of a meta-analysis of several dozen papers focused on silica particles, that “after over a decade of research, answers for the most basic questions are still lacking” and suggested that more coherence in the experimental methods and materials used is needed (2).
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Ten years have passed since the review by Oberdörster et al. defining and outlining the emerging discipline of nanotoxicology (3); in their review, which is now a ‘citation classic’, the authors defined nanotoxicology as a “science of engineered nanodevices and nanostructures that deals
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with their effects in living organisms” and they pointed out that nanotoxicology research also will advance the field of nanomedicine by providing information on the undesirable properties of nanomaterials and means to avoid them. Indeed, this is sometimes referred to as ‘safe-by-design’
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(4).
In another very pertinent review, also published in 2005, Oberdörster et al. summarized the views of an international expert group convened to develop a screening strategy for the hazard identification of engineered nanomaterials (5). Hence, the authors stated that “there is a strong
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likelihood that biological activity of nanoparticles will depend on physicochemical parameters not routinely considered in toxicity screening [of chemicals]” and put forward a list of physicochemical properties that may be important in understanding the toxicity of nanomaterials
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namely: particle size and size distribution, agglomeration state, particle shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge, and porosity (5). Similar
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suggestions for minimal material characterization requirements in nanotoxicology have been proposed in recent years, as we shall discuss in the present essay. The question is: are we ready to adopt such requirements as an international standard(s)? Indeed, can we afford not to do so? Furthermore, are there any examples of ‘good’ nanotoxicological studies, or has all been for naught?
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2. International harmonization efforts in nanotoxicology Warheit asked in an editorial several years ago “how meaningful are the results of nanotoxicity
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studies in the absence of adequate material characterization?” (6). He also noted that while most nanotoxicological studies are conducted under in vitro conditions (i.e., in the wet phase), the physicochemical characterization is frequently carried out on the “just-received” nanomaterials in the dry phase, which has limited relevance for the test conditions. He also professed a list of
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minimal characterization requirements prior to conducting hazard assessment studies, similar to
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the one cited above. In a follow-up, Sayes and Warheit discussed three phases of material characterization (7). Primary characterization is performed on particles as-synthesized or asreceived, in its dry state or powder form. Secondary characterization, on the other hand, is performed on particles in the wet phase as a solution or suspension in aqueous media, eg. in water or cell culture medium. Finally, tertiary characterizations are performed on particles following
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interactions with biological systems in vitro or in vivo, and may include characterization of particles in blood, or lung fluid. The tertiary characterization of particles in the actual test system is certainly non-trivial, but it is the most relevant for the interpretation of the toxicological data
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(7). The authors offered a list of physicochemical properties relevant to nanotoxicological testing,
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and they concluded that “no single technique can accurately describe a specific property of a material” (7). Thus, all material characterization should be performed using more than one method.
The journal Nature Nanotechnology recently invited the nanotoxicology community to ‘join the dialogue’ on whether guidelines should be implemented on the types of information that are
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required to improve the quality and relevance of the published papers (8). It was further stated in the editorial that nanomaterial characterization “should be done based on relevance to the study” (8). Indeed, different types of information (and consequently, the use of different methods) may
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be needed depending on the purpose of the study (9). Hence, nanomaterial characterization should be fit-for-purpose. Furthermore, the methods that are used for characterization need to be standardized and validated. Fubini et al. emphasized that while there are obvious properties that
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should be assessed before any in vivo or in vitro testing is conducted, the “choice of characteristics to be measured more accurately should be tailored to the end-point investigated in
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that particular study” (10). Others have highlighted that, “for regulatory purposes, the standards applied and data generation required must be more prescriptive, whereas for research, these must be primarily based on the hypothesis to be tested” (11). Furthermore, while it would be laudable to characterize every aspect of a test material, both at synthesis and in the test system, this is
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certainly impractical (5) and it would therefore be advisable for the scientific community to agree on a number of common parameters that should be measured for all studies, in order to describe what the material looks like, what it is made of, and what factors govern how it interacts with its
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study (13).
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biological surroundings (12), while other characteristics should be based on relevance to the
The Seventh Framework Programme of the European Commission started in 2007 and projects funded in the final round will run until 2017. Among these projects, 50 have focused on nanosafety. The NanoImpactNet project played an important role in the integration of nanosafety research in the early phase of FP7 through the organization of workshops and conferences. In one such workshop, experts provided
recommendations for minimal
characterization of
nanomaterials, and a distinction was made between ‘essential metrics’, including size and size 6
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distribution, chemical composition and surface charge, and ‘often important metrics’, including shape and solubility (14). The need for methods to determine interactions of nanomaterials with the surrounding biological matrix was also highlighted. The research infrastructure project
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QualityNano has emphasized the importance of standardization and harmonization of procedures in all aspects of nanosafety assessment and also highlighted the need for in situ characterization (15). The NANOREG project, with close to 70 partner institutes and a total of 50 million Euro in
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funding (provided by the European Commission and the participating member states), is designed to facilitate a common approach to regulatory testing of nanomaterials and aims to develop a
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regulatory framework for nanomaterials, in close cooperation with international organisations involved in standardization and regulation of nanomaterials, such as ECHA, OECD, CEN and ISO. In the NANOREG project, several characterization protocols based on OECD guidelines for analysis of physicochemical properties of nanomaterials are being verified and validated,
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including quantitative analysis of surface coatings, and size distribution analysis in the dry state or powder form as well as in the wet state, i.e., in liquids for compliance with the EU definition
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(16).
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The FP7 project ITS-NANO (for ‘intelligent testing strategy’) developed a framework of future research priorities in cooperation with all the major stakeholders (i.e., government, industry, academia, funding agencies and NGOs), and emphasized the importance of physicochemical characterization, along with exposure identification, hazard identification and modelling approaches (17). In the large FP7 project, MARINA (for ‘managing risks of nanomaterials’), first steps are taken towards developing an intelligent testing strategy for nanomaterials. Notably, in the project, a common panel of representative nanomaterials from the Joint Research Centre
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(JRC) nanomaterial repository are being applied. Each type of material in the repository has been sourced as a large single batch which has been sub-sampled into individual vials to produce a collection of thoroughly characterized nanomaterials available for benchmarking in research and
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regulatory studies. In a recent study, 6 metal oxide nanomaterials were evaluated using 10 different toxicity assays in 9 different laboratories using 12 cellular models representing 6 different target organs (18). The nanomaterials were all subjected to detailed physicochemical
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characterization. With this approach, a hazard ranking of the metal oxides could be established and cell-specific responses were noted. The NANOREG project also uses nanomaterials from the
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JRC repository. In another recent study, US researchers belonging to the ‘engineered nanomaterials grand opportunity’ (Nano GO) consortium conducted so-called round robin or interlaboratory comparisons of a total of 7 metal oxides and multi-walled carbon nanotubes using 3 different cell lines (19). These and other studies (20) point to the importance of conducting
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studies with multiple relevant cell types in order to perform accurate in vitro evaluations of nanomaterials. Moreover, applying extensively characterized nanomaterials for benchmarking will allow for comparisons across studies and may prevent the generation of “low-value” results
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(1).
For a discussion of material characterization in the context of nanomedicines, see the excellent papers by McNeil and co-workers from the US Nanomaterial Characterization Laboratory, NCL (21,22).
3. The issue of dose and dosimetry in nanotoxicology 8
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The purposeful characterization of nanomaterials is thus of paramount importance in nanotoxicology, but so is dosimetry, i.e., the accurate measurement of the dose, or the amount of the nanomaterial which comes into contact with the biological target. Several different metrics
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have been proposed in for in vitro nanotoxicology, including µg/ml, cm2/mL, µg/cm2, or particle number/mL (23). However, defining the biologically effective dose is not a trivial issue, as highlighted in a recent editorial in Particle & Fibre Toxicology (24). In nanotoxicology, the dose
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is commonly defined as the nominal dose, i.e., the amount of nanomaterial introduced into the culture medium, but as pointed out, it remains possible to measure both the deposited, cell-
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associated or intracellular doses (24). Lison et al. concluded that information about the mechanism of toxicity, obtained experimentally or deduced by analogy with similar nanomaterials, “is essential to guide investigators when selecting the most relevant dose to characterize dose-effect/response relationships” (24). But a priori knowledge of the mechanism
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is not always, or very seldom, available, and we are not yet able to perform grouping of nanomaterials, although attempts are being made (see below). In order to better define the dose that is actually delivered to cells (i.e., the “relevant” dose), computational models have been
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developed in recent years that take into account in vitro particokinetics (sedimentation and diffusion) for individual particles and their agglomerates (25). With this approach, it is possible
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to calculate the total mass, surface area, or particle number delivered to cells as a function of exposure time in an in vitro system (26). However, while the latter approach may be suited for spherical nanoparticles, the methodology needs to be validated also for high aspect ratio materials such as carbon nanotubes. In a recent study, the authors showed that the in vitro dosimetry has implications for hazard ranking of nanomaterials, using THP.1 cells as a model (27). For carbonbased nanomaterials, the deposited doses varied from 20-50% of the administered doses at 24 h, whereas for the metal/metal oxides with larger effective densities of their formed agglomerates, 9
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the deposited doses were much closer to the administered doses. Overall, discrepancies between administered and delivered doses were more evident during the first few hours. Interestingly, the slope of the dose-response relationship for cell viability and cytokine production changed for
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some, but not all nanomaterials, when delivered dose was taken into account (27). This may have implications for the toxicity screening of different nanomaterials. Furthermore, and closely related to the issue of the (relevant) dose, is the matter of nanoparticle dispersion protocols,
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which will influence particle agglomeration, which in turn will affect sedimentation and diffusion in a cell culture system. There is probably no one-size-fits-all recipe for nanomaterial dispersions
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and various approaches are being applied in nanotoxicology. However, reliable methods with which to characterize nanomaterial dispersions are needed. Pal et al. provided one example using so-called tunable resistive pulse sensing (TRPS) technology and found that this technology yielded higher resolution and sensitivity when compared to conventional dynamic light scattering
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(DLS), which is currently the preferred technique (28). Unlike DLS, the TRPS technology does not rely on light scattering properties. Instead, it monitors changes in ionic current as individual nanoparticles or agglomerates pass through an elastomeric membrane; the TRPS technology may
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(28).
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complement DLS measurements for nanomaterial and nanomaterial-biomolecule characterization
The synthetic and biological identities
It follows from the previous discussion that nanomaterial characterization is a crucial element of nanotoxicology. It is also clear that there are numerous material physicochemical properties that one could characterize, although there seems to be an emerging consensus regarding the most essential ones. However, as we have discussed in a previous review, understanding which of the
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physicochemical properties that are driving toxicity remains a key challenge; if one could connect material properties (such as, composition, size, shape, surface charge, porosity, colloidal stability, purity/degree of contamination, and so on) with toxicity, then this might enable the prediction of
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potential hazards and support safe-by-design approaches to produce nanomaterials with minimal toxicity (29).
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The intrinsic physicochemical properties may be viewed as the ‘synthetic identity’ of the nanomaterial. However, there is an emerging realization in recent years that nanomaterials are not
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‘naked’ following their introduction into a biological system, and, moreover, that “the organic and synthetic worlds merge into a new science” at the interface between nanomaterials and biological systems (30). In fact, as we and others have discussed previously, the ‘biological identity’ is shaped, in part, by the adsorption of biomolecules including proteins and lipids, or
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both, onto the surface of the nanoparticles (29,31), and nanomaterials may therefore be viewed as – and should perhaps also be regulated as – ‘biological entities’ (32). The composition of the biocorona depends on the portal of entry into the body and on the particular biofluid (eg., blood,
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lung fluid, gastro-intestinal fluid, etc) that the nanomaterials encounter and may exhibit dynamic changes as the nanoparticle crosses from one biological compartment to another (33). In a recent,
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comprehensive study, Tenzer et al. reported that plasma protein adsorption occurs very rapidly on the surface of nanoparticles and that this could significantly affect biological outcomes relevant for the hematocompatibility of the particles, including lysis of red blood cells, and thrombocyte activation (34). Notably, the authors found that none of the physicochemical properties alone could account for the formation, composition and evolution of the protein corona (34). Nonetheless, as explained by Walkey and Chan (35), “once fully mapped, the relationships between synthetic identity, biological identity, and physiological response will enable researchers 11
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to predict the physiological response of a nanomaterial by characterizing its synthetic identity” (and see Figure 1). Thus, when nanomaterial characterization is performed, attention should also be devoted to the biological surroundings, meaning that nanomaterials should be characterized
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not only in their pristine state, but also in the relevant biological system or fluid (i.e., wet state), and that the adsorbed biomolecules also need to be taken into account. At present, most biocorona studies have focused on the identification of adsorbed proteins (in some cases, also on the
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adsorption of lipids and other biomolecules), without specifically connecting individual proteins in the corona to biological outcomes. However, there are some emerging and illustrative
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examples. Hence, Walkey et al. conducted so-called serum protein corona 'fingerprinting' of a library of 105 surface-modified gold nanoparticles and found that the corona fingerprint predicts cellular uptake more accurately than a model that uses parameters describing nanoparticle size, aggregation state, and surface charge, with A549 human lung epithelial carcinoma cells as a
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model (36). Interestingly, the study also revealed that the core material (gold or silver) exerted a greater influence on protein corona composition than core size or surface functional group. As the authors pointed out that, even if the ligand-protected core does not make direct contact with
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proteins in the biological environment, it determines the density, arrangement, and orientation of the associated ligands (36). Thus, the synthetic and biological identities of nanoparticles are
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connected. However, it remains to be understood whether these observations can be generalized for other classes of nanomaterials. The above dataset has recently been explored further using both linear and non-linear quantitative structure-activity relationship (QSAR) analysis and these studies revealed a small set of serum proteins, including apolipoprotein B-100 (ApoB-100), along with the nanomaterial zeta potential (as synthesized), as being significant descriptors correlating with cell association/cell uptake (37). In another recent study, Ritz et al. first determined the corona composition on a set of polymeric nanoparticles with different surface functionalization, 12
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then correlated the relative abundance of identified proteins with cellular uptake, and, finally, verified the role of candidate proteins by decorating nanoparticles with specific proteins (38). By this approach, they could demonstrate that the apolipoproteins ApoA4 and ApoC3 significantly
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decreased cellular uptake, while ApoH (also known as β2 glycoprotein-I) increased uptake by human mesenchymal stem cells (38). In synopsis, understanding the nano-bio interface is important and methods to investigate the nano-bio interface – qualitatively and quantitivately –
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are needed.
5. Naming and categorizing nanomaterials
The so-called ‘grouping of substances’ or category approach has been recognized as an important means to avoid unnecessary testing of new chemicals (39). For chemicals in general, technical
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guidance documents on grouping are available, from the Organisation for Economic Cooperation and Development (OECD) or the European Chemicals Agency (ECHA). For nanomaterials, specific guidance is not yet available. However, members of the European Centre for
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Ecotoxicology and Toxicology of Chemicals (ECETOC) recently reviewed available concepts for the grouping of nanomaterials for human health risk assessment (39). The authors argued in favor
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of a comprehensive, ‘multiple perspective’ framework that would take into account the different stages of the life cycle of the nanomaterial or nanomaterial-enabled product. In a more recent installment, a decision-making framework for grouping of nanomaterials was proposed (40). The authors noted that most nanomaterials will not be accurately grouped based solely on their intrinsic material properties as it is not yet understood how intrinsic material properties relate to toxic effects (40). Therefore, the ECETOC proposal follows a functionality-driven approach
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instead of being predominantly based upon intrinsic nanomaterial properties. In a recent report on a workshop convened by the University of California Center for Environmental Implications of Nanotechnology (UC-CEIN) and the UCLA Center for Nanobiology and Predictive Toxicology,
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Godwin et al. discussed how categorization of nanomaterials coupled with alternative (nonanimal based) testing strategies might be used to expedite hazard characterization, allowing for integrated environmental and occupational health and safety decision-making (41). The authors
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suggested that grouping of nanomaterials could be helpful during the early stages of risk assessment to identify ‘nanomaterials of concern’, which could then be selected for more detailed
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testing. They also concluded that physicochemical properties alone are not currently sufficient for nanomaterial categorization and that categorization methods for regulatory purposes should include indicators of both hazard and exposure (41), in line with the approach advocated by
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ECETOC.
In order to categorize nanomaterials, one first needs to study (many) nanomaterials. To this end,
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high-throughput screening of libraries of well-defined nanomaterials with systematic variation of different physicochemical properties is a useful tool, to help establish structure-activity
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relationships (42). This also highlights the need for high-throughput characterization methods, especially those methods that could be applied for so-called secondary phase characterization, i.e., characterization performed on particles in the wet phase as a dispersion in an aqueous medium relevant to the test system (7). In a recent publication, Meng and Ugaz demonstrated a novel, real-time screening approach in order to perform instantaneous physicochemical analysis of suspension-based nanomaterials (43). The authors exploited surface complexation interactions that emerge when a micron-scale chemical discontinuity is established between suspended 14
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nanoparticles and a fluorescent molecular tracer in a laminar flow environment that removes limitations associated with convective transport and mixing (43). The resulting fluorescence signature is detected and can be used to extract information – via image processing and a
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developed algorithm – about composition, quantity, size, and morphology of nanoparticles in suspension, independent of their agglomeration state. The authors successfully demonstrated the use of this approach for continuous sizing of ZnO nanoparticles, and for quantification of the
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anatase and rutile composition of TiO2 nanoparticle mixtures (43). The method is highly specific for surface complexation of different tracers with the metal ions in the nanoparticles, and the
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model is based on the number of available surface binding sites in the suspended nanoparticles. However, this method could perhaps be applied to other nanoparticle systems, once a good tracer
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is identified.
Naming supports categorization (of nanomaterials). To this end, members of the nanotechnology working group of the US National Institutes of Health (NIH) National Cancer Informatics
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Program have developed ‘ISA-TAB-Nano’, a general framework for representing and integrating data pertaining to the description and characterization of nanomaterials using spreadsheet files
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(44). The EU-funded FP7-eNANOMAPPER project aims to develop a computational infrastructure for data management of toxicological data to enable a more effective approach to research in nanotoxicology, and the first steps towards an integrated ontology were recently reported (45). Other attempts to codify nanomaterials as a function of their physicochemical properties have been presented (46). Of course, any nomenclature system based on characterization of nanomaterial properties presupposes that the characterization can be executed in an unambiguous manner across different laboratories. The most ambitious proposal by far is 15
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the nanomaterials classification roadmap developed by Tomalia, which points towards a periodic table(s) of nanomaterials with the aim of predicting and defining risk-benefit boundaries in the nanotechnology field (47). The biological identity of nanomaterials might add one more
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dimension to the periodic table(s), although it could be argued that the formation and composition of a bio-corona will depend on the intrinsic physicochemical properties of the nanomaterials (see
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6. Conclusions
The ‘nanomaterial characterization bottleneck’ is a concern not only in basic research, but also has significant implications for the commercialization of nano-enabled products, and for their regulation (48). In the present essay, we highlighted the importance of thorough material characterization in order to improve the quality and relevance of nanotoxicological studies. We
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emphasized that physicochemical characterization needs to be fit-for-purpose and of relevance for the hypothesis that is being addressed, although there should also be minimal characterization requirements in order to understand what the material looks like, and what it is made of (12).
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International guidelines for (minimal) material characterization are needed not least for
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regulatory purposes, and the methods used for characterization need to be standardized and validated.
We also discussed the so-called synthetic ‘identity’ of nanomaterials which is defined by the material-intrinsic properties versus the biological ‘identity’ which is manifested in a living system and can be viewed as the sum of the context-dependent properties of the nanomaterial.
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Both identities are connected and should be taken into consideration in nanotoxicological
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investigations.
7. Acknowledgements
We gratefully acknowledge the financial support of the European Commission (FP7-NANOREG,
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Grant No. 310584; FP7-eNANOMAPPER, Grant No. 604134; COST Action MODENA, TD1204) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial
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Planning.
References
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1. H.F. Krug, Nanosafety research--are we on the right track? Angew Chem Int Ed Engl. 53 (2014) 12304-12319.
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2. F. Schrurs, D. Lison, Focusing the research efforts, Nat Nanotechnol. 7 (2012) 546-548. 3. G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline
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evolving from studies of ultrafine particles, Environ Health Perspect. 113 (2005) 823-839. 4. B. Fadeel, Nanosafety: towards safer design of nanomedicines, J Intern Med. 274 (2013) 578-580.
5. G. Oberdörster, A. Maynard, K. Donaldson, V. Castranova, J. Fitzpatrick, K. Ausman, J. Carter, B. Karn, W. Kreyling, D. Lai, S. Olin, N. Monteiro-Riviere, D. Warheit, H. Yang, Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy, Part Fibre Toxicol. 2 (2005) 8. 17
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6. D.B. Warheit, How meaningful are the results of nanotoxicity studies in the absence of adequate material characterization? Toxicol Sci. 101 (2008) 183-185. 7. C.M. Sayes, D.B. Warheit, Characterization of nanomaterials for toxicity assessment,
8. Editorial, Join the dialogue, Nat Nanotechnol. 7 (2012) 545.
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Wiley Interdiscip Rev Nanomed Nanobiotechnol. 1 (2009) 660-670.
9. B. Fadeel, K. Savolainen, Broaden the discussion, Nat Nanotechnol. 8 (2013) 71.
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10. B. Fubini, M. Ghiazza, I. Fenoglio, Physico-chemical features of engineered nanoparticles relevant to their toxicity, Nanotoxicology 4 (2010) 347-363.
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11. J. Lead, S. Holgate, Regulatory and research needs, Nat Nanotechnol. 8 (2013) 72. 12. D.R. Boverhof, R.M. David, Nanomaterial characterization: considerations and needs for hazard assessment and safety evaluation, Anal Bioanal Chem. 396 (2010) 953-961. 13. KA. Dawson, Leave the policing to others, Nat Nanotechnol. 8 (2013) 73.
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14. H. Bouwmeester, I. Lynch, H.J. Marvin, K.A. Dawson, M. Berges, D. Braguer, H.J. Byrne, A. Casey, G. Chambers, M.J. Clift, G. Elia, T.F. Fernandes, L.B. Fjellsbø, P. Hatto, L. Juillerat, C. Klein, W.G. Kreyling, C. Nickel, M. Riediker, V. Stone, Minimal
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analytical characterization of engineered nanomaterials needed for hazard assessment in biological matrices, Nanotoxicology 5 (2011) 1-11.
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15. K.A. Dawson, S. Anguissola, I. Lynch, The need for in situ characterisation in nanosafety assessment: funded transnational access via the QNano research infrastructure, Nanotoxicology 7 (2013) 346-349.
16. E.A. Bleeker, W.H. de Jong, R.E. Geertsma, M. Groenewold, E.H. Heugens, M. KoersJacquemijns, D. van de Meent, J.R. Popma, A.G. Rietveld, S.W. Wijnhoven, F.R. Cassee, A.G. Oomen, Considerations on the EU definition of a nanomaterial: science to support policy making, Regul Toxicol Pharmacol. 65 (2013) 119-125. 18
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17. V. Stone, S. Pozzi-Mucelli, L. Tran, K. Aschberger, S. Sabella, U. Vogel, C. Poland, D. Balharry, T. Fernandes, S. Gottardo, S. Hankin, M.G. Hartl, N. Hartmann, D. Hristozov, K. Hund-Rinke, H. Johnston, A. Marcomini, O. Panzer, D. Roncato, A.T. Saber, H.
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Wallin, J.J. Scott-Fordsmand, ITS-NANO--prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy, Part Fibre Toxicol. 11 (2014) 9.
18. L. Farcal, F.T. Andón, L. Di Cristo, B.M. Rotoli, O. Bussolati, E. Bergamaschi, A. Mech,
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N.B. Hartmann, K. Rasmussen, J. Riego-Sintes, J. Ponti, A. Kinsner-Ovaskainen, F. Rossi, A. Oomen, P. Bos, R. Chen, R. Bai, C. Chen, L. Rocks, N. Fulton, B. Ross, G.
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Hutchison, L. Tran, S. Mues, R. Ossig, J. Schnekenburger, L. Campagnolo, L. Vecchione, A. Pietroiusti, B. Fadeel, Comprehensive in vitro toxicity testing of a panel of representative oxide nanomaterials: first steps towards an intelligent testing strategy, PLoS One 10 (2015) e0127174.
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19. T. Xia, R.F. Hamilton, J.C. Bonner, E.D. Crandall, A. Elder, F. Fazlollahi, T.A. Girtsman, K. Kim, S. Mitra, S.A. Ntim, G. Orr, M. Tagmount, A.J. Taylor, D. Telesca, A. Tolic, C.D. Vulpe, A.J. Walker, X. Wang, F.A. Witzmann, N. Wu, Y. Xie, J.I. Zink, A. Nel, A.
EP
Holian, Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium, Environ Health Perspect.
AC C
121 (2013) 683-690.
20. A. Kroll, C. Dierker, C. Rommel, D. Hahn, W. Wohlleben, C. Schulze-Isfort, C. Göbbert, M. Voetz, F. Hardinghaus, J. Schnekenburger, Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays, Part Fibre
Toxicol. 8 (2011) 9. 21. J.B. Hall, M.A. Dobrovolskaia, A.K. Patri, S.E. McNeil, Characterization of nanoparticles for therapeutics, Nanomedicine (Lond). 2 (2007) 789-803. 19
ACCEPTED MANUSCRIPT
22. P.P. Adiseshaiah, J.B. Hall, S.E. McNeil, Nanomaterial standards for efficacy and toxicity assessment, Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2 (2010) 99-112.
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23. M. Hull, A.J. Kennedy, C. Detzel, P. Vikesland, M.A. Chappell, Moving beyond mass: the unmet need to consider dose metrics in environmental nanotoxicology studies, Environ Sci Technol. 46 (2012) 10881-10882.
24. D. Lison, G. Vietti, S. van den Brule, Paracelsus in nanotoxicology, Part Fibre Toxicol.
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11 (2014) 35.
25. P.M. Hinderliter, K.R. Minard, G. Orr, W.B. Chrisler, B.D. Thrall, J.G. Pounds, J.G.
M AN U
Teeguarden, ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies, Part Fibre Toxicol. 7 (2010) 36. 26. J.M. Cohen, J.G. Teeguarden, P. Demokritou, An integrated approach for the in vitro dosimetry of engineered nanomaterials, Part Fibre Toxicol. 11 (2014) 20.
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27. A.K. Pal, D. Bello, J. Cohen, P. Demokritou, Implications of in vitro dosimetry on toxicological ranking of low aspect ratio engineered nanomaterials, Nanotoxicology 12 (2015) 1-15. [Epub ahead of print].
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28. A.K. Pal, I. Aalaei, S. Gadde, P. Gaines, D. Schmidt, P. Demokritou, D. Bello, High resolution characterization of engineered nanomaterial dispersions in complex media
AC C
using tunable resistive pulse sensing technology, ACS Nano 8 (2014) 9003-9015.
29. B. Fadeel, N. Feliu, C. Vogt, A.M. Abdelmonem, W.J. Parak, Bridge over troubled waters: understanding the synthetic and biological identities of engineered nanomaterials,
Wiley Interdiscip Rev Nanomed Nanobiotechnol, 5 (2013) 111-129.
20
ACCEPTED MANUSCRIPT
30. A.E. Nel, L. Mädler, D. Velegol, T. Xia, E.M. Hoek, P. Somasundaran, F. Klaessig, V. Castranova, M. Thompson, Understanding biophysicochemical interactions at the nanobio interface, Nat Mater. 8 (2009) 543-557.
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31. M.P. Monopoli, C. Åberg, A. Salvati, K.A. Dawson, Biomolecular coronas provide the biological identity of nanosized materials, Nat Nanotechnol. 7 (2012) 779-786.
32. I. Lynch, A. Ahluwalia, D. Boraschi, H.J. Byrne, B. Fadeel, P. Gehr, A.C. Gutleb, M.
SC
Kendall, M.G. Papadopoulos, The bio-nano-interface in predicting nanoparticle fate and behaviour in living organisms: towards grouping and categorising nanomaterials and
M AN U
ensuring nanosafety by design, BioNanoMaterials 14 (2013) 195-216. 33. A. Pietroiusti, L. Campagnolo, B. Fadeel, Interactions of engineered nanoparticles with organs protected by internal biological barriers, Small 9 (2013) 1557-1572. 34. S. Tenzer, D. Docter, J. Kuharev, A. Musyanovych, V. Fetz, R. Hecht, F. Schlenk, D.
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Fischer, K. Kiouptsi, C. Reinhardt, K. Landfester, H. Schild, M. Maskos, S.K. Knauer, R.H. Stauber, Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology, Nat Nanotechnol. 8 (2013) 772-781.
EP
35. C.D. Walkey, W.C. Chan, Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment, Chem Soc Rev. 41 (2012) 2780-2799.
AC C
36. C.D. Walkey, J.B. Olsen, F. Song, R. Liu, H. Guo, D.W. Olsen, Y. Cohen, A. Emili, W.C. Chan, Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles, ACS Nano 8 (2014) 2439-2455.
37. R. Liu, W. Jiang, C.D. Walkey, W.C. Chan, Y. Cohen, Prediction of nanoparticles-cell association based on corona proteins and physicochemical properties, Nanoscale 7 (2015) 9664-9675.
21
ACCEPTED MANUSCRIPT
38. S. Ritz, S. Schöttler, N. Kotman, G. Baier, A. Musyanovych, J. Kuharev, K. Landfester, H. Schild, O. Jahn, S. Tenzer, V. Mailänder, Protein corona of nanoparticles: distinct proteins regulate the cellular uptake, Biomacromolecules 16 (2015) 1311-1321.
RI PT
39. J.H. Arts, M. Hadi, A.M. Keene, R. Kreiling, D. Lyon, M. Maier, K. Michel, T. Petry, U.G. Sauer, D. Warheit, K. Wiench, R. Landsiedel, A critical appraisal of existing concepts for the grouping of nanomaterials, Regul Toxicol Pharmacol. 70 (2014) 492-
SC
506.
40. J.H. Arts, M. Hadi, M.A Irfan, A.M. Keene, R. Kreiling, D. Lyon, M. Maier, K. Michel,
M AN U
T. Petry, U.G. Sauer, D. Warheit, K. Wiench, W. Wohlleben, R. Landsiedel, A decisionmaking framework for the grouping and testing of nanomaterials (DF4nanoGrouping), Regul Toxicol Pharmacol. (2015). [Epub ahead of print].
41. H. Godwin, C. Nameth, D. Avery, L.L. Bergeson, D. Bernard, E. Beryt, W. Boyes, S.
TE D
Brown, A.J. Clippinger, Y. Cohen, M. Doa, C.O. Hendren, P. Holden, K. Houck, A.B. Kane, F. Klaessig, T. Kodas, R. Landsiedel, I. Lynch, T. Malloy, M.B. Miller, J. Muller, G. Oberdorster, E.J. Petersen, R.C. Pleus, P. Sayre, V. Stone, K.M. Sullivan, J.
EP
Tentschert, P. Wallis, A.E. Nel, Nanomaterial categorization for assessing risk potential to facilitate regulatory decision-making, ACS Nano 9 (2015) 3409-3417.
AC C
42. A. Nel, T. Xia, H. Meng, X. Wang, S. Lin, Z. Ji, H. Zhang, Nanomaterial toxicity testing in the 21st century: use of a predictive toxicological approach and high-throughput screening, Acc Chem Res. 46 (2013) 607-621.
43. F. Meng, V.M. Ugaz, Instantaneous physico-chemical analysis of suspension-based nanomaterials, Sci Rep. 5 (2015) 9896. 44. D.G. Thomas, S. Gaheen, S.L. Harper, M. Fritts, F. Klaessig, E. Hahn-Dantona, D. Paik, S. Pan, G.A. Stafford, E.T. Freund, J.D. Klemm, N.A. Baker, ISA-TAB-Nano: a 22
ACCEPTED MANUSCRIPT
specification for sharing nanomaterial research data in spreadsheet-based format, BMC Biotechnol. 13 (2013) 2. 45. J. Hastings, N. Jeliazkova, G. Owen, G. Tsiliki, C.R. Munteanu, C. Steinbeck, E.
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Willighagen, eNanoMapper: harnessing ontologies to enable data integration for nanomaterial risk assessment, J Biomed Semantics. 6 (2015) 10.
nanostructures, Small 5 (2009) 426-431.
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46. D.J. Gentleman, W.C. Chan, A systematic nomenclature for codifying engineered
47. D.A. Tomalia, In quest of a systematic framework for unifying and defining nanoscience,
M AN U
J Nanopart Res. 11 (2009) 1251-1310.
48. E.K. Richman, J.E. Hutchison, The nanomaterial characterization bottleneck, ACS Nano
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EP
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3 (2009) 2441-2446.
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Figure legend
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Figure 1. Predicting responses to nanomaterials: schematic illustration of the relationship between the synthetic ‘identity’, the biological ‘identity’, and the physiological or toxicological response to nanomaterials in a living system. Reprinted from Walkey CD, Chan WC.
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Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chem Soc Rev. 2012;41(7):2780-99 with permission from The Royal Society of
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Chemistry.
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ACCEPTED MANUSCRIPT Highlights More coherence is needed in the way that nanotoxicology is conducted.
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Thorough physicochemical characterization is necessary in nanotoxicology.
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The biological ‘identity’ of nanomaterials in a living system is also important.
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Material characterization should be fit-for-purpose, and relevant for the study.
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Categorization of nanomaterials based on functionality may support regulation.
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S0006-291X(15)30207-2
DOI:
10.1016/j.bbrc.2015.06.178
Reference:
YBBRC 34267
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 4 June 2015 Accepted Date: 23 June 2015
Please cite this article as: B. Fadeel, A. Fornara, M.S. Toprak, K. Bhattacharya, Keeping it real: the importance of material characterization in nanotoxicology, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.06.178. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Keeping it real: the importance of material characterization in nanotoxicology
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet,
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Bengt Fadeel1,*, Andrea Fornara2, Muhammet S. Toprak3, and Kunal Bhattacharya1
171 77 Stockholm, Sweden;
Unit for Chemistry, Materials and Surfaces, SP Technical Research Institute of Sweden, 114 86
Stockholm, Sweden; 3
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2
Functional Materials Division, Department of Materials and Nano Physics, Royal Institute of
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Technology, 164 40 Stockholm, Sweden.
*Corresponding author: Institute of Environmental Medicine, Nobels väg 13, Karolinska
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[email protected]
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Institutet, 171 77 Stockholm, Sweden; Tel. +46 8 524 877 37; Fax: +46 8 34 38 49; E-mail:
Running title: Nanomaterial characterization for safety assessment.
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Abstract Nanomaterials are small and the small size and corresponding large surface area of nanomaterials confers specific properties, making these materials desirable for various applications, not least in
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medicine. However, it is pertinent to ask whether size is the only property that matters for the desirable or detrimental effects of nanomaterials? Indeed, it is important to know not only what the material looks like, but also what it is made of, as well as how the material interacts with its
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biological surroundings. It has been suggested that guidelines should be implemented on the types of information required in terms of physicochemical characterization of nanomaterials for
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toxicological studies in order to improve the quality and relevance of the published results. This is certainly a key issue, but it is important to keep in mind that material characterization should be fit-for-purpose, that is, the information gathered should be relevant for the end-points being
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studied.
Key words: engineered nanomaterials; nanotoxicology; physicochemical characterization; bio-
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corona.
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“When things are large, they are what they are. When they are small, it’s a different game: they
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are what our measurements make them.”
1.
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George M. Whitesides, No Small Matter. Science on the Nanoscale. [2009]
Introduction
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Engineered nanomaterials have become the focus of extensive research in many areas including biomedical applications due to the novel and unique properties arising at the nanoscale. In addition, during the past decade, there has been an exponential increase in the number of papers on the toxicological effects of nanomaterials. However, while this certainly shows that the potential risks of nanomaterials are being considered, it has been argued that numerous poorly
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controlled studies have been published, offering little insight into any ‘nanospecific’ effects (1). Indeed, Krug concluded in a recent overview of the field that while 10.000 papers have been produced on environmental and health effects of nanomaterials in the last 15 years, we are left
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with “a plethora of low-value results” due to the lack of harmonized experimental protocols, poor
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or nonexistent characterization of the nanomaterials, a lack of reference materials, the frequent reliance on unrealistically high doses both for in vitro and in vivo studies, and so on (1). Others have also complained, on the basis of a meta-analysis of several dozen papers focused on silica particles, that “after over a decade of research, answers for the most basic questions are still lacking” and suggested that more coherence in the experimental methods and materials used is needed (2).
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Ten years have passed since the review by Oberdörster et al. defining and outlining the emerging discipline of nanotoxicology (3); in their review, which is now a ‘citation classic’, the authors defined nanotoxicology as a “science of engineered nanodevices and nanostructures that deals
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with their effects in living organisms” and they pointed out that nanotoxicology research also will advance the field of nanomedicine by providing information on the undesirable properties of nanomaterials and means to avoid them. Indeed, this is sometimes referred to as ‘safe-by-design’
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(4).
In another very pertinent review, also published in 2005, Oberdörster et al. summarized the views of an international expert group convened to develop a screening strategy for the hazard identification of engineered nanomaterials (5). Hence, the authors stated that “there is a strong
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likelihood that biological activity of nanoparticles will depend on physicochemical parameters not routinely considered in toxicity screening [of chemicals]” and put forward a list of physicochemical properties that may be important in understanding the toxicity of nanomaterials
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namely: particle size and size distribution, agglomeration state, particle shape, crystal structure, chemical composition, surface area, surface chemistry, surface charge, and porosity (5). Similar
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suggestions for minimal material characterization requirements in nanotoxicology have been proposed in recent years, as we shall discuss in the present essay. The question is: are we ready to adopt such requirements as an international standard(s)? Indeed, can we afford not to do so? Furthermore, are there any examples of ‘good’ nanotoxicological studies, or has all been for naught?
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2. International harmonization efforts in nanotoxicology Warheit asked in an editorial several years ago “how meaningful are the results of nanotoxicity
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studies in the absence of adequate material characterization?” (6). He also noted that while most nanotoxicological studies are conducted under in vitro conditions (i.e., in the wet phase), the physicochemical characterization is frequently carried out on the “just-received” nanomaterials in the dry phase, which has limited relevance for the test conditions. He also professed a list of
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minimal characterization requirements prior to conducting hazard assessment studies, similar to
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the one cited above. In a follow-up, Sayes and Warheit discussed three phases of material characterization (7). Primary characterization is performed on particles as-synthesized or asreceived, in its dry state or powder form. Secondary characterization, on the other hand, is performed on particles in the wet phase as a solution or suspension in aqueous media, eg. in water or cell culture medium. Finally, tertiary characterizations are performed on particles following
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interactions with biological systems in vitro or in vivo, and may include characterization of particles in blood, or lung fluid. The tertiary characterization of particles in the actual test system is certainly non-trivial, but it is the most relevant for the interpretation of the toxicological data
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(7). The authors offered a list of physicochemical properties relevant to nanotoxicological testing,
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and they concluded that “no single technique can accurately describe a specific property of a material” (7). Thus, all material characterization should be performed using more than one method.
The journal Nature Nanotechnology recently invited the nanotoxicology community to ‘join the dialogue’ on whether guidelines should be implemented on the types of information that are
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required to improve the quality and relevance of the published papers (8). It was further stated in the editorial that nanomaterial characterization “should be done based on relevance to the study” (8). Indeed, different types of information (and consequently, the use of different methods) may
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be needed depending on the purpose of the study (9). Hence, nanomaterial characterization should be fit-for-purpose. Furthermore, the methods that are used for characterization need to be standardized and validated. Fubini et al. emphasized that while there are obvious properties that
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should be assessed before any in vivo or in vitro testing is conducted, the “choice of characteristics to be measured more accurately should be tailored to the end-point investigated in
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that particular study” (10). Others have highlighted that, “for regulatory purposes, the standards applied and data generation required must be more prescriptive, whereas for research, these must be primarily based on the hypothesis to be tested” (11). Furthermore, while it would be laudable to characterize every aspect of a test material, both at synthesis and in the test system, this is
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certainly impractical (5) and it would therefore be advisable for the scientific community to agree on a number of common parameters that should be measured for all studies, in order to describe what the material looks like, what it is made of, and what factors govern how it interacts with its
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study (13).
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biological surroundings (12), while other characteristics should be based on relevance to the
The Seventh Framework Programme of the European Commission started in 2007 and projects funded in the final round will run until 2017. Among these projects, 50 have focused on nanosafety. The NanoImpactNet project played an important role in the integration of nanosafety research in the early phase of FP7 through the organization of workshops and conferences. In one such workshop, experts provided
recommendations for minimal
characterization of
nanomaterials, and a distinction was made between ‘essential metrics’, including size and size 6
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distribution, chemical composition and surface charge, and ‘often important metrics’, including shape and solubility (14). The need for methods to determine interactions of nanomaterials with the surrounding biological matrix was also highlighted. The research infrastructure project
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QualityNano has emphasized the importance of standardization and harmonization of procedures in all aspects of nanosafety assessment and also highlighted the need for in situ characterization (15). The NANOREG project, with close to 70 partner institutes and a total of 50 million Euro in
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funding (provided by the European Commission and the participating member states), is designed to facilitate a common approach to regulatory testing of nanomaterials and aims to develop a
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regulatory framework for nanomaterials, in close cooperation with international organisations involved in standardization and regulation of nanomaterials, such as ECHA, OECD, CEN and ISO. In the NANOREG project, several characterization protocols based on OECD guidelines for analysis of physicochemical properties of nanomaterials are being verified and validated,
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including quantitative analysis of surface coatings, and size distribution analysis in the dry state or powder form as well as in the wet state, i.e., in liquids for compliance with the EU definition
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(16).
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The FP7 project ITS-NANO (for ‘intelligent testing strategy’) developed a framework of future research priorities in cooperation with all the major stakeholders (i.e., government, industry, academia, funding agencies and NGOs), and emphasized the importance of physicochemical characterization, along with exposure identification, hazard identification and modelling approaches (17). In the large FP7 project, MARINA (for ‘managing risks of nanomaterials’), first steps are taken towards developing an intelligent testing strategy for nanomaterials. Notably, in the project, a common panel of representative nanomaterials from the Joint Research Centre
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(JRC) nanomaterial repository are being applied. Each type of material in the repository has been sourced as a large single batch which has been sub-sampled into individual vials to produce a collection of thoroughly characterized nanomaterials available for benchmarking in research and
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regulatory studies. In a recent study, 6 metal oxide nanomaterials were evaluated using 10 different toxicity assays in 9 different laboratories using 12 cellular models representing 6 different target organs (18). The nanomaterials were all subjected to detailed physicochemical
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characterization. With this approach, a hazard ranking of the metal oxides could be established and cell-specific responses were noted. The NANOREG project also uses nanomaterials from the
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JRC repository. In another recent study, US researchers belonging to the ‘engineered nanomaterials grand opportunity’ (Nano GO) consortium conducted so-called round robin or interlaboratory comparisons of a total of 7 metal oxides and multi-walled carbon nanotubes using 3 different cell lines (19). These and other studies (20) point to the importance of conducting
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studies with multiple relevant cell types in order to perform accurate in vitro evaluations of nanomaterials. Moreover, applying extensively characterized nanomaterials for benchmarking will allow for comparisons across studies and may prevent the generation of “low-value” results
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(1).
For a discussion of material characterization in the context of nanomedicines, see the excellent papers by McNeil and co-workers from the US Nanomaterial Characterization Laboratory, NCL (21,22).
3. The issue of dose and dosimetry in nanotoxicology 8
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The purposeful characterization of nanomaterials is thus of paramount importance in nanotoxicology, but so is dosimetry, i.e., the accurate measurement of the dose, or the amount of the nanomaterial which comes into contact with the biological target. Several different metrics
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have been proposed in for in vitro nanotoxicology, including µg/ml, cm2/mL, µg/cm2, or particle number/mL (23). However, defining the biologically effective dose is not a trivial issue, as highlighted in a recent editorial in Particle & Fibre Toxicology (24). In nanotoxicology, the dose
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is commonly defined as the nominal dose, i.e., the amount of nanomaterial introduced into the culture medium, but as pointed out, it remains possible to measure both the deposited, cell-
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associated or intracellular doses (24). Lison et al. concluded that information about the mechanism of toxicity, obtained experimentally or deduced by analogy with similar nanomaterials, “is essential to guide investigators when selecting the most relevant dose to characterize dose-effect/response relationships” (24). But a priori knowledge of the mechanism
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is not always, or very seldom, available, and we are not yet able to perform grouping of nanomaterials, although attempts are being made (see below). In order to better define the dose that is actually delivered to cells (i.e., the “relevant” dose), computational models have been
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developed in recent years that take into account in vitro particokinetics (sedimentation and diffusion) for individual particles and their agglomerates (25). With this approach, it is possible
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to calculate the total mass, surface area, or particle number delivered to cells as a function of exposure time in an in vitro system (26). However, while the latter approach may be suited for spherical nanoparticles, the methodology needs to be validated also for high aspect ratio materials such as carbon nanotubes. In a recent study, the authors showed that the in vitro dosimetry has implications for hazard ranking of nanomaterials, using THP.1 cells as a model (27). For carbonbased nanomaterials, the deposited doses varied from 20-50% of the administered doses at 24 h, whereas for the metal/metal oxides with larger effective densities of their formed agglomerates, 9
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the deposited doses were much closer to the administered doses. Overall, discrepancies between administered and delivered doses were more evident during the first few hours. Interestingly, the slope of the dose-response relationship for cell viability and cytokine production changed for
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some, but not all nanomaterials, when delivered dose was taken into account (27). This may have implications for the toxicity screening of different nanomaterials. Furthermore, and closely related to the issue of the (relevant) dose, is the matter of nanoparticle dispersion protocols,
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which will influence particle agglomeration, which in turn will affect sedimentation and diffusion in a cell culture system. There is probably no one-size-fits-all recipe for nanomaterial dispersions
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and various approaches are being applied in nanotoxicology. However, reliable methods with which to characterize nanomaterial dispersions are needed. Pal et al. provided one example using so-called tunable resistive pulse sensing (TRPS) technology and found that this technology yielded higher resolution and sensitivity when compared to conventional dynamic light scattering
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(DLS), which is currently the preferred technique (28). Unlike DLS, the TRPS technology does not rely on light scattering properties. Instead, it monitors changes in ionic current as individual nanoparticles or agglomerates pass through an elastomeric membrane; the TRPS technology may
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(28).
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complement DLS measurements for nanomaterial and nanomaterial-biomolecule characterization
The synthetic and biological identities
It follows from the previous discussion that nanomaterial characterization is a crucial element of nanotoxicology. It is also clear that there are numerous material physicochemical properties that one could characterize, although there seems to be an emerging consensus regarding the most essential ones. However, as we have discussed in a previous review, understanding which of the
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physicochemical properties that are driving toxicity remains a key challenge; if one could connect material properties (such as, composition, size, shape, surface charge, porosity, colloidal stability, purity/degree of contamination, and so on) with toxicity, then this might enable the prediction of
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potential hazards and support safe-by-design approaches to produce nanomaterials with minimal toxicity (29).
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The intrinsic physicochemical properties may be viewed as the ‘synthetic identity’ of the nanomaterial. However, there is an emerging realization in recent years that nanomaterials are not
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‘naked’ following their introduction into a biological system, and, moreover, that “the organic and synthetic worlds merge into a new science” at the interface between nanomaterials and biological systems (30). In fact, as we and others have discussed previously, the ‘biological identity’ is shaped, in part, by the adsorption of biomolecules including proteins and lipids, or
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both, onto the surface of the nanoparticles (29,31), and nanomaterials may therefore be viewed as – and should perhaps also be regulated as – ‘biological entities’ (32). The composition of the biocorona depends on the portal of entry into the body and on the particular biofluid (eg., blood,
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lung fluid, gastro-intestinal fluid, etc) that the nanomaterials encounter and may exhibit dynamic changes as the nanoparticle crosses from one biological compartment to another (33). In a recent,
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comprehensive study, Tenzer et al. reported that plasma protein adsorption occurs very rapidly on the surface of nanoparticles and that this could significantly affect biological outcomes relevant for the hematocompatibility of the particles, including lysis of red blood cells, and thrombocyte activation (34). Notably, the authors found that none of the physicochemical properties alone could account for the formation, composition and evolution of the protein corona (34). Nonetheless, as explained by Walkey and Chan (35), “once fully mapped, the relationships between synthetic identity, biological identity, and physiological response will enable researchers 11
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to predict the physiological response of a nanomaterial by characterizing its synthetic identity” (and see Figure 1). Thus, when nanomaterial characterization is performed, attention should also be devoted to the biological surroundings, meaning that nanomaterials should be characterized
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not only in their pristine state, but also in the relevant biological system or fluid (i.e., wet state), and that the adsorbed biomolecules also need to be taken into account. At present, most biocorona studies have focused on the identification of adsorbed proteins (in some cases, also on the
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adsorption of lipids and other biomolecules), without specifically connecting individual proteins in the corona to biological outcomes. However, there are some emerging and illustrative
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examples. Hence, Walkey et al. conducted so-called serum protein corona 'fingerprinting' of a library of 105 surface-modified gold nanoparticles and found that the corona fingerprint predicts cellular uptake more accurately than a model that uses parameters describing nanoparticle size, aggregation state, and surface charge, with A549 human lung epithelial carcinoma cells as a
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model (36). Interestingly, the study also revealed that the core material (gold or silver) exerted a greater influence on protein corona composition than core size or surface functional group. As the authors pointed out that, even if the ligand-protected core does not make direct contact with
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proteins in the biological environment, it determines the density, arrangement, and orientation of the associated ligands (36). Thus, the synthetic and biological identities of nanoparticles are
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connected. However, it remains to be understood whether these observations can be generalized for other classes of nanomaterials. The above dataset has recently been explored further using both linear and non-linear quantitative structure-activity relationship (QSAR) analysis and these studies revealed a small set of serum proteins, including apolipoprotein B-100 (ApoB-100), along with the nanomaterial zeta potential (as synthesized), as being significant descriptors correlating with cell association/cell uptake (37). In another recent study, Ritz et al. first determined the corona composition on a set of polymeric nanoparticles with different surface functionalization, 12
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then correlated the relative abundance of identified proteins with cellular uptake, and, finally, verified the role of candidate proteins by decorating nanoparticles with specific proteins (38). By this approach, they could demonstrate that the apolipoproteins ApoA4 and ApoC3 significantly
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decreased cellular uptake, while ApoH (also known as β2 glycoprotein-I) increased uptake by human mesenchymal stem cells (38). In synopsis, understanding the nano-bio interface is important and methods to investigate the nano-bio interface – qualitatively and quantitivately –
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are needed.
5. Naming and categorizing nanomaterials
The so-called ‘grouping of substances’ or category approach has been recognized as an important means to avoid unnecessary testing of new chemicals (39). For chemicals in general, technical
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guidance documents on grouping are available, from the Organisation for Economic Cooperation and Development (OECD) or the European Chemicals Agency (ECHA). For nanomaterials, specific guidance is not yet available. However, members of the European Centre for
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Ecotoxicology and Toxicology of Chemicals (ECETOC) recently reviewed available concepts for the grouping of nanomaterials for human health risk assessment (39). The authors argued in favor
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of a comprehensive, ‘multiple perspective’ framework that would take into account the different stages of the life cycle of the nanomaterial or nanomaterial-enabled product. In a more recent installment, a decision-making framework for grouping of nanomaterials was proposed (40). The authors noted that most nanomaterials will not be accurately grouped based solely on their intrinsic material properties as it is not yet understood how intrinsic material properties relate to toxic effects (40). Therefore, the ECETOC proposal follows a functionality-driven approach
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instead of being predominantly based upon intrinsic nanomaterial properties. In a recent report on a workshop convened by the University of California Center for Environmental Implications of Nanotechnology (UC-CEIN) and the UCLA Center for Nanobiology and Predictive Toxicology,
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Godwin et al. discussed how categorization of nanomaterials coupled with alternative (nonanimal based) testing strategies might be used to expedite hazard characterization, allowing for integrated environmental and occupational health and safety decision-making (41). The authors
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suggested that grouping of nanomaterials could be helpful during the early stages of risk assessment to identify ‘nanomaterials of concern’, which could then be selected for more detailed
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testing. They also concluded that physicochemical properties alone are not currently sufficient for nanomaterial categorization and that categorization methods for regulatory purposes should include indicators of both hazard and exposure (41), in line with the approach advocated by
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ECETOC.
In order to categorize nanomaterials, one first needs to study (many) nanomaterials. To this end,
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high-throughput screening of libraries of well-defined nanomaterials with systematic variation of different physicochemical properties is a useful tool, to help establish structure-activity
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relationships (42). This also highlights the need for high-throughput characterization methods, especially those methods that could be applied for so-called secondary phase characterization, i.e., characterization performed on particles in the wet phase as a dispersion in an aqueous medium relevant to the test system (7). In a recent publication, Meng and Ugaz demonstrated a novel, real-time screening approach in order to perform instantaneous physicochemical analysis of suspension-based nanomaterials (43). The authors exploited surface complexation interactions that emerge when a micron-scale chemical discontinuity is established between suspended 14
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nanoparticles and a fluorescent molecular tracer in a laminar flow environment that removes limitations associated with convective transport and mixing (43). The resulting fluorescence signature is detected and can be used to extract information – via image processing and a
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developed algorithm – about composition, quantity, size, and morphology of nanoparticles in suspension, independent of their agglomeration state. The authors successfully demonstrated the use of this approach for continuous sizing of ZnO nanoparticles, and for quantification of the
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anatase and rutile composition of TiO2 nanoparticle mixtures (43). The method is highly specific for surface complexation of different tracers with the metal ions in the nanoparticles, and the
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model is based on the number of available surface binding sites in the suspended nanoparticles. However, this method could perhaps be applied to other nanoparticle systems, once a good tracer
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is identified.
Naming supports categorization (of nanomaterials). To this end, members of the nanotechnology working group of the US National Institutes of Health (NIH) National Cancer Informatics
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Program have developed ‘ISA-TAB-Nano’, a general framework for representing and integrating data pertaining to the description and characterization of nanomaterials using spreadsheet files
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(44). The EU-funded FP7-eNANOMAPPER project aims to develop a computational infrastructure for data management of toxicological data to enable a more effective approach to research in nanotoxicology, and the first steps towards an integrated ontology were recently reported (45). Other attempts to codify nanomaterials as a function of their physicochemical properties have been presented (46). Of course, any nomenclature system based on characterization of nanomaterial properties presupposes that the characterization can be executed in an unambiguous manner across different laboratories. The most ambitious proposal by far is 15
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the nanomaterials classification roadmap developed by Tomalia, which points towards a periodic table(s) of nanomaterials with the aim of predicting and defining risk-benefit boundaries in the nanotechnology field (47). The biological identity of nanomaterials might add one more
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dimension to the periodic table(s), although it could be argued that the formation and composition of a bio-corona will depend on the intrinsic physicochemical properties of the nanomaterials (see
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above).
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6. Conclusions
The ‘nanomaterial characterization bottleneck’ is a concern not only in basic research, but also has significant implications for the commercialization of nano-enabled products, and for their regulation (48). In the present essay, we highlighted the importance of thorough material characterization in order to improve the quality and relevance of nanotoxicological studies. We
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emphasized that physicochemical characterization needs to be fit-for-purpose and of relevance for the hypothesis that is being addressed, although there should also be minimal characterization requirements in order to understand what the material looks like, and what it is made of (12).
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International guidelines for (minimal) material characterization are needed not least for
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regulatory purposes, and the methods used for characterization need to be standardized and validated.
We also discussed the so-called synthetic ‘identity’ of nanomaterials which is defined by the material-intrinsic properties versus the biological ‘identity’ which is manifested in a living system and can be viewed as the sum of the context-dependent properties of the nanomaterial.
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Both identities are connected and should be taken into consideration in nanotoxicological
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investigations.
7. Acknowledgements
We gratefully acknowledge the financial support of the European Commission (FP7-NANOREG,
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Grant No. 310584; FP7-eNANOMAPPER, Grant No. 604134; COST Action MODENA, TD1204) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial
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Planning.
References
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1. H.F. Krug, Nanosafety research--are we on the right track? Angew Chem Int Ed Engl. 53 (2014) 12304-12319.
EP
2. F. Schrurs, D. Lison, Focusing the research efforts, Nat Nanotechnol. 7 (2012) 546-548. 3. G. Oberdörster, E. Oberdörster, J. Oberdörster, Nanotoxicology: an emerging discipline
AC C
evolving from studies of ultrafine particles, Environ Health Perspect. 113 (2005) 823-839. 4. B. Fadeel, Nanosafety: towards safer design of nanomedicines, J Intern Med. 274 (2013) 578-580.
5. G. Oberdörster, A. Maynard, K. Donaldson, V. Castranova, J. Fitzpatrick, K. Ausman, J. Carter, B. Karn, W. Kreyling, D. Lai, S. Olin, N. Monteiro-Riviere, D. Warheit, H. Yang, Principles for characterizing the potential human health effects from exposure to nanomaterials: elements of a screening strategy, Part Fibre Toxicol. 2 (2005) 8. 17
ACCEPTED MANUSCRIPT
6. D.B. Warheit, How meaningful are the results of nanotoxicity studies in the absence of adequate material characterization? Toxicol Sci. 101 (2008) 183-185. 7. C.M. Sayes, D.B. Warheit, Characterization of nanomaterials for toxicity assessment,
8. Editorial, Join the dialogue, Nat Nanotechnol. 7 (2012) 545.
RI PT
Wiley Interdiscip Rev Nanomed Nanobiotechnol. 1 (2009) 660-670.
9. B. Fadeel, K. Savolainen, Broaden the discussion, Nat Nanotechnol. 8 (2013) 71.
SC
10. B. Fubini, M. Ghiazza, I. Fenoglio, Physico-chemical features of engineered nanoparticles relevant to their toxicity, Nanotoxicology 4 (2010) 347-363.
M AN U
11. J. Lead, S. Holgate, Regulatory and research needs, Nat Nanotechnol. 8 (2013) 72. 12. D.R. Boverhof, R.M. David, Nanomaterial characterization: considerations and needs for hazard assessment and safety evaluation, Anal Bioanal Chem. 396 (2010) 953-961. 13. KA. Dawson, Leave the policing to others, Nat Nanotechnol. 8 (2013) 73.
TE D
14. H. Bouwmeester, I. Lynch, H.J. Marvin, K.A. Dawson, M. Berges, D. Braguer, H.J. Byrne, A. Casey, G. Chambers, M.J. Clift, G. Elia, T.F. Fernandes, L.B. Fjellsbø, P. Hatto, L. Juillerat, C. Klein, W.G. Kreyling, C. Nickel, M. Riediker, V. Stone, Minimal
EP
analytical characterization of engineered nanomaterials needed for hazard assessment in biological matrices, Nanotoxicology 5 (2011) 1-11.
AC C
15. K.A. Dawson, S. Anguissola, I. Lynch, The need for in situ characterisation in nanosafety assessment: funded transnational access via the QNano research infrastructure, Nanotoxicology 7 (2013) 346-349.
16. E.A. Bleeker, W.H. de Jong, R.E. Geertsma, M. Groenewold, E.H. Heugens, M. KoersJacquemijns, D. van de Meent, J.R. Popma, A.G. Rietveld, S.W. Wijnhoven, F.R. Cassee, A.G. Oomen, Considerations on the EU definition of a nanomaterial: science to support policy making, Regul Toxicol Pharmacol. 65 (2013) 119-125. 18
ACCEPTED MANUSCRIPT
17. V. Stone, S. Pozzi-Mucelli, L. Tran, K. Aschberger, S. Sabella, U. Vogel, C. Poland, D. Balharry, T. Fernandes, S. Gottardo, S. Hankin, M.G. Hartl, N. Hartmann, D. Hristozov, K. Hund-Rinke, H. Johnston, A. Marcomini, O. Panzer, D. Roncato, A.T. Saber, H.
RI PT
Wallin, J.J. Scott-Fordsmand, ITS-NANO--prioritising nanosafety research to develop a stakeholder driven intelligent testing strategy, Part Fibre Toxicol. 11 (2014) 9.
18. L. Farcal, F.T. Andón, L. Di Cristo, B.M. Rotoli, O. Bussolati, E. Bergamaschi, A. Mech,
SC
N.B. Hartmann, K. Rasmussen, J. Riego-Sintes, J. Ponti, A. Kinsner-Ovaskainen, F. Rossi, A. Oomen, P. Bos, R. Chen, R. Bai, C. Chen, L. Rocks, N. Fulton, B. Ross, G.
M AN U
Hutchison, L. Tran, S. Mues, R. Ossig, J. Schnekenburger, L. Campagnolo, L. Vecchione, A. Pietroiusti, B. Fadeel, Comprehensive in vitro toxicity testing of a panel of representative oxide nanomaterials: first steps towards an intelligent testing strategy, PLoS One 10 (2015) e0127174.
TE D
19. T. Xia, R.F. Hamilton, J.C. Bonner, E.D. Crandall, A. Elder, F. Fazlollahi, T.A. Girtsman, K. Kim, S. Mitra, S.A. Ntim, G. Orr, M. Tagmount, A.J. Taylor, D. Telesca, A. Tolic, C.D. Vulpe, A.J. Walker, X. Wang, F.A. Witzmann, N. Wu, Y. Xie, J.I. Zink, A. Nel, A.
EP
Holian, Interlaboratory evaluation of in vitro cytotoxicity and inflammatory responses to engineered nanomaterials: the NIEHS Nano GO Consortium, Environ Health Perspect.
AC C
121 (2013) 683-690.
20. A. Kroll, C. Dierker, C. Rommel, D. Hahn, W. Wohlleben, C. Schulze-Isfort, C. Göbbert, M. Voetz, F. Hardinghaus, J. Schnekenburger, Cytotoxicity screening of 23 engineered nanomaterials using a test matrix of ten cell lines and three different assays, Part Fibre
Toxicol. 8 (2011) 9. 21. J.B. Hall, M.A. Dobrovolskaia, A.K. Patri, S.E. McNeil, Characterization of nanoparticles for therapeutics, Nanomedicine (Lond). 2 (2007) 789-803. 19
ACCEPTED MANUSCRIPT
22. P.P. Adiseshaiah, J.B. Hall, S.E. McNeil, Nanomaterial standards for efficacy and toxicity assessment, Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2 (2010) 99-112.
RI PT
23. M. Hull, A.J. Kennedy, C. Detzel, P. Vikesland, M.A. Chappell, Moving beyond mass: the unmet need to consider dose metrics in environmental nanotoxicology studies, Environ Sci Technol. 46 (2012) 10881-10882.
24. D. Lison, G. Vietti, S. van den Brule, Paracelsus in nanotoxicology, Part Fibre Toxicol.
SC
11 (2014) 35.
25. P.M. Hinderliter, K.R. Minard, G. Orr, W.B. Chrisler, B.D. Thrall, J.G. Pounds, J.G.
M AN U
Teeguarden, ISDD: A computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies, Part Fibre Toxicol. 7 (2010) 36. 26. J.M. Cohen, J.G. Teeguarden, P. Demokritou, An integrated approach for the in vitro dosimetry of engineered nanomaterials, Part Fibre Toxicol. 11 (2014) 20.
TE D
27. A.K. Pal, D. Bello, J. Cohen, P. Demokritou, Implications of in vitro dosimetry on toxicological ranking of low aspect ratio engineered nanomaterials, Nanotoxicology 12 (2015) 1-15. [Epub ahead of print].
EP
28. A.K. Pal, I. Aalaei, S. Gadde, P. Gaines, D. Schmidt, P. Demokritou, D. Bello, High resolution characterization of engineered nanomaterial dispersions in complex media
AC C
using tunable resistive pulse sensing technology, ACS Nano 8 (2014) 9003-9015.
29. B. Fadeel, N. Feliu, C. Vogt, A.M. Abdelmonem, W.J. Parak, Bridge over troubled waters: understanding the synthetic and biological identities of engineered nanomaterials,
Wiley Interdiscip Rev Nanomed Nanobiotechnol, 5 (2013) 111-129.
20
ACCEPTED MANUSCRIPT
30. A.E. Nel, L. Mädler, D. Velegol, T. Xia, E.M. Hoek, P. Somasundaran, F. Klaessig, V. Castranova, M. Thompson, Understanding biophysicochemical interactions at the nanobio interface, Nat Mater. 8 (2009) 543-557.
RI PT
31. M.P. Monopoli, C. Åberg, A. Salvati, K.A. Dawson, Biomolecular coronas provide the biological identity of nanosized materials, Nat Nanotechnol. 7 (2012) 779-786.
32. I. Lynch, A. Ahluwalia, D. Boraschi, H.J. Byrne, B. Fadeel, P. Gehr, A.C. Gutleb, M.
SC
Kendall, M.G. Papadopoulos, The bio-nano-interface in predicting nanoparticle fate and behaviour in living organisms: towards grouping and categorising nanomaterials and
M AN U
ensuring nanosafety by design, BioNanoMaterials 14 (2013) 195-216. 33. A. Pietroiusti, L. Campagnolo, B. Fadeel, Interactions of engineered nanoparticles with organs protected by internal biological barriers, Small 9 (2013) 1557-1572. 34. S. Tenzer, D. Docter, J. Kuharev, A. Musyanovych, V. Fetz, R. Hecht, F. Schlenk, D.
TE D
Fischer, K. Kiouptsi, C. Reinhardt, K. Landfester, H. Schild, M. Maskos, S.K. Knauer, R.H. Stauber, Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology, Nat Nanotechnol. 8 (2013) 772-781.
EP
35. C.D. Walkey, W.C. Chan, Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment, Chem Soc Rev. 41 (2012) 2780-2799.
AC C
36. C.D. Walkey, J.B. Olsen, F. Song, R. Liu, H. Guo, D.W. Olsen, Y. Cohen, A. Emili, W.C. Chan, Protein corona fingerprinting predicts the cellular interaction of gold and silver nanoparticles, ACS Nano 8 (2014) 2439-2455.
37. R. Liu, W. Jiang, C.D. Walkey, W.C. Chan, Y. Cohen, Prediction of nanoparticles-cell association based on corona proteins and physicochemical properties, Nanoscale 7 (2015) 9664-9675.
21
ACCEPTED MANUSCRIPT
38. S. Ritz, S. Schöttler, N. Kotman, G. Baier, A. Musyanovych, J. Kuharev, K. Landfester, H. Schild, O. Jahn, S. Tenzer, V. Mailänder, Protein corona of nanoparticles: distinct proteins regulate the cellular uptake, Biomacromolecules 16 (2015) 1311-1321.
RI PT
39. J.H. Arts, M. Hadi, A.M. Keene, R. Kreiling, D. Lyon, M. Maier, K. Michel, T. Petry, U.G. Sauer, D. Warheit, K. Wiench, R. Landsiedel, A critical appraisal of existing concepts for the grouping of nanomaterials, Regul Toxicol Pharmacol. 70 (2014) 492-
SC
506.
40. J.H. Arts, M. Hadi, M.A Irfan, A.M. Keene, R. Kreiling, D. Lyon, M. Maier, K. Michel,
M AN U
T. Petry, U.G. Sauer, D. Warheit, K. Wiench, W. Wohlleben, R. Landsiedel, A decisionmaking framework for the grouping and testing of nanomaterials (DF4nanoGrouping), Regul Toxicol Pharmacol. (2015). [Epub ahead of print].
41. H. Godwin, C. Nameth, D. Avery, L.L. Bergeson, D. Bernard, E. Beryt, W. Boyes, S.
TE D
Brown, A.J. Clippinger, Y. Cohen, M. Doa, C.O. Hendren, P. Holden, K. Houck, A.B. Kane, F. Klaessig, T. Kodas, R. Landsiedel, I. Lynch, T. Malloy, M.B. Miller, J. Muller, G. Oberdorster, E.J. Petersen, R.C. Pleus, P. Sayre, V. Stone, K.M. Sullivan, J.
EP
Tentschert, P. Wallis, A.E. Nel, Nanomaterial categorization for assessing risk potential to facilitate regulatory decision-making, ACS Nano 9 (2015) 3409-3417.
AC C
42. A. Nel, T. Xia, H. Meng, X. Wang, S. Lin, Z. Ji, H. Zhang, Nanomaterial toxicity testing in the 21st century: use of a predictive toxicological approach and high-throughput screening, Acc Chem Res. 46 (2013) 607-621.
43. F. Meng, V.M. Ugaz, Instantaneous physico-chemical analysis of suspension-based nanomaterials, Sci Rep. 5 (2015) 9896. 44. D.G. Thomas, S. Gaheen, S.L. Harper, M. Fritts, F. Klaessig, E. Hahn-Dantona, D. Paik, S. Pan, G.A. Stafford, E.T. Freund, J.D. Klemm, N.A. Baker, ISA-TAB-Nano: a 22
ACCEPTED MANUSCRIPT
specification for sharing nanomaterial research data in spreadsheet-based format, BMC Biotechnol. 13 (2013) 2. 45. J. Hastings, N. Jeliazkova, G. Owen, G. Tsiliki, C.R. Munteanu, C. Steinbeck, E.
RI PT
Willighagen, eNanoMapper: harnessing ontologies to enable data integration for nanomaterial risk assessment, J Biomed Semantics. 6 (2015) 10.
nanostructures, Small 5 (2009) 426-431.
SC
46. D.J. Gentleman, W.C. Chan, A systematic nomenclature for codifying engineered
47. D.A. Tomalia, In quest of a systematic framework for unifying and defining nanoscience,
M AN U
J Nanopart Res. 11 (2009) 1251-1310.
48. E.K. Richman, J.E. Hutchison, The nanomaterial characterization bottleneck, ACS Nano
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EP
TE D
3 (2009) 2441-2446.
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Figure legend
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Figure 1. Predicting responses to nanomaterials: schematic illustration of the relationship between the synthetic ‘identity’, the biological ‘identity’, and the physiological or toxicological response to nanomaterials in a living system. Reprinted from Walkey CD, Chan WC.
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Understanding and controlling the interaction of nanomaterials with proteins in a physiological environment. Chem Soc Rev. 2012;41(7):2780-99 with permission from The Royal Society of
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ACCEPTED MANUSCRIPT Highlights More coherence is needed in the way that nanotoxicology is conducted.
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Thorough physicochemical characterization is necessary in nanotoxicology.
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The biological ‘identity’ of nanomaterials in a living system is also important.
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Material characterization should be fit-for-purpose, and relevant for the study.
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Categorization of nanomaterials based on functionality may support regulation.
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