Towards a New Paradigm in Nano-Genotoxicology: Facing Complexity of Nanomaterials' Cellular Interactions and Effects.

Changes in paradigm contribute to advances in research. The current paradigms for the evaluation of toxicity of chemicals refer to linear or curvilinear dose-response curves with or without threshold and to surface-dependent induction of oxidative damage for particles. The unique physicochemical properties and biological/genotoxic activity of engineered nanomaterials (NMs) require the development of a new paradigm. Because of their unusual dosimetry and their multiple interactions at NM level (agglomeration/aggregation) and at different cellular and extracellular levels, NMs are likely to have complex modes of action (multiple hits at multiple targets) leading to complex thresholded-non-thresholded dose-response curves. Understanding their cellular targets and their modes of action will contribute to the production of safe-by-design NMs. An integrative, cell-by-cell approach for genotoxic effects should be applied to tackle this emerging paradigm in nano-genotoxicology.

Reprint of "Biomonitoring of genotoxic effects for human exposure to nanomaterials: The challenge ahead".

Exposures to nanomaterials (NMs), with their specific physico-chemical characteristics, are likely to increase over the next years, as their production for industrial, consumer and medical applications is steadily rising. Therefore, there is an urgent need for the implementation of human biomonitoring studies of genotoxic effects after NM exposures in order to monitor and assure safety for workers and the general population. In this review, most commonly used biomarkers of early genetic effects were analyzed for their adequacy after NM exposures. A more in depth analysis of the ex vivo/in vitro lymphocyte MN assay was performed, although, in literature no studies are available using this assay for NM exposures. Therefore, the known factors determining the NMs tissue/cellular targets and the multiplicity of modes of action of NMs were summarized. The main pending questions are whether (1) lymphocytes are a NM target or an adequate surrogate tissue, (2) whether the buccal MN assay might be more suitable for NM exposures via inhalation or ingestion, as buccal cells might be exposed more directly. While the current state-of-the-art does not allow for drawing firm conclusions, major research gaps are identified and some cautious recommendations can be formulated. Therefore in vitro and in vivo studies should be conducted comparing methodologies side-by-side in the same subjects and for different types of NMs. The ex vivo/in vitro MN assay in its automated version, allowing objective analysis of large cohorts and detection of direct and indirect genotoxic effects, remains a valuable candidate for human biomonitoring to NM exposure. Considering the potential cancer risk from exposure to NMs and previous dramatic experiences with too late surveillance of occupational exposures to similar substances (e.g. to asbestos), there is an urgent need to define and implement adequate scientifically sound biomonitoring methods and programme for exposure to NMs.

Biomonitoring of genotoxic effects for human exposure to nanomaterials: The challenge ahead.

Exposures to nanomaterials (NMs), with their specific physico-chemical characteristics, are likely to increase over the next years, as their production for industrial, consumer and medical applications is steadily rising. Therefore, there is an urgent need for the implementation of human biomonitoring studies of genotoxic effects after NM exposures in order to monitor and assure safety for workers and the general population. In this review, most commonly used biomarkers of early genetic effects were analyzed for their adequacy after NM exposures. A more in depth analysis of the ex vivo/in vitro lymphocyte MN assay was performed, although, in literature no studies are available using this assay for NM exposures. Therefore, the known factors determining the NMs tissue/cellular targets and the multiplicity of modes of action of NMs were summarized. The main pending questions are whether (1) lymphocytes are a NM target or an adequate surrogate tissue, (2) whether the buccal MN assay might be more suitable for NM exposures via inhalation or ingestion, as buccal cells might be exposed more directly. While the current state-of-the-art does not allow for drawing firm conclusions, major research gaps are identified and some cautious recommendations can be formulated. Therefore in vitro and in vivo studies should be conducted comparing methodologies side-by-side in the same subjects and for different types of NMs. The ex vivo/in vitro MN assay in its automated version, allowing objective analysis of large cohorts and detection of direct and indirect genotoxic effects, remains a valuable candidate for human biomonitoring to NM exposure. Considering the potential cancer risk from exposure to NMs and previous dramatic experiences with too late surveillance of occupational exposures to similar substances (e.g. to asbestos), there is an urgent need to define and implement adequate scientifically sound biomonitoring methods and programme for exposure to NMs.

Tetraploid cells produced by absence of substrate adhesion during cytokinesis are limited in their proliferation and enter senescence after DNA replication.

Tetraploidy has been proposed as an intermediate state in neoplastic transformation due to the intrinsic chromosome instability of tetraploid cells. Despite the identification of p53 as a major factor in growth arrest of tetraploid cells, it is still unclear whether the p53-dependent mechanism for proliferation restriction is intrinsic to the tetraploid status or dependent on the origin of tetraploidy. Substrate adherence is fundamental for cytokinesis completion in adherent untransformed cells. Here we show that untransformed fibroblast cells undergoing mitosis in suspension produce binucleated tetraploid cells due to defective cleavage furrow constriction that leads to incomplete cell abscission. Binucleated cells obtained after loss of substrate adhesion maintain an inactive p53 status and are able to progress into G1 and S phase. However, binucleated cells arrest in G2, accumulate p53 and are not able to enter mitosis as no tetraploid metaphases were recorded after one cell cycle time. In contrast, tetraploid metaphases were found following pharmacological inhibition of Chk1 kinase, suggesting the involvement of the ATR/Chk1 pathway in the G2 arrest of binucleated cells. Interestingly, after persistence in the G2 phase of the cell cycle, a large fraction of binucleated cells become senescent. These findings identify a new pathway of proliferation restriction for tetraploid untransformed cells that seems to be specific for loss of adhesion-dependent cytokinesis failure. This involves Chk1 and p53 activation during G2. Inhibition of growth and entrance into senescence after cytokinesis in suspension may represent an important mechanism to control tumor growth. In fact, anchorage independent growth is a hallmark of cancer and it has been demonstrated that binucleated transformed cells can enter a cycle of anchorage independent growth.

Causes of genome instability: the effect of low dose chemical exposures in modern society.

Genome instability is a prerequisite for the development of cancer. It occurs when genome maintenance systems fail to safeguard the genome's integrity, whether as a consequence of inherited defects or induced via exposure to environmental agents (chemicals, biological agents and radiation). Thus, genome instability can be defined as an enhanced tendency for the genome to acquire mutations; ranging from changes to the nucleotide sequence to chromosomal gain, rearrangements or loss. This review raises the hypothesis that in addition to known human carcinogens, exposure to low dose of other chemicals present in our modern society could contribute to carcinogenesis by indirectly affecting genome stability. The selected chemicals with their mechanisms of action proposed to indirectly contribute to genome instability are: heavy metals (DNA repair, epigenetic modification, DNA damage signaling, telomere length), acrylamide (DNA repair, chromosome segregation), bisphenol A (epigenetic modification, DNA damage signaling, mitochondrial function, chromosome segregation), benomyl (chromosome segregation), quinones (epigenetic modification) and nano-sized particles (epigenetic pathways, mitochondrial function, chromosome segregation, telomere length). The purpose of this review is to describe the crucial aspects of genome instability, to outline the ways in which environmental chemicals can affect this cancer hallmark and to identify candidate chemicals for further study. The overall aim is to make scientists aware of the increasing need to unravel the underlying mechanisms via which chemicals at low doses can induce genome instability and thus promote carcinogenesis.

Amorphous silica nanoparticles alter microtubule dynamics and cell migration.

Amorphous silica nanoparticles (SiO2-NPs) have been studied for their toxic and genotoxic potential. Although contradictory data have been reported and the possible modes of action are not fully elucidated, aneugenic events have been reported, indicating the microtubule (MT) network as a potential target. To investigate this, we examined the effects of 59 nm (10 µg/ml) and 174 nm (7.5 µg/ml) SiO2-NPs on MTs in mitotic and interphase A549 human lung carcinoma cells. No gross morphological changes of the mitotic spindle or induction of multipolar spindles were observed upon SiO2-NPs treatment. The influence of SiO2-NPs on the interphase MTs network dynamics was investigated by in situ depolymerisation/repolymerisation experiments. Results showed a clear increase in MT dynamics after SiO2-NP treatment. Consistent with this, reduced levels of MT acetylation were observed. In addition, live cell microscopy demonstrated that SiO2-NP treatment reduced A549 cell motility. The SiO2-NP doses and conditions (serum-free) used in this study did not induce significant cell toxicity or MN frequencies. Therefore, the effects on MT dynamics, MT acetylation and migration observed, are direct effects of the SiO2-NPs and not a consequence of NP overload or toxic or genotoxic effects.

Co-assessment of cell cycle and micronucleus frequencies demonstrates the influence of serum on the in vitro genotoxic response to amorphous monodisperse silica nanoparticles of varying sizes.

Serum proteins have been shown to modulate the cytotoxic and genotoxic responses to nanomaterials. The aim was to investigate the influence of serum on the induction of micronuclei (MN) by nanoparticles (NPs) of different sizes. Therefore, A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles (SNPs) were used as models. Assessment of the cell viability, cell cycle changes and induction of MN by SNPs ranging from 12 to 174 nm was performed in presence or absence of serum, applying the in vitro flow cytometry-based MN assay. Here, it has been demonstrated that serum has an influence on these end points, with a lower cell viability in absence of serum compared with the presence of serum. Further, cell cycle changes, specifically, G1 and S-phase arrest, were observed in absence of serum for four out of six SNPs tested. A size-dependent MN induction was observed: larger SNPs being more active in absence of serum. In addition, the serum influence was characterised by a size-dependency for cytotoxic and genotoxic effects, with a higher influence of serum for smaller particles. The data indicate that the in vitro micronucleus assay in presence and absence of serum could be advised for hazard assessment because it demonstrates a higher sensitivity in serum-free conditions than in conditions with serum. However, this recommendation applies only if the cell line used is able to proliferate under serum-free conditions because cell division is a prerequisite for MN expression.

Oxidative stress induced by pure and iron-doped amorphous silica nanoparticles in subtoxic conditions.

Amorphous silica nanoparticles (SiO2-NPs) have found broad applications in industry and are currently intensively studied for potential uses in medical and biomedical fields. Several studies have reported cytotoxic and inflammatory responses induced by SiO2-NPs in different cell types. The present study was designed to examine the association of oxidative stress markers with SiO2-NP induced cytotoxicity in human endothelial cells. We used pure monodisperse amorphous silica nanoparticles of two sizes (16 and 60 nm; S16 and S60) and a positive control, iron-doped nanosilica (16 nm; SFe), to study the generation of hydroxyl radicals (HO·) in cellular-free conditions and oxidative stress in cellular systems. We investigated whether SiO2-NPs could influence intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) levels, increase lipid peroxidation (malondialdehyde (MDA) and 4-hydroxyalkenal (HAE) concentrations), and up-regulate heme oxygenase-1 (HO-1) mRNA expression in the studied cells. None of the particles, except SFe, produced ROS in cell-free systems. We found significant modifications for all parameters in cells treated with SFe nanoparticles. At cytotoxic doses of S16 (40-50 µg/mL), we detected weak alterations of intracellular glutathione (4 h) and a marked induction of HO-1 mRNA (6 h). Cytotoxic doses of S60 elicited similar responses. Preincubation of cells being exposed to SiO2-NPs with an antioxidant (5 mM N-acetylcysteine, NAC) significantly reduced the cytotoxic activity of S16 and SFe (when exposed up to 25 and 50 µg/mL, respectively) but did not protect cells treated with S60. Preincubation with NAC significantly reduced HO-1 mRNA expression in cells treated with SFe but did not have any effect on HO-1 mRNA level in cell exposed to S16 and S60. Our study demonstrates that the chemical composition of the silica nanoparticles is a dominant factor in inducing oxidative stress.

Influence of serum on in situ proliferation and genotoxicity in A549 human lung cells exposed to nanomaterials.

In this work in situ proliferation of A549 human lung epithelial carcinoma cells exposed to nanomaterials (NMs) was investigated in the presence or absence of 10% serum. NMs were selected based on chemical composition, size, charge and shape (Lys-SiO(2), TiO(2), ZnO, and multi walled carbon nanotubes, MWCNTs). Cells were treated with NMs and 4h later, cytochalasin-B was added. 36 h later, cell morphology was analyzed under a light microscope. Nuclearity was scored to determine the cytokinesis-block proliferation index (CBPI). CBPI, based on percentage of mono-, bi- and multi-nucleated cells, reflects cell toxicity and cell cycle delay. For some conditions depending on NM type (TiO(2) and MWCNT) and serum concentration (0%) scoring of CBPI was impossible due to overload of agglomerated NMs. Moreover, where heavy agglomeration occurs, micronuclei (MN) detection and scoring under microscope was prevented. A statistically significant decrease of CBPI was found for ZnO NM suspended in medium in the absence or presence of 10% serum at 25 µg/ml and 50 µg/ml, respectively and for Lys-SiO(2) NM at 3.5 µg/ml in 0% serum. Increase in MN frequency was observed in cells treated in 10% serum with 50 µg/ml ZnO. In 0% serum, the concentrations tested led to high toxicity. No genotoxic effects were induced by Lys-SiO(2) both in the absence or presence of serum up to 5 µg/ml. No toxicity was detected for TiO(2) and MWCNTs in both 10% and 0% serum, up to the dose of 250 µg/ml. Restoration of CBPI comparable to untreated control was shown for cells cultured without serum and treated with 5 µg/ml of Lys-SiO(2) NM pre-incubated in 100% serum. This observation confirms the protective effect of serum on Lys-SiO(2) NM cell toxicity. In conclusion in situ CBPI is proposed as a simple preliminary assay to assess both NMs induced cell toxicity and feasibility of MN scoring under microscope.

The in vitro MN assay in 2011: origin and fate, biological significance, protocols, high throughput methodologies and toxicological relevance.

Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids lagging behind in anaphase and are not included in the daughter nuclei at telophase. The mechanisms of MN formation are well understood; their possible postmitotic fate is less evident. The MN assay allows detection of both aneugens and clastogens, shows simplicity of scoring, is widely applicable in different cell types, is internationally validated, has potential for automation and is predictive for cancer. The cytokinesis-block micronucleus assay (CBMN) allows assessment of nucleoplasmic bridges, nuclear buds, cell division inhibition, necrosis and apoptosis and in combination with FISH using centromeric probes, the mechanistic origin of the MN. Therefore, the CBMN test can be considered as a "cytome" assay covering chromosome instability, mitotic dysfunction, cell proliferation and cell death. The toxicological relevance of the MN test is strong: it covers several endpoints, its sensitivity is high, its predictivity for in vivo genotoxicity requires adequate selection of cell lines, its statistical power is increased by the recently available high throughput methodologies, it might become a possible candidate for replacing in vivo testing, it allows good extrapolation for potential limits of exposure or thresholds and it is traceable in experimental in vitro and in vivo systems. Implementation of in vitro MN assays in the test battery for hazard and risk assessment of potential mutagens/carcinogens is therefore fully justified.

Methodological approaches influencing cellular uptake and cyto-(geno) toxic effects of nanoparticles.

Methods are needed to assess cytotoxicity and genotoxicity of nanoparticles (NPs). The influence of serum and the use of cytochalasin-B were assessed on the cellular uptake of amorphous silica NPs (SNPs) and their biological effects. Our observations indicate that some methodological approaches may modulate the outcome of the assay. Therefore the experimental design and choice of the assays are of great importance in nanotoxicology.

Induction of chromosome malsegregation by nanomaterials.

NMs (nanomaterials), defined as materials with at least one dimension smaller than 100 nm, are able to induce genotoxic effects. One of the hypotheses of the mode-of-action in which they exert their genotoxic potential is to mechanically interfere with subcellular structures, in particular the microtubules. In the present paper, we review studies exploring interactions between NMs and tubulin; therefore a PubMed literature search was performed. From this search 12 studies, applying both acellular and cellular assays, were retrieved and are summarized according to endpoint and particle type. These studies show that there are interactions between different types of NMs and tubulins in both acellular and cellular systems. For several types of NMs, the multi-walled carbon nanotubes, amorphous SiO(2), TiO(2) and CoCr, an induction of aneuploidy was observed in vitro. There is, therefore, a critical need to assess the capacity of NMs to interfere with the cytoskeleton, and in particular the tubulins. This might require definition of relevant dosimetry, adaptations of some testing protocols, possibly development of new methodologies and studies on a larger size-range of NMs.

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models.

We explored how to assess the genotoxic potential of nanosize particles with a well validated assay, the in vitro cytochalasin-B micronucleus assay, detecting both clastogens and aneugens. Monodisperse Stöber amorphous silica nanoparticles (SNPs) of three different sizes (16, 60 and 104 nm) and A549 lung carcinoma cells were selected as models. Cellular uptake of silica was monitored by ICP-MS. At non-cytotoxic doses the smallest particles showed a slightly higher fold induction of micronuclei (MNBN). When considering the three SNPs together, particle number and total surface area appeared to account for MNBN induction as they both correlated significantly with the amplitude of the effect. Using nominal or cellular dose did not show statistically significant differences. Likewise, alkaline comet assay and FISH-centromeric probing of MNBN indicated a weak and not statistically significant induction of oxidative DNA damage, chromosome breakage and chromosome loss. This line of investigation will contribute to adequately design and interpret nanogenotoxicity assays.

Influence of size, surface area and microporosity on the in vitro cytotoxic activity of amorphous silica nanoparticles in different cell types.

Identifying the physico-chemical characteristics of nanoparticles (NPs) that drive their toxic activity is the key to conducting hazard assessment and guiding the design of safer nanomaterials. Here we used a set of 17 stable suspensions of monodisperse amorphous silica nanoparticles (SNPs) with selected variations in size (diameter, 2-335 nm), surface area (BET, 16-422 m(2)/g) and microporosity (micropore volume, 0-71 microl/g) to assess with multiple regression analysis the physico-chemical determinants of the cytotoxic activity in four different cell types (J774 macrophages, EAHY926 endothelial cells, 3T3 fibroblasts and human erythrocytes). We found that the response to these SNPs is governed by different physico-chemical parameters which vary with cell type: In J774 macrophages, the cytotoxic activity (WST1 assay) increased with external surface area (alphas method) and decreased with micropore volume (r(2) of the model, 0.797); in EAHY926 and 3T3 cells, the cytotoxic activity of the SNPs (MTT and WST1 assay, respectively) increased with surface roughness and small diameter (r(2), 0.740 and 0.872, respectively); in erythrocytes, the hemolytic activity increased with the diameter of the SNP (r(2), 0.860). We conclude that it is possible to predict with good accuracy the in vitro cytotoxic potential of SNPs on the basis of their physico-chemical characteristics. These determinants are, however, complex and vary with cell type, reflecting the pleiotropic interactions of nanoparticles with biological systems.

Eco-, geno- and human toxicology of bio-active nanoparticles for biomedical applications.

Gene delivery has become an increasingly important strategy for treating a variety of human diseases, including infections, genetic disorders and tumours. To avoid the difficulties of using viral carriers, more and more non-viral gene delivery nanoparticles are developed. Among these new approaches polyethylene imine (PEI) is currently considered as one of the most effective polymer based method solution and considered as the gold standard. The toxicity of nanoparticles is a major concern when used for medical application. In this study we chose two nanoparticles for an in depth toxicological and ecotoxicological evaluation, one well characterized, PEI, and another novel polymer, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA). In the present study we have assessed the toxicity of these cation nanoparticles as such and of the polyplexes - nanoparticles covered with DNA. As these nanoparticles are also frequently used in high volumes in various industries and as such may enter in the environment, we also made an initial assessment of ecotoxicological effects assessment. The following nanoparticles related aspects have been studied during the project: development and characterization, ecotoxicity, general toxicity and specific toxicity. To this end a battery of different tests was used. The conclusion of these tests is that toxicity is varying between different nanoparticles and between different DNA covering ratios. In general, in the different systems tested, the PEI polymer is more toxic than the PDMAEMA polymer. The same difference is seen for the polyplexes and the higher the charge ratio, the more toxic are the polyplexes. Our study also clearly shows the need for a broad spectrum of toxicity assays for a comprehensive risk assessment. Our study has performed such a comprehensive analysis of two biomedical nanoparticles.

Synthesis and characterization of stable monodisperse silica nanoparticle sols for in vitro cytotoxicity testing.

For the investigation of the interaction of nanoparticles with biomolecules, cells, organs, and animal models there is a need for well-characterized nanoparticle suspensions. In this paper we report the preparation of monodisperse dense amorphous silica nanoparticles (SNP) suspended in physiological media that are sterile and sufficiently stable against aggregation. SNP sols with various particle sizes (2-335 nm) were prepared via base-catalyzed hydrolysis and polymerization of tetraethyl orthosilicate under sterile conditions using either ammonia (Stober process (1) ) or lysine catalyst (Lys-Sil process (2) ). The series was complemented with commercial silica sols (Ludox). Silica nanoparticle suspensions were purified by dialysis and dispersed without using any dispersing agent into cell culture media (Dulbecco's Modified Eagle's medium) containing antibiotics. Particle sizes were determined by dynamic light scattering. SNP morphology, surface area, and porosity were characterized using electron microscopy and nitrogen adsorption. The SNP sols in cell culture medium were stable for several days. The catalytic activity of the SNP in the conversion of hydrogen peroxide into hydroxyl radicals was investigated using electron paramagnetic resonance. The catalytic activity per square meter of exposed silica surface area was found to be independent of particle size and preparation method. Using this unique series of nanoparticle suspensions, the relationship between cytotoxicity and particle size was investigated using human endothelial and mouse monocyte-macrophage cells. The cytotoxicity of the SNP was strongly dependent on particle size and cell type. This unique methodology and the collection of well-characterized SNP will be useful for further in vitro studies exploring the physicochemical determinants of nanoparticle toxicity.

Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells.

The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EAHY926 cell line) is investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (as indicated by lactate dehydrogenase (LDH) release) and a decrease in cell survival (as determined by the tetrazolium reduction, MTT, assay) in the EAHY926 cell line in a dose-related manner. Concentrations leading to a 50% reduction in cell viability (TC(50)) for the smallest particles tested (14-, 15-, and 16-nm diameter) ranging from 33 to 47 microg cm(-2) of cell culture differ significantly from values assessed for the bigger nanoparticles: 89 and 254 microg cm(-2) (diameter of 19 and 60 nm, respectively). Two fine silica particles with diameters of 104 and 335 nm show very low cytotoxic response compared to nanometer-sized particles with TC(50) values of 1095 and 1087 microg cm(-2), respectively. The smaller particles also appear to affect the exposed cells faster with cell death (by necrosis) being observed within just a few hours. The surface area of the tested particles is an important parameter in determining the toxicity of monodisperse amorphous silica nanoparticles.

Nominal and effective dosimetry of silica nanoparticles in cytotoxicity assays.

Because of their small size and large specific surface area (SA), insoluble nanoparticles are almost not affected by the gravitational force and are generally formulated in stable suspensions or sols. This raises, however, a potential difficulty in in vitro assay systems in which cells adhering to the bottom of a culture vessel may not be exposed to the majority of nanoparticles in suspension. J. G. Teeguarden et al., 2007, Toxicol. Sci. 95, 300-312 have recently addressed this issue theoretically, emphasizing the need to characterize the effective dose (mass or number or SA dose of particles that affect the cells) which, according to their model based on sedimentation and gravitation forces, might only represent a very small fraction of the nominal dose. We hypothesized, in contrast, that because of convection forces that usually develop in sols, the majority of the particles may reach the target cells and exert their potential toxicity. To address this issue, we exposed three different cell lines (A549 epithelial cells, EAHY926 endothelial cells, and J774 monocyte-macrophages) to a monodisperse suspension of Stöber silica nanoparticles (SNP) in three different laboratories. Four different end points (lacticodehydrogenase [LDH] release, LDH cell content, tetrazolium salt (MTT), and crystal violet staining) were used to assess the cell response to nanoparticles. We found, in all cell lines and for all end points, that the cellular response was determined by the total mass/number/SA of particles as well as their concentration. Practically, for a given volume of dispersion, both parameters are of course intimately interdependent. We conclude that the nominal dose remains the most appropriate metric for in vitro toxicity testing of insoluble SNP dispersed in aqueous medium. This observation has important bearings on the experimental design and the interpretation of in vitro toxicological studies with nanoparticles.