DEVELOPMENT OF AN AUTOMATED HIGH-THROUGHPUT

SCREENING PROCEDURE FOR NANOMATERIALS

GENOTOXICITY ASSESSMENT

August 2013

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Acknowledgements This research was undertaken at Flinders University, at the University of South Australia’s Mawson Institute and at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) Animal, Food and Health Sciences (Adelaide), under commission from Safe Work Australia. The personnel involved were Prof. Nico Voelcker, Dr. Michael Fenech, Dr. Giuseppe Vecchio, Dr. Emily Anglin, Dr. Barbara Sanderson, Ms. Clare Cooksley, Mr. Martin Sweetman, Ms. Josefine Splinter and Dr Howard Morris. Funding for this proof-of-principle project was provided by the Department of Industry, Innovation, Science and Research under the National Enabling Technologies Strategy. The authors would like to thank Dr Vladimir Murashov (United States National Institute for Occupational Safety and Health, NIOSH), Dr Miriam Baron (German Federal Institute for Occupational Safety and Health, BAuA), Associate Professor Paul Wright (RMIT University), Mr Stephen Thomas (Anglicare SA) and Dr Maxine McCall (CSIRO) for their review comments and input on the report. Disclaimer The information provided in this document can only assist you in the most general way. This document does not replace any statutory requirements under any relevant state and territory legislation. Safe Work Australia is not liable for any loss resulting from any action taken or reliance made by you on the information or material contained on this document. Before relying on the material, users should carefully make their own assessment as to its accuracy, currency, completeness and relevance for their purposes, and should obtain any appropriate professional advice relevant to their particular circumstances. The views in this report should not be taken to represent the views of Safe Work Australia unless otherwise expressly stated.

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Creative Commons With the exception of the Safe Work Australia logo, this report is licensed by Safe Work Australia under a Creative Commons 3.0 Australia Licence. To view a copy of this licence, visit http://creativecommons.org/licenses/by/3.0/au/deed.en In essence, you are free to copy, communicate and adapt the work, as long as you attribute the work to Flinders University, the University of South Australia, CSIRO and Safe Work Australia and abide by the other licensing terms. The report should be attributed as the Development of an automated high-throughput screening procedure for nanomaterials genotoxicity assessment. Enquiries regarding the licence and any use of the report are welcome at: Copyright Officer Safe Work Australia GPO Box 641 Canberra ACT 2601 Email: copyrightrequests@safeworkaustralia.gov.au ISBN 978-1-74361-172-2 [Online PDF] ISBN 978-1-74361-173-9 [Online DOCX]

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Executive Summary In this project, an automated procedure for high-throughput screening of the genotoxicity of synthetic nanoparticles has been developed. Surface-engineered microarrays were used in order to provide in situ cell sorting, localisation, and immobilisation of various subsets of human primary lymphocytes, suitable for on-chip bioassays. The microarray platform was then integrated with the CBMN assay for analysis of the genotoxicity of specific subsets of human peripheral lymphocytes exposed to nanoparticles. Protocols suitable for automated scanning, imaging and analysis of microarrays for the assessment of micronuclei in binucleated (BN) cells (to examine genotoxicity) and nuclear division index (NDI) (an indicator of cytotoxicity) were developed. The genotoxic effect of silver nanoparticles (AgNPs), with different size and surface coating, were assessed. The genotoxic impact of nanoparticles on human B lymphocyte cell line was examined successfully using the screening method. Preliminary investigations of primary human lymphocyte genotoxicity has indicated that short exposure time and low nanoparticle concentrations need to be used to examine genotoxicity effects using the CBMN assay. The key features of the approach described are that it requires very small volumes of reagents, allows sorting of lymphocyte subsets in situ, increases throughput of cell assays and is amenable to high content microscopy analysis, which is important when considering the growing number of different nanoparticle formulations already on the market and in development. In summary, this proof-of-principle project has been successful in developing procedures for lymphocyte separation, nanoparticle incubation, fluorescence staining and automated scoring of microarray experiments to determine the extent of genotoxicity and make comparison between the different nanoparticle concentrations, particle chemistries and particle sizes. In ongoing work, the authors will use these procedures for the genotoxicity assessment of different type of nanomaterials on human primary lymphocytes.

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Contents Glossary of terms 5 Background 6 Objectives 7 Key Results 8 PART 1 – Details of evaluation of cell sorting specificity on microarray 8

PART 2 – Examining cytotoxicity: Effect of concentration of different nanoparticles on cell viability 11

PART 3 – CBMN assay after treatment of lymphocytes with silver nanoparticles 12

PART 4 – Implementation of an automated procedure for cell microarray imaging and genotoxicity scoring 14

PART 5 – AgNP genotoxicity assessment using microarray-based high-throughput screening 16

Discussion 23 Conclusion 25 Further work 25 References 26 Appendix 1 – Experimental protocols 27

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Glossary of Terms AgNPs - Silver Nanoparticles BN - Binucleated Cells CBMN - Cytokinesis-Block Micronucleus CD - Cluster of Differentiation; Cluster Designation CTRL - Control Sample Cyt-B - Cytochalasin-B DiO - Fluorescent Cell Membrane Stain (Vybrant™) ENM - Engineered Nanomaterials FBS - Fetal Bovine Serum FDA - Fluorescein Diacetate HPRT – Hypoxanthine Phosphorybosyl Transferase MN - Micronuclei MNed BN- Micronucleated Binucleated Cells MonoN – Mononucleated Cells MultiN – Multinucleated Cells Nbud - Nuclear Buds NDCI - Nuclear Division Cytotoxic Index NDI - Nuclear Division Index NPB - Nucleoplasmic Bridges PBS - Phosphate-Buffered Saline PI - Propidium Iodide PHA - Phytohemagglutinin PVP - Polyvinyl Pyrrolidone SWNT - Single Walled Carbon Nanotubes

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Background

Synthesis and commercialisation of engineered nanomaterials (ENMs) is continuing to grow due to their particular physical and chemical characteristics and their potential applications in a wide range of fields. The increased production of ENMs also involves potential risk for the environment and human health and safety. People may come into contact with nanomaterials in a number of ways. Nanomaterials may be deliberately introduced into the human body, for example in the case of nanomaterials used in the production of pharmaceutical formulations or otherwise used in the biomedical fields. The probability that nanomaterials may enter into our body involuntarily is also increased also due to their increased presence in our environment1-4. The toxicological screening of engineered nanomaterials has not kept up with the growth in their development and use. This situation represents a bottleneck between the synthesis of new nanomaterials and toxicological characterisation before their commercialisation, and impacts on capability of hazard classification of nanomaterials prior to use. Some nanomaterials potentially have high hazards and recent findings demonstrate the presence of genotoxic and mutagenic effects related to the administration of ENMs both in vivo and in vitro5, 6. A number of issues impact on capability of accurately determining nanomaterial hazards. Variations in nanotoxicity results have been observed that could be attributed to the vast number of toxicity assays and biological model systems available to determine toxicity and the diverse fabrication processes used to build nanoscale materials. Furthermore, the low throughput and the high cost associated with many conventional toxicity assays also prevent the full potential of these studies from being realised. A solution to these problems is represented by the use of high-volume screening approaches for the safety evaluation of ENMs. High-throughput screening, that makes use of automated platforms to conduct assays and to generate data for their hazard ranking, has the advantage of reduced reagent requirements, rapidity of analysis, reduced manpower needs, and providing a cost-effective screening approach that can also be used to prioritize materials for in vivo testing7, 8. A novel high-throughput cell-based microarray platform has recently been developed, and then successfully integrated with toxicological screening methods, including the cytokinesis-block micronucleus (CBMN) assay. This surface engineered on-chip cytometer was used to determine the extent and location of genetic damage present within primary human lymphocyte subsets9. In this study, this microarray platform technology has been developed in order to develop a fully automated analysis procedure for the screening of toxicity of different nanoparticles, in order to design the best procedure for the genotoxicity assessment on human primary lymphocytes. This project was commissioned by Safe Work Australia as part of the Nanotechnology Work Health and Safety Program.

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Objectives

The overall aim of this project is to develop an automated high-throughput screening procedure for nanomaterials genotoxicity assessment that is capable of discriminating between the effects induced by ENMs on different primary human lymphocyte subsets that are separated on a microarray chip. The specific objectives of the project are: Objective 1. To evaluate the in situ cell sorting platforms (‘on-chip cytometer’) to study genetic damage caused by exposure to ENM in human lymphocytes by automated analysis of CBMN assay. Objective 2. To develop an automated procedure for ‘scoring’ and quantifying biomarkers for genetic damage (such as the appearance of micronuclei) on the microarray platform. Objective 3. To evaluate the genotoxic effect induced by silver nanoparticles (AgNPs) with different surface capping and different sizes.

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Key Results Experimental protocols are described in Appendix 1. PART 1 – Details of evaluation of cell sorting specificity on microarray In the classic CBMN assay protocol, cells are treated with cytochalasin-B (cyt-B) and then harvested by cytocentrifugation using cytospin centrifuge. Here, in order to develop a new protocol for the automated high-throughput screening of nanomaterials genotoxicity, we modified this procedure introducing the step of cell separation and immobilisation onto antibody microarrays. 1.1 On-chip separation of human B and T lymphocytes In order to develop an automated high-throughput screening procedure for nanomaterials genotoxicity assessment that is capable of discriminating between the effect induced by ENMs in different primary human lymphocyte subsets on a microarray platform, specificity in the cell sorting on the microarray is needed. Cell sorting and separation is achieved by using different antibodies to capture different cells. Experimental details

- Anti-CD2, anti-CD20 and anti-CD45 microarray - HR1K (human B lymphocyte cell line) and Jurkat (human T lymphocytes cell line) - Cells collected in exponential growth phase, stained separately and mixed - HR1K cells stained with Vybrant™ DiO (membrane), Jurkat cells stained with Hoechst

33342 (nuclei) Three monoclonal antibodies that are capable of differentiating between human leukocytes, the antigens CD2, CD20, and CD45, were printed in a 3x3 pattern onto epoxy-silane modified surface of glass microscopy slides as shown in Figure 1 (top). As shown in Figure 1 (bottom), the three different antibodies sort the B and T lymphocytes with specificity. HR1K and Jurkat cell lines, cultured separately at density of 1x106 cells/mL were differently stained. HR1K cells were labelled using Vybrant™ DiO Cell-labelling (specific for cell membrane), while Jurkat cells nuclei were stained using Hoechst-33342. Equal portions (1 mL) of the stained cell suspensions were combined and 1 mL of the mixed cell population was incubated on the microarray. After incubation, the microarray was gently washed using PBS and cells were fixed with formaldehyde solution at 3.7% v/v for 10 min. Images of cells separated on the microarray were collected by Operetta® High Content Imaging System (Perkin Elmer Inc.).

Figure 1: Representative scheme of microarray slide consisting of three different antibody arrays printed in 3x3 patterns (top), and description of respective correspondence between cells and antigens (bottom).

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Results As shown in Figure 2, cells are present only in correspondence to the antibody pattern, demonstrating the correct and high specificity of on-chip cell sorting. Thus, the results demonstrate the high specificity in the capturing capability of the immobilised antibodies. B Lymphocytes (HR1K cells) were recognised only in the correspondence of anti-CD20 and anti-CD45 microarray spots, while T lymphocytes (Jurkat cells) were captured specifically by anti-CD2 and anti-CD45 antibodies.

Figure 2: Image of B and T lymphocytes captured on microarray. HR1K cell membranes were stained with Vibrant™ DiO Cell-labelling (green); Jurkat cell nuclei were stained with Hoechst 33342 (blue). 1.2 Effect of cytochalasin-B (cyt-B) on separation of cells on-chip The CBMN assay technique is based on the use of cyt-B in order to induce the cytokinesis block in cells that allow the scoring of micronuclei in bi-nucleated (BN) cells. The frequency of micronuclei (MN) in BN cells represents an index of genotoxicity. However, after the treatment with cyt-B, mono-nucleated (MonoN) and multi-nucleated (MultiN) cells are also observed. Thus, it is important to evaluate the effect of cyt-B treatment on the cells captured by antibody immobilised onto the glass surface. Experimental details

- HR1K (human B lymphocyte cell line) and Jurkat (human T lymphocytes cell line) - Cell division blocked by addition of Cyt-B at 4.5 µg/mL - Anti-CD2, anti-CD20 and anti-CD45 microarray

Figure 3 shows a representative image of HR1K cells captured onto microarray after the cytokinesis block. As shown in Figure 3, the antibody microarray was able to capture MonoN, BN, and MultiN cells permitting the scoring of micronuclei in BN cells.

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Figure 3: Representative fluorescence image of HR1K cells immobilised on-chip after treatment with cyt-B. The total number of BN cells represents an important factor in order to evaluate genotoxicity, as a minimum of 500 BN cells must be scored. The number of microarray spots required to score for MN using a sufficient number of BN cells needs to be determined. The number of cells per microarray spot is shown in Figure 4. Comparing the number of untreated (control) and cyt-B treated cells immobilised on the spots of antibodies, a 35% reduction was observed in the number of cyt-B treated cells with respect of the untreated cells. Evaluating the frequency of MonoN, BN and MultiN cells with respect of the total number of cells, there is about 30% of BN cells, 10% of MultiN cells and 60% of MonoN cells. Using this information, the number of microarray spots required to score for MN, using a sufficient number of BN cells can be calculated. This reduction in number of cells captured is due to the increased cell footprint (area occupied) of the cyt-B treated cells. The footprint of the lymphocyte cells is constituted mainly by the nuclei and thus, subsequent to the block of cell division, the area each cell occupies on the chip is approximately proportional to the number of nuclei.

Figure 4: Number of untreated (CTRL - control) and cyt-B treated HR1K cells immobilised on single microarray spot. Data are expressed as the mean and standard error of measurement (SEM) (n = 3 experiments).

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PART 2 – Examining cytotoxicity: Effect of concentration of different nanoparticles on cell viability Having determined that cells can be separated and captured on the microarray chip with specificity, the next step was to examine the effect of different nanoparticles on cell viability, i.e. to examine the cytotoxicity of the nanoparticles. This was undertaken for two reasons:

1. To choose suitable nanoparticles for subsequent genotoxicity testing, and 2. To determine suitable doses of nanoparticles to use for genotoxicity testing

Experimental details

- HRIK, human B lymphocyte cell line - All NP tested at 100 and 150 µg/mL concentrations - 24 h treatment - Live/dead staining with fluorescein diacetate (FDA) and propidium iodide (PI)

Results The effect of eight different, commonly used nanoparticles and 180 nm titanium dioxide particles on the viability of the lymphocyte cell line HR1K was investigated. The particles used were 25 nm and 180nm titanium dioxide, 10nm and 70 nm citrate-capped silver nanoparticles, 10 nm and 70 nm polyvinyl pyrrolidone- (PVP-) capped silver nanoparticles, P2 and P3 single-walled carbon nanotubes (SWNT) and carbon black. Initial investigation looked at the effect of nanoparticle concentration, over a 24 h exposure period. Figure 5 displays the cell viability results for all eight nanoparticles, along with a negative media control and positive ethanol control. It can be seen in most cases that as the nanoparticle concentration is increased from 100 to 150 µg/mL the cell viability drops, which is expected as the amount of toxic material present is increased. There is however no statistically significant difference in viability between the two nanoparticle concentrations for most cases. For a number of materials, there was little difference in the cell viability of the nanoparticle treated samples compared to the media control. The samples that showed significantly lower cell viability to the control were the 70 nm citrate- and polyvinyl pyrrolidone- (PVP-) capped silver nanoparticles at 150 µg/mL, along with the 10 nm citrate and PVP silver nanoparticles at both concentrations. The 10 nm silver nanoparticles showed the highest levels of toxicity of all the nanoparticles, with the citrate functionalised silver being more toxic than the PVP functionalised particles. In relation to the data on cytotoxicity and particle size, it is noted that there was little difference in cell viability between 180 nm and 25 nm titanium dioxide particles. However, for silver nanoparticles, the smaller size nanoparticles (10 nm) are more toxic.

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Figure 5: Effect of nanoparticle type and concentration on lymphocyte cell viability for a 24 h exposure time. Data are expressed as the mean and SEM (n = 5 experiments). PART 3 – CBMN assay after treatment of lymphocytes with silver nanoparticles Further information on cytotoxicity and an initial investigation of cell genotoxicity was undertaken by using a CBMN assay after AgNP treatment of cells. This set of experiments did not use the microarray chip. Instead, cells were spun onto slides. Based on the cytotoxicity results, the 10 nm citrate-capped AgNPs were selected as the most appropriate material for further experiments because of their pronounced cytotoxicity. A positive genotoxic result was expected to be observed in primary lymphocytes treated with this material. Exposure periods of 3 – 6 days were selected, with nanoparticle concentrations of 12.5 – 25 µg/mL deemed most appropriate to induce a genotoxic response. The lower nanoparticle dosage was chosen to reduce acute cytotoxicity effects and give cells time to potentially show signs of genotoxicity. Experimental details

- 10 nm citrate AgNPs - 12.5 and 25 µg/mL concentration - Primary human lymphocytes - 3 and 6 day treatments - Phytohemagglutinin (PHA) and nanoparticles were both added on day 1 for each

experiment - Cytospin cells onto slides (not microarray) - Cells stained with Diffquick brightfield stains

Cytotoxicity data Primary lymphocyte cells treated with the silver nanoparticles were harvested and scored according to the scoring protocol for the CBMN assay10. Figure 6 (a) and (b) displays the nuclear

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division index (NDI) and the nuclear division cytotoxic index (NDCI) respectively, for the control sample and the nanoparticle treated samples for the 3 and 6 day exposure periods. The NDI provides a measure of the proliferative status (i.e. degree of cell division) of all viable cells in a sample, while the NDCI is a measure of the proliferative status of viable and non-viable cells (apoptotic and necrotic). The NDI and NDCI can be used to compare cytotoxicity between different samples. It can be observed that for the 3 day nanoparticle treated cell samples the NDI is around 1.2 – 1.3. This is lower than the NDI for the control sample of approximately 1.5 and indicates that there was a lower number of BN and MultiN cells in the nanoparticle treated samples. This result is indicative of nanoparticle cytotoxicity impacting on lymphocyte proliferation. However, it does not appear that the results are dependent on the dosage of AgNPs (12.5 vs. 25 µg/mL). Cytotoxicity can be an issue when examining genotoxicity, i.e. when it comes to scoring the DNA damage in these samples, as only viable BN cells can be used to score the damage. The problem that can arise from the use of cytotoxic concentrations is that there may be insufficient BN cells to score.

Figure 6: Nuclear division index (NDI) (a) and nuclear division cytotoxic index (NDCI) (b), determined by CBMN assay for 10 nm citrate AgNP treated primary lymphocytes. Data are expressed as the mean and SEM (n = 3 experiments). Genotoxicity data DNA damage was scored for the 3 day sample only, as there were not enough BN cells in the 6 day sample to accurately score the damage. Figure 7 displays the observed frequency of the markers that indicate DNA damage for the control and nanoparticle treated cells. These markers are:

• BN cells with a micronucleus • Total number of micronuclei • BN cells with a nucleoplasmic bridge • BN cells with a nuclear bud

The graph displays the frequency of markers per 1000 BN cells. Only for the 12.5 µg/mL sample, an increase in micronuclei and multinucleated cells was observed. No significant differences in other genotoxic indicators were observed between the three samples.

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Figure 7: Frequency of DNA damage markers for 10 nm citrate silver nanoparticle treated primary lymphocytes (BN = binucleated cell, MNed BN= micronucleated bi-nucleated cells, MN = micronuclei, NPB = nucleoplasmic bridges, Nbud = nuclear buds). Data are expressed as the mean and SEM (n = 3 experiments). PART 4 – Implementation of an automated procedure for cell microarray imaging and genotoxicity scoring Development of the procedure was initially undertaken with hydrogen peroxide, a known genotoxic chemical, as a positive control. This experiment series focused on demonstrating the ability to automatically acquire fluorescence microscopy images and perform automated image analysis after the CBMN assay. Experimental details

- Anti-CD20 microarray - WIL2-NS (Human B lymphocyte cell line) - Cells treated with different concentration of hydrogen peroxide - Cells stained with Vybrant™ DiO (cell membrane) and Hoechst 33342 (nuclei)

Based on the results in Part 1, which showed that the microarray system permits the separation of the different human lymphocytes, we used the Operetta® High Content Imaging System equipped with Harmony software version 3.0 (Operetta imaging system, PerkinElmer, Hamburg, Germany) for the collection of fluorescence images and automated image analysis. For proof-of-principle, we used a microarray of anti-CD20 antibodies, the WIL2-NS cells line and hydrogen peroxide as a genotoxic control. We chose this particular cell line for the implementation of automated analysis procedure because it is a human B lymphoblastoid cell line that presents an excellent morphology and a high nuclear division index due to the suppressed apoptotic activity, which is ideal for the CBMN assay. WIL2-NS cells were treated for 1 h with different concentration of hydrogen peroxide (0, 10, and 20 µM), then were washed by centrifugation and cultured in presence of cyt-B for 24h. The images of cell microarrays were collected at magnification of 20x and 40x. The analysis of microarrays was carried out using the implemented procedure described in Figure 8.

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Figure 8: Step sequence implemented in Harmony software for the automatised analysis of genotoxicity. Images were collected using the filter combination for the FITC and DAPI fluorescence, in order to acquire the emission spectra of cell membrane (Vybrant) and nuclei (Hoechst 33342). After the recognition of nuclei, cytoplasm and micronuclei, different building blocks were added for the automatic recognition of MonoN, BN, MultiN and MNed BN cells. In the analysis block, we added formulae to also calculate the Nuclear Division Index (NDI) and the frequency of BN cells carrying MN. As shown in Figure 9, the number of MN located in BN cells is increased in the cells treated with hydrogen peroxide and the genotoxic effect is clearly related to the concentration of the genotoxic agent. On the other hand, the different concentrations of hydrogen peroxide induce only slight differences in the NDI, i.e. cytotoxicity is less significant. These results were validated using the manual scoring procedure and were found to be comparable with results reported in the literature11. The genotoxic and cytotoxic data were as predicted for hydrogen peroxide, thus providing validation of the procedure for cell microarray imaging and genotoxicity scoring.

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Figure 9: Number of micronucleated binucleated (MNed BN) cells in 500 BN cells scored (top) and NDI (bottom). Data obtained by automatised analysis of WIL2-NS cells untreated (control) and treated with hydrogen peroxide at concentrations of 10 and 20 µM. Data are expressed as the mean and SEM (n = 3 experiments). PART 5 – AgNP genotoxicity assessment using microarray-based high-throughput screening From Parts 1-4:

• Cells can be sorted on the microarray, • Conditions for AgNP assessment were established, • Microarray imaging and genotoxicity scoring was validated.

Using this preparatory work, genotoxicity of AgNPs was now examined using the microarray technique. 5.1 Genotoxic effect induced by AgNPs in human B lymphocyte cell line Experimental details

- Anti-CD20 microarray - WIL2-NS (Human B lymphocyte cell line) - Cells treated at 12.5 µg/mL concentration for 24 h with different AgNPs (10 and 70 nm,

citrate- and PVP-capped) - Cells stained with Vybrant™ DiO (cell membrane) and Hoechst 33342 (nuclei)

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WIL2-NS cells were treated with different AgNPs in order to investigate the importance of size and surface coating in the genotoxicity assessment of this nanomaterial. In this experiment, WIL2-NS cells were seeded at a density of 0.5x106 cells/mL in complete cell medium containing cyt-B at 4.5 μg/mL and 10 and 70 nm citrate- and PVP-capped AgNPs for 24 h. Hydrogen peroxide at 20 µM concentration was used as positive control while untreated cells represented the negative control. Cells were incubated on anti-CD20 microarrays, fixed and stained. Fluorescence images of cells immobilised onto microarray slides were collected by the Operetta system and analyzed by Harmony software following the automated procedure previously described. Results are reported in Figure 10. Results As shown in the Figure 10, all the different AgNPs induced an increase in the number of BN cells featuring MN, and in particular the highest number of MNed BN cells was observed in the cells treated with 10 nm citrate-capped AgNPs demonstrating a strong genotoxic effect induced by these nanoparticles. We observed that the genotoxicity induced by 10 nm citrate-capped AgNPs after 24 h is higher that the genotoxicity induced by treatment for 1 h with 20 µM of hydrogen peroxide, a well known genotoxic agent.11, 12 Size and surface coating play a key role in the modulation of genotoxicity induced by AgNPs. Regarding size, the genotoxic effect induced by 70 nm citrate-capped AgNPs was about 50% less compared to the 10 nm citrate-capped AgNPs. In terms of the nature of the surface coating, cells treated with 10 nm PVP-capped AgNPs show about a 40% decreased number of MNed BN cells compared to the cells treated with 10 nm citrate-capped AgNPs. Also PVP-capping had minimal effect on the genotoxic response to 70 nm AgNPs. Examining the relationship between NDI and micronuclei, we observed that a high number of MN corresponded to a low NDI. Thus for the particles tested, highest genotoxicity is seen for particles that also have the highest cytotoxicity. This is consistent with the cytotoxicity results shown in Figure 5 in terms of the relative toxicity of the different AgNP species.

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Figure 10: Number of BN cells featuring MN per 500 BN cells (top) and NDI (bottom) determined by automatised analysis in WIL2-NS cells treated with AgNPs of different size (10 and 70 nm) and surface coating (citrate- and PVP-capped). Data are expressed as the mean and SEM (n = 3 experiments).

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5.2 Genotoxicity of AgNPs in primary human lymphocytes

Ultimately, the aim is to develop a screening tool for use with primary human lymphocytes. Therefore, the next set of experiments investigates genotoxicity of AgNPs in primary human lymphocytes captured and separated on the microarray chip. Primary lymphocytes often behave differently to lymphocyte cell lines and differences between the two sets of experiments were expected. Experimental details

- Anti-CD2, -CD4, -CD8, -CD20 and -CD45 microarray - Primary human lymphocytes - HR1K (human B lymphocyte cell line) - Cells treated at 12.5 µg/mL concentration for 72h with different AgNPs (10 and 70 nm,

citrate and PVP capped) - Cells stained with Vybrant™ DiO (cell membrane) and Hoechst 33342 (nuclei)

Separation of primary human lymphocytes on the microarray chip after CBMN assay We tested the efficiency of our microarray platform in the recognition and separation of different primary human lymphocytes by 5 different antibodies immobilised on the microarray slides. As shown in Figure 11, microarray slides were prepared by printing 3 arrays with 3x3 patterns for each different antibody in order to increase the number of scored cells. The microarray pattern was specifically designed in order to permit the automatic acquisition of fluorescent images using the 348-well plate assay integrated on the Operetta system. This microarray pattern simplifies the finding of the slide regions in which the different separated cells are located.

Figure 11: Microarray pattern: 5 different antibodies printed in 3 arrays of 3x3 spots each. Primary lymphocyte cell division was activated by PHA and after 44 h cyt-B was added to the cell cultures following the classical CBMN assay procedure10. Cells were incubated for 1 h onto microarray slides and fluorescence images of cells immobilised onto antibody spots were acquired using the Operetta system (Figure 12). Thus it was confirmed that the microarray platform can recognise and separate primary human lymphocyte cells after performing a CBMN assay.

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Figure 12: Representative fluorescence image of primary lymphocytes separated on microarray. Examining the genotoxicity of AgNPs Initial experiments The above experiment was repeated, but now including the nanoparticle treatments in order to establish the genotoxic effect induced by the differently sized and surface-coated AgNPs. All nanoparticles were incubated at a concentration of 12.5 µg/mL with primary lymphocytes for 72 h. At the same time, as a control, cells were stimulated with PHA followed by cyt-B treatment. In this experiment, the analysis of cells immobilised onto microarray slides was impossible due to the high cytotoxic effect induced by AgNPs. In particular, the number of BN cells was very low (about 20%), the presence of nuclear debris, and cells with morphologically-deformed nuclei caused several problems in the recognition of nuclei, micronuclei and cells immobilised onto the microarray. In Figure 13, representative images collected by Operetta system of anti-CD45 spots are shown. As shown in Figure 13A, primary human lymphocytes treated with PHA and cyt-B (control) present a good morphology of nuclei with a high presence of BN and MultiN cells. Figure 13B and Figure 13C are human primary lymphocytes treated with 12.5 µg/mL of 10 nm citrate- and PVP-capped AgNPs respectively, and show the presence of nuclear debris and morphologically-malformed nuclei. The same effects were observed in cells treated with 70 nm citrate- and PVP-capped AgNPs. This result was probably due to the previously observed cytotoxic effect induced by AgNPs. Apoptotic and necrotic events can induce the presence of cellular and nuclear debris and it is well known that AgNPs induce changes in nuclear morphology of cells13. These last results highlighted the necessity to adjust the concentration and the time of AgNPs exposure in order to assess the genotoxicity of this nanomaterial by the implemented automatised procedure.

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Figure 13: Representative fluorescence image of primary human lymphocytes on CD45 spot. A) Control; B) 10 nm citrate-capped AgNPs; C) 10 nm PVP-capped AgNPs. Reduction in exposure time to nanoparticles The time that cells are exposed to nanoparticles was then reduced from 72 h to 48 h (10 nm citrate –capped AgNPs were added to cells culture 24 h after the addition of PHA) in order to limit the extent of cytotoxic effects that could prevent BN cell formation.

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This change in the procedure reduced the cytotoxic effect induced by AgNPs and the immobilised cells showed a slight improvement of their aspect. Figure 14 displays two representative microarray spots for each nanoparticle exposure time treatment of the lymphocyte cells. For each spot, a selection of BN cells is circled to indicate the presence and proportion over the whole spot. When the number of BN cells is compared for each spot, between the two experiments it is clear that the time reduction of AgNPs treatment yielded a far greater percentage of BN cells. This is an important result in terms of scoring DNA damage in the treated cells, since only BN cells can be scored for MN formation and since a high number of scorable cells are required for accurate scoring. However, the automated analysis was still difficult to perform in this experiment.

Figure 14: Representative fluorescence microscopy images on CD45 microarray spots of lymphocytes treated for 72 h (A) and 48 h (B). Cells stained with Cell Tracker Orange (cytoplasm) and Hoechst 33342 (nuclei). BN cells are circled in yellow. Reduction in nanoparticle concentration In subsequent experiments, we examined the use of HR1K cells with a 100-fold lower concentration of 10 nm citrate-capped AgNPs at 48 h exposure time. HR1K is a cell line but is more similar in behaviour to primary lymphocytes than WIL2-NS. In this case, 10 nm citrate- and PVP-capped AgNPs at final concentration of 12.5 µg/mL (as previously) and 125 ng/mL were used. As previously, the automated analysis of micronuclei was difficult to perform for the cells treated with 12.5 µg/mL, due to the presence of cellular and nuclear debris and the presence of morphologically deformed cells. However, cells treated with AgNPs diluted by a factor of 100 to 125 ng/mL show a good morphological aspect and the presence of nuclear and cellular debris were comparable to the control. The automated analysis of MN of this sample revealed the persisting presence of genotoxicity induced by the differently capped 10 nm AgNPs at a low concentration as shown in the histogram reported in Figure 15A, with the number of MNed BN cells being higher with citrate-capped compared with PVP-capped AgNPs. In Figure 15B, the NDI is similar for the treatment with both differently coated 10 nm AgNPs and significantly lower than the NDI of untreated cells. Thus both genotoxic and cytotoxic effects are shown at this lower concentration, but the cytotoxicity is not so severe as to prevent evaluation of genotoxicity. Thus by decreasing both exposure time and concentration, the genotoxic effects can be examined effectively.

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Figure 15: Number of MNed BN cells in 1000 BN cells scored (A) and NDI (B) determined by automatised analysis in HR1K cells treated with 125 ng/mL of 10 nm citrate- and PVP-capped AgNPs. Data are expressed as the mean and SEM (n = 3 experiments). Discussion The first stage of this work confirmed that specificity in cell recognition and separation can be achieved using antibodies in the microarray chip platform9. Next, the effect induced by treatment of cyt-B on the number of cells immobilised onto microarray spots was determined and a reduction in the number of cells immobilised onto microarrays was observed. Using the mean number of cells observed per spot and the frequency of BN cells amongst these cells, microarray slides were designed with a sufficient number of antibody spots in order to screen at least 500 BN cells for each treatment group. The effect of nanoparticle treatments in the specificity of cell sorting on the microarray was examined, and it was found that incubation with nanoparticles does not seem to have an effect on the recognition of the lymphocyte types by the printed antibodies. Following this work, an automated procedure for nanomaterial genotoxicity assessment of cells immobilised on-chip was developed, a key step in the development of an automated high-throughput screening platform. This new procedure was validated by evaluating the genotoxicity induced by two different concentrations of hydrogen peroxide (10 and 20 µM), obtaining results comparable with results reported in literature11. Using the validated automated procedure, the genotoxicity effect induced by 4 different AgNPs (10 and 70 nm, citrate- and PVP-capped) was examined. In preliminary experiments, 10 nm citrate capped AgNPs showed the highest levels of genotoxicity inducing a greatly increased number of MNed BN cells with the respect of negative and positive control. Results indicated that 10 nm citrate capped AgNPs at 12.5 µg/mL are more genotoxic than hydrogen peroxide, which was used as the positive control at 20 µM of concentration and is well known to induce oxidative stress and DNA damage in cells11, 12. The high toxicity induced by 10 nm citrate-capped AgNPs with respect to other sizes is probably due to differential nanoparticle uptake and bioaccumulation in cells and it is known that smaller nanoparticles enter cells more easily than larger ones14. Surface coatings play a key role in the stability, uptake and toxicity of AgNPs15 and differing results were obtained for citrate-capped and PVP-capped AgNPs.

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Finally, the genotoxic effect induced by the four differently sized and surface-coated AgNPs in primary human lymphocytes was assessed. Experiments demonstrated the capability of our microarray to recognise the different primary human lymphocytes subsets after the treatment with PHA, cyt-B and AgNPs. At long exposure times and high concentrations of AgNPs, the automated analysis of microarray was impossible due the presence of nuclear and cellular debris and the morphological aberration of the treated cells. In fact, as reported in recent studies, human cells treated with AgNPs show an increased level of apoptotic and in particular necrotic events13, as well as the presence of deformed cells and cells with damaged membranes and large vacuolation16. Furthermore, in these studies the genotoxic effect induced by AgNPs was evaluated by manual scoring of COMET assay and micronucleus test, demonstrating the presence of genotoxicity induced by AgNPs.

The above result with long exposure time and high concentration of AgNPs shows that to investigate genotoxic effects of ENM, it is important to avoid acute cytotoxicity since that impedes the study of chromosomal abnormalities induced by genotoxicity. Preliminary results using HR1K cells treated with a significantly lower concentration, 125 ng/mL, of the two differently coated 10 nm AgNPs demonstrated the presence of genotoxic effects induced by this nanomaterial and the decreased cytotoxic stress induced by the low concentration permit the automatic analysis of samples using the Operetta system. This last observation is consistent with published works where the persistence of DNA damage induced by AgNPs at noncytotoxic doses was demonstrated17.

This in vitro study using the high throughput screening procedure, has indicated that AgNPs may be both cytotoxic and genotoxic. Similar results have been observed by other authors when examining the effect on cells using in vitro methods13, 16-18

Screening tests are a first step in examining whether a material is potentially toxic and are useful in advising whether further evaluation is needed. However, test results cannot in isolation be taken as conclusive evidence that a material is toxic. A low concordance has previously been reported between in vitro and in vivo toxicity for nanoparticles19.

The OECD test guidelines contain a number of tests relating to the examination of genotoxicity20. The validity of the current OECD in vitro genotoxicity test battery for nanomaterials has been examined and in regard to the optimum strategy required for the safety assessment of nanomaterials, it was found there are a number outstanding questions21. However in the interim, the authors recommend that the following approach may be used as a first-stage in vitro assessment to screen nanomaterials for further in vivo genotoxicity evaluation:

• Complete description of the physico-chemical features of the material under investigation • Mammalian cell chromosome aberration test, which could be a micronucleus assay • Mammalian cell point mutation test, e.g. HPRT assay • Extended dose responses

Further work is required on the screening methods described to establish excursion criteria for when to take further action based on the results. The criteria would define:

• No significant genotoxicity - no further testing needed • Biologically significant evidence of genotoxicity - further testing needed

In relation to the data on cytotoxicity and particle size, little difference was found in the viability of cells exposed to 180nm and 25nm titanium dioxide particles. This contrasts with in vivo data for titanium dioxide particles, which showed the potential for formation of lung tumours in rats was dependent on particle size and specifically on surface area22. This is the basis of the United States

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NIOSH’s Recommended Exposure Limits (RELs) of 0.3mg/m3 for ultrafine titanium dioxide and 2.4mg/m3 for fine-sized titanium dioxide22.

Silver is known to be potentially cytotoxic if inhaled. The Australian Workplace Exposure Standard (8-hour Time-weighted average, TWA) for silver metal is 0.1 mg/m3 and for soluble silver compounds is 0.01 mg/m3 (measured as Ag)23. Prolonged inhalation exposure tests in rats have indicated that lung function changes and inflammation can occur at lower concentrations of silver nanoparticles when compared with sub-micrometer silver particles24. In this research 10 nm sized AgNPs were found to be more toxic than 70nm AgNPs. This finding that smaller nanoparticles show higher toxicity in in vitro tests is in agreement with other authors14, 25.

The absence of genotoxic responses to AgNPs has been reported in a number of in vivo studies. AgNPs did not induce genetic toxicity in male and female rat bone marrow in vivo in 28 day oral toxicity tests26 and in 90 day inhalation toxicity tests27, and were not found to induce genotoxicity in a bacterial reverse mutation test and chromosomal aberration test, although some cytotoxicity was observed28.

This work has set the stage for automated scoring of cytogenetic damage to human lymphocytes using synthetic nanoparticles and for determining the sub-types of lymphocytes in which the damage is most pronounced. Conclusion In summary, this proof-of-principle project has been successful in developing protocols and procedure for automated high-throughput screening on-chip genotoxicity assays of nanomaterials. Further Work In ongoing work, the authors will use these procedures to score microarray experiments to determine the extent of genotoxicity and make comparison between the different incubation times, nanoparticle concentrations, particle chemistries and particle sizes on primary human lymphocytes. The proof-of-principle data obtained during this study has led to a grant funded by the South Australian Government (Regione Puglia-SA Awards for Research Collaboration) to investigate the nanotoxicity of synthetic nanoparticles produced at the Italian Institute of Technology using this microarray platform. This work will also involve validation of the microarray technology using an in vivo genotoxicity assay in use at the Italian Institute of Technology.

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Appendix 1 Experimental Protocols Antibody microarray slide preparation Glass microscopy slide surfaces were cleaned and etched by immersion for 1 h in a 3:1 mixture of concentrated sulfuric acid (H2SO4) with hydrogen peroxide (H2O2). Subsequently, the slides were washed thoroughly with milliQ water. After drying in an oven at 60 °C for 2 h, the substrates were immediately modified by immersing pretreated glass slides in a solution of 10% v/v (3-Glycidyloxypropyl) trimethoxysilane in anhydrous toluene for 25 min. After rinsing with toluene and acetone several times, the modified slides were dried under nitrogen gas. Antibody solutions at 0.2 mg/mL were printed onto the silane modified surface using XactII™ Compact Microarray System equipped with 350 µM diameter Xtend™ Capillary Microarray Pins. Printed slides were incubated at 4 °C for 24 h in a humidified chamber, then washed with PBS and incubated at 4°C for 24 h with a 5% solution of bovine serum albumin (BSA) in PBS. Finally, they were washed in PBS and immediately used for cell incubation. Cell Culture WIL2-NS, HR1K and Jurkat cells were routinely cultivated in RPMI 1640 with 50 μM glutamine, supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were incubated in a humidified controlled atmosphere with a 95% to 5% ratio of air/CO2, at 37 °C. The medium was changed every 3 days. Fluorescein Di-Acetate/Propidium Iodide (live/dead) Staining 375 µL of cell cultures were transferred to 1.5 mL Eppendorf tubes and centrifuged 10 min at 200 g. The cell pellet was resuspended in 50 µL of staining solution (5 mL PBS containing 5 µL FDA (1.5 mg/mL in acetone) and 4 µL PI (20 mM in DMSO)) and incubated at room temperature for 10 min. 20 µL of stained cells were added to microscopy slide, covered with a coverslip and counted by fluorescence microscopy. Hydrogen peroxide treatments One day before the CBMN assay, the WIL2-NS cells were seeded at a density of 0.3×106 cells/mL. On the assay day, cells were washed once with HBSS by centrifugation at 180 g for 5 min. For hydrogen peroxide exposure, cells were resuspended at a density of 0.5x106 cells/mL in HBSS with 10 and 20 µM of hydrogen peroxide and incubated for 1 h at 37 °C in a humidified atmosphere with 5% CO2 (CO2 incubator). Cells were then washed with HBSS by centrifugation at 180 g for 5 min and then resuspended in complete RPMI medium containing 4.5 μg/mL cyt-B and incubated for 24 h in CO2 incubator. The experimental procedure was concluded following the steps reported below in the “Cell immobilisation, fixation, and staining on microarray slides” section. AgNPs treatments AgNPs aqueous solution at 1 mg/mL of concentration (BioPure) used in this work were purchased from nanoComposix (San Diego, CA). BioPure silver nanoparticles are fabricated and processed using the same methods as OECD silver nanoparticle standards. AgNPs were diluted to a final concentration of 0.1 mg/mL in RPMI 1640 with 50 μM glutamine, supplemented with 10% FBS, 100 U/mL penicillin and 100 mg/mL streptomycin. Working solutions were obtained by appropriate dilution of this solution in complete cell medium. WIL2-NS and HR1K cells were seeded at a density of 0.3×106 cells/mL and cultured for 24 h under normal conditions, then cells were washed once with HBSS by centrifugation at 180 g for 5 min, resuspended at a density of 0.5x106 cells/mL in complete RPMI medium containing 4.5 μg/mL cyt-B and AgNPs at desired concentration, and incubated for 24 h in a CO2 incubator. The experimental procedure was concluded following the steps reported in “Cell immobilisation, fixation, and staining on microarray slides” section.

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Primary human lymphocytes were collected following the procedure described by M. Fenech10. Briefly, fresh blood collected by venepuncture into vacutainer blood tubes with heparin anticoagulant was diluted 1:1 with HBSS, gently mixed and overlayed onto Ficoll-Paque using a ratio 1:3 (Ficoll-Paque : diluted blood). After centrifugation at 400 g for 30 min at 18-20 °C, the leucocyte layer was collected and diluted with 3x its volume of HBSS at room temperature and centrifuged at 180 g for 10 min at 18-22 °C. The pellet was diluted with 2x the volume removed of HBSS and centrifuged at 100 g for 10 min at 18-20 °C. The leucocyte pellet was resuspended in 1 mL culture medium at room temperature, cells were counted and seeded at density of 1x106 cells/mL in 750 µL. Mitotic division was stimulated by adding PHA at final concentration of 30 µg/mL and cells were incubated in a CO2 incubator. AgNPs solutions were added immediately after the adding of PHA at final concentration of 12.5 µg/mL. Cell cultures were returned to the CO2 incubator and after 44 h cyt-B was added at final concentration of 4.5 µg/mL and incubated for a further 28 h. The experimental procedure was concluded following the steps reported in “Cell immobilisation, fixation, and staining on microarray slides” section. Cell immobilisation, fixation, and staining on microarray slides After incubation for 24 h with cyt-B at 4.5 μg/mL, cells were washed with HBSS by centrifugation at 180 g for 5 min, resuspended in HBSS with 5mL Vybrant® DiO cell-labeling solution (Life Technologies) or in a solution of CellTracker Orange (Invitrogen) at final concentration of 25 µM in a serum-free medium, and incubated at 37°C for 15 min, then centrifuged with HBSS at 180 g for 5 min, resuspended in RPMI 1640 without FBS and incubated for 1 h on microarrays in a CO2 incubator. After incubation, microarray slides were washed with PBS to eliminate cells not captured by antibody microarray spots, immobilised cells were fixed with 3.7% v/v formaldehyde for 10 min, permeabilised by incubation with 0.3% v/vTriton-X 100 in PBS solution for 10 min. Finally, nuclei were stained with 0.12 µg/mL of Hoechst 33342 in PBS, washed with PBS and then microarray slides were allowed to dry. Fluorescence imaging acquisition Fluorescence images of cells immobilised on microarray slides were recorded using Operetta® High Content Imaging System equipped with Harmony software version 3.0 (Perkin Elmer Inc.). Automatised analysis of microarray by Harmony software Fluorescence images acquired by Operetta system were analysed by Harmony software version 3.0. The procedure of analysis consists in the automatic detection of nuclei, cytoplasm and micronuclei. The number of cytoplasmic regions, corresponding to the number of cells, was used to define the number on MonoN, BN and MultiN cells by the analysis of the number of detected nuclei in each cytoplasmic region. Finally, the number of cytoplasmic regions containing 2 nuclei and micronuclei (>0) was categorised as MNed BN cells. In the analysis block was inserted the formulas to calculate the index of genotoxicity (number of MNed BN cells in 500 BN cells scored) and NDI [(MonoN cells + 2*BN Cells + 3*MultiN Cells) / (total number of cells)]. Data Analysis Results collected from 3 or 5 different experiments were used in order to calculate the mean value and standard error of measurement (SEM).