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NanoSafetyCluster - Compendium 2012 Figure 4. The stability of a 10 mg/mL titania suspension stabilised with citrate (excess citrate was dialysed out). (Ramirez-Garcia 2011) This is a very simple but effective method that can be easily implemented in a very reproducible manner in any dispersions laboratory. Moreover, the resulting suspensions were stable in cell media with different concentrations of plasma. The protein corona formed on this titania and also onto titania with other crystal structures is currently under investigation in our laboratory. Alternative routes to oxidative stress? The classical understanding of oxidative stress is that it represents an imbalance between the production of reactive oxygen species and a biological system's ability to readily detoxify the reactive intermediates or to repair the resulting damage. Disturbances in the normal redox state of tissues can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids, and DNA. Some reactive oxidative species can act as messengers through a phenomenon called redox signaling. Key observations from NeuroNano regarding the role of nanoparticles in the induction of oxidative stress responses by cells represent significant departures from conventional understanding. The full consequences of these observations are still under consideration, and will be reported on in detail in due course. These observations include: 1. The way nanoparticles are prepared and dispersed, and what they pick up from their surroundings into their biomolecule corona, determines so much of their fate and behaviour. This is reflected in the fact that amine modified polystyrene nanoparticles with different degrees of amination of the surface induced quite different responses - one induces catalases while the other does not. 2. There is emerging evidence that oxidative stress may not be caused only by the formation of Reactive Oxygen Species, and thus, attempts to rank the toxicity of nanoparticles on that basis only will fail. Thus, the FIBROS map requires more detailed mechanisms to identify the oxidative damage, such as the formation of protein lesions such as carbonyls or thiols. The approach of redox proteomics is shedding important light on some very subtle protein lesions, even using particles that are considered fully benign by all other methods, e.g. COOH-polystyrene nanoparticles. This suggests that nanoparticles that accumulate could induce some effects, even if the particles themselves are entirely passive. 3. Oxidative stress may have signalling origins, and again the biomolecule corona effect on this may greatly affect signalling. For example, a recent paper suggested that binding of fibrinogen to nanoparticles in an orientation that allows it to engage with the MAC-1 cell receptor initiated a signalling cascade resulting in oxidative stress, whereas the same protein bound to large nanoparticles of identical composition did not induce oxidative stress.(Deng 2010) These findings might also explain some of the differences in data reported in the literature. A key outcome from NeuroNano will be a comprehensive review of the sources and types of nanoparticleinduced oxidative stress. There is always the concern with in vitro studies that the use of immortalised or cancer-derived cells introduces anomalies that are absent from equivalent primary cells or in vivo. However, within NeuroNano we have used a high content analysis approach and found no evidence of differential responses to nanoparticles of primary or cultured astrocytes in terms of impacts including oxidative stress. Nanoparticle access to the brain In the conception of NeuroNano, the focus was on determining which nanoparticles could potentially reach the brain, and what it was about those nanoparticles, or their protein/biomolecule coronas that enabled them to cross the Blood-Brain barrier or reach the brain via the olfactory nerve. However, it turns out that this too was somewhat simplistic, as it has emerged from work outside NeuroNano, and using the foetal barrier as a model, that nanoparticles have the potential to signal across the biological barriers, without crossing themselves.(Bhabra 2009) Based on the work within NeuroNano on the in vitro blood brain barrier, another more pertinent question may be whether crossing of the barrier is actually not the key issue, but rather whether poisoning of the barrier cells due to endocytosis of nanoparticles and their localisation in the lysosomes of the barrier cells, with consequent signalling impacts, may in fact not be the greater potential harm. The logic for this is that for each nanoparticle that crosses the biological barrier, there are very many more nanoparticles trapped in the lysosomes of the barrier cells, as this has been shown to be the final location for large numbers of nanoparticles in both these and other cell lines. Thus, crossing of the biological barrier will be an extremely rare event, and instead accumulation in the endothelium is potentially the major sink for nanoparticles. This scenario of accumulation of the nanoparticles in the lysosomes of the barrier cells is consistent with everything we know at cell level. For that not to happen in barrier would require some very significant biological signalling to re-direct the nanoparticles from the endo-lysosomal pathway to the transcytotic pathways. In the same way that we have been unable 230 Compendium of Projects in the European NanoSafety Cluster
to identify exit pathways for nanoparticles from a range of cell types (e.g. lung epithelial cells), we have also However, discussions at the joint NeuroNano-ESF EpitopeMap workshop on nanoparticle interactions with biological barriers (6 th and 7 th December 2011) suggested that such large numbers of lysosomes have not been observed in vivo. Indeed most in vivo dosimetric studies consider the nanoparticle load in the brain endothelium as being in the brain (as distinct from being in the blood). This raises the need for some additional approaches to distinguish nanoparticles contained in the barrier tissue itself from nanoparticles that have actually crossed through the biological barrier into the brain tissue. It also suggests that further identification of the sub-cellular localisation of nanoparticles in tissue in vivo might shed some important light on this question. In vivo data showed that unconjugated Au (citrate stabilised) nanoparticles were rapidly cleared and within 30 minutes were almost all in liver following IV injection. Apo-E functionalised Au nanoparticles showed a different biodistribution in vivo, where the particle load in the liver decreased from 90 to 70% and the particle load in the spleen increased to close to 20%. In contrast, for Albumin-functionalised Au nanoaprticles, distribution in both the lungs and spleen was significant, while the particle load in the liver was dramatically low (Kreyling et al, manuscript in preparation). This links well with the findings that the nature of the adsorbed proteins has a dramatic role in determining the uptake and biodistribution of nanoparticles. In terms of access of these nanoparticles to the brain, Apo-E functionalised Au nanoparticles reached the brain by a factor of 10 more than citrate stabilised Au nanoparticles, and the nanoparticle load in the brain was observed to decrease over the subsequent 10 hours. The effect was even more prominent for the Albumin-Au NPs, suggesting that albumin plays a role in determining retention in endothelium or penetration into brain. Behavioural effects of nanomaterials A key element of the project was to connect exposure to nanomaterials with any observed behavioural changes. While much of the data is still under evaluation, and as such it is too early to draw significant conclusions, some behavioural effects were observed in two species studied – mice and ragworm. In the ragworm, exposure to both CuO2 (1000 ppb) and TiO2 (2000 ppb) nanoparticles resulted in a distinct decrease in burrowing behaviour, after 10 days of exposure, with some mortality also observed. Histology effects are currently being analysed. The full study is being repeated to determine the dose-response curve and redox proteomics is also underway. It is worth noting that the route of exposure for worms is oral, and that the worm is a model for Parkinson’s disease. Learning, memory, attention, and performance are elements of cognitive function that can be assessed in a variety of tests , such as the water maze or the lever-press response. Behavioural effects following direct injection of relatively high doses of TiO2 nanoparticles was assessed, and while the data are still being processed, prelimiary results suggest that here also effects were persisting over 10 days, which is an unusual response that is never observed with chemicals. NanoSafetyCluster - Compendium 2012 5 Significant outcomes / conclusions Radiolabelled nanoparticles synthesised by JRC A key output from NeuroNano is a range of radio-labelled nanoparticles for use in dosimetric / biokinetics or environmental fate and behaviour studies, or other studies where tracking of nanoparticles over specific time periods is required. Pre-synthesised Au, CeO2, Fe3O4, and TiO2 nanoparticles can be radiolabelled using ion-beam methods, and Au and Ag electrodes can be activated for subsequent nanoparticle synthesis. Significant progress towards activation of carbon-based nanoparticles has been made, with direct high-energy proton bombardment and a novel recoil method both achieving good levels of activity in MWCNTs. Some further work has to be performed to check for radiation damage effects. The recoil technique can be used to radiolabel nearly any type of nanoparticle, and successful initial experiments have been performed on SiO2 and nanodiamonds. Radiochemical synthesis methods can also be used to synthesise labelled nanoparticles starting from precursors with trace amounts of radioisotopes. In this way, SiO2 nanoparticles labelled with a Co- 56 radiotracer have been successfully created. This route can potentially be used to synthesise many different types of radiolabelled nanoparticle under carefully controlled laboratory conditions. Access to radiolabelled nanomaterials is possible via the QNano Research Infrastructure (www.qnano-ri.eu), or by contacting the JRC (Neil.Gibson@jrc.ec.europa.eu), assuming that all necessary safety provisions and permissions for handling radioactive materials are in place, that the irradiation/labelling process can be appropriately included in the JRC cyclotron’s schedule, and that the requesting institute provides the necessary input (knowledge, assistance, etc.) in the case of new experiments. Unique protocols developed covering a range of aspects Protocols covering a variety of aspects, from nanomaterials synthesis and characterisation, surface functionalisation and dispersion, to all aspects of nanoparticle uptake and localisation, apoptosis, assessment of changes in gene expression, oxidative stress and redox proteomics, dosimetrics and biokinetics and assessment of behavioural impacts of nanomaterials, have been developed within NeuroNano. Many have already been published as part of scientific manuscripts, and many others are currently in final stages of preparation for publication. As part of the wrap-up of the NeuroNano project, these protocols are currently being re-formatted into the template designed by NanoImpactNet and will be made available to the community via the NanoImpactNet protocols database, which will be taken over by QNano and/or the NanoSafety Cluster. Recommendations for further research As part of the NeuroNano final meeting (5th December 2011), and in collaboration with the ESF EpitopeMap Research Networking Compendium of Projects in the European NanoSafety Cluster 231
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Compendium of Projects in the Europ
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EDITED BY Michael Riediker, PD Dr.s
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CONTENTS NanoSafetyCluster - Compen
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Project Acronym ENNSATOX ENPRA EURO
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Foreword NanoSafetyCluster - Compen
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ENNSATOX ENgineered Nanoparticle Im
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The biological membrane and its dep
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distinguishes between the interacti
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Figure 10. Schematic representation
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WP5: • Permeability of NPs in hyd
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NanoSafetyCluster - Compendium 2012
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ENPRA RISK ASSESSMENT OF ENGINEERED
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vitro findings with in vivo models;
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protocols with real biological samp
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and it was organised in the frame o
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First Name Last Name Affiliation Ad
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EURO-NanoTox European Center for Na
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silver nanoparticles, iron nanopart
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The core functions of the Center, h
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8 Directory Table 1 Directory of pe
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HINAMOX Health Impact of Engineered
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High Resolution Electron Microscopy
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integrated risk assessment. Integra
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powders: What is the difference in
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InLiveTox Intestinal, Liver and End
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- to develop and validate a novel m
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environments, IEEE Industrial Elect
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INSTANT Innovative Sensor for the f
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complementary research which will l
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7 Copyright NanoSafetyCluster - Com
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ITS-NANO Intelligent testing strate
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approach to conveying the appropria
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in the discussion. The integration
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MARINA MANAGING RISKS of NANOMATERI
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for risk analysis, these tools need
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environmental ENM toxicity, but tox
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propose it as a Method Validation F
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and SiO2-NM200 and NM203, and a SiO
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ModNanoTox Modelling nanoparticle t
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3) To model environmental exposure
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Nanodetector European Network on th
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measurement interval. Measurements
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5 Directory Table 1 Directory of pe
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NANODEVICE Novel Concepts, Methods,
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ENP in order to be sure of their sa
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4.3 Exploring the association betwe
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NanoFATE Nanoparticle Fate Assessme
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or proteins associated with particu
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sludge. Information on rates of slu
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The exposures to be conducted will
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NanoFATE progression beyond the “
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Timing, hosts and locations of (grouped) events of NanoImpactNet

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