Abstract book. 3 rd NanoImpactNet Conference Building a bridge from NanoImpactNet to nanomedical research. Lausanne, Switzerland February 2011

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1 The European Network on the Health and Environmental Impact of Nanomaterials Abstract book 3 rd NanoImpactNet Conference Building a bridge from NanoImpactNet to nanomedical research Lausanne, Switzerland February 2011 Hosted by the Institute for Work and Health, Lausanne, Switzerland

2 NanoImpactNet is a European Commission-sponsored FP7 project The NanoImpactNet s 2011 Integrating Conference Gold sponsor is: Our Silver sponsor is: Our media partner is: Members of the NanoImpactNet consortium: Abstract Book - 2 / 121-3rd NanoImpactNet Conference

3 Content 1 NANO-PHARMACOLOGICAL INPUT TO RESEARCH ON THE HUMAN AND ENVIRONMENTAL IMPACT OF NANOMATERIALS 1.1 Oral presentations Blood clearance and tissue distribution of PEGylated and non-pegylated gold nanorods after intravenous administration in rats Identification of Protein-Nanoparticle Interaction Site Nose to brain delivery of nanostructured lipid carriers (NLC) loaded with an antidepressant drug Poster presentations Mechanistic studies of in vitro cytotoxicity of Polyamidoamine dendrimers in mammalian cells In vitro cytotoxicity assessment of nano-particulate silver in mammalian cell lines Nanostructured carrier based formulation of curcumin for bioavailability enhancement Novel food contact materials and the in vitro toxicity of low dose nano ZnO exposures to human intestinal cells Integration and Analysis of Available Information for Building Exposure Scenarios for Nanomaterials Genotoxicity of inhaled nanosized TiO 2 in mice Interaction of nanoparticles used in medical applications with lung epithelial cells: uptake, cytotoxicity, genotoxicity, oxidant stress and proinflammatory response Internalisation and transcytosis of SiO 2 and TiO 2 nanoparticles by lung epithelial cells Nanoparticles in Food Analytical methods for detection and characterisation Nanoparticles as potential cytostatic agents for treatment of leukemia Deposition of CNTs in the Respiratory Tract for two Industrial Exposure Scenarios Secondary characterization of TiO 2 nanoparticles in biological media by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) techniques Oxidative potential of fine and ultrafine particles in occupational situations Biogenic synthesis of Silver nanoparticles from aqueous extract of Solanum nigram and its characterization Migration of silver from plastic food containers 24 2 LESSONS FROM NANO-IMMUNOLOGY ON THE IMPACT OF NANOMATERIALS 2.1 Oral presentations Understanding Interactions of Engineered Nanomaterials with the Immune System The Effect of Two Iron Oxide Nanoparticles on Immune Response of Lymphocytes Identification of immune-related gene markers following interaction of engineered nanoparticles with human intestinal epithelial cells Immunosafety of engineered nanoparticles:methods implementation for the development of nanomedicines Poster presentations Responses of lung cell cultures after realistic exposure to secondary organic aerosols Biocompatibility of Zeolite-MFI nanoparticles in Human lung cells Viral ligands potentiate the human alveolar epithelial innate immune response to silver nanoparticles and carbon nanotubes Effect of sonication and serum proteins on copper release from copper nanoparticles and the toxicity towards lung epithelial cells Medical application of nano-sized magnetite and silica during pregnancy: in vitro studies assessing placental transport and toxicity Effects of nanoparticles on hepatocyte survival, mitochondrial function, antioxidant levels and cellular function Translocation of engineered and nanoscaled by-products from environment to human body 35 Abstract Book - 3 / 121-3rd NanoImpactNet Conference

4 2.2.8 Double-walled carbon nanotubes: suitable containers for biomedical applications A large local nanotoxicolgy project reveals old and new problems requiring strict interdisciplinarity Genotoxicity testing of titanium dioxide, iron oxide and silica nanoparticles in human lymphocytes and lymphyblastoid cells using micronucleus assay In vitro exposure of human macrophages to different functionalized multi-walled carbon nanotubes: what is the role of the pulmonary surfactant? Testing the toxicological profile of therapeutic nanoparticles: the example of a blood-brain barrier model Silver Wires Significantly Affect Cell Viability and Induce Immune Activation of A549 Cells NANOMMUNE: Comprehensive Assessment of Hazardous Effects of Engineered Nanomaterials on the Immune System Genotoxicity of nanocellulose whiskers in human bronchial epithelial cells measured by the micronucleus assay The retention of long, but not short, carbon nanotubes leads to inflammation and progressive fibrosis in the pleural space of mice The role of nanoparticle-protein interactions in determining the toxic consequences of nanoparticle exposure Biocompatibility of poly-n-isopropylacrylamide (PNIPAM) nanoparticles with human keratinocyte (HaCaT) and colon cells (SW 480) Comparing the interaction of Ag and Au nanoparticles with a 3D in vitro model of the epithelial airway barrier Assessment of cytotoxicity and genotoxicity of uncoated and oleic acid coated magnetite nanoparticles Molecular insight of the interaction between surface-tailored Si/SiO 2 wafers and fibrinogen Genotoxicity of zinc oxide nanoparticles in human mesothelial and bronchial epithelial cells in vitro 50 3 HUMAN IMPACT OF ENGINEERED NANOMATERIALS AND LESSONS FOR THE NANOMEDICAL FIELD Oral presentations Silicon nitride porous membranes for nanoparticle translocation in vitro assay Exposure of lung cells in vitro to zinc oxide: A comparison between suspension and aerosol exposure scenarios CuO nanoparticles act via a Trojan horse type mechanism A Screening Tool for Nanoparticles in Toxicity Experiments Poster presentations Use of fluorescent amorphous silica to study the intracellular fate of nanoparticles Kinetics of chitosan nanoparticles in mice Preliminary Eco-nanotoxicity results of C60 and Carbon Black assessed by established tests over a range of trophic levels in the Aquatic environment Ecotoxicity of fluorescent silica nanoparticles in a battery of freshwater test species Fate and behaviour of TiO 2 Nanomaterials in the environment influenced by their shape, size and surface area IMPLICATIONS FROM ENVIRONMENTAL FATE & BEHAVIOUR RESEARCH FOR THE FIELD OF NANOMEDICINE INVOLVING NANOMATERIALS Oral presentations Biological Interactions of Gold Nanoparticles: A Model System for Nanotoxicity? The assessment of exposure risk, persistence and accumulation of nanoparticle silver in the aquatic and marine environment A simple route to highly fluorescent silica nanoparticles for tracing the intracellular fate of nanoparticles 63 Abstract Book - 4 / 121-3rd NanoImpactNet Conference

5 4.1.4 Nanoparticles and their behaviour in biological fluid Poster presentations Bivalve immunocytes as a model for studying NP toxicity Effect of Dissolved Copper and NanoCopper on Histopathology and Haematopoietic Organs of Rainbow Trout (Oncorhynchus mykiss) Electrochemical Modelling of Nanoparticle Toxicity Establishment of a High Content Analysis (HCA) platform to assess nano-toxicology and explore nanoparticle-induced cell death SiO 2 nanoparticle trafficking across in vitro human blood-brain barrier Uptake of TiO 2 nanoparticles across the isolated perfused intestine of rainbow trout (Oncorhynchus mykiss) 70 5 STAKEHOLDER SESSION What benefits might nanomedicine offer? Involving stakeholders in setting research priorities - Reflections from consumers 6 SYMPOSIUMS 6.1 Immunosafety Task Force Kickoff The need for concerted action toward immunosafety of nanomaterials Immunosafety of nanomedicines: an introduction Nanoparticles in paints Risks of nanoparticle handling and sanding nanoparticle-containing paints Inflammatory and genotoxic effects of nanoparticles and dust generated from nanoparticlecontaining paints and lacquers Cardiovascular health effects of paint dust with and without nanoparticles compared to the primary nanoparticles Developmental and reproductive toxicity of nanoparticles Emission of Nanoparticles from Painted Surfaces Coatings and Nanoparticles Activities of the German Paint Industry on Workers Safety and Consumer Protection in the field of Smart Coatings 80 7 OTHER Poster presentations Harmonization of Measurement Strategies for the Assessment of Exposure to Manufactured Nano object; Report of a workshop Microvascular Distribution and Effects of Surface-modified Quantum Dots in Postischemic Tissues 82 U7.1.3U UNanoRiskCat A Conceptual Decision Support Tool for NanomaterialsU 83 U7.1.4U UEffect of nanoparticle morphology on the detection efficiency of condensation particle counters (CPCs)U 84 U7.1.5U UIn vitro evaluation of silver nanoparticles of different sizes in assays for cytotoxicity, inflammation and developmental toxicityu 85 U7.1.6U UAssessing the toxicological impact of a panel of engineered nanoparticles for risk assessment purposesu 86 U7.1.7U UCritical analysis of frameworks and approaches to assess the environmental risks of nanomaterialsu87 U7.1.8U UNANOGENOTOX: European Joint action on «Safety evaluation of manufactured nanomaterials by characterisation of their potential genotoxic hazard»u 88 U7.1.9U UDevelopment of a control banding tool adapted to nanomaterialsu 89 U7.1.10U UNanotubes of imogolite do not activate macrophages and modestly perturb the barrier properties of airway epithelial cells in vitrou 90 Abstract Book - 5 / 121-3rd NanoImpactNet Conference

6 Nanotoxicology at European Center for the Sustainable Impact of Nanotechnology-ECSIN and its research approach-an overview The basic requirement for comparable results between laboratories from in vitro tests remains a significant challenge Bioavailability of silver nanoparticles orally administered to rats IANH assessment of Nano-particle Cytotoxicity Cytotoxicity and genotoxicity induced in human and murine cell assays by copper oxide nanoparticles Forecasting Nano Law: The Small Matter of Big Risks NANODEVICE: Novel Concepts, Methods, and Technologies for the Production of Portable, Easyto-use Devices for the Measurement and Analysis of Airborne Engineered Nanoparticles in Workplace Air Social and Ethical Aspects of Nanomedicine: Sharing Benefits and Risks Cellular effects of nanosilver in human macrophages: Uptake, oxidative stress responses, lipid alterations and functional impairment Polystyrene: A potential standard for developing In Vitro cellular tracking methods for nanotoxicology Protective effect of biosynthesized AgNPs from Melia azedarach against Dalton s Ascites Lymphoma Ag and TiO 2 Nanoparticles: Effects on Model Aquatic Organisms Low-dose Single Wall Carbon Nanotubes affect embryonic development: an in vitro and in vivo study Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology Ingested metal nanoparticles pass through intestine epithelia and enter immune cells and gonads of the sea urchin Paracentrotus lividus Critical exposure to ultrafine particles during highway maintenance work Development of polysaccharide-based nanocarriers for drug delivery applications NanoImpactNet s Stakeholder Engagement Median lethal dose of titanium dioxide and oleic acid coated magnetite nanoparticles after single intravenous injection to adult rats Novel Hydrophilic Ce(III)-Doped Maghemite (γ-fe 2 O 3 ) Nanoparticles - Preliminary Toxicity Studies in Relation to the Nanoparticle Aggregation Level Development of Novel Nanotechnology Based Diagnostic Systems for Rheumatoid Arthritis and Osteoarthritis (NanoDiaRA) Biological responses induced in bronchial epithelial cells by carbon black and titanium dioxide nanoparticles: similar outcomes but distinct molecular pathways Toxicology of iron oxide nanoparticles: impact of the size and surface modifications In vitro Assessment of the Cellular Toxicity of Nanotubes An in vitro integrated ultrasensitive approach to biocompatibility analysis of silver nanowires Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic fields CNT-mediated wireless cell permeabilisation: drug and gene uptake Quantification of risk assessment in nanosafety: Determination of run-off effect of metallic nanoparticles in simulated body fluids AUTHOR INDEX 119 NanoImpactNet is a Coordination Action under the European Commission's 7 th Framework Programme. The 24 institutes organising the NanoImpactNet activities are leading European research groups active in the fields of nanosafety, nanorisk assessment and nanotoxicology. Contact Information: Michael Riediker, PD Dr.sc.nat., Coordinator NanoImpactNet Institute for Work and Health (Institut universitaire romand de Santé au Travail) Rue du Bugnon 21 / CH-1011 Lausanne / SWITZERLAND Phone: Fax: Web: Abstract Book - 6 / 121-3rd NanoImpactNet Conference

7 Session 1 1 Nano-pharmacological input to research on the human and environmental impact of nanomaterials 1.1 Oral presentations Blood clearance and tissue distribution of PEGylated and non-pegylated gold nanorods after intravenous administration in rats Daniëlle P.K. Lankveld 1, Raja G. Rayavarapu 2, Petra Krystek 3, Agnes G. Oomen 1, Hennie W. Verharen 1, Robert Geertsma 1, Ton G. van Leeuwen 2,4, Wim H. de Jong 1, Srirang Manohar 2 1 RIVM, Bilthoven, The Netherlands 2 University of Twente, Enschede, The Netherlands 3 MiPlaza Material Analysis, Philips Research, Eindhoven, The Netherlands 4 University of Amsterdam, Amsterdam, The Netherlands Aims: To develop and determine safety of gold nanorods whose aspect ratios can be tuned to obtain plasmon peaks between 650 nm and 850 nm, as contrast enhancing agents for diagnostic and therapeutic applications. Materials and methods: In this study we compared the blood clearance and tissue distribution of cetyl trimethyl ammonium bromide (CTAB) capped and poly ethylene glycol (PEG) coated gold nanorods after intravenous injection in the tail vein of rats. The gold content in blood and various organs was measured quantitatively with inductively coupled plasma mass spectrometry (ICP-MS). Results and discussion: The CTAB capped gold nanorods were almost immediately (< 15 minutes) cleared from the blood circulation whereas the PEGylation of gold nanorods resulted in a prolonged blood circulation with a half life time (t 1/2 ) of 19 h and more wide spread tissue distribution. While for the CTAB capped gold nanorods the tissue distribution was limited to liver, spleen and lung, the PEGylated gold nanorods also distributed to kidney, heart, thymus, brain and testes. PEGylation of the gold nanorods resulted in the spleen being the organ with the highest exposure whereas for the non-pegylated CTAB capped gold nanorods the liver was the organ with the highest exposure, per gram organ. Conclusions: The PEGylation of gold nanorods resulted in a prolongation of the blood clearance and the highest organ exposure in the spleen. In view of the time frame (up to 48 hours) of the observed presence in blood circulation PEGylated gold nanorods can be considered to be promising candidates for therapeutic and diagnostic imaging purposes. Abstract Book - 7 / 121-3rd NanoImpactNet Conference

8 1.1.2 Identification of Protein-Nanoparticle Interaction Site Luigi Calzolai, Fabio Franchini, Douglas Gilliland, François Rossi European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy AuNP The interaction of nanoparticles with proteins is a key parameter in nanomedicine and nanotoxicology. When nanoparticles (NP) interact with proteins, they might alter protein conformation, expose new epitopes on the protein surface or perturb the normal protein function, which could induce unexpected biological reactions and lead to toxicity. Here we show that is possible to characterize the interaction of gold nanoparticles to proteins at atomic level resolution. For the first time, by using state of the art Nuclear Magnetic Resonance techniques, it has been possible to identify the amino acids of the ubiquitin protein (shown in red in the figure) that bind to gold nanoparticles. Using NMR, chemical shift perturbation analysis, and dynamic light scattering we have identified a specific domain of human ubiquitin that interacts with gold nanoparticles.. The ubiquitin proteins interact with the gold surface via a limited number of amino acids that form a well-defined Au-binding area on the protein surface. These results open up the possibility of characterizing at atomic level resolution a large variety of nanoparticles-protein complexes in physiological conditions with a great potential impact in the nanotoxicology field and in nanomedicine. [1] Calzolai et al. Nano Lett. 2010, 10: Abstract Book - 8 / 121-3rd NanoImpactNet Conference

9 1.1.3 Nose to brain delivery of nanostructured lipid carriers (NLC) loaded with an antidepressant drug M. Intakhab Alam 1, Sanjula Baboota 1, Mushir Ali 1, Javed Ali 1, Alka Ahuja 2 1 Department of Pharmaceutics, Hamdard University, New Delhi, India 2 Oman Medical College, Azaiba, Muscat, Sultanate of Oman Nanostructured lipid carriers (NLC) loaded with duloxetine HCl (DLX) were prepared for intranasal application using glyceryl monostearate as solid core, capryol PGMC as liquid lipid material, and sodium taurocholate and pluronic F68 as stabilizers. NLC were prepared by homogenization followed by ultrasonication. These were characterized for surface morphology (TEM, SEM), particle size and distribution, drug loading and in vitro drug release. The pharmacodynamic evaluation (forced swimming test) was performed in albino Wistar rats after intranasal administration of NLC dispersion in chitosan gel (0.5% w/v). The average particle size of the NLC dispersion was estimated to be 125 nm with a polydispersity index of indicating a narrow particle size distribution. The TEM micrographs revealed that DLX loaded NLC were spherical in shape with smooth surfaces and uniformly distributed below 200 nm in diameter (Fig. 1). The nanoparticulate nature of the NLC dispersion particles was further confirmed by SEM studies (Fig. 2). The drug loading was found to be 2% w/w. The sustained release of DLX was observed from different formulations of NLC up to 24h of the study. In pharmacodynamic evaluation DLX-loaded NLC (NLC-DLX) treatment reduced immobility by 24% (p = 0.06) and 58% (p < 0.05) and increased climbing by 46% (p < 0.05) and 84% (p < 0.05) in comparison to the treatment with DLX dispersed in gel (Gel- DLX) and control (untreated) respectively (Fig. 3). DLX was effectively delivered to the brain by intranasal administration of formulated NLC dispersed in mucoadhesive gel. The study conducted in rats clearly demonstrated effectiveness of intranasal delivery of DLX as an antidepressant agent, however clinical data is needed to evaluate the risk vs. benefit ratio. Number of counts Immobility Climbing NLC-DLX Gel-DLX Control Fig.1: SEM image of NLC Fig.2: Mean counts of immobility and climbing in the test swim of rats (n= 6) Fig.3: TEM image of NLC Abstract Book - 9 / 121-3rd NanoImpactNet Conference

10 1.2 Poster presentations Mechanistic studies of in vitro cytotoxicity of Polyamidoamine dendrimers in mammalian cells Sourav Prasanna Mukherjee 1*, Fiona M. Lyng 1, Amaya Garcia 1, Maria Davoren 1, Hugh J. Byrne 2 1 Radiation and Environmental Science Centre, Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland 2 Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland The in vitro cytotoxic response of human dermal and colon cell lines to structurally well defined full generation cationic dendritic polyamidoamine (PAMAM) nanoparticles was investigated. Dendrimers of generations G4, G5, G6 were chosen for this study. PAMAM dendrimers have been demonstrated to elicit a well defined cytotoxicological response from Alamar Blue, Neutral Red and MTT assays, where the response increases systematically with dendrimer generation and number of surface amino groups 1. A good correlation was found between the EC 50 values of these assays 2. This systematic response is furthermore demonstrated for the generation of reactive oxygen species, inflammatory responses, lysosomal activity, caspase activation, onset of apoptosis and levels of DNA damage 2. The molecular mechanism of endosomal escape of PAMAM by the so-called proton-sponge effect was also studied. The results are consistent with a pathway of the endosomal uptake of PAMAM, followed by the endosomal rupture and subsequent localisation of PAMAM dendrimers in the mitochondria, leading to PAMAM generation, dose and time dependant biphasic ROS production and caspase- 8 and 3 activation, inflammatory responses (TNF-α, IL-6 and IL-8 expression), apoptosis and DNA damage (by TUNEL assay). Overall, significant differences are observed between the responses of the dermal and colon cell lines, and it is suggested that these can be understood in terms of differing intrinsic antioxidant levels 2. [1] Mukherjee, S.P., Davoren, M., Byrne, H.J., In vitro mammalian cytotoxicological study of PAMAM dendrimers Towards quantitative structure activity relationships. Toxicol. In Vitro 24, [2] Mukherjee, S.P., Lyng, F.M., Garcia, A., Davoren, M., Byrne, H.J., 2010, Mechanistic studies of in vitro cytotoxicity of Poly(amidoamine) dendrimers in mammalian cells, TAAP 248, Fig1. ROS localization in Mitochondria after 24 h PAMAM exposure. Selected as cover art of TAAP 2010, 248, Abstract Book - 10 / 121-3rd NanoImpactNet Conference

11 1.2.2 In vitro cytotoxicity assessment of nano-particulate silver in mammalian cell lines Sanchali Gupta Mukherjee 1,2, Niall Ó Claonadh 1, Gordon Chambers 1,2 and Alan Casey 1,2 1 Focas Institute, Dublin Institute of Technology, Dublin, Ireland 2 School of Physics, Dublin Institute of Technology, Dublin, Ireland In this study the cytotoxic effect of commercially available silver (Ag) nanopowder was evaluated using four different cell lines, namely SW480 (ATCC, CCL-228), HT29 (ATCC, HTB-38 TM ), HeLa (ATCC, CCL-2 TM ) and HaCaT. Prior to the cellular studies a full particle size characterisation was carried out using Dynamic Light Scattering, Transmission Electron Microscopy and Atomic Force Microscopy. The surface charge and Zeta Potential associated with the nano Ag was also determined in order to assess its stability in solution. The toxic effects of Ag nanopowder were then evaluated using five cytotoxic endpoints namely the lysosomal activity, mitochondrial metabolism, basic cellular metabolism, cellular protein content and cellular proliferative capacity. The cytotoxic effect of Ag nanoparticle was dependant on dose, exposure time and on the cell line tested. Further investigation was carried out on HeLa and HaCaT cell lines to elucidate the mechanism of its cytotoxicity. The Ag nanopowder was noted to induce elevated levels of oxidative stress and apoptosis. Overall, significant differences are observed between the responses of the four different cell lines. Abstract Book - 11 / 121-3rd NanoImpactNet Conference

12 1.2.3 Nanostructured carrier based formulation of curcumin for bioavailability enhancement Sanjula Baboota 1, Anil Kumar 1, Javed Ali 1, Alka Ahuja 1,2 1 Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India 2 Department of Pharmacy, Oman Medical College, Azaiba, Muscat Curcumin has surprisingly a wide range of beneficial properties, including anti-inflammatory, antioxidant, chemopreventive, radiosensitizing, wound healing activities, antiviral, antifungal and chemotherapeutic activity. The use of curcumin is limited due to low aqueous solubility under acidic or neutral conditions and high decomposition rate in alkaline media. Low aqueous solubility leads to poor bioavailability of curcumin [1]. The purpose of the present work was to formulate nanocarrier based nanoemulsion (NE) of curcumin for solubility and bioavailability enhancement. The solubility of curcumin in various oils including fish oil, sesame oil, Caprol 10 G 100, Labrafac 1349 and Captex GTO were determined by dissolving excess amount of curcumin in 2 ml of each oil in 5 ml stoppered vials and after solubility selection, the pseudoternary phase diagram by spontaneous emulsification method were constructed. To overcome the problem of metastable formulation, physical stability tests including heating cooling cycle, centrifugation and freeze thaw cycle were performed. The formulations were characterized for the droplet size, zeta potential, transmission electron microscopy (TEM). The stability of ethanolic curcumin and nanoemulsion were performed in phosphate buffer (ph 6.8) for 1 month. In vitro release studies for NEs and control (curcumin was dispersed in water and sonicated for 5 min) were performed in phosphate buffer (ph 6.8), (900 ml) at 100 rpm using basket type apparatus. The NE and dispersed curcumin were filled in excised duodenum part of intestinal tract of rat and were placed in beaker. The samples were withdrawn at predetermined time intervals and analysed with UV spectrophotometer. The solubility of curcumin was found to be highest in Labrafac 1349 (18.87 ±0.82 mg/ml) so it was used as oil phase. Unitop FFT 40 and PEG 400 were selected as surfactant and cosurfactant due to good miscibility with Labrafac After physical stability study the most stable nanoemulsion formulation were selected. The average particle size, polydispersity index and zeta potential of the nanoemulsions were in the range of nm, and -5 to -32 mv respectively. The particles were spherical in morphology as observed by TEM. In alkaline stability study, after 24 hours only 12% drug was remained in the ethanolic solution containing phosphate buffer while no significant degradation was observed in case of curcumin loaded NE up to one month. It was also observed that there was no effect of light on the degradation of curcumin in the formulation. During in vitro study the release of curcumin from intestine was not detected with control while with nanoemulsion formulations the drug release was in the range of 74-94% in phosphate buffer (ph 6.8) at 4 hr. The enhanced releases of the curcumin with nanoemulsions were attributed to the small particle size leading to large surface area of drug. The study demonstrates that nanoemulsion formulation can be employed to improve the bioavailability of a poorly water soluble drug like curcumin. [1] Anil Kumar, Ahuja et al Conundrum and therapeutic potential of curcumin in drug delivery. Crit Rev Therap Drug Carrier Syst: 27(4), Abstract Book - 12 / 121-3rd NanoImpactNet Conference

13 1.2.4 Novel food contact materials and the in vitro toxicity of low dose nano ZnO exposures to human intestinal cells Niall Ó Claonadh, Alan Casey, Gordon Chambers Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland Nano Zinc Oxide (nzno) has been shown to display antimicrobial effects which have lead to its application in a number of areas such as antimicrobial surface coatings, anti bacterial wound dressings and more recently in polymer composite systems for use in food contact materials. Concerns have been raised due to the incorporation of nanoparticles in food packaging stemming from the possibility of repeated low dose direct exposure, through ingestion, primarily due to degradation and nanoparticle leaching from the polymer composite. To address these concerns, composites consisting of nzno and polyethylene were formed using twin screw extrusion to mimic commercial methods of food contact material production. A leaching study was performed using Atomic Absorbtion Spectroscopy in order to determine the concentration of nzno leached from the composite. In this study two human colorectal carcinoma cell lines, HT29 (ATCC No: HTB-38) and SW480 (ATTC No: CCL-228), were employed as an intestinal model. These lines were exposed to a concentration range of nzno which incorporated the concentration leached from the composites. Prior to any cellular studies a full particle size characterisation was carried out using Dynamic Light Scattering, Transmission Electron Microscopy and Atomic Force Microscopy. The Zeta Potential associated with nzno was also determined in order to assess its stability in solution along with its surface charge. The cytotoxic effects of nzno were then evaluated using five cytotoxic endpoints namely the Neutral Red, Alamar Blue, Coomassie Blue, MTT and Clonogenic assays. An initial investigation into the mechanism by which nzno causes cytotoxicity was also performed. This consisted of a simple colorimetric determination of reactive oxygen species formed. Direct exposure of both cell lines to the nanoparticles ZnO revealed a cytotoxic effect which was dependant on dose, exposure time and on cell line tested. The results of these studies are presented and their implications for the use on nano ZnO in direct food contact surfaces will be discussed. Abstract Book - 13 / 121-3rd NanoImpactNet Conference

14 1.2.5 Integration and Analysis of Available Information for Building Exposure Scenarios for Nanomaterials Katherine Clark 1, Rob Aitken 2, Derk Brouwer 3, Frans Christensen 4, Rianda Gerritsen 3, Christian Micheletti 4, Kaspar Schmid 2, Martie van Tongeren 2, the NANEX Consortium 5, Michael Riediker 1 1 Institute for Work and Health, Lausanne, Switzerland 2 Institute of Occupational Medicine, Edinburgh, United Kingdom 3 TNO Quality of Life, Zeist, The Netherlands 4 Joint Research Centre, Ispra, Italy 5 NANEX partner institutions, pan-europe The goal of the EU FP7 NANEX project was to develop a catalog of generic and specific exposure scenarios (ES) covering the life cycle of certain uses of nano-tio 2, nano-ag, and carbon nanotubes. Leading scientists from twelve partner institutions in Europe compiled exposure information from a variety of sources that was relevant to occupational exposure, consumer exposure, and environmental release, including literature, industry case studies and exposure estimation models. This information was then used to develop ES in a format similar to that outlined by the European Chemicals Agency for compliance with the REACH regulation. Both the information used to build the ES and the ES themselves were evaluated for quality and completeness, and research needs were identified. The ES developed in NANEX should not be considered final exposure scenarios due to the limited information available in the public domain and as the ES have not been 'validated' vs. no-effect levels (outside the scope of Nanex). Although several studies describe uses of manufactured nanomaterials (MNM) in consumer products, very few studies contained specific or quantitative information on amount in and release from such products, making it difficult to build reasonable ES. Over 75% of the occupational studies reviewed that contained quantitative exposure information were associated with primary manufacture of MNM (largely lab/pilot scale information), and very little exposure information was found on exposure to downstream users. Lack of information on context or sampling strategy made it difficult to compare the results of these studies. It was demonstrated that current models to estimate worker or consumer exposure are not accurate since they are neither calibrated nor validated for specific nano exposure features. Due to lack of detection methods and knowledge of use volumes for MNMs, modeling is currently the best method available to estimate MNM release to the environment. Overall, the ES that were developed were often missing information or could only be completed using highly uncertain information. The development of ES is challenged by both limited availability of exposure information and lack of standardization for interpreting and reporting information relevant to exposure conditions and exposure levels. The aim of future research should be to determine which factors (e.g., activity, material characteristics, operational conditions and risk management measures) are the greatest determinants of exposure and which types of information are most useful for describing exposure level. In the short term, while waiting for more precise exposure and hazard information on MNM, attention should be on risk management strategies. Abstract Book - 14 / 121-3rd NanoImpactNet Conference

15 1.2.6 Genotoxicity of inhaled nanosized TiO 2 in mice Hannu Norppa 1, Hanna K Lindberg 1, Ghita C-M Falck 1, Joonas Koivisto 1, Elina Rossi 1, Lea Pylkkänen 1, Heli Nykäsenoja 1, Hilkka Järventaus 1, Satu Suhonen 1, Minnamari Vippola 1,2, Julia Catalán 1,3, Kai Savolainen 1 1 Finnish Institute of Occupational Health, Helsinki, Finland 2 Tampere University of Technology, Tampere, Finland 3 University of Zaragoza, Zaragoza, Spain In vitro studies have suggested that nanosized TiO 2 has genotoxic properties in various cell systems. The significance of these findings with respect to in vivo effects is presently unclear, since very few in vivo genotoxicity studies on TiO 2 exist. Recently, nanosized TiO 2 administered in drinking water was reported to be genotoxic in mice, including induction of micronuclei (MN) in peripheral blood polychromatic erythrocytes (PCEs), among other effects. The apparent systemic genotoxic effect, observed in a tissue remote from the exposure route, was proposed to be reflect secondary genotoxicity of TiO 2 nanoparticles due to inflammation. We studied the in vivo genotoxicity of nanosized TiO 2 in C57BL/6J mice after a 5-day inhalation exposure (4 h/day) to 0.8, 7.2, and 28.5 mg/m 3 (respective average particle sizes 86, 76, and 116 nm) of anatase (74%) and brookite (26%) from a gas-to-particle aerosol generator. DNA damage was assessed by the comet assay in lung cells sampled immediately following the exposure. Micronuclei were analyzed by acridine orange staining in peripheral blood PCEs collected 48 h after the exposure. A dose-dependent deposition of Ti in lung tissue was seen. Although the highest exposure level resulted in a clear increase in neutrophils in bronchoalveolar lavage fluid, suggesting an inflammatory effect, no significant increase in the level of micronucleated cells in blood or DNA damage in lungs was observed. Our findings indicate no genotoxic effects by the 5-day inhalation exposure to nanosized TiO 2 anatase under the experimental conditions applied. On the other hand, systemic TiO 2 doses were probably much lower in our inhalation experiment than in the previous drinking water study. Funded by the European Commission (NANOSH, NMP4-CT ) and the Academy of Finland Abstract Book - 15 / 121-3rd NanoImpactNet Conference

16 1.2.7 Interaction of nanoparticles used in medical applications with lung epithelial cells: uptake, cytotoxicity, genotoxicity, oxidant stress and proinflammatory response Rina Guadagnini 1, Sonja Boland 1, Sandra Vranic 1, Salik Hussain 1, Kevin Moreau 1, Caroline Borot 1, Francelyne Marano 1 1 Laboratory of Functional and Adaptative Biology, unit of Réponses Moléculaires et Cellulaires aux Xénobiotiques (RMCX), CNRS EAC, University Paris Diderot, Paris, France In view of the considerable development of nanotechnologies and nanomedicine it s important to evaluate the potential risk of NPs for human health. Our goal was to determine the effects of nanoparticles (NPs) on the lung as first target during inhalation of NPs. We investigated the effects of different NPs [titanium dioxide (TiO 2 ), poly (lactic-co-glycolic acid) (PLGA), non coated Fe 3 O 4 (N Fe 3 O 4 ), Fe 3 O 4 coated with oleic acid (C Fe 3 O 4 ) and Fluorescent Silica oxides (SiO 25nm and 50nm)] on human bronchial (16HBE line) and human alveolar type II cells (A549 line). We evaluated the cytotoxicity of these NPs by WST-1 assay and propidium iodide incorporation showing that toxicity depends on particle type, size and coating. We investigated the genotoxic potential of NPs by Comet Assay. We determined the ability of NPs to enter cells measuring by flow cytometry the right angle scattering of the laser and we notice that they can be internalized by cells. We measured also the induction of oxidative stress in 16HBE and A549 cells after 24 and 48h of treatment with NPs by dihydroethidium oxidation assay (flow cytometry) seeing that they have different ability to induce oxidant stress. Finally we investigated whether NPs have capacity to induce inflammatory reponse evaluating the thiol content of A549 and 16HBE cells after treatment with N-ethyl-maleimide, buthionine sulfoximine, NPs by monobromobimane (mbbr) assay (flow cytometry). We determined the production of cytokines (GM-CSF, IL-8, IL-6, IL-1beta) by A549 and 16HBE cells after 24 and 48 hours of treatment with NPs by ELISA test and by RT- qpcr. Results show that at non toxic concentration NPs can induce inflammation in cells. Supported by FP7 program NanoTEST Abstract Book - 16 / 121-3rd NanoImpactNet Conference

17 1.2.8 Internalisation and transcytosis of SiO 2 and TiO 2 nanoparticles by lung epithelial cells Sandra Vranic 1, Rina Guadagnini 1, Armelle Baeza 1, M. Caroline Borot 1, Francelyne Marano 1, Sonja Boland 1 1 Laboratoire de Biologie Fonctionnelle et Adaptative, équipe Réponses Moléculaires et Cellulaires aux Xénobiotiques (RMCX), CNRS EAC 7059, Université Paris Diderot, Paris, France In view of the considerable development of nanotechnologies it is important to evaluate their potential risk for human health. Our goal was to determine the cytotoxic effects of nanoparticles (NPs) in the lung, which is the first target after inhalation of NPs. We further studied the endocytosis of NPs by respiratory epithelial cells and their capacity to cross the epithelial barrier. We investigated the effects of different NP [fluorescently labelled or non fluorescent titanium dioxide (TiO 2 ) and silicium dioxide (SiO 2 )] on human bronchial epithelial cells (16HBE14O-), bronchial glandular adenocarcinoma cells (Calu-3) and human mucoepidermoid carcinoma cells (NCI-H292). First we evaluated the cytotoxicity of NPs by WST-1 assay. TiO 2 NPs are cytotoxic for 16HBE and NCI-H292 cell lines at high concentrations inducing apoptosis. SiO 2 NPs are cytotoxic in a size-dependent manner. We also evaluated quantitatively and qualitatively the endocytosis of NPs by epithelial cells. We determined the ability of NPs to enter 16HBE and NCI-H292 cells by measuring with a flow cytometer the right angle scattering of the laser or intensity of fluorescence of the cells treated with fluorescent NPs. We further studied the endocytosis of NPs by confocal microscopy to determine which of the three major endocytotic pathways (clathrin dependent, caveolin dependent or macropinocytosis) is involved in the internalisation of TiO 2 NPs. For this study we used specific inhibitors for each pathway after evaluating the specificity of each inhibitor using positive controls for each endocytotic pathway. TiO 2 and SiO 2 NPs are internalized by respiratory epithelial cells using predominantly clathrin dependent cellular machinery, but we have shown a poor specificity of the inhibitors used. Regarding the transcytosis of NPs we examined the possibility of NPs to pass through pulmonary epithelial barriers. First we compared the capacity of different cell lines to develop a tight epithelial layer by measuring the transepithelial electric resistance (TEER), passage of fluorescent molecule Lucifer Yellow, marker of paracellular passage and regarding by confocal microscopy the expression of proteins specific for tight junctions. After establishing a model by comparing different cell lines and culture conditions (Transwells with pore size of 0.4µm and 3µm) we evaluated the possibility of transcytosis of NPs. TiO 2 and SiO 2 NPs are able to cross the epithelial barrier but the percentage of particles crossing the epithelium is very low. In conclusion, NPs are cytotoxic at high concentrations, depending on the cell line used and on their size. However, at non cytotoxic concentrations these NPs are taken up by respiratory epithelial cells but have poor capacity to cross the epithelial barrier by transcytosis. This work was supported by grant from EC FP (Nanotest) and EC FP (ENPRA), National Grant Nanotrans. Abstract Book - 17 / 121-3rd NanoImpactNet Conference

18 1.2.9 Nanoparticles in Food Analytical methods for detection and characterisation Stefan Weigel, Ruud Peters, Hans Bouwmeester RIKILT Institute of Food Safety, Wageningen UR, Wageningen, The Netherlands The Scientific Committee [of EFSA] makes a series of recommendations; in particular, actions should be taken to develop methods to detect and measure ENMs [engineered nanomaterials] in food/feed and biological tissues, to survey the use of ENMs in the food/feed area, to assess the exposure in consumers and livestock, and to generate information on the toxicity of different ENMs. [1]. The above citation illustrates well the current situation with view to the analysis of engineered nanoparticles (ENP) in food. At the moment, nanotechnology applications for the food sector are intensively investigated and developed. A number of nanomaterials are already in use as food additives or in food contact materials. At the same time, very limited knowledge is available on the potential impact of ENP on consumers health. Exposure of the consumer to ENP cannot be determined due to the lack of appropriate analytical methods. This gap is addressed by the FP7 project NanoLyse. The NanoLyse project focusses on the development of validated methods and reference materials for the analysis of engineered nano-particles (ENP) in food and beverages. The developed methods will cover relevant classes of ENP with reported or expected food and food contact material applications, i.e. metal, metal oxide/silicate, and encapsulate ENP. Priority ENPs have been selected out of each class as model particles to demonstrate the applicability of the developed approaches, e.g. nano-silver for the metal NPs. Priority is given to methods which can be implemented in existing food analysis laboratories. A dual approach is followed. Rapid imaging and screening methods will allow the distinction between samples which contain ENP and those that do not. These methods will be characterised by minimal sample preparation, costefficiency and high throughput. More sophisticated, hyphenated methods will allow the unambiguous characterisation and quantification of ENP. These will include elaborate sample preparation, separation by field flow fractionation and chromatographic techniques as well as mass spectrometric and electron microscopic characterisation techniques. The developed methods will be validated using the well characterised food matrix reference materials that will be produced within the project. Small-scale interlaboratory method performance studies and the analysis of a few commercially available products claiming or suspect to contain ENP will demonstrate the applicability and soundness of the developed methods. [1] EFSA 2009, Scientific Opinion of the Scientific Committee on the Potential Risks Arising from Nanoscience and Nanotechnologies on Food and Feed Safety, The EFSA Journal 958, 1-39) Abstract Book - 18 / 121-3rd NanoImpactNet Conference

19 Nanoparticles as potential cytostatic agents for treatment of leukemia Ylva Rodhe and Lennart Möller Karolinska Institutet, Stockholm, Sweden Background: The malignant disease leukaemia affects the blood and bone marrow, causing an abnormal accumulation of blood cells. Both adults and children are affected and as many as 100,000 people in the US and in the EU were expected to develop leukaemia in Despite the improved survival rates in cancer in recent years, many survivors treated with cytostatic agents will experience long-term side effects including cardiotoxic effects, secondary tumours, growth retardation in children, hormonal disturbances and infertility. The average mean survival rate of leukaemia in the EU is today 34 % and it is highly important to improve leukaemia treatment and to find alternatives to the methods used today. The fast developing nanotechnology reveals new possibilities in cancer treatment. Nanoparticles are particles smaller than 100 nm and can have different chemical composition, including metals and metal oxides. Due to their small size with a large surface area per unit weight, they exert special characteristics different to larger particles, e.g. altering biological activity, reactivity, colour and magnetic properties. Studies have shown that some metal nanoparticles can cause increased levels of oxidative stress, inflammation and DNA damage in human cells. Nanoparticles can be used in cancer treatment as carriers of anticancer drugs, as a tool in radiation therapy or as a cytostatic agent itself. The aim with this project is to investigate the toxicology and biocompability of different metal nanoparticles, both on normal cells and leukaemia cells. The approach is to find a metal-based nanoparticle with a more potent anticancer activity but less system toxicity, compared to what is used today in leukaemia treatment. Methods: The leukaemia cell lines HL60, K562 and Jurkat cells are used to evaluate toxic effects of a wide range of metal nanoparticles. As reference cells, general human cell lines and fresh lymphocytes from healthy donors are used. The cells are incubated with different nanoparticles and the toxicity is assessed in terms of cytotoxicity using trypan blue staining, morphology studies, general DNA damage and oxidative DNA damage using the comet assay, mitochondrial damage using flow cytometry and protein expression using Western blot analysis. Results and conclusions: Preliminary results from screening of nanoparticles show that the cytotoxicity varies greatly in the human lung cell line A549. Gold and ferrous nanoparticles did not generate cytotoxicity, whereas zinc nanoparticles generated up to 100% cytotoxicity after an exposure of 80 microg/ml for 18 hours. Differences are also seen when comparing cytotoxicity between A549, three leukaemia cell lines and fresh lymphocytes. This indicates that certain nanoparticles are potential cytostatic agents. Further research is required to find a candidate with high anti-cancer activity and low systemic toxicity. Abstract Book - 19 / 121-3rd NanoImpactNet Conference

20 Deposition of CNTs in the Respiratory Tract for two Industrial Exposure Scenarios Marika Pilou 1,5, Gaelle Uzu 2, Vasilis Gkanis 1, Derk Brouwer 3, Martie van Tongeren 4 and Christos Housiadas 1 1 Thermal Hydraulics & Multiphase Flow Laboratory, NCSR Demokritos, Agia Paraskevi, Greece 2 NanoChemistry and NanoSafety Laboratory, CEA, Grenoble, France 3 TNO Quality of Life, Zeist, The Netherlands 4 Institute of Occupational Medicine, Edinburgh, UK 5 Laboratory of Biofluid Mechanics & Biomedical Engineering, School of Mechanical Engineering, Athens, Greece Carbon nanotubes (CNTs) are a diverse group of materials, which have various attractive physicochemical properties for use in many industrial and biomedical applications. Nevertheless, there are indications that chronic occupational inhalation of CNTs may lead to adverse health effects [1]. In the present study, a mechanistic dosimetry model [2] was used to calculate particle deposition along the human respiratory tract during exposure to CNTs. The model solves numerically the general dynamic equation (GDE) of the aerosol population in an Eulerian framework. Moreover, the simultaneous action of different mechanisms, such as sedimentation, diffusion, and inertial impaction, on the inhaled aerosol is assumed to result in particle deposition. Interception was not modelled in the present calculations because only the aerodynamic characteristics of the CNTs were known. Measurements had been performed inside an industrial site in France, during the handling and pouring of CNTs. A CPC Grimm spectrometer was used to measure particles number concentration based on the aerodynamic diameter, ranging between 5.5 nm and 350 nm. Simulations were carried out to estimate CNTs deposition in different regions of the respiratory tract during the aforementioned processes. The physiological parameters used in the model were those of an adult worker undergoing light activity, proposed by ICRP [3], under the assumption that no protective measures were taken (worst case scenario). Acknowledgements: This work is partially supported by project NanEx under Contract No. NMP of FP7 of the European Commission [1] Aschberger, K. et al Review of carbon nanotubes toxicity and exposure-appraisal of human health risk assessment based on open literature. Critical Reviews in Toxicology 40: [2] Mitsakou, C. et al Eulerian modelling of lung deposition with sectional representation of aerosol dynamics. Journal of Aerosol Science 36 :75-94 [3] Annals of the ICRP, 1994, Vol.24(1-3) Abstract Book - 20 / 121-3rd NanoImpactNet Conference

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