The European Network on the Health and
Environmental Impact of Nanomaterials
Abstract book
3rd NanoImpactNet Conference
Building a bridge from NanoImpactNet to nanomedical research
Lausanne, Switzerland
14-17 February 2011
Hosted by the Institute for Work and Health, Lausanne, Switzerland
The European Network on the Health and Environmental Impact of Nanomaterials
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:
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Content
1 NANO-PHARMACOLOGICAL INPUT TO RESEARCH ON THE HUMAN AND
ENVIRONMENTAL IMPACT OF NANOMATERIALS
1.1
Oral presentations
1.1.1 Blood clearance and tissue distribution of PEGylated and non-PEGylated gold nanorods after
intravenous administration in rats
1.1.2 Identification of Protein-Nanoparticle Interaction Site
1.1.3 Nose to brain delivery of nanostructured lipid carriers (NLC) loaded with an antidepressant drug
7
7
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8
9
Poster presentations
10
1.2
10
1.2.1 Mechanistic studies of in vitro cytotoxicity of Polyamidoamine dendrimers in mammalian cells
11
1.2.2 In vitro cytotoxicity assessment of nano-particulate silver in mammalian cell lines
12
1.2.3 Nanostructured carrier based formulation of curcumin for bioavailability enhancement
1.2.4 Novel food contact materials and the in vitro toxicity of low dose nano ZnO exposures to human
13
intestinal cells
1.2.5 Integration and Analysis of Available Information for Building Exposure Scenarios for Nanomaterials
14
15
1.2.6 Genotoxicity of inhaled nanosized TiO2 in mice
1.2.7 Interaction of nanoparticles used in medical applications with lung epithelial cells: uptake,
16
cytotoxicity, genotoxicity, oxidant stress and proinflammatory response
17
1.2.8 Internalisation and transcytosis of SiO2 and TiO2 nanoparticles by lung epithelial cells
18
1.2.9 Nanoparticles in Food Analytical methods for detection and characterisation
Nanoparticles as potential cytostatic agents for treatment of leukemia
19
1.2.10
Deposition of CNTs in the Respiratory Tract for two Industrial Exposure Scenarios
20
1.2.11
Secondary characterization of TiO2 nanoparticles in biological media by Dynamic Light Scattering
1.2.12
21
(DLS) and Transmission Electron Microscopy (TEM) techniques
Oxidative potential of fine and ultrafine particles in occupational situations
22
1.2.13
Biogenic synthesis of Silver nanoparticles from aqueous extract of Solanum nigram and its
1.2.14
23
characterization
Migration of silver from plastic food containers
24
1.2.15
2
LESSONS FROM NANO-IMMUNOLOGY ON THE IMPACT OF NANOMATERIALS 25
2.1
Oral presentations
2.1.1 Understanding Interactions of Engineered Nanomaterials with the Immune System
2.1.2 The Effect of Two Iron Oxide Nanoparticles on Immune Response of Lymphocytes
2.1.3 Identification of immune-related gene markers following interaction of engineered nanoparticles
with human intestinal epithelial cells
2.1.4 Immunosafety of engineered nanoparticles:Methods implementation for the development of
nanomedicines
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Poster presentations
29
2.2
29
2.2.1 Responses of lung cell cultures after realistic exposure to secondary organic aerosols
30
2.2.2 Biocompatibility of Zeolite-MFI nanoparticles in Human lung cells
2.2.3 Viral ligands potentiate the human alveolar epithelial innate immune response to silver
31
nanoparticles and carbon nanotubes
2.2.4 Effect of sonication and serum proteins on copper release from copper nanoparticles and the
32
toxicity towards lung epithelial cells
2.2.5 Medical application of nano-sized magnetite and silica during pregnancy: in vitro studies assessing
33
placental transport and toxicity
2.2.6 Effects of nanoparticles on hepatocyte survival, mitochondrial function, antioxidant levels and
34
cellular function
35
2.2.7 Translocation of engineered and nanoscaled by-products from environment to human body
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2.2.8 Double-walled carbon nanotubes: suitable containers for biomedical applications
36
2.2.9 A large local nanotoxicolgy project reveals old and new problems requiring strict interdisciplinarity37
Genotoxicity testing of titanium dioxide, iron oxide and silica nanoparticles in human lymphocytes
2.2.10
38
and lymphyblastoid cells using micronucleus assay
In vitro exposure of human macrophages to different functionalized multi-walled carbon
2.2.11
39
nanotubes: what is the role of the pulmonary surfactant?
Testing the toxicological profile of therapeutic nanoparticles: the example of a blood-brain barrier
2.2.12
40
model
Silver Wires Significantly Affect Cell Viability and Induce Immune Activation of A549 Cells
41
2.2.13
NANOMMUNE: Comprehensive Assessment of Hazardous Effects of Engineered Nanomaterials on
2.2.14
42
the Immune System
Genotoxicity of nanocellulose whiskers in human bronchial epithelial cells measured by the
2.2.15
43
micronucleus assay
The retention of long, but not short, carbon nanotubes leads to inflammation and progressive
2.2.16
44
fibrosis in the pleural space of mice
The role of nanoparticle-protein interactions in determining the toxic consequences of
2.2.17
45
nanoparticle exposure
Biocompatibility of poly-N-isopropylacrylamide (PNIPAM) nanoparticles with human keratinocyte
2.2.18
46
(HaCaT) and colon cells (SW 480)
Comparing the interaction of Ag and Au nanoparticles with a 3D in vitro model of the epithelial
2.2.19
47
airway barrier
Assessment of cytotoxicity and genotoxicity of uncoated and oleic acid coated magnetite
2.2.20
48
nanoparticles
Molecular insight of the interaction between surface-tailored Si/SiO2 wafers and fibrinogen
49
2.2.21
Genotoxicity of zinc oxide nanoparticles in human mesothelial and bronchial epithelial cells in
2.2.22
50
vitro
3 HUMAN IMPACT OF ENGINEERED NANOMATERIALS AND LESSONS FOR THE
NANOMEDICAL FIELD
51
3.1
Oral presentations
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51
3.1.1 Silicon nitride porous membranes for nanoparticle translocation in vitro assay
3.1.2 Exposure of lung cells in vitro to zinc oxide: A comparison between suspension and aerosol exposure
52
scenarios
53
3.1.3 CuO nanoparticles act via a Trojan horse type mechanism
54
3.1.4 A Screening Tool for Nanoparticles in Toxicity Experiments
Poster presentations
3.2
3.2.1 Use of fluorescent amorphous silica to study the intracellular fate of nanoparticles
3.2.2 Kinetics of chitosan nanoparticles in mice
3.2.3 Preliminary Eco-nanotoxicity results of C60 and Carbon Black assessed by established tests over a
range of trophic levels in the Aquatic environment
3.2.4 Ecotoxicity of fluorescent silica nanoparticles in a battery of freshwater test species
3.2.5 Fate and behaviour of TiO2 Nanomaterials in the environment influenced by their shape, size and
surface area
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55
56
57
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4 IMPLICATIONS FROM ENVIRONMENTAL FATE & BEHAVIOUR RESEARCH FOR
THE FIELD OF NANOMEDICINE INVOLVING NANOMATERIALS
61
4.1
Oral presentations
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61
4.1.1 Biological Interactions of Gold Nanoparticles: A Model System for Nanotoxicity?
4.1.2 The assessment of exposure risk, persistence and accumulation of nanoparticle silver in the aquatic
62
and marine environment
4.1.3 A simple route to highly fluorescent silica nanoparticles for tracing the intracellular fate of
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nanoparticles
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4.1.4
Nanoparticles and their behaviour in biological fluid
64
Poster presentations
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4.2
65
4.2.1 Bivalve immunocytes as a model for studying NP toxicity
4.2.2 Effect of Dissolved Copper and NanoCopper on Histopathology and Haematopoietic Organs of
66
Rainbow Trout (Oncorhynchus mykiss)
67
4.2.3 Electrochemical Modelling of Nanoparticle Toxicity
4.2.4 Establishment of a High Content Analysis (HCA) platform to assess nano-toxicology and explore
68
nanoparticle-induced cell death
69
4.2.5 SiO2 nanoparticle trafficking across in vitro human blood-brain barrier
4.2.6 Uptake of TiO2 nanoparticles across the isolated perfused intestine of rainbow trout (Oncorhynchus
mykiss) 70
5
STAKEHOLDER SESSION
5.1.1
5.1.2
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What benefits might nanomedicine offer?
Involving stakeholders in setting research priorities - Reflections from consumers
SYMPOSIUMS
71
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73
6.1
Immunosafety Task Force Kickoff
6.1.1 The need for concerted action toward immunosafety of nanomaterials
6.1.2 Immunosafety of nanomedicines: an introduction
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Nanoparticles in paints
75
6.2
75
6.2.1 Risks of nanoparticle handling and sanding nanoparticle-containing paints
6.2.2 Inflammatory and genotoxic effects of nanoparticles and dust generated from nanoparticle76
containing paints and lacquers
6.2.3 Cardiovascular health effects of paint dust with and without nanoparticles compared to the primary
77
nanoparticles
78
6.2.4 Developmental and reproductive toxicity of nanoparticles
79
6.2.5 Emission of Nanoparticles from Painted Surfaces
6.2.6 Coatings and Nanoparticles – Activities of the German Paint Industry on Workers’ Safety and
80
Consumer Protection in the field of Smart Coatings
7
OTHER
81
7.1
Poster presentations
81
7.1.1 Harmonization of Measurement Strategies for the Assessment of Exposure to Manufactured Nano
81
object; Report of a workshop
7.1.2 Microvascular Distribution and Effects of Surface-modified Quantum Dots in Postischemic Tissues 82
7.1.3 NanoRiskCat••• •• – A Conceptual Decision Support Tool for Nanomaterials
83
7.1.4 Effect of nanoparticle morphology on the detection efficiency of condensation particle counters
(CPCs) 84
7.1.5 In vitro evaluation of silver nanoparticles of different sizes in assays for cytotoxicity, inflammation
85
and developmental toxicity
7.1.6 Assessing the toxicological impact of a panel of engineered nanoparticles for risk assessment
86
purposes
7.1.7 Critical analysis of frameworks and approaches to assess the environmental risks of nanomaterials87
7.1.8 NANOGENOTOX: European Joint action on «Safety evaluation of manufactured nanomaterials by
88
characterisation of their potential genotoxic hazard»
7.1.9 Development of a control banding tool adapted to nanomaterials
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7.1.10
Nanotubes of imogolite do not activate macrophages and modestly perturb the barrier properties
90
of airway epithelial cells in vitro
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7.1.11
Nanotoxicology at European Center for the Sustainable Impact of Nanotechnology-ECSIN and its
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research approach-an overview
The basic requirement for comparable results between laboratories from in vitro tests remains a
7.1.12
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significant challenge
Bioavailability of silver nanoparticles orally administered to rats
93
7.1.13
IANH assessment of Nano-particle Cytotoxicity
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7.1.14
Cytotoxicity and genotoxicity induced in human and murine cell assays by copper oxide
7.1.15
95
nanoparticles
Forecasting Nano Law: The Small Matter of Big Risks
96
7.1.16
NANODEVICE: Novel Concepts, Methods, and Technologies for the Production of Portable, Easy7.1.17
to-use Devices for the Measurement and Analysis of Airborne Engineered Nanoparticles in Workplace Air 97
Social and Ethical Aspects of Nanomedicine: Sharing Benefits and Risks
98
7.1.18
Cellular effects of nanosilver in human macrophages: Uptake, oxidative stress responses, lipid
7.1.19
99
alterations and functional impairment
Polystyrene: A potential standard for developing In Vitro cellular tracking methods for
7.1.20
100
nanotoxicology
Protective effect of biosynthesized AgNPs from Melia azedarach against Dalton’s Ascites
7.1.21
101
Lymphoma
Ag and TiO2 Nanoparticles: Effects on Model Aquatic Organisms
102
7.1.22
Low-dose Single Wall Carbon Nanotubes affect embryonic development: an in vitro and in vivo
7.1.23
103
study
Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology
104
7.1.24
Ingested metal nanoparticles pass through intestine epithelia and enter immune cells and gonads
7.1.25
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of the sea urchin Paracentrotus lividus
Critical exposure to ultrafine particles during highway maintenance work
106
7.1.26
Development of polysaccharide-based nanocarriers for drug delivery applications
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7.1.27
NanoImpactNet’s Stakeholder Engagement
108
7.1.28
Median lethal dose of titanium dioxide and oleic acid coated magnetite nanoparticles after single
7.1.29
109
intravenous injection to adult rats
7.1.30
Novel Hydrophilic Ce(III)-Doped Maghemite (γ-Fe2O3) Nanoparticles - Preliminary Toxicity Studies
110
in Relation to the Nanoparticle Aggregation Level
Development of Novel Nanotechnology Based Diagnostic Systems for Rheumatoid Arthritis and
7.1.31
111
Osteoarthritis (NanoDiaRA)
7.1.32
Biological responses induced in bronchial epithelial cells by carbon black and titanium dioxide
112
nanoparticles: similar outcomes but distinct molecular pathways
Toxicology of iron oxide nanoparticles: impact of the size and surface modifications.
113
7.1.33
In vitro Assessment of the Cellular Toxicity of Nanotubes
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7.1.34
An in vitro integrated ultrasensitive approach to biocompatibility analysis of silver nanowires 115
7.1.35
Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic
7.1.36
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fields
CNT-mediated wireless cell permeabilisation: drug and gene uptake
117
7.1.37
Quantification of risk assessment in nanosafety: Determination of “run-off” effect of metallic
7.1.38
118
nanoparticles in simulated body fluids
8
AUTHOR INDEX
119
th
NanoImpactNet is a Coordination Action under the European Commission's 7 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: +41 - 21 314 74 21 Fax: +41 - 21 314 74 30
e-mail: [email protected]
http://www.nanoimpactnet.eu/
Web:
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Session 1
1 Nano-pharmacological input to research on the human
and environmental impact of nanomaterials
1.1 Oral presentations
1.1.1 Blood clearance and tissue distribution of PEGylated and non-PEGylated
gold nanorods after intravenous administration in rats
Daniëlle P.K. Lankveld1, Raja G. Rayavarapu2, Petra Krystek3, Agnes G. Oomen1, Hennie W.
Verharen1, Robert Geertsma1, Ton G. van Leeuwen2,4, Wim H. de Jong1, Srirang Manohar2
1
RIVM, Bilthoven, The Netherlands
University of Twente, Enschede, The Netherlands
3
MiPlaza Material Analysis, Philips Research, Eindhoven, The Netherlands
4
University of Amsterdam, Amsterdam, The Netherlands
Email: [email protected]
2
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 (t1/2) 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.
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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
Email: [email protected]
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, 10:3101-5
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1.1.3 Nose to brain delivery of nanostructured lipid carriers (NLC) loaded with
an antidepressant drug
M. Intakhab Alam1, Sanjula Baboota1, Mushir Ali1, Javed Ali1, Alka Ahuja2
1
Department of Pharmaceutics, Hamdard University, New Delhi, India
2
Oman Medical College, Azaiba, Muscat, Sultanate of Oman
Email: [email protected]
Number of counts
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 0.217 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 (GelDLX) 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.
150
NLC-DLX
100
Gel-DLX
50
Control
0
Immobility
Climbing
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
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1.2 Poster presentations
1.2.1 Mechanistic studies of in vitro
dendrimers in mammalian cells
cytotoxicity of
Polyamidoamine
Sourav Prasanna Mukherjee1*, Fiona M. Lyng1, Amaya Garcia1, Maria Davoren1, Hugh J.
Byrne2
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
Email: [email protected]
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 EC50 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., 2010. In vitro mammalian cytotoxicological
study of PAMAM dendrimers –Towards quantitative structure activity relationships. Toxicol.
In Vitro 24, 1169-177.
[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, 259–268.
Fig1. ROS localization in Mitochondria
after 24 h PAMAM exposure. Selected
as ‘cover art’ of TAAP 2010, 248, 259–
268.
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1.2.2 In vitro cytotoxicity assessment of nano-particulate silver in mammalian
cell lines
Sanchali Gupta Mukherjee1,2, Niall Ó Claonadh1, Gordon Chambers1,2 and Alan Casey1,2
1
Focas Institute, Dublin Institute of Technology, Dublin, Ireland
School of Physics, Dublin Institute of Technology, Dublin, Ireland
Email: [email protected]
2
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-38TM), HeLa (ATCC, CCL-2TM) 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.
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1.2.3 Nanostructured carrier based formulation of curcumin for bioavailability
enhancement
Sanjula Baboota1, Anil Kumar1, Javed Ali1, Alka Ahuja1,2
1
Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India
2
Department of Pharmacy, Oman Medical College, Azaiba, Muscat
Email: [email protected]
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 1349. 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 58 -123 nm, 0.313-0.625
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. 2010. Conundrum and therapeutic potential of curcumin in drug
delivery. Crit Rev Therap Drug Carrier Syst: 27(4), 279-320.
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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
Email: [email protected]
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.
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1.2.5 Integration and Analysis of Available Information for Building Exposure
Scenarios for Nanomaterials
Katherine Clark1, Rob Aitken2, Derk Brouwer3, Frans Christensen4, Rianda Gerritsen3,
Christian Micheletti4, Kaspar Schmid2, Martie van Tongeren2, the NANEX Consortium5,
Michael Riediker1
1
Institute for Work and Health, Lausanne, Switzerland
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
Email: [email protected]
2
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-TiO2, 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.
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1.2.6 Genotoxicity of inhaled nanosized TiO2 in mice
Hannu Norppa1, Hanna K Lindberg1, Ghita C-M Falck1, Joonas Koivisto1, Elina Rossi1, Lea
Pylkkänen1, Heli Nykäsenoja1, Hilkka Järventaus1, Satu Suhonen1, Minnamari Vippola1,2,
Julia Catalán1,3, Kai Savolainen1
1
Finnish Institute of Occupational Health, Helsinki, Finland
2
Tampere University of Technology, Tampere, Finland
3
University of Zaragoza, Zaragoza, Spain
Email: [email protected]
In vitro studies have suggested that nanosized TiO2 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 TiO2 exist. Recently, nanosized TiO2
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 TiO2 nanoparticles due
to inflammation.
We studied the in vivo genotoxicity of nanosized TiO2 in C57BL/6J mice after a 5-day
inhalation exposure (4 h/day) to 0.8, 7.2, and 28.5 mg/m3 (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 TiO2
anatase under the experimental conditions applied. On the other hand, systemic TiO2 doses
were probably much lower in our inhalation experiment than in the previous drinking water
study.
Funded by the European Commission (NANOSH, NMP4-CT-2006-032777) and the
Academy of Finland
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1.2.7 Interaction of nanoparticles used in medical applications with lung
epithelial cells: uptake, cytotoxicity, genotoxicity, oxidant stress and
proinflammatory response
Rina Guadagnini1, Sonja Boland1, Sandra Vranic1, Salik Hussain1, Kevin Moreau1, Caroline
Borot1, Francelyne Marano1
1
Laboratory of Functional and Adaptative Biology, unit of Réponses Moléculaires et
Cellulaires aux Xénobiotiques (RMCX), CNRS EAC, University Paris Diderot, Paris, France
Email: [email protected]
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 (TiO2), poly (lactic-co-glycolic acid)
(PLGA), non coated Fe3O4 (N Fe3O4), Fe3O4 coated with oleic acid (C Fe3O4) 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
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1.2.8 Internalisation and transcytosis of SiO2 and TiO2 nanoparticles by lung
epithelial cells
Sandra Vranic1, Rina Guadagnini1, Armelle Baeza1, M. Caroline Borot1, Francelyne Marano1,
Sonja Boland1
1
Laboratoire de Biologie Fonctionnelle et Adaptative, équipe Réponses Moléculaires et Cellulaires
aux Xénobiotiques (RMCX), CNRS EAC 7059, Université Paris Diderot, Paris, France
Email: [email protected]
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 (TiO2) and silicium dioxide (SiO2)] 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. TiO2 NPs are cytotoxic for 16HBE
and NCI-H292 cell lines at high concentrations inducing apoptosis. SiO2 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 TiO2 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. TiO2 and SiO2 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. TiO2 and SiO2 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 FP7 201335 (Nanotest) and EC FP7 228789
(ENPRA), National Grant Nanotrans.
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1.2.9 Nanoparticles in
characterisation
Food
Analytical
methods
for
detection
and
Stefan Weigel, Ruud Peters, Hans Bouwmeester
RIKILT – Institute of Food Safety, Wageningen UR, Wageningen, The Netherlands
Email: [email protected]
“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)
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1.2.10 Nanoparticles as potential cytostatic agents for treatment of leukemia
Ylva Rodhe and Lennart Möller
Karolinska Institutet, Stockholm, Sweden
Email: [email protected]
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 2009.
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.
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1.2.11 Deposition of CNTs in the Respiratory Tract for two Industrial Exposure
Scenarios
Marika Pilou1,5, Gaelle Uzu2, Vasilis Gkanis1, Derk Brouwer3, Martie van Tongeren4 and
Christos Housiadas1
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
Email: [email protected]
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-2009-1.3-2 of FP7 of the European Commission
[1] Aschberger, K. et al. 2010. Review of carbon nanotubes toxicity and exposure-appraisal
of human health risk assessment based on open literature. Critical Reviews in Toxicology
40:759-790
[2] Mitsakou, C. et al. 2005. 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)
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1.2.12 Secondary characterization of TiO2 nanoparticles in biological media by
Dynamic Light Scattering (DLS) and Transmission Electron Microscopy
(TEM) techniques
Dagmar Bilaničová, Giulio Pojana, Davide Cristofori, Antonio Marcomini
University Ca’ Foscari of Venice, Venice, Italy
Email: [email protected]
Advantages and limits of Dynamic Light Scattering (DLS) and Transmission Electron
Microscopy (TEM) techniques for secondary size characterization of nanoparticles in
biological media were addressed and discussed. Both techniques were employed to
investigate size distribution of nano-TiO2 (Degussa-Evonik P25) in biological media
commonly employed for in-vitro toxicological studies. Titanium dioxide nanoparticles were
dispersed at physiological pH up to 5 mg/ml, stabilized with foetal bovine serum, a
biologically compatible dispersant, according to specifically developed protocols, and their
size distributions and stability vs. agglomeration were investigated in a wide set of commonly
employed biological media. The DLS technique showed to be a reliable technique for
secondary size characterization of nanoparticles in biological media. This technique
permitted also to highlight phenomena, such as agglomeration processes and hydrodynamic
diameter increase with time due to protein, as well as of other species, sorption, which would
need careful evaluation during nanotoxicological studies. The TEM technique was instead
demonstrated to be a recommendable supporting technique for agglomeration size and
shape visualization, but formation of aggregates during grid preparation step has to be taken
into careful consideration because it can lead to misleading conclusions about actual size
distribution of investigated nanoparticles.
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1.2.13 Oxidative potential of fine and ultrafine particles in occupational
situations
Jean-Jacques Sauvain1, Simon Deslarzes1, Ferdinand Storti1, Michael Riediker1
1
Institute for Work and Health, Lausanne, Switzerland
Email: [email protected]
Introduction: Evaluation of the exposure to fine/ultrafine particulate (PM) and manufactured
nanomaterials (MNM) is challenging. In particular, exposure levels to MNM in occupational
situations are lacking. For risk assessment, the mass is often used to evaluate the biological
dose, whereas it is recognised that such a metric is not ideal. The PM redox properties on
the other hand are suggested to be more important to explain their biological activity. Such a
parameter could constitute a novel, integrative and more refined metric for hazard evaluation.
Different acellular in vitro assays are developed for such measurements. In this study, we
selected the dithiothreitol (DTT) assay and applied it in different occupational situations.
Objectives: (1) to evaluate sampling requirements for fine/ultrafine particle allowing
measurement of their oxidative potential (2) to apply the methodology to occupational
situations where fine/ultrafine particle from combustion sources are generated (3) to
determine which particle constituents are associated with such an oxidative potential.
Material and method: Sampling parameters (type of filters and loaded amount) and storage
duration affecting the DTT measurements were evaluated.
Based on these results, a methodological approach was defined and applied in two
occupational situations where diesel and other combustion particles are present (toll station
in a tunnel and mechanical yard for bus reparation). In parallel, the particle bulk content for
organic/elemental carbon and metals (iron and copper) as well as adsorbed organics (six
polyaromatic hydrocarbons and four quinones) were determined.
Results: Teflon filters loaded with diesel particles were found more suitable for the DTT
assay, due to their better chemical inertness compared to quartz filters: after storage
durations larger than 150 hours, an increased reactivity toward DTT was observed only with
quartz filters. Reactivity was linearly correlated to the loaded mass until about 1000 µg/filter.
Different redox reactivities were determined in both working places, with the mechanical yard
presenting a higher DTT consumption rate.
Associations between DTT consumption rate and iron as well as organic carbon were
observed for both working situations. In addition, the sum of four quinones, copper,
elemental carbon and surface area (SMPS measurements) were also observed to be
associated with DTT reactivity, but only for the toll station.
Discussion and conclusions: These results demonstrate the feasibility of this methodology
to determine the oxidative potential of fine/ultrafine particles in occupational situations. The
particle oxidative potential has been observed to be very variable depending on the working
day and is function of the physico-chemical characteristics of the particles. In particularly, the
total iron and organic content are associated with such reactivity.
This approach could be very useful for hazard assessment of workplaces where exposure to
MNM is expected.
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1.2.14 Biogenic synthesis of Silver nanoparticles from aqueous extract of
Solanum nigram and its characterization
Kamalakkannan S1, Sukirtha R2, Jacob Joe Antony2, Siva D2, Achiraman S2
1
Institute of Physiology, University of Lausanne, Lausanne, Switzerland
Department of Environmental Biotechnology, Bharathidasan University, Tiruchirappalli, India
Email: [email protected]
2
Discovering of new molecules from nature and manipulating those in nanosize with greater
potential to improve health care. Silver nanoparticles (AgNPs), an emerging nanomedicine is
renowned for its promising therapeutic possibilities, due to its significant properties such as
biocompatibility and plasmon resonance. The present study aimed at the synthesis of AgNPs
from aqueous extract of Solanum nigram at various temperatures. The synthesis of AgNPs at
30°C, 60°C, 90°C and 95°C for an incubation of 10 min. Exposure to 95°C conferred the
maximum AgNPs synthesis compared to other temperatures. Presence of specific plasmon
resonance at 430nm confirmed the synthesis of AgNPs from UV-vis spectrum bands.
Structural characterization of AgNPs by Scanning Electron Microscopic (SEM) analysis
confirmed formation of cubical and spherical shaped nanoparticles. Further, the average
particle size of 40nm was confirmed from Transmission Electron Microscopic analysis (TEM).
An immediate reduction of silver ions in the present investigation might have resulted due to
the presence of water soluble heterocyclic compounds in the S. nigrum aqueous extract,
which were confirmed with their Fourier Transform Infrared Spectroscopic (FTIR) results.
Hence, we conclude the efficient synthesis of AgNPs from aqueous extract of S. nigram.
Keywords: Silver nanoparticles (AgNPs), Solanum nigram, Scanning Electron Microscopic
(SEM), Transmission Electron Microscopic analysis (TEM), Fourier Transform Infrared
Spectroscopic (FTIR).
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1.2.15 Migration of silver from plastic food containers
Lars Fabricius1,2, Natalie von Goetz1, Reto Glaus3, Detlef Günther3, Konrad Hungerbühler1
1
Institute for Chemical and Bioengineering, ETH Zurich, Zurich, Switzerland
Norwegian University of Science and Technology (NTNU), Trondheim, Norway
3
Laboratory of Inorganic Chemistry, ETH Zurich, Zurich, Switzerland
Email: [email protected]
2
Silver (Ag) in nanoparticular or ionic form is already introduced as an antimicrobial additive to
a large quantity of consumer products, including plastic food storage containers. In most
cases, the silver is only physically bound in the plastic polymer. It can therefore migrate to
the food items, as is observed for other plastic additives (e.g. the phthalates). The aim of our
study is to quantify human exposure to silver from food stored in different types of silverdoped plastic containers.
Table 1: Silver content in plastic containers
Silver content
in μg/g
Standard
deviation in μg/g
Kinetic Go Green Nano
Silver Basic
18.7
0.6
FresherLonger
37.1
1.2
Kinetic Go Green Nano
Silver Premium
<0.1
-
NanosilberFrischhaltedosen
<0.1
-
120.4
2.7
Type
Lens
container
comparison)
(for
We bought commercially available plastic
containers labelled “containing silver”. The
silver content of the plastic containers was
determined with ICP-MS after microwave
digestion. Table 1 shows the silver content in
the polymers. Three out of five tested
products contained silver at concentrations
of approx. 20-120 μg/g. The limit of detection
was 0.1 μg/g, respectively.
Ag concentration in ng/g
Furthermore, silver migration from the containers into food simulants was studied after 6h,
10d and 20d by ICP-MS. The experiments showed an initial migration after 6h. However,
after about 10 days no further increase in silver concentration was observed (see Figure 1).
Further and more detailed migration experiments are under way and will be discussed in the
presentation. They follow the Commission Directive 97/48/EC [1] by investigating migration
to four food simulants at 20°C with 7 measuring points within 10 days.
Silver migration into Water
16
14
12
10
8
6
4
2
0
Figure 1: Silver concentration transferred
from the “Kinetic Go Green Nano Silver
Basic” container into water measured
after 6h, 10 and 20 days. The
concentrations are related to the silver
migration from individual samples of 100
mg plastic to 1.8 g of water each.
0
10
Days
20
[1] The commission of the European communities. Basic rules for overall and specific
migration testing. Commission Directive 97/48/EC. Official Journal of the European
Communities. Brussels, 29th of July, 1997
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Session 2
2 Lessons from Nano-Immunology on the impact of
nanomaterials
2.1 Oral presentations
2.1.1 Understanding Interactions of Engineered Nanomaterials with the
Immune System
Bengt Fadeel
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet,
Stockholm, Sweden
Engineered nanoparticles may affect the innate or adaptive immune system; such
interactions, in turn, could result in adverse outcomes or could potentially be exploited for
therapeutic gain. The recognition or non-recognition of engineered nanomaterials by
immune-competent cells may determine not only the toxic potential of such materials but also
their biodistribution. However, understanding the physico-chemical properties that drive
cellular interactions of nanoparticles remains a key challenge. Lessons may be learned from
studies of natural nano-scale objects such as viruses and from decades of research on
micron-sized particles and fibres. The assessment of nanoparticle effects on the immune
system also requires validated assays designed to capture relevant endpoints; in this
context, the applicability of animal models to the human condition should be carefully
evaluated. When human subjects are deliberately exposed to engineered nanomaterials, for
diagnostic or therapeutic purposes (or both), it becomes critically important to determine the
ultimate fate of the nanoparticles: are engineered nanomaterials excreted from the body, or
biodegraded by cells of the immune system, or do the bioaccumulate, thereby leading to
potentially harmful long-term effects? The surface of nanoparticles can be modified using
targeting moieties, etc but as these particles enter into a biological system, for instance via
inhalation or through injection into the bloodstream, it is likely that the surface of the particles
are covered with biomolecules – proteins and lipids – that modify the properties of the
nanoparticles and the way in which the particles interact with cells, including immunecompetent cells. Moreover, the binding of proteins to nanoparticles may also induce
modifications of the proteins. Understanding such nano-bio-interactions is critical for the safe
application of nanoparticles in medicine.
[1] Feliu N, Fadeel B. Nanotoxicology: no small matter. Nanoscale. 2010 Dec 1;2(12):251420.
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2.1.2 The Effect of Two Iron Oxide Nanoparticles on Immune Response of
Lymphocytes
Jana Tulinska1, Miroslava Kuricova1, Aurelia Liskova1, Eva Neubauerova1, Katarina
Volkovova1, Dagmar Bilanicova2, Giulio Pojana2, Maria Dusinska1,3
1
Slovak Medical University, Bratislava, Slovakia
University Ca’ Foscari of Venice, Venice, Italy
3
Norwegian Institute for Air Research, Kjeller, Norway
Email: [email protected]
2
Introduction: Nanoparticles (NPs) are promising source for new medical diagnostic and
therapeutic possibilities therefore nanomaterial safety and the risk assessment of newly
engineered NPs are crucial. Important part of toxicity testing is assessment of the effect of
NPs on the immune response. In vitro studies using human peripheral whole blood or
isolated blood products can be utilized in evaluating the effect of NP on circulating blood.
However, the effects of NP on immune functions in vitro require the development of
appropriate tests.
Material and method: Iron oxide NPs as promising imaging contrast agents for magnetic
resonance imaging have been selected for in vitro testing. Human peripheral whole blood
cultures (n=10) were treated with bare iron oxide (Fe3O4) (IO) and oleic acid coated iron
oxide (OA IO) nanoparticles in three different concentrations: 0.12 μg/cm2, 3 μg/cm2 and 75
μg/cm2 and four time intervals: 72h, 48h, 24h and 4h. Crystallite size distribution of iron
oxides NPs by TEM: IO 5-13 nm, OA IO: 5-12 nm, crystal structure octahedral, shape
oblong. Lymphocyte transformation assay was used to assess the effect of NPs on
lymphocyte function. Lymphocytes were stimulated with mitogens concanavalin A,
phytohaemmagglutinin, pokeweed mitogen and antigen CD3. Cell proliferation was
quantified by [3H]-thymidine incorporation into DNA. Fluorescence was measured by
scintillation spectrophotometer.
Results and conclusions: Our findings indicate significant differences in immunotoxicity of
bare and oleic acid coated iron oxide nanoparticles. Meanwhile bare oxide significantly
diminished function of lymphocytes exposed to high and middle dose of NPs, suppressive
effect of oleic acid coated iron oxide on proliferative activity was seen only in lymphocyte
cultures treated with high dose of NPs. These effects of IO and OA IO on proliferative
response of lymphocytes were found in peripheral blood cultures in vitro stimulated with all
three mitogens (Con A, PHA and PWM). Similar pattern of lymphocyte response to different
mitogens refers to similar sensitivity of T-cell response and T-dependent B-cell response of
lymphocytes to iron oxide nanoparticles exposure. No clear influence of treatment time
interval related to the immunosuppressive effects of iron oxide NPs was observed.
In conclusion, proliferation of lymphocytes in vitro might be one of the relevant endpoints to
evaluate for NPs. Bare iron oxide is candidate for nanoparticle immunosuppressive control
for in vitro testing.
Acknowledgement: Supported by EC FP7 [Health-2007-1.3-4], Contract: 201335. We thank
Helena Nagyova, Edita Mrvikova, and Marta Postrkova for their excellent technical help.
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2.1.3 Identification of immune-related gene markers following interaction of
engineered nanoparticles with human intestinal epithelial cells
Sandra Verstraelen1, Patrick De Boever1, Eudald Casals 2, Victor F. Puntes2, Hilda Witters1,
Inge Nelissen1
1
VITO N.V. (Flemish Institute for Technological Research), Environmental Risk and Health
Unit, Mol, Belgium
2
Institut Català de Nanotecnologia (ICN), Barcelona, Spain
Email: [email protected]
Nanotechnologies offer a variety of possibilities for application in the food area, such as in
food additives, nutritional supplements, packaging, and food storage devices. Besides
ingestion, unintended human exposure to nanoparticles (NP) may occur when NP are
released to the atmosphere during industrial production processes, or in derivates of
agricultural products discharged to waste waters or soils. Ingestion of NP may pose human
health risks, but data on oral exposure to specific NP and any consequent toxicity are scarce,
and the implicated biological and molecular processes are largely unexplored.
In this study, the human adenocarcinoma Caco-2 cell line, widely used as an in vitro model
of the intestinal barrier, was exposed to suspensions of monodispersed, spherical cobalt (7
nm) and cerium dioxide (4 nm) NP at non-cytotoxic concentrations. A genome-wide
transcriptomics study was performed to reveal the genes and processes involved in immunerelated effects after NP exposure. Parallel experiments were set up involving cobalt chloride
and cerium nitrate exposures to correct NP-specific responses by subtracting those induced
by the corresponding ions. Statistically significant changes in gene expression as compared
to solvent-treated cells (mean |fold-change|>1.5 (n=3), p<0.05) were evaluated after 3, 6, 10,
and 24 hours of exposure.
Nanoparticle exposure mainly induced downregulation of gene expression in the Caco-2 cell
line. The cell model showed NP-dependent kinetics in its transcriptional response, with the
number of differentially expressed genes (DEG) being highest after 3 hours of exposure to
cobalt NP (# 1410), and then gradually decreasing up to 24 hours. In contrast, cerium dioxide
NP induced a sustained high number of DEG from 6 hours of exposure onward (# 3372). For
both NP, approximately 6% of DEG was related to immune function, and this percentage
remained similar over time. In contrast to cerium dioxide NP which induced on average 0.4%
up- and 5.4% downregulated immune genes over the entire exposure duration, cobalt NP
induced an increasing portion of upregulated genes with a maximum of 2.3% after 10 hours.
To allow for identifying candidate gene markers of cell-NP interaction independent of NP
type, immune-related DEG which were significantly affected by both cobalt and cerium
dioxide NP in the Caco-2 cell line were selected after correction for ion-induced gene
responses. At the different exposure times, twenty three immune-related DEG were
observed which each showed a transient response to NP exposure. The gene encoding
protein tyrosine phosphatase, receptor type C (PTPRC or CD45) was the only one being
significantly induced over a prolonged time period (at 6 and 10 hours), and therefore may
constitute a promising marker.
Our data suggest that cobalt and cerium dioxide NP give rise to a distinct immunological
response in intestinal epithelial cells, with only few molecular players in common. We
identified PTPRC gene as a candidate marker that can be used for more targeted toxicity
testing. The investigation triggers off additional research to validate the results using different
technologies and to test an extended set of NP and/or other cell models.
This work was partly funded by the EU-FP6 project DIPNA (Contract #STRP 032131).
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2.1.4 Immunosafety of engineered nanoparticles:Methods implementation for
the development of nanomedicines
Diana Boraschi1, Gertie J. Oostingh2, Paola Italiani1, Eudald Casals3, Inge Nelissen4,
Hagen Thielecke5, Victor F. Puntes3 and Albert Duschl2
1
Institute of Biomedical Technologies, National Research Council, Pisa, Italy
Department of Molecular Biology, University of Salzburg, Salzburg, Austria
3
Institut Català de Nanotecnologia, Campus de la UAB, Bellaterra, Spain
4
VITO NV, Mol, Belgium
5
Fraunhofer Institute for Biomedical Engineering, St. Ingbert, Germany
Email: [email protected]
2
Safety assessment of nanomaterials, and of nanomedicines in particular, should include
evaluation of the possible effects on the immune system. Alterations of the normal functions
of the immune system, a possible consequence of interaction with nanoparticles, can cause
severe pathological derangements. The unprecedented benefits expected from novel
nanomedicines and health-targeted nano-devices will be greatly increased by the availability
of robust and representative safety methods, able to predict the immune-related risk of
developing diseases. In most cases, standard immunological assays are not suited for
detecting nanoparticle effects, and should therefore be custom-adapted or re-designed.
A major issue in immunosafety assay design is the need of standardisation and ease of
applicability of the validated assays. In vitro assays are greatly preferred because of their
high reproducibility, in addition to avoiding the use of experimental animals. The possibility of
employing in vitro models of human primary cells, rather than animal tissues or
transformed/tumour cell lines, will further increase relevance.
The following topics will be addressed:
• Interaction of nanoparticles with biological systems: particle size, shape, and chemical
composition define the features of interaction with immune cell and consequent effects.
• Risk-predicting immunomarkers: how to select phenotypic/functional endpoints relevant to
immunosafety evaluation.
• Representative in vitro assays: how to select systems, cells, types and times of exposure.
• Assay validation and standardisation: assays should represent the real-life situation, and
artefacts should be identified and avoided.
• Applicability to different human populations: need for addressing the problem of
immunologically weak/altered groups (elderly, babies, sick people, pregnant women) and
genetically/geographically distinct populations (residing in climatically diverse areas and/or
with a different genetic background).
The conclusions will establish the following points:
a. immunosafety of engineered nanoparticles, with special reference to nanomedicines, is a
highly relevant health issue that should be addressed with appropriate investigation tools;
b. extensive controls are essential to set up reliable and robust assays;
c. representative in vitro assays with human cells, including primary cells, are possible and
strongly recommended;
d. implementing an array of relevant immunosafety tests will contribute to the sustainable
knowledge-based development of nanotechnologies applied to medicine, up to including
novel tools specifically directed to modifying immune response.
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2.2 Poster presentations
2.2.1 Responses of lung cell cultures after realistic exposure to secondary
organic aerosols
Lisa Künzi1, Sarah Schneider1, Peter Mertes2, Markus Kalberer3, Josef Dommen2, Urs
Baltensperger2, Marianne Geiser1
1
Institute of Anatomy, University of Berne, Switzerland
Laboratory of Atmospheric Chemistry, Paul Scherrer Institut, Villigen, Switzerland
3
Centre for Atmospheric Sciences, University of Cambridge, UK
2
The interaction of particles with the inner surface of the lungs, the main pathway of undesired
particle uptake, is still poorly understood [1]. Commonly used model systems for in vitro
studies deviate significantly from the situation in vivo such that responses possibly are not
representative of those induced by the particles in vivo. To contribute to the closure of this
information gap we examined the responses of lung cell cultures to primary and secondary
organic aerosols (POA and SOA) originating from diesel and wood burning under conditions
replicating the in vivo situation. The particles were applied to the cell cultures under realistic
ambient air and physiological conditions in a novel particle deposition chamber [2]. The cell
cultures, representing the inner surface of airways and alveoli, were microdissected epithelia
from pig tracheae and redifferentiated human airway epithelia both with established air liquid
interface (ALI), porcine lung surface macrophages, the humane bronchial epithelial cell line
BEAS-2B, as well as the rat alveolar epithelial cell line R3/1. Cells were cultured on
microporous filter inserts and exposed to the aerosol for 2 hours at ALI conditions. Control
cell cultures were (i) exposed to filtered air or (ii) left untreated in the incubator. Cellular
responses were measured 24 hours after exposure to the aerosol. Biological endpoint
measurements included cytotoxicity, cell and tissue integrity, phagocytic activity of
macrophages, release of inflammatory mediators such as interleukin-6 (IL-6), IL-8, IL-10,
tumor necrosis factor alpha (TNF-α) and monocyte chemotactic protein-1 (MCP-1).
The results demonstrate that an acute exposure of the various lung cell types to the aerosols
at ambient-air concentrations of about 104 particles/cm3 during 2 hours leads to only
moderate cellular responses. However, there is evidence for i) different effects of POA and
SOA and for ii) different effects of aerosols originating from diesel and wood burning. The
data indicate that a short time exposure to realistic aerosol concentration does not induce
changes in cell and tissue integrity, but leads to subtle changes in cellular functions that are
essential for lung homoeostasis. A slightly increased cytotoxicity for the various cell cultures
after SOA exposure was found. The phagocytic activity was increased after exposure to SOA
from diesel exhaust and tended to be decreased after exposure to SOA from wood burning.
The release of inflammatory mediators is currently under evaluation.
Supported by the Swiss National Science Foundation grant K-32K1-120524
[1] Geiser, M, Kreyling, WG 2010. Deposition and biokinetics of inhaled nanoparticles.
Particle Fibre Toxicol 7:2
[2] Savi, M et al. 2008. A novel exposure system for the efficient and controlled deposition
of aerosol particles onto cell cultures. Environ Sci Techn 42: 5667-5674
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2.2.2 Biocompatibility of Zeolite-MFI nanoparticles in Human lung cells
Kunal Bhattacharya1, Izabela Naydenova2, Svetlana Mintova3, Hugh J. Byrne1
1
Nanolab research centre, FOCAS Institute, Dublin Institute of Technology, Dublin, Ireland
Centre for Industrial and Engineering Optics, Focas Research Institute, Dublin Institute of
Technology, Dublin, Ireland
3
Laboratoire Catalyse & Spectrochimie, University of Caen, Caen, France
Email: [email protected]
2
The Zeolite-MFI used in this study is a silica based mesoporous nanoparticulate material
having the chemical formula of SiO2 and average size of 50 and 100 nm. They samples were
compared to amorphous silicon dioxide nanoparticles which have a primary size of
approximately 14nm but form secondary/tertiary chained structures of a few microns in
length. Zeta potential and particle size distribution measurements in water showed all the
nanoparticles to have a high negative surface charge which is reduced on suspension in
RPMI-1640 media with 5% FBS. This was attributed to the high rate of salvation/opsonization
of ions and proteins on the surface of the nanoparticles using UV/Visible absorption
spectroscopy, Bradford assay and phase contrast microscopy. The cell lines studied for the
uptake and toxicity of the nanoparticles were human based SV40 transformed bronchial
epithelial cells (BEAS-2B) and alveolar carcinoma epithelial cells (A549). Through phase
contrast monitoring of live cells it was observed that the agglomerated nanoparticles were
actively phagocytosed through filopodia by both the cell lines immediately following their
precipitation. Cellular proliferation and cytotoxicity assays (alamar blue and neutral red)
demonstrated that amorphous SiO2 reduced both the cellular proliferation and cell viability of
the A549 cells while it had no effect on the BEAS-2B cells. Zeolite-MFI nanoparticles,
however, had no effect on the viability of the A549 cells at lower concentrations but
effectively reduced the cellular proliferation capability depending upon their size and
concentration. The extracellular reactive oxygen species (ROS) detection assay Eu(III)-TC
dye showed that the nanoparticles had a non-reactive surface but they induced significant
levels of intracellular ROS immediately after uptake. This effect was however reduced at
longer exposure times. Also, during this short period of increased ROS activity, a significant
reduction in the mitochondrial membrane potential was observed, which however was
recoverable. Through this study it was found that even though the nanoparticles themselves
were non-reactive, they easily provoked a short duration but intense intracellular response.
This might be the cause for the variety of individual responses observed through the
cytotoxicity and cellular proliferation assays in the two different cell lines. The study so far
points towards the capability of these non-reactive nanoparticles to cause intracellular
damage through the process of long term biopersistence and bioaccumulation. Further
studies are in progress to understand these long term effects.
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2.2.3 Viral ligands potentiate the human alveolar epithelial innate immune
response to silver nanoparticles and carbon nanotubes
Andrew J Thorley1, Gareth L Evans1, Teresa D Tetley1
1
National Heart & Lung Institute, Imperial College, London, UK
Email: [email protected]
Epidemiological studies have previously shown a correlation between increases in the
concentration of ambient particulate matter and increased hospital admissions for cardiorespiratory morbidity and mortality, particularly in those with underlying respiratory diseases
such as asthma, COPD. In addition studies have shown that there is an increase in
admissions for respiratory infections following periods of increased atmospheric PM2.5. This
has led to the hypothesis that engineered nanoparticles may have the same effect.
We hypothesise that engineered nanoparticles (NPs) will drive a pro-inflammatory response
in the alveolar epithelium through oxidative stress which will synergise with the innate
immune responses initiated through activation of Toll-like receptor-3 by virus-associated
ligands, leading to increased pulmonary epithelial inflammation and cell death.
Confluent monolayers of human alveolar type I epithelial cells were exposed to increasing
doses of carbon nanotubes (CNT) and silver nanoparticles (Ag NP) in the presence and
absence of Poly I:C, a TLR-3 ligand for 24 hours. Following this, cell viability was measured
by MTT assay, oxidative stress by fluorescence microscopy and cytokine release by ELISA.
CNT and Ag NP (10µg/ml) induced a 35% and 50% loss in cell viability respectively. Coincubation with Poly I:C significantly reduced cell viability in both exposure groups by a
further 15%. Poly I:C alone did not affect cell viability. Co-exposure of epithelial cells to Poly
I:C and CNT or Ag NP potentiated the oxidative stress induced by exposure to the
nanoparticles alone. Poly I:C, CNT and Ag nanoparticles all induced significant IL-6 and IL-8
release over 24 hours exposure. Co-incubation of Poly I:C with either 25µg/ml CNT or Ag NP
potentiated IL-6 release 1.5 and 2.2 fold respectively; IL-8 release was not potentiated by coexposure. Electron microscopy analysis showed that incubation of CNT and Ag NP with Poly
I:C decreased agglomeration of the nanoparticles.
In conclusion, we demonstrate that co-exposure to microbial ligands and engineered
nanoparticles results in an exaggerated innate immune response and increase cytotoxicity.
We hypothesise that this may be due to increased dispersion of the nanoparticles which may
improve their ability to enter epithelial cells and elicit adverse effects.
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2.2.4 Effect of sonication and serum proteins on copper release from copper
nanoparticles and the toxicity towards lung epithelial cells
Pontus Cronholm1, Klara Midander2, Hanna L Karlsson1, Karine Elihn3, Inger Odnevall
Wallinder2, Lennart Möller1
1
Karolinska Institutet, Stockholm, Sweden
Royal Institute of Technology, Stockholm, Sweden
3
Stockholm University, Stockholm, Sweden
Email: [email protected]
2
To understand how different methodological settings can influence particle characteristics
and toxicity is important in nanotoxicology. The aim of this study was to investigate how
serum proteins and sonication of Cu nanoparticle suspensions influence the properties of the
nanoparticles and toxicological responses on human lung epithelial cells. This was
investigated by using methods for particle characterisation (photon correlation spectroscopy
and TEM) and Cu release (atomic absorption spectroscopy) in combination with assays for
analysing cell toxicity (MTT-, trypan blue- and Comet assay). The results showed that
sonication of Cu nanoparticles caused decreased cell viability and increased Cu release
compared to non-sonicated particles. Furthermore, serum in the cell medium resulted in less
particle agglomeration and increased Cu release compared with medium without serum, but
no clear difference in toxicity was detected. Few cells showed intracellular Cu nanoparticles
due to fast release/dissolution processes of Cu. In conclusion; sonication can affect the
toxicity of nanoparticles.
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2.2.5 Medical application of nano-sized magnetite and silica during
pregnancy: in vitro studies assessing placental transport and toxicity
Sara Correia Carreira1, Laura Cartwright1, Kai Paul1, John Schwieso2, Faryal Henry2, Stuart
Ferguson2, Margaret Saunders1
1
BIRCH, Biophysics Research Unit, University Hospitals Bristol NHS Foundation Trust,
Bristol, UK
2
University of the West of England, Bristol, UK
E-mail: [email protected]
Nano-sized magnetite and silica used in imaging and drug delivery have significantly
improved medical diagnostics and therapies. Due to the enhanced sensitivity of the
developing foetus, it is not yet known whether these materials are safe for use in pregnancy.
Pregnant women may be denied most appropriate treatment due to the lack of research in
this field. Hence, there is a need to assess toxicity and transport of these particles across the
placenta.
We have established an in vitro model of the placental barrier using BeWo b30 cells. Using a
range of bioassays, we have assessed the toxicological profile of nano-sized magnetite
(uncoated or coated with oleic acid) and silica (25nm and 50nm) in vitro in order to estimate
toxicity to the placental barrier. Furthermore, by growing BeWo b30 as a monolayer on
permeable Transwell inserts, we have sought to investigate placental transport kinetics in
order to estimate potential foetal exposure.
We will present data on dose dependent toxicity of both particles, as well as their transport
across BeWo b30 monolayers. No toxicity to BeWo b30 cells was found at low, clinically
relevant concentrations. However, coated magnetite and both types of nano sized silica were
able to cross BeWo monolayers to some extent (approx. 10-20%).
In addition, we will discuss the challenges of screening the toxicological profile of
nanoparticles in colorimetric bioassays and suggest strategies to overcome technical
difficulties.
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2.2.6 Effects of nanoparticles on hepatocyte survival, mitochondrial function,
antioxidant levels and cellular function
Birgit Gaiser, Vicki Stone
1
Heriot-Watt University, Edinburgh, UK
Email: [email protected]
As nanoparticles (NPs) are increasingly used in a large number of consumer products,
particle toxicity testing is becoming essential. Some mechanisms by which NPs cause
toxicity, such as generation of oxidative stress [1] and up-regulation of inflammatory markers
[2], have been well-established for some time.
To investigate both toxic and sub-toxic effects of NPs on hepatocytes, a set of particles of
different composures and sizes was used, including polystyrene beads (Fluoresbrite 50 and
200 nm, Polysciences Inc), silver (Ag, nominal diameter <25 nm, Mercator) and titanium
dioxide (TiO2 rutile-anatase, nominal diameter 7 nm, Mercator). Experiments with gold
particles (15 and 80 nm) are soon to follow. All studies were performed on the human
hepatocyte cell line C3A.
We found, using the LDH assay for cytotoxicity and the Alamar Blue assay for mitochondrial
function, that the fluorescent beads of both sizes only caused significant reduction in viability
at an extremely high concentration of 625 µg/cm2, and TiO2 NPs had no toxic effects at any
concentration up to 625 µg/cm2. In contrast, Ag NPs were highly toxic with an LC50 between
2.5 µg/cm2 (LDH) and 15 µg/cm2 (Alamar Blue).
Initial experiments showed that Ag NPs compromised cellular function by reducing the
secretion of albumin into the medium at doses of and below the LC50, whereas TiO2 did not
reduce albumin secretion. Another preliminary study examining the effects of particles on
levels of the intracellular antioxidant glutathione showed a reduction in reduced glutathione 2
and 6 hours after exposure to Ag nanoparticles at concentrations below the LC50.
These preliminary experiments will be repeated and extended to the other particles in the
test set over the next few months. So far our results seem to confirm the toxicity of Ag NPs
previously reported [3], and to show that even at non-toxic concentrations, cellular function
and antioxidant levels are compromised, potentially providing a means to test for adverse
effects of NPs at low concentrations which is specific for hepatocytes. In contrast, cellular
function does not appear to be impaired even at high concentrations of the low-toxicity TiO2
NPs.
[1] Brown, D et al. 2004. Calcium and ROS-mediated activation of transcription factors and
TNF-alpha cytokine gene expression in macrophages exposed to ultrafine particles. Am. J.
Physiol. Lung Cell Mol. Physiol. 286: L344-L353.
[2] Duffin, R et al. 2007. Pro-inflammatory effects of low toxiity and metal nanoparticles in
vivo and in vitro: Highlighting the role of particle surface area and surface reactivity. Inhal.
Toxicol. 19 : 849-856.
[3] Gaiser, B et al. 2009. Assessing exposure, uptake and toxicity of silver and cerium
dioxide nanoparticles from contaminated environments. Environ Health 8 Suppl 1:S2.
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2.2.7 Translocation of engineered
environment to human body
and
nanoscaled
by-products
from
Antonietta Gatti1,Federico Capitani2, Stefano Montanari,2
1
University of Modena and Reggio Emilia, Modena, Italy
2
Nanodiagnostics srl, San Vito, Modena, Italy
Email: [email protected]
Nanotechnologies are rampant disciplines, growing exponentially beyond any expectation,
but worldwide concern is arising about the possible toxicity of nanoparticles. The appearance
on the market of nanoparticle-containing products represents a possible, unintentional
source of exposure of end-users and to nanoparticles. Besides, those products must be
disposed of at the end of their life-cycle, causing a possible dispersion of their nanosized
content in the environment. Because of that, scientists are called to verify the possible impact
of nanoparticles on humans, animals and the environment.
The present study started from the observation that engineered nanosized by-products can
already be found in the environment, as shown by the EC DIPNA Project (FP6-03231- 200609), and be ingested with contaminated food or inhaled.
About 1,200 pathological samples from humans suffering from cancer of soft tissues,
lymphoma, and leukemia were analyzed according to a protocol developed within the EC
Nanopathology Project (FP5 QOL-147-2002-05) meant to identify morphologically and
chemically submicronic, inorganic particulate matter in such samples. A novel
ultramicroscopic technique using a Field Emission Gun Environmental Scanning Electron
Microscopy (Quanta 250, FEI Company, the Netherlands), was carried out on bioptic,
autoptic or surgical samples in order to identify inorganic foreign bodies whose elemental
composition was assessed by means of an Energy Dispersive System, (EDS by EDAX,
USA). Two reference groups, a positive and a negative one, were selected: 140 samples
from Italian soldiers who served in war theatres and who, after a 6-month mission, developed
lethal diseases, [they were exposed to unintended nanopollution generated by hightemperature combustions due to high-technology weapons (Decree of the Italian
Governmental Commission, 3-3-2009)] and 10 samples from the internal organs of aborted
fetuses. The results showed a constant presence of micro- and nano-sized foreign bodies
(about 96% of the cases) in the soldiers‘pathological tissues, while all fetal specimens were
“clean”. This proved the capacity of submichronic particulate matter to negotiate the
physiological barriers, to reach the innermost districts of the organism (1), to accumulate
there and to interact directly with the internal part of the cells. The particles ranged from a
size of 10nm up to few microns (smaller than a red cell’s size) and the elements most
frequently found were Iron, Chromium, Silicon, Calcium and Titanium, even if rare elements
like Gold, Tungsten or Zirconium were occasionally detected (2). Some of those particles
were recognized as engineered, others were nano-sized by-products generated by non
controlled combustions. Specific anamnestic studies about possible exposures to
nanopollution or analyses of the clinical records helped in tracing the internal particulate
matter to the environmental working pollution. These results represent the basic step to the
identification of pathological threshold concentrations of nanoparticle exposure.
[1] Nemmar et al.: Passage of 100nm sized particles in the blood and in the liver ,Circulation
2002, 105:411-15.
[2] Gatti, A et al. 2008. “Nanopathology: The health impact of nanoparticles” book,
PanStanford Publishing Pte.Ltd, Singapore, 1-298
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2.2.8 Double-walled carbon nanotubes: suitable containers for biomedical
applications
Carmen-Mihaela Tîlmaciu1, Brigitte Soula1, Anne-Marie Galibert1, Petar Lukanov1,
Ruediger Klingeler2, Emmanuel Flahaut3
1
Université de Toulouse, UPS, INP, Institut Carnot Cirimat, Toulouse, France
Kirchhoff Institute for Physics, University of Heidelberg, Heidelberg, Germany
3
CNRS, Institut Carnot Cirimat,Toulouse, France
Email: [email protected]
2
In this work, performed within the Marie Curie RTN CARBIO (http://www.carbio.eu), narrow
double-walled CNT (DWNT) were prepared by catalytic chemical vapour deposition, using a
MgO-based catalyst, which was reduced at 1000 ˚C in a mixture of H 2 and CH4, containing
18 mol % of CH4. The selectivity towards DWNT is ca. 80% [1]. Before and after purification
in air, these tubes with inner diameters ≤ 2 nm were filled by capillary action with melted iron
and cobalt precursors (FeI2, FeCl2, FeCp2 or CoI2), followed by reduction in H2, in order to
prepare magnetic nanowires inside the DWNT for hyperthermia application [2]. The
Mössbauer characterizations after reduction of the iron [email protected] in H2, have
evidenced the presence of superparamagnetic nanoparticles of Fe(III) oxides (SPION), which
present very high interest, as they are sensitive to magnetic fields, without retaining
magnetization after removal of the latter [3]. In parallel, after reduction of the [email protected],
AGM and SQUID measurements revealed the presence of ferromagnetic nanowires of cobalt
confined in DWNT.
Using the same method of filling in melted phase,
gadonanotubes ([email protected]) were synthesized for MRI
imaging [4] (Fig. 1). Preliminary measurements of
relaxation times and control (if possible leaks) were
achieved
on
several
samples
with
different
concentrations of gadolinium. The results are
encouraging: a good stability in time (over six months)
and high relaxivities of the [email protected] (about fourty
times greater than the current main clinical agents).
Fig. 1: HRTEM image of DWNT filled with
GdCl3 for MRI application.
Filling in solution with chloroquine diphosphate salt (an antimalarian drug) was also
successfully achieved. Luciferase assay, MTT toxicity test, as well as HRTEM, EDX and
elemental analysis were performed, in order to prove the filling and to quantify the percent of
the drug in the sample.
[1] Flahaut, E et al. Chemical Communications, (2003) 1442
[2] Tîlmaciu, C.-M. et al. Chemical Communications, (2009) 6664
[3] Barnes, A.L. et al., Biomagnetic research and technology, (2007) 5:1
[4] Sitharaman, B. et al., International Journal of Nanomedicine, (2006) 1:291
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2.2.9 A large local nanotoxicolgy project reveals old and new problems
requiring strict interdisciplinarity
Francesco Turci, Mara Ghiazza, Maura Tomatis, Ivana Fenoglio, Bice Fubini
Dip. Chimica IFM, "G. Scansetti" Interdepartmental Centre for Studies on Asbestos and
other Toxic Particulates and NIS - Nanostructured Interfaces and Surfaces
University of Torino, Italy
Email: [email protected]
NanoSAFE, a four years multidisciplinary project (www.centroscansetti.unito.it/nanosafe)
involving local industries and a large variety of research units operating in different fields,
from nanomaterial synthesis to nanotoxicology, founded by local government of Regione
Piemonte, Italy, will conclude on September 2011. Two major Interdepartmental Centers
from the University of Torino, namely the Scansetti Center for the Studies on Asbestos and
other Toxic Particulates and the Nanostructured Interfaces and Surfaces Center (NIS) as
well as two other universities (Turin Polytechnic and the University of Piemonte orientale)
and four industrial partners have been involved.
The project concerns the possible detrimental health effects induced by some nanoparticles
employed by the industrial partners or released in air by incinerators. The NPs and
nanomaterials considered in this project are: i) carbon nanotubes (CNT) and CNT-based
composites possibly employed in the manufacture of friction materials; ii) variously sized and
coated TiO2 NPs for cosmetic usage iii) SiO2, Fe2O3 and C NPs prepared in controlled sizes,
both to mimic the particle size found in incinerator off-gases and to build up a repository of
model particles.
The projects have been organized as follows:
1. synthesis, preparation or gathering of the NP and NP-based materials ;
2. physico chemical characterisation of NP and tests for the presence of adverse
toxicochemical features ;
3. selection, on the basis of findings at point 2, of the most appropriate samples to
proceed with biological tests and trials ;
4. analysis of viability, function, uptake, inflammatory response and genotoxicity of NP
when in contact with selected cell cultures;
5. selection, on the basis of findings at point 4, of the most appropriate samples to
proceed with in vivo trials;
6. test NPs selected at point 4 on adult mice in view of their possible translocation
through the nervous system;
7. some NPs (mainly TiO2) have be also tested on pig- and human-reconstructed skin
tissues
The interactions among the various groups highlighted some relevant problems in
nanotoxicology as well as the requirement of interdisciplinarity and continuous mutual
exchange between the various units.
Taking advantage from this experience, which will be coarsely described, and from traditional
particle toxicology studies, some points/results relevant to well-designed studies on
nanoparticles and HARNS will be reported. The crucial role of several physico-chemical
aspects, beyond classical “characterization” will be highlighted, namely:




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Interparticle forces, agglomeration and dispersion in biological fluids
Free radical generation and quenching
Metal particles and particle associated metals: the iron story
Coatings role and stability
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2.2.10 Genotoxicity testing of titanium dioxide, iron oxide and silica
nanoparticles in human lymphocytes and lymphyblastoid cells using
micronucleus assay
Alena Kazimirova1, Magdalena Barancokova1; Ladislava Wsolova1; Maria Dusinska1, 2
1
Slovak Medical University, Bratislava, Slovakia
Norwegian Institute for Air Research, NILU, Kjeller, Norway
E‐mail: [email protected]
2
Our aim was to test genotoxic potential of selected nanoparticles (NP) (PLGA-PEO, oleic
acid coated and uncoated iron oxide, TiO2, silica) on human peripheral lymphocytes (from 10
volunteers) and on TK6 lymphoblastoid cells. The micronucleus (MN) assay is one of the
most widely used methods for measuring DNA damage. Micronuclei (MNi) originate from
chromosome fragments or whole chromosomes that lag behind at anaphase during nuclear
division. The cytokinesis-block micronucleus assay (CBMN) is the preferred method for
measuring micronuclei in cultured human and/or mammalian cells because scoring is
specifically restricted to once-divided binucleated cells.
Cells were exposed to three NP doses (75, 15 and 3 μg/cm2) for 24 hours (lymphocytes)
and 4, 24, 48 and 72 hours (TK6 cells). Cytochalasin B (6 μg/ml; Sigma-Aldrich) was added
to the cell cultures to induce binucleation of dividing cells. Both, untreated control and
positive control treated with mitomycin C (0.05μg/ml) were used in each experiment. Two
cultures of each sample were set up. At the end of cultivation the cells were harvested and
microscopic slides were prepared. MN analysis was performed on 2000 binucleated cells
with preserved cytoplasm. MNi were evaluated according to the accepted criteria of HUMN
project (www.humn.org). To assess cell proliferation, the cytokinesis – block proliferation
index (CBPI) was determined from 500 cells per culture.
Preliminary data show that PLGA-PEO (75 μg/cm2) and oleic acid coated iron oxide NPs (3
μg/cm2) increased the number of cells with micronuclei while uncoated iron oxide and TiO2
NPs did not show any genotoxic effect in peripheral human lymphocytes. No statistically
significant difference in micronucleus frequency with PLGA-PEO or uncoated iron oxide NPs
was observed (except PLGA-PEO NPs 3 µg/cm2 at 48 h) in TK6 cells.
Supported by EC FP7 [Health-2007-1.3-4], Contract no: 201335; authors thank Giulio
Pojana, Dagmar Bilanicova and Antonio Marcomini for NP characterization.
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2.2.11 In vitro exposure of human macrophages to different functionalized
multi-walled carbon nanotubes: what is the role of the pulmonary
surfactant?
M. Gasser1,2, H.F. Krug1, P. Gehr2, P. Wick1, B. Rothen-Rutishauser2
1
Empa, Swiss Federal Laboratories for Materials Testing and Research, Laboratory for
Materials Biology Interactions, St. Gallen, Switzerland
2
Institute of Anatomy, Division of Histology, University of Bern, Bern, Switzerland
Email: [email protected]
During production and processing of multi-walled carbon nanotubes (MWCNTs) the lung may
be the primary organ affected after aerosol exposure [1]. In the alveoli, the structural units of
gas exchange, MWCNTs first come into contact with the pulmonary surfactant. This barrier
structure at the air-liquid interface, which mainly consists of lipids, prevents the alveoli from
collapsing by reducing the surface tension. Recently we have shown that lipids of the
pulmonary surfactant bind to the MWCNTs and thus affect their surface characteristics [2].
This was shown by a typical pattern of bound plasma proteins on MWCNTs which were precoated with surfactant – this pattern was different to the one on bare MWCNTs. In addition
the functionalization of MWCNTs with positively (-NH2) and negatively (-COOH) charged
side groups alters the pattern of bound proteins.
The aim of the present study was to correlate the different surface configurations of the
MWCNTs to their potential adverse effects on human macrophages in vitro. Thus the role of
the functionalization and the surfactant pre-coating were examined by the exposure of
primary human monocyte-derived macrophages to MWCNTs under the various conditions.
First experiments have shown that pre-coating of the MWCNTs with surfactant reduces the
release of the pro-inflammatory cytokine tumor necrosis factor alpha compared to control
cultures. Furthermore, the quantification of reactive oxygen species and data on cytotoxicity
(Lactate dehydrogenase release) will be presented.
[1] Kaiser et al., Nanomedicine, Vol. 4, 2009
[2] Gasser et al., in preparation, 2010
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2.2.12 Testing the toxicological profile of therapeutic nanoparticles: the
example of a blood-brain barrier model
Lucienne Juillerat-Jeanneret, Catherine Chapuis-Bernasconi, Blanka Halamoda Kenzaoui
University Institute of Pathology, CHUV-UNIL, Lausanne, Switzerland
Email: [email protected]
Nanoparticles are becoming a very interesting option for both medical diagnosis and targeted
drug delivery. Their large biomedical application in the future is probable; however detailed
studies have to be performed to exclude any potential toxicity of therapeutic nanoparticles to
living organisms. Our objectives [1] are to evaluate the interaction of several types of
nanoparticles with cells of different origins: cell uptake and release, transport across
biological barriers and potential cytotoxicity. Five different types of nanoparticles have been
selected for this study: titanium dioxide nanoparticles, iron oxide nanoparticles, either
uncoated and coated with oleic acid, poly(lactic-co-glycolic) acid (PLGA-PEO) nanoparticles
and 2 sized (25 nm and 50 nm) fluorescent silica nanoparticles.
An in vitro model of the blood–brain barrier has been developed using human brain–derived
HCEC endothelial cells in order to study the mechanisms and consequences of the transport
of nanoparticles across this barrier, using optical and transmission electron microscopy.
The results of the transport of iron oxide nanoparticles as well as cellular uptake and toxicity
of tested nanoparticles were influenced by the size and the coating of the nanoparticles
(Fig.1). We found that whereas human brain-derived endothelial cells were able to internalize
nanoparticles, they neither released them after uptake nor transported them across the
endothelial layer, even in the presence of a strong magnetic field. The mechanism of
possible cytotoxicity of these nanoparticles was studied focusing on oxidative stress and
genotoxicity.
The information gained by these approaches will then be useful to design efficient
standardized in vitro methods for testing therapeutic nanoparticles of potential future interest.
Figure 1: Left: cellular uptake of iron oxide nanoparticles (uncoated (A) and oleic acid coated (B)) by HCEC cells
was evaluated after 24 h exposure using the Prussian Blue reaction. Right: TEM image of HCEC cells after 24 h
exposure to uncoated (up) and oleic acid coated (down) iron oxide nanoparticles.
Funded by the FNRS and the european FP7 projects NanoTEST and NanoImpactNet
[1] Dusinska, M and the NanoTest Consortium 2009.Testing strategies for the safety of
nanoparticles used in medical applications. Nanomedicine 4: 605-607
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2.2.13 Silver Wires Significantly Affect Cell Viability and Induce Immune
Activation of A549 Cells
Linda C. Stöhr1, Zoe Megson2, Edgar Gonzalez2, Victor Puntes2, Albert Duschl1, Gertie J.
Oostingh1
1
University of Salzburg, Salzburg, Austria
Institut Català de Nanotecnologia (ICN), Bellaterra, Spain
Email: mailto:[email protected]
2
At current, silver nanowires are rarely used in products, therefore only very few data about
possible health effects exist. The potential toxicity of silver nanoparticles is well-studied and
these particles were described to be cytotoxic. In the presented study, human bronchial
epithelial cells were used and exposed to silver nanoparticles and nanowires. The effects of
these nanomaterials on the viability of the cells and eventual immunomodulatory effects of
these particles were analysed.
Different samples of polyvinylpyrrolidone coated silver nanowires were used, with a diameter
of <100 nm and a length ranging from 1.5 – 20 µm. In addition, spherical silver nanoparticles
(30 nm) and large silver microparticles (<45 µm) were used. These nanomaterials were
tested for their capacity to induce cytotoxicity in A549 cells by employing the CellTiter-Blue®
(analysis of cell metabolism) and the LDH release (analysis of membrane porosity, i.e. cell
death) assays. Moreover, the immunotoxic effects of these nanomaterials were tested using
various stably transfected A549 reporter cell lines, which possess a luciferase reporter
construct under regulation of the promoter of a certain cytokine (IL-6, IL-8 or TNF-α) or of
multiple copies of the NF-B response element. These cell lines have previously been used
for the analysis of metal(oxide) nanoparticle-induced immunomodulatory effects [1]. To
compare the response against the particles in a naive and a stimulated immune system, cells
were treated with recombinant human TNF-α, or left untreated. Close care was taken to
ensure that the concentration curves of the different nanomaterials had an overlap in the total
surface area as well as the number of particles added to the cells.
The results showed that the spherical nanoparticles did not induce cytotoxicity and no altered
immune responses were observed. In contrast, the silver nanowires did significantly reduce
the cell viability and increased the LDH release in a concentration dependent manner after
24 – 48 hours of incubation. Immunomodulatory effects were similar to those observed when
analysing toxicity, although the immune response was generally affected at lower particle
concentrations. A small, but not significant difference was observed for the nanowires with
different lengths, and there is an indication that the smallest nanowires affected the cells to a
larger extent than the larger nanowires.
In conclusion, we found that spherical nanoparticles have a lower impact on the cells
compared to small wire-shaped particles, suggesting shape to be one of the important
factors that determine toxicity.
[1] Pfaller, T et al. 2009. In vitro investigation of immunomodulatory effects caused by
engineered inorganic nanoparticles – the impact of experimental design and cell choice.
Nanotoxicology, March 2009; 3(1): 46-59
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2.2.14 NANOMMUNE: Comprehensive Assessment of Hazardous Effects of
Engineered Nanomaterials on the Immune System
Erika Witasp1, Bengt Fadeel1
1
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet,
Stockholm, Sweden
Email: [email protected]
Despite the tremendous opportunities of engineered nanomaterials (ENs) there are
considerable knowledge gaps concerning the potential hazardous effects of ENs on human
health and the environment. The NANOMMUNE partnership aims to fill these gaps through a
comprehensive assessment of ENs, with particular focus on effects on the immune system.
The recognition and uptake of ENs by immune cells are central questions in our studies. Our
team has demonstrated that surface coating with the phospholipid phosphatidylserine (PS)
promotes the uptake of single-walled carbon nanotubes (SWCNT) by macrophages and this
attenuates pro-inflammatory cytokine secretion by activated macrophages (Konduru et al.,
PLOS ONE 2009). Moreover, human neutrophils can engulf and biodegrade
immunoglobulin-coated SWCNT through a myeloperoxidase-mediated reaction (Kagan et al.,
Nat Nanotechnol 2010). Importantly, the biodegraded nanotubes aspirated into the lungs of
mice caused no inflammatory response. Controlled biodegradation of engineered
nanomaterials is relevant to their safe management and is also of significance for biomedical
applications.
The NANOMMUNE project also intends to assess the response of immune-competent cells
to ENs using a global transcriptomics approach. TiO2 and ZnO nanoparticles were thoroughly
characterized and thereafter the cellular uptake, subcellular localization, and toxic effects
were studied in primary human macrophages and dendritic cells, and a human T cell
leukemia-derived cell line (Jurkat). We observed that ZnO but not TiO2 nanoparticles induced
a dose-dependent toxicity and ROS induction in all three cell types. Global gene expression
identified 11 genes which were significantly differentially expressed in all three cell types
after ZnO stimulation (unpublished observations). We plan to study additional classes of
engineered nanoparticles to explore whether common “nanotoxicogenomic“ signatures exist.
Our consortium is also conducting transcriptomics studies of lung tissue from mice exposed
via pharyngeal aspiration to carbon-based nanomaterials (fullerenes, SWCNT) versus
crocidolite asbestos in order to ascertain whether common signatures exists at the gene
expression level (ongoing collaboration between EU and US partners).
Overall, the NANOMMUNE project results will enhance the understanding of possible
adverse effects of nanomaterials and will thus contribute to a continuous and sustainable
growth of the nanotechnologies. The NANOMMUNE project was launched on September 1st
2008 and will run for 3 years. The project has a total budget of 3.358.500 EURO and is
funded by the European Commission through the 7th Framework Programme (NMP4-SL2008-214281). The consortium consists of 13 research groups at 10 institutes/agencies in
Europe and the United States. The US partners are funded through US funding sources
including National Institutes of Health (NIH). For further information, please visit the project
website: www.nanommune.eu
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2.2.15 Genotoxicity of nanocellulose whiskers in human bronchial epithelial
cells measured by the micronucleus assay
Julia Catalán1,2, Hilkka Järventaus1, Kati Hannukainen1, Eero Kontturi3, Esa Vanhala1, Kai
Savolainen1, Hannu Norppa1
1
Finnish Institute of Occupational Health, Helsinki, Finland
2
University of Zaragoza, Zaragoza, Spain
3
Aalto University School of Science and Technology, Espoo, Finland
Email: [email protected]
Nanocellulosics are among the most promising innovations for wide-variety applications in
materials science. Although nanocellulose is presently prepared and applied only in
laboratory scale, its possible impacts on public health and the environment should be
investigated at an early stage. The aim of the present study was to examine in vitro the
potential genotoxicity of two celluloses: Avicel, a commercially available microcrystalline
cellulose (Fluka) used as a model of a non-nanoscale material (particle size ~50 µm), and
nanocellulose whiskers produced by the Aalto University School of Science and Technology
(mean length 152.2 nm, mean diameter 15.7 nm). Cytotoxicity was analyzed at three
different exposure times (4, 24 and 48 h) by the propidium iodide exclusion technique and
luminometric detection of ATP in human bronchial epithelial BEAS 2B cells. Cytotoxicity
reached the 50% level at about the 100 µg/ml dose for both celluloses. Genotoxicity was
assessed by the analysis of micronuclei (MN) in BEAS 2B cells using various doses (2.5-100
µg/ml) of Avicel and nanocellulose whiskers. The induction of MN was examined by the
cytokinesis-block method after a 48-h treatment with the materials. Our results indicated no
induction of micronucleated binucleate or mononucleate cells by Avicel or nanocellulose
whiskers. No linear dose-dependent response could neither be found for the materials. In
conclusion, our results from the MN assay show that nanocellulose whiskers are not
genotoxic under the conditions tested
Supported by SUNPAP, NMP-2008-1.2-1, The Finnish Centre for Nanocellulosic
Technologies and the Spanish Ministry of Science and Innovation
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2.2.16 The retention of long, but not short, carbon nanotubes leads to
inflammation and progressive fibrosis in the pleural space of mice
Fiona A. Murphy1, Craig A. Poland1, Rodger Duffin1, Khuloud T. Al-Jamal2, Hanene AliBoucetta2, Antonio Nunes2, Fiona Byrne3, Adriele Prina-Mello3, Shouping Li4, Stephen J.
Mather5, Alberto Bianco6, Maurizio Prato4, William MacNee1, Kostas Kostarelos2,*, Ken
Donaldson1,*
1
MRC/University of Edinburgh, Centre for Inflammation Research, Edinburgh, UK
2
Nanomedicine Laboratory, University of London, London, UK
3
School of Physics and Centre for Research on Adaptive Nanostructures and Nanodevices
(CRANN), Trinity College, Dublin, Ireland
4
Center of Excellence for Nanostructured Materials, University of Trieste, Trieste, Italy
5
Department of Nuclear Medicine, St Bartholomew's Hospital, London, U.K
6
CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France
The fibrous shape of carbon nanotubes (CNT) has raised concern that they may pose an
asbestos-like inhalation hazard which may lead to the development of diseases like pleural
mesothelioma. We developed a method to directly instill CNT into the pleural space of mice,
which we utilised to assess the inflammatory and fibrotic effect of CNT at the pleural
mesothelium up to 24 weeks post instillation. By exposing the pleural mesothelium to long
and short CNT we show a length dependent pathogenicity for CNT similar to that seen with
asbestos. The response to long CNT is characterised by an initial acute inflammatory
reaction, typified by an influx of granulocytes and an increase in pleural fluid protein levels,
followed by progressive fibrosis. Histological examination and SEM analysis of the chest
wall show the formation of inflammatory lesions along the parietal pleura which become
increasingly collagenous over time. Conversely, the response seen with short nanotubes is
characterised by a low level inflammatory response which appears to be resolving by 7 days.
We propose the differing responses to long and short CNT is mediated by size-restricted
clearance of particles from the pleural space where short fibres and particles are efficiently
cleared while long fibres are retained at the parietal pleura at points of egress of the lymph.
We confirm this by visualising the migration of short CNT from the pleural space by
SPECT/CT imaging and also demonstrating the clearance of short but not long CNT and
nickel nanowires to the mediastinal lymph nodes. Our data lead us to the conclusion that the
sustained pathogenic response to CNT arises as a result of length-dependent retention in the
pleural cavity.
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2.2.17 The role of nanoparticle-protein interactions in determining the toxic
consequences of nanoparticle exposure
David Brown1, Marco Monpoli2, Iseult Lynch2, Vicky Stone 1, Eva Gubbins1
1
School of Life Sciences, Edinburgh Napier University, Edinburgh, Scotland
Center for BioNano Interactions, University college Dublin, Dublin, Ireland
Email: [email protected]
2
At present considerable uncertainty exists regarding risks from nanoparticles (NPs) and their
corresponding products/applications. Once NPs enter the body they immediately come into
contact with biological fluids such as airway mucus, alveolar lining fluid or blood
components1. These fluids contain, amongst other molecules, an abundance of proteins and
lipids. It has been proposed2 that an area of particular interest in nanosafety assessment
studies is this interaction layer between the particle and the biological fluid particularly the
associated proteins (the ‘protein corona’). Understanding these interactions in depth will
allow a more relevant understanding of NP induced toxicity.
In this study, we focused on two different chemical species of iron oxide NPs (IONPs), Fe203
(280nm and 22nm) and Fe304 (40nm), which are the most commonly found iron oxides in
nature and are finding increasing applications in nanoparticulate form. Initial studies
investigated some basic properties of these particles (size, shape, charge, metal analysis
etc) and cytotoxicity studies were performed using a murine Macrophage J774 cell line.
These initial results were followed up by NP-protein binding studies looking particularly at NP
binding to key proteins such as cytokines and albumin, and more complex fluids such as lung
lining fluid ( LLF) and foetal calf serum.
Initial results have revealed the particle characteristics and the exposure studies found that
the IONPs cause cell death only at high concentrations (500-250μg/ml LDH/24 hour
exposure). The IONPs also seem to be selective in which proteins they bind. These
preliminary results will be followed up by in-depth protein binding studies to investigate how
NPs with different surface characteristics vary in their ability to bind proteins, to investigate
the dynamic process by which specific proteins bind to IONPs and to investigate the binding
affinities, equilibrium constants, and stoichiometries of specific proteins in complex fluids
(such as lung lining fluid) to IONPs
[1] Brain, J.D., et al., Biologic responses to nanomaterials depend on exposure, clearance,
and material characteristics. Nanotoxicology, 2009. 3(3): p. 174-180.
[2] Lynch, I., Dawson, K.A., Linse, S., Detecting crytpic epitopes in proteins adsorbed onto
nanoparticles. Science STKE, 2006. 327: p. pp pe 14.
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2.2.18 Biocompatibility of poly-N-isopropylacrylamide (PNIPAM) nanoparticles
with human keratinocyte (HaCaT) and colon cells (SW 480)
Pratap C. Naha1,2; Tiziana Tenuta3, Kenneth A. Dawson3; Iseult Lynch3; Fiona M. Lyng1,
Hugh J. Byrne2
1
DIT Centre for Radiation and Environmental Science Centre, Focas Research Institute,
Dublin Institute of Technology, Dublin, Ireland
2
Nanolab, Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland
3
Centre for BioNano Interactions, School of Chemistry and Chemical Biology and Conway
Institute, University College Dublin, Dublin, Ireland
Email: [email protected]
Polymeric nanoparticles are widely used in different aspects of the medical field in terms of
diagnosis, tissue engineering and as drug delivery devices. As nanomaterials are currently
being widely used in modern technology, there is an increasing need for information
regarding the human health and environmental implications of these nanomaterials. To date
the human health impacts of this class of materials have received the greatest attention and
more recently studies of their environmental effects are being reported in the literature. This
study focussed on interaction of the PNIPAM nanoparticles with mammalian cells. The cytoand genotoxicity of PNIPAM nanoparticles were analysed in two representative mammalian
cell lines, SW480, a colon, and HaCaT, a dermal cell line. Physical characterisation in terms
of particle size and zeta potential of the PNIPAM nanoparticles was carried out both in
aqueous solution and in the appropriate cell culture media. Uptake and co-localisation of
fluorescently labelled PNIPAM nanoparticles was monitored in both cell lines using confocal
laser scanning microscopy. Genotoxicity analysis using the Comet assay was performed in
both cell lines to evaluate any DNA damage. It was observed that the PNIPAM nanoparticles
were internalized and localised in lysosomes within 24 hrs. No significant cytotoxic response
(p ≤ 0.05) was observed in either cell line over concentration ranges from 12.5 mg/l to 1000
mg/l for all exposure time periods. Furthermore, no significant genotoxic response (p ≤ 0.05)
was observed in either cell line over concentration ranges from 12.5 mg/l to 800 mg/l for all
exposure time periods. The results suggest that the PNIPAM nanoparticles show excellent
biocompatibility in vitro.
Key words: PNIPAM, Nanotoxicology, HaCaT, SW 480, Lysosomes, Genotoxicity, In Vitro.
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2.2.19 Comparing the interaction of Ag and Au nanoparticles with a 3D in vitro
model of the epithelial airway barrier
Fabian Herzog1, Martin J. D. Clift1, Christina Brandenberger2, Barbara Rothen-Rutishauser1
1
University of Bern, Institute of Anatomy, Bern, Switzerland
Email: [email protected]
Due to their potent antibacterial properties, Silver (Ag) nanoparticles (NPs) are currently the
fastest growing product category in the nano-size range [1]. Despite this, a clear
understanding of their interaction with biological systems is lacking, hence increased
attention has begun to focus upon the potential human health effects following Ag NP
exposure. It is imperative therefore, that increased, in-depth research is performed in order to
assess if the potential advantageous properties of Ag NPs can be realised safely. The aim of
this study is to investigate the potential adverse effects of Ag NPs on a triple cell co-culture
model of the human epithelial airway barrier; composed of epithelial cells, human monocytederived macrophages and dendritic cells [2]; in combination with a recently developed
exposure system which enables the study of NP-lung interactions at the air-liquid interface in
vitro [3]. To validate any obtained results we will compare the Ag particles with a previous
study which assessed gold (Au) NPs with a similar experimental setup [4].
In the previous study [4], the triple cell co-culture was exposed to a well-characterised
aerosol of 15nm Au NPs (61ng Au/cm2 and 561ngAu/cm2 deposition) and post-incubated for
4h and 24h. The mRNA induction of pro-inflammatory (TNFα, IL-8, iNOS) and oxidative
stress markers (HO-1, SOD2) was measured. A pre-stimulation of the triple cell co-culture
with lipopolysacharide (LPS) was also performed to further study the effects of the Au NPs
upon the in vitro system when under inflammatory conditions. In addition, Au NP deposition
and cellular uptake were qualitatively analysed by transmission electron microscopy (TEM).
A homogeneous deposition was observed, and Au NPs were found to enter the cells in a
concentration dependent manner. Au NPs were localised in vesicles but not in the nucleus
and mitochondria. No mRNA induction following Au NP exposure was observed for all
markers (TNFα, IL-8, iNOS, HO-1, SOD2) tested. The cell culture system was sensitive to
LPS but Au NPs did not cause any additional effect.
Initial experiments using Ag NPs have been performed. TEM has shown a homogenous
deposition of Ag NPs, in comparison with the deposition of the Au NPs, when using the
ALICE system [3,4]. The interaction between the in vitro triple cell co-culture and Ag NPs, as
well as to the contribution of Ag ions to this interaction, is currently under investigation.
This work is supported by the Swiss Federal Office of Public Health.
[1] Woodrow Wilson International Center for Scholars. 2009. Nanotechnology Consumer
Products Inventory. www.nanotechproject.org/consumerproducts. Accessed: 01.11.2010
[2] Rothen-Rutishauser, B. et al. 2005. A three-dimensional cellular model of the human
respiratory tract to study the interaction with particles. Am J Respir Cell Mol Biol 32(4): 281-9.
[3] Lenz, A.G. et al. 2009. A dose-controlled system for air-liquid interface cell exposure and
application to zinc oxide nanoparticles. Part Fibre Toxicol. 6: 32.
[4] Brandenberger, C. et al. 2010. Effects and uptake of gold nanoparticles deposited at the
air-liquid interface of a human epithelial airway model. Toxicol Appl Pharmacol. 242: 56-65
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2.2.20 Assessment of cytotoxicity and genotoxicity of uncoated and oleic acid
coated magnetite nanoparticles
Zuzana Magdolenova1, Alessandra Rinna1, Lise Fjellsbø1, Martina Drlickova2, Dagmar
Bilanicova3, Giulio Pojana3, Antonio Marcomini3, Maria Dusinska1,2
1
Norwegian Institute for Air research, Kjeller, Norway
2
Slovak Medical University, Bratislava, Slovakia
3
University Ca’ Foscari of Venice, Venice, Italy
Email: [email protected]
Magnetite nanoparticles (NPs) are used in various biomedical applications, such as
diagnostic agents for cancer therapy, although their toxicity depending on the particle coating
has been demonstrated. Surface properties are among features which may influence
behaviour and toxicity of NPs. We investigated the cytotoxic and genotoxic effects of
uncoated and coated magnetite NPs (both 9 nm core, manufactured by PlasmaChem) in
vitro. The comet assay is one of the most promising methods for genotoxicity testing due to
its simplicity, versatility, and the ability to detect different DNA lesions. The alkaline comet
assay and its modification for detection of oxidized lesions with lesion specific enzyme
formamidopyrimidine DNA glycosylase (FPG), were used to examine strand breaks and
oxidized bases (FPG sites) together with proliferation (growth activity) and plating efficiency
(colony forming ability) assays to assess cytotoxicity in two in vitro models - TK6
lymphoblastoid and Cos-1 monkey kidney cell lines As the different coating (surface
modification) of NPs may exhibit different effect in cyto- and genotoxicity, we used, both
uncoated magnetite (TEM: 5-13 nm; chemical composition Fe,O; particle concentration
2.8%; zeta poenial -2.8mV; crystal structure octahedral; shape oblong) and oleic acid coated
magnetite (TEM: 5-12 nm; particle concentration 26%; zeta potential -31.9mV; crystal
structure octahedral; shape oblong). Our results indicate that bare magnetite NPs cause no
cytotoxic and no genotoxic effects using the comet assay in both Cos-1 and TK6 cells after 2
and 24h treatment. Oleic acid coated magnetite NPs increased levels of strand breaks and
oxidized bases after 2h exposure to the highest concentration (75 µg/cm2) in both Cos-1 and
TK6 cells. The highest concentrations of the oleic acid coated magnetite showed also
cytotoxicity measured by plating efficiency or relative proliferation activity, respectively. The
cytotoxic and genotoxic effects were time dependent. These results suggest that cyto- and
geno- toxicological profile of magnetite NPs is dependent on their surface coating.
Supported by EC FP7 [Health-2007-1.3-4], Contract no: 201335.
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2.2.21 Molecular insight of the interaction between surface-tailored Si/SiO2
wafers and fibrinogen
Arianna Marucco1, Emanuele Carella1, Ivana Fenoglio1, Bice Fubini1, Francesco Turci1, Luke
O’Neill2, Hugh J. Byrne2, Giacomo Ceccone3, François Rossi3
1
Dip. Chimica IFM, "G. Scansetti" Interdepartmental Centre for Studies on Asbestos and
other Toxic Particulates, and NIS - Nanostructured Interfaces and Surfaces, University of
Torino, Italy
2
Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland
3
European Commission, Joint Research Centre, Institute for Health and Consumer
Protection, Ispra, Italy
Email: [email protected]
The interaction with proteins appears to be among the early events occurring when a nano
particle contacts biological fluids and is a crucial consideration governing the fate of and the
responses to a particle inside a living organism. The amount of proteins, the composition of
the protein “corona” and the possible conformational changes or rearrangements of the
proteins on the particle surface have a profound influence on the biological events following
penetration and ultimately determine the response of the tissues towards exogenous
materials.
An integrated picture of the adsorption process has recently emerged, whereby some
proteins having distinct resistance to conformational change, net charge and charge
distribution were adsorbed on amorphous nanosized silica powders having a hydrophilic
character [1]. The aim of this continuation study was to elucidate the effect of silica
substrates with differing amount of surface silanols/siloxanes, hence different hydrophilicity,
on the molecular mechanism of adsorption of fibrinogen. Fibrinogen was selected among
other plasma proteins because of its relevance to blood clotting processes. Silicon dioxide
layers were grown on the surface of silicon wafers using thermal oxidation. Some of these
wafers were further treated in order to decrease (heating in vacuum at 800°C) or increase
(cold plasma in H2O vapour) the surface hydrophilicity.
Tailored Si/SiO2 surfaces were thoroughly characterized by means of several physicochemical techniques (contact angle, Raman, XPS) and the adsorption of fibrinogen was
monitored with confocal micro-Raman spectroscopy, AMF, and in situ ellipsometry.
We have shown that different molecular portions of fibrinogen interact with the Si/SiO2
surface when the surface hydrophilicity of tailored wafers is modified. This means that
surface hydrophilicity likely governs fibrinogen adsorption on silica substrates and can alter
to different extent fibrinogen biological function. We believe that these results are relevant for
both toxicity and material biocompatibility studies.
[1] Turci, F. et al. 2010. An Integrated Approach to the Study of the Interaction between
Proteins and Nanoparticles. Langmuir. DOI: 10.1021/la904758j
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2.2.22 Genotoxicity of zinc oxide nanoparticles in human mesothelial and
bronchial epithelial cells in vitro
Hanna K Lindberg1, Kirsi Siivola1, Ghita C-M Falck1, Satu Suhonen1, Hilkka Järventaus1,
Minnamari Vippola1,2, Kai Savolainen1, Julia Catalán1,3, Hannu Norppa1
1
Finnish Institute of Occupational Health, Helsinki, Finland
2
Tampere University of Technology, Tampere, Finland
3
University of Zaragoza, Zaragoza, Spain
Email: [email protected]
ZnO nanoparticles, used, e.g., in cosmetics, are genotoxic and strongly cytotoxic in various
in vitro systems, but the underlying mechanisms are not well understood. ZnO is partly
soluble, and its solubility is increased in acidic conditions and in the presence of chelators.
The genotoxic effects of ZnO in vitro may be caused by Zn2+ ions released from the particles
(a) to the cell culture medium or (b) inside the cell, after particle uptake. In the former case,
the genotoxicity of ZnO nanoparticles should be comparable to soluble zinc compounds at
similar ion concentrations, while Zn ion release inside the cell could be expected to result in a
differential effect.
We compared the genotoxicity of the slightly soluble ZnO (zincite; 30-35 nm; Umicore)
nanoparticles and soluble ZnCl2 in human mesothelial cells (MeT 5A) and bronchial epithelial
cells (BEAS 2B) in vitro. The single cell gel electrophoresis (comet) assay was applied to
study the induction of DNA damage (4-h, 24-h, and 48-h exposures) in MeT 5A and BEAS
2B cells. The induction of micronuclei (MN) was examined by the cytokinesis-block technique
in BEAS 2B cells.
ZnO was clearly more cytotoxic than ZnCl2 in both cell types, as measured by viable cell
number relative to control using the Trypan blue exclusion technique. In MeT 5A cells, a
clear DNA-damaging effect was observed after the 4-h ZnO exposure but not after the longer
treatments, whereas ZnCl2 induced DNA damage only in the 24-h and 48-h treatments. In
BEAS 2B cells, DNA damage induction occurred at 4 h by ZnO and at 4 h and 24 h by ZnCl2.
However, ZnO caused a clear dose-dependent induction of MN in BEAS 2B cells at doses
that were not cytotoxic. Centromeric and telomeric fluorescence in situ hybridization
suggested that ZnO induced MN by both clastogenic and aneugenic mechanisms. ZnCl2 did
not affect the number of MN.
Our results show that slightly soluble ZnO nanoparticles induce DNA damage in human
BEAS 2B and MeT 5A cells at shorter exposure times than the soluble ZnCl2. Furthermore,
ZnO but not ZnCl2 induces MN in BEAS 2B cells. These findings probably reflect a fast
uptake and intracellular solubilisation of the ZnO nanoparticles.
Funded
by
the
European
Commission
(NANOSH,
NANOGENOTOX, 2009 21 01) and Academy of Finland
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Session 3
3 Human impact of engineered nanomaterials and lessons
for the nanomedical field
3.1 Oral presentations
3.1.1 Silicon nitride porous membranes for nanoparticle translocation in vitro
assay
Silvia Angeloni1, Mélanie Favre1, Marta Giazzon1, Nadège Matthey1 and Martha Liley1
1
CSEM SA Centre Suisse d’Electronique et Microtechnique, Neuchâtel, Switzerland
Email: [email protected]
The risk assessment of engineered nanomaterials may benefit from on purpose designed in
vitro tests for nanoparticle uptake investigation. These tests must respond to two major
requirements. Firstly they are supposed to mimic the in vivo conditions and possibly be
representative of human biological barriers (BBs). Secondly their design requires well
controlled components because the significance of the data strongly depends on the
reproducibility of the conditions under which they are collected.
A model BB involves a cellular layer supported by a porous membrane. Recently at CSEM
we have been developing thin, transparent, mechanically robust microporous membranes
using microfabrication technology. They are made of silicon nitride, they are thinner than the
commercially available insert membranes, thus they exhibit improved translocation
properties. They display 1 cm2 of surface available for cell growth featuring ordered pores
whose diameter and density can be tuned: from 1 to 3 microns for the pore size and from 5
to 20% for the typical pore surface fraction. This in house technology allows for the
production of highly reproducible membranes. This is an advantage in the direction of
standardised procedures for cell culture. The same technology allows tuning all the above
listed features according to the specific application.
Different epithelial cell lines have been successfully grown to tightness on the membranes.
Miniaturized platinum microelectrodes can be embedded in the chip for TEER (Trans
Epithelial Electrical Resistance) measurements. This feature makes the silicon nitride
membranes suitable to ensure the quality control of the tight junction of the BB model in a
non invasive way.
Specifically designed holders allow them to be used with standard laboratory equipment and
consumables. Nevertheless they are especially suitable to be imagined as a component of
more complex systems, eventuality leading to automated microfluidic devices, which
combined with NP readouts will contribute to the still missing set up of benchmark tests,
domain where the scientific community is investing a major effort. Smart chips for the
evaluation of simple nanoparticle translocation screening or preliminary immune response
screening according to the complexity of the hosted model can be conceived.
Figure 1: (left to right)
Membrane array chips with
contact pads. Membrane wells,
and membrane wells blow-up.
Pores area (1um pores).
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3.1.2 Exposure of lung cells in vitro to zinc oxide: A comparison between
suspension and aerosol exposure scenarios
David O. Raemy1, Ludwig K. Limbach2, Robert N. Grass2, Wendelin J. Stark2, Peter Gehr1,
B. Rothen-Rutishauser1
1
Institute of Anatomy, University of Bern, Bern, Switzerland
Functional materials laboratory,ETH Zurich, Zurich, Switzerland
Email: [email protected]
2
Most current in vitro approaches in nanotoxicology base on exposure of submerged cell
cultures to particle suspensions. Such an approach does not reflect the pulmonary
compartment, where aerosolized particles are inhaled and might be deposited on the surface
of lung cells. As a more realistic simulation of this scenario, efforts were made towards direct
delivery of aerosols to air-liquid cultivated cell cultures [1].
The aim ot this study is to provide a direct comparison of the in vitro toxicology of a reference
particle under both exposure conditions. The biological experiments have been carried out
with a 3D model of the human epithelial airway barrier [2], cultivated either under submerged
conditions or at the air-liquid interface. Analysis include endpoints as cytotoxicity (LDH
assay) the release of (pro)-inflammatory cytokines / chemokines (TNFαIL-8), antioxidant
status (GSH) and cellular morphology assessed by microscopy methods. Zinc oxide (ZnO)
was chosen, because it is already produced in high tonnage (as e.g. pigment, sunscreen),
and can be synthesized in an aerosolized state by flame spray pyrolysis (FSP) [3].
A mayor challenge of this study is comparing the dosimetry of the aerosol and suspension
exposure scenario, to ensure identical cellular doses in each case. A series of ZnO aerosols
was generated by adjusting the runtime of the FSP-reactor, and deposition per area was
measured over 30 min by atomic absorption spectroscopy (AAS). Additionally, the aerosol
was characterized in terms of number concentration and agglomeration properties, to get an
impression of the deposited agglomerate size. The obtained deposition data in the aerosol
provide the dose range which has to be covered in further suspension experiments. As
comparison, ZnO deposition onto submerged cell cultures was determined by AAS.
First results show that cells exposed to a ZnO suspension with comparable mass deposition
as in the aerosol, revealed increase TNFα release after 4h exposure. Further experiments
will clarify if such an effect can also be associated to a corresponding aerosol scenario.
We could show that the deposition of ZnO on cell cultures in suspension and in an air-liquid
exposure system could be adjusted in such a way that the same particle concentration (mass
per area) is applied to the cell surface. This approach will allow us to compare the toxicity of
ZnO in two different exposure scenarios.
This work is supported by the Lungenliga Bern.
[1] Rothen-Rutishauser, et al. 2009. . Environ. Sci. Technol. 43: 2634–2640
[2] Blank et al. 2007 Am J. Mol. Cell. Biol. 36: 669-677
[3] Brunner, et al. 2006.. Environ. Sci. Technol., 40: 4374–4381
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3.1.3 CuO nanoparticles act via a Trojan horse type mechanism
Hanna L. Karlsson1,2, Pontus Cronholm1, Klara Midander4, Lennart Möller1, Inger Odnevall
Wallinder4
1
Karolinska Institutet, Dep. of Biosciences and Nutrition, Stockholm, Sweden
Karolinska Institutet, The Institute of Environmental Medicine, Stockholm, Sweden
4
Royal Institute of Technology, Div. Surface and Corrosion Science, Stockholm, Sweden
Email: [email protected]
2
The increased use of nanoparticles raises concern about their toxic properties. We showed
recently that CuO nanoparticles were the most toxic among different metal oxide
nanoparticles investigated [1] and that the toxicity of nano-sized Cu and CuO is higher
compared to micron-sized particles [2]. From a risk assessment perspective, it is important to
understand whether the toxicity is due to the particles as such or to released ionic species.
We hypothesize that the toxicity of CuO nanoparticles is highly dependent on the particles
structure and that one reason is due to increase cellular uptake via the so-called Trojan
horse type mechanism. The aim of this study was to test this hypothesis. The toxicity of CuO
nanoparticles was assessed in terms of cell death and DNA damage following exposure of
human lung epithelial cells. The toxicity was compared to that of a) Cu ions from a Cu salt
(CuCl2) and of b) the released ionic species from CuO nanoparticles. Furthermore, the
intracellular concentration of Cu was analyzed using atomic absorption spectrophotometry
(AAS). The results showed that when cells were exposed to the same mass of Cu, the
toxicity of the CuO nanoparticles was much higher compared to that of the ions or of the
released fraction from CuO. In addition, higher amount of intracellular Cu was also detected.
It was concluded that CuO nanoparticles likely act via a Trojan horse type mechanism. The
particle structure seems to increase the cellular uptake compared to that of metal ions. Once
inside the cell, the toxicity is likely caused by a combination of metal ions and a reactive
surface of the CuO nanoparticles.
[1] Karlsson, HL, et al. 2008. Copper oxide nanoparticles are highly toxic: a comparison
between metal oxide nanoparticles and carbon nanotubes. Chem Res Toxicol. 21:1726-32
[2] Midander, K et al 2009. Surface characteristics, copper release, and toxicity of nano- and
micrometer-sized copper and copper(II) oxide particles: a cross-disciplinary study.
Small. 5:389-99
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3.1.4 A Screening Tool for Nanoparticles in Toxicity Experiments
Ruud Peters, Elly Wijma, Agata Walczak, Meike van der Zande and Hans Bouwmeester
RIKILT – Institute of Food Safety, AE Wageningen, The Netherlands
Email: [email protected]
The potential benefits for consumers and producers of the application of nanotechnology are
widely recognized. Products based on nanotechnology or containing engineered
nanoparticles are already manufactured in the field of electronics, consumer products and
pharmaceutical industry, and are beginning to impact the food associated industries. As a
consequence direct and indirect consumer exposure to nanoparticles is likely. Detection and
characterization of nanoparticles in biological and toxicological tests is an essential part of
understanding the potential benefits as well as the potential risks of the application of
nanoparticles [1]. Currently, size separation techniques in combination with ICPMS are used
to determine nanoparticles [2]. However, sample preparation remains problematic. As an
alternative single particle ICPMS was studied and applied as a screening method for the
determination and characterization of nanoparticles, allowing for fast analysis with limited
sample preparation.
Single particle ICPMS is a time modulated method that is able to detect individual
nanoparticles. Nanoparticles introduced into the ICPMS produce a plume of ions that is
detected as a signal spike in the mass spectrometer. This allows for determining the mass of
the particle. From that a particle size can be calculated. In addition, ionic concentrations of
the same element can be distinguished from particles and determined in the same data. The
method was validated and used as a screening method for the determination of silver
(currently sizes >20nm) and silica nanoparticles in samples from an in vitro digestion model.
The digestion model consists of three steps; a short incubation with saliva; addition of gastric
juice followed by a two hour incubation; addition of duodenal and bile juice followed by a two
hour incubation [3]. Since the sensitivity of the single particle ICPMS method is very high (in
the nanogram per /liter range), sample preparation is generally limited to dilution and the
diluted sample is directly introduced into the ICPMS. The results indicate that nanoparticles
do survive the saliva incubation, but do agglomerate during the gastric juice incubation. In
addition, for silver nanoparticles an increase of the silver ion concentration was found. This
may indicate that the availability of nanoparticles for uptake is limited. The presence of silver
nanoparticles in biological samples like blood and liver was also studied. Typically, samples
were enzymatically digested and the liquid digest diluted to reach a concentration range
suitable for the single particle ICPMS method. The first results indicate that, depending on
the particle size, there is an uptake of silver nanoparticles from the food into the body.
[1] Bouwmeester H, et al. 2010. Minimal analytical characterisation of engineered
nanomaterials needed for hazard assessment in biological matrices. Nanotoxicology DOI:
10.3109/17435391003775266.
[2] Dekkers S. et al. 2010. Presence and risks of nanosilica in food products. Nanotoxicology,
DOI: 10.3109/17435390.2010.519836
[3] Oomen AG et al., 2003. Development of an in vitro digestion model for estimating the
bioaccessibility of soil contaminants. Arch Environ Contam Toxicol. 2003 Apr;44(3):281-7.
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3.2 Poster presentations
3.2.1 Use of fluorescent amorphous silica to study the intracellular fate of
nanoparticles
Chiara Uboldi, Guido Giudetti, Douglas Gilliland, François Rossi
European Commission, DG-JRC, IHCP-Nanobiosciences Unit, Ispra, Italy
Email: [email protected]
Due to their low or absent toxicity and their versatility, amorphous Silica (SiO2) nanoparticles
(NPs) are nowadays widely used in many industrial applications, such as glass production,
cosmetics, dentistry and telecommunications. Moreover, silica NPs are candidate tools in
several biomedical applications, such as gene therapy and cancer diagnosis.
We have investigated the uptake and the intracellular localization of 85 nm-sized fluorescent
amorphous silica NPs, labelled with Tris(2,2′-bipyridyl)-dichlororuthenium(II) hexahydrate
(Ru(II)(bipy)3Cl2). The internalization has been studied in four cell lines of human origin,
which are representative for different body compartments (A549, lung; Caco-2, intestine;
HaCaT, skin; HepG2, liver). By fluorescence microscopy and at different exposure times (224h), we have examined a panel of markers for the endosomal pathway (EEA-1 for early
endosomes; LAMP-1 for late endosomes; LAMP-2 for lysosomes), as well as the Golgi
complex and the endoplasmic reticulum. We have observed that the SiO2-Ru(II)(bipy)3Cl2
NPs show little or no toxicity, are easily internalized and stored in vesicles located in the perinuclear region. Our current results will be the starting point for future inhibition experiments
to specifically address the pathway that is implicated in the internalization of these specific
nanoparticles.
Figure 1: HaCaT cells exposed for 24h to fluorescent silica nanoparticles. Blue: nuclei; Green: lysosomes; Red:
SiO2- Ru(II)(bipy)3Cl2 nanoparticles.
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3.2.2 Kinetics of chitosan nanoparticles in mice
Mina Choi1, Wan-Seob Cho2
1
Division of Toxicological Research, Korea Food and Drug Administration, Korea
ELEGI/Colt Laboratory, Centre for Inflammation Research, University of Edinburgh
Email: [email protected]
2
Recently pulmonary delivery of nanoparticles (NP) loading therapeutic agents has been
considered recently for both lung disorders and systemic circulation. Hydrophobically
modified glycol chitosan (HGC) NP as an organic NP have advantages compared to
inorganic NP including bio-compatibility and bio-degradability inside of the body. Here, we
evaluated the kinetics and toxicity of HGC NP by intratracheal instillation to mice. HGC NP
showed a positive charge and average hydrodynamic size was around 350 nm (Figure 1).
The half-life of NP in the lung was determined as 131.97 ± 50.51 h (Figure 1). NP showed
rapid uptake into systemic circulation and excretion via urine which was peaked at 6 h after
instillation. The levels of NP in the several extrapulmonary organs were extremely low and
transient. Acute neutrophilic inflammation was induced by HGC NP from 6 h to day 3 postinstillation. Expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and chemokine
(MIP-1α) in lung showed an increase from 1 h to 24 h after instillation and recovered
thereafter. Our findings suggest that HGC NP can be successful candidates for use as
pulmonary delivery vehicles, owing to their excellent biocompatibility, transiency, and low
pulmonary toxicity, and property of rapid elimination without accumulation.
Figure 1: The structure of HGC NP and the pulmonary kinetics.
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3.2.3 Preliminary Eco-nanotoxicity results of C60 and Carbon Black assessed
by established tests over a range of trophic levels in the Aquatic
environment
K. Carey1, K. Bhattacharya1, H.J.Byrne1, G.Chambers2, A. Casey1
1
Focas Research Institute, DIT, Dublin, Ireland
2
Department of Physics, Dublin, Ireland
Email: [email protected]
According to a project carried out on emerging nanomaterials in 2008, its estimated there are
more than 800 consumer products on the market that contain Nano Particles, (NPs), and that
number is increasing. The use of NPs in industrial and household applications will very likely
lead to the release of these into the environment. Aquatic environments may be particularly
vulnerable due to the potential for rapid mixing and dispersal of nanomaterials. There is an
urgent need for eco-nanotoxicology studies on the affects of NPs on a range of aquatic
species at all levels of the aquatic ecosystem. The most commonly employed type of Nano
material is carbon, its uses ranging from cosmetics to car tyres. The current study shows
preliminary eco-nanotoxicology results for C60 and carbon black from 100ppm that merit
further investigation. These studies include 24 hour Thamnocephalus platyurus toxicity test,
acute Daphnia magna toxicity study and growth inhibition of Pseudokirchneriella subcapitata.
In the current study C60 showed no significant toxicity in the tests employed, Carbon Black
showed significant toxicity in both the Daphnia magna acute toxicity study and the
Pseudokirchneriella subcapitata growth inhibition test. Future work for this study includes NP
characterisation and expansion of trophic levels studied.
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3.2.4 Ecotoxicity of fluorescent silica nanoparticles in a battery of freshwater
test species
Fiona Lyng, Ailbhe Macken, Hugh J. Byrne, Maria Casado
Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland
Email: [email protected]
The anticipated increase in nanoparticle production makes exposure of the environment to
these materials more and more likely. Assessing the benefits and risks of nanomaterials
requires a better understanding of their chemistry, mobility, bioavailability, and ecotoxicity in
the environment. Nanoparticles currently in use, or nearly so, in industry are studied as a
priority. Amorphous silica nanoparticles are already used commercially in foods, have
significant industrial relevance to Ireland via their use in the IT sector, and are thus likely to
have an early appearance in the environment, making them a strategic starting point. By
labelling these particles, representative biological and environmental fate as a function of
size will be studied by tracking and imaging methods.
In this project, the ecotoxiciticy of well-characterized 50 nm and 100 nm plain and
fluorescently labelled amorphous silica nanoparticles on a test battery of aquatic organisms
representing four trophic levels are investigated. The tests used are validated and
standardized short-term methods for estimating the acute and chronic toxicity of toxicants to
bacteria, algae, invertebrates and fish. Preliminary results show no acute toxicity of
concentrations up to 1000 ppm of the different types and sizes of silica nanoparticles to the
different species and further testing is on-going to predict the chronic effects of silica
nanoparticles on Daphnia magna reproduction.
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3.2.5 Fate and behaviour of TiO2 Nanomaterials in the environment influenced
by their shape, size and surface area
Carmen Nickel1, Bryan Hellack1, Stefan Gartiser2, Stephan Gabsch3, Michael Stintz3, Hanna
Maes4, Stefanie Damme4, Lothar Erdinger5, Thomas A.J. Kuhlbusch1
1
Institute of Energy and Environmental Technology IUTA e.V., Duisburg, Germany
2
Hydrotox GmbH, Freiburg, Germany
3
TU Dresden - Dresden, Germany
4
RWTH Aachen University, Aachen, Germany
5
Universitätsklinikum Heidelberg, Heidelberg, Germany
Email: [email protected]
Engineered Nanomaterials (ENM) are commonly used in production processes and materials
and consequently already exist in many everyday life products. Due to this they can be
released into the environment during their life cycle and the possibility of exposure to these
materials also increases [1]. Especially the knowledge of fate, behaviour, exposure routes
and concentration of these ENM in the environment is still lacking.
The aim of this study was to generate information about the fate and behaviour of TiO2
Nanomaterials [NM] at two specific points of their life cycle. Therefore the behaviour of nanoTiO2 in two compartments with possible exposure was investigated. Experiments with the
titania P25 in a laboratory sewage treatment plant (according to OECD Test Guideline No.
303A) and three different TiO2 materials for leaching experiments in soil (according to OECD
Test Guideline No. 312) in a laboratory scale were conducted.
A first objective was to generate stable nanomaterial suspensions for the tests. The effect of
ultrasonication, material concentration, test volume and pH-value were investigated. Results
show no concentration but pH value dependent differences of the (functionalised and nonfunctionalised) TiO2 Materials P25, PC 105 and UV Titan M262. For example P25 showed no
influence on the average size by varying the pH, whereas PC105 and UV Titan M262
showed a pH dependency, by increased stability at lower pH values.
The fate and behaviour of three different concentrations of P25 in a laboratory sewage
treatment plant were studied. The suspension was characterised before it was applied using
a Dynamic light scattering system. During the experiment the sludge and the effluent of the
laboratory treatment plant was collected and analysed by ICP-MS and SEM-EDX. First
results are expected in near future and will be presented at the conference.
The mobility, fate and behaviour of different TiO2 materials (P25, PC105, UV TitanM262) in
different types of soils were studied, to also get information about the effect of different
parameters like pH-value, organic matter or cation exchange capacity. Pre-tests indicate no
mobility of the TiO2 NM if dry powder of the material is applied to the soil surface, in contrast
to results from other studies [2] where a high mobility of TiO2 NM in soils was detected. Due
to this further tests were conducted applying suspensions of the NM to generate smaller
particles (compared with the powder) so that a higher mobility can be expected. The eluate
was collected and the soil was sectioned in segments for ICP-MS and SEM-analyses. First
results from this test will be presented.
[1] Nowak, et al. (2007): Occurrence, behaviour and effects of nanoparticles in the
environment. Environmental Pollution 150, 5-20
[2] Fang, J. et al. (2009): Stability of titania nanoparticles in soil suspensions and transport in
saturated homogeneous soil columns, Environmental Pollution 157(4), 1101-1109
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Session 4
4 Implications from environmental fate & behaviour
research for the field of nanomedicine involving
nanomaterials
4.1 Oral presentations
4.1.1 Biological Interactions of Gold Nanoparticles: A Model System for
Nanotoxicity?
Mathias Brust
Department of Chemistry, University of Liverpool, Liverpool, UK
Email: [email protected]
In this lecture I will summarise the experience we and many other groups have had with
studying the interactions of gold nanoparticles with biological systems. These are
predominantly studies of cellular uptake and intracellular fate, mainly addressing the question
to what extent these phenomena depend on controllable parameters such as the surface
chemistry of the nanoparticles. Gold nanoparticles do not represent a serious concern for the
environment or for human health, chiefly because gold is relatively rare and will never be
present in the environment at concentrations where its potential toxicity may become a
problem. This is different in the case of medical applications for imaging or therapy, where
individuals are exposed to higher doses of gold nanoparticles that may indeed exhibit toxic
effects. On the other hand, humans have used gold in its metallic form in close contact with
the body since prehistoric times, and later also in dentistry, without ever experiencing
significant toxic effects. While it is less obvious that metallic gold in the form of nanoparticles
is similarly harmless there is at present little or no evidence for toxic effects that arise
primarily from the nanoscale dimensions of a material. Indeed, anecdotal evidence from
alchemists suggests that the prolonged ingestion of large amounts of colloidal gold leads to
its deposition in the skin as blue stains, an indirect evidence for the absence of acute high
toxicity. Although, for the above reasons, nanoscale gold itself does not pose a serious risk, it
may serve as a model system to investigate if reducing the dimensions of a material to the
nanoscale leads to the emergence of new parameters that determine its toxicity. Gold
nanoparticles of different shapes, sizes and surface chemistries are readily prepared, can
easily be stored and handled under ambient conditions, and their interactions with biological
cells can be monitored by a range of techniques with single particle resolution. The absence
of high toxicity of the metal itself makes it possible to immediately detect toxic affects of
shape, size or surface chemistry and hence to determine whether toxicity can arise alone
from the nanoscale dimensions of a material. While the latter is widely assumed with
frequent references to the asbestos analogy on the micrometre scale, little evidence exists to
date to support this notion of nanotoxicity. Being an outsider to the field of toxicology I will
neither attempt to provide such evidence nor to argue against it, but I will instead speculate
about ways in which real nanotoxicity could arise and how this phenomenon could be studied
using gold nanoparticles as a model.
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4.1.2 The assessment of exposure risk, persistence and accumulation of
nanoparticle silver in the aquatic and marine environment
S. Cunningham1, M. Brennan-Fournet2, D. Ledwith3, M. Voisin4,
L. Byrnes5, A. Boyd6, G. Fleming6 and L. Joshi1
1
Glycoscience Group, NUI Galway, Ireland
School of Physical Sciences, Dublin City University, Dublin, Ireland
3
School of Physics and School of Chemistry, Trinity College, Dublin, Ireland
4
Ireland Dept of Physics, NUI Galway, Ireland
5
Dept of Biochemistry, NUI Galway, Ireland
6
Dept of Microbiology, NUI Galway, Ireland
Email: [email protected]; [email protected]
2
Though the potential benefits and applications of nanotechnology continue to expand, there
is continued concern regarding exposure risk with human health and environmental
consequences. This risk has been attributed to their unique physicochemical properties
which have been demonstrated to alter the properties of nanoparticles from their
characterised bulk forms. Along with this risk areas of bio-accumulation, bio-persistence, and
environmental fate and exposure effects require further monitoring and assessment.
Within this study, focusing primarily on silver (Ag), the most commonly used metal oxide
particle currently, we have performed continuous characterisation from synthesis to post
exposure testing examining size, shape, polydispersity, concentration, state surface area,
surface charge, surface function and purity. Using Zebrafish (Danio rerio) as an exposure
model system, the relationships between physicochemical properties of silver nanoparticles
(electrostatic, polymer, biocompatible and thiol stabilised) and their biological effects in
relation to developmental and morphological effects have been observed. Comparison to
bulk and ionic forms of silver has also been performed, to permit determination of metal,
nano or ionic effects. Parallel testing of these nanoparticles have been performed using two
marine Vibrio bacteria species in accordance with ISO testing protocols.
Exposure tested ranges have been assessed from ppb (10-9) to ppm (10-6) concentrations,
examining the both model systems and at points in accordance with OCED ISO testing
procedures implemented for such testing. During exposure testing the physicochemical
properties have been monitored for fate in marine and aquatic conditions.
Findings, using a bulk metal reference have indicated that silver nanoparticles have the
ability to generate a variety of embryonic morphological malformations and may accumulate
within the embryo. The rate of ion release and its role in these effects is currently being
assessed. The stability of nanoparticle silver under environmental conditions is dependent
upon a number of factors including initial surface modifications and release concentration.
These assessments using zebrafish embryo model may lead to the identification of
physicochemical attributes which afford minimal or no toxicity in vivo aiding in the safe
designs of nanomaterials and may provide further information towards regulatory decisions.
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4.1.3 A simple route to highly fluorescent silica nanoparticles for tracing the
intracellular fate of nanoparticles
Douglas Gilliland, Giacomo Ceccone, Chiara Uboldi, Guido Giudetti, François Rossi
European Commission, DG-JRC, IHCP-Nanobiosciences Unit, Ispra, Italy
Email: [email protected]
The increasing use of engineered nanoparticles is resulting in concerns for potential
undesirable effects in living organisms and the environment as a whole. For studies of
nanoparticle accumulation and eventually potential toxicity one of the greatest challenges is
to detect and locate nanoparticles in complex environments such soil and water, alimentary
products, cells and living organisms. A first step towards meeting this challenge is to develop
and test effective protocols for the detection, extraction and quantification using specifically
labelled nanoparticle systems. Although engineered nanoparticle can be labelled by a variety
of means (magnetic, radioactivity or enriched stable isotopes) one of the most sensitive and
flexible methods for detection is to study particles which have been modified to incorporate
fluorescent molecules. The presence of fluorophores permits the detection, localization and
quantification using relatively routine laboratory practices and equipment. The main
challenge in this area is to produce chemically stable nanoparticles which combine the
properties of strong, low bleaching fluorescence with a flexible synthesis method which
permits the production of particles with controllable size and easily modifiable surface
chemistry.
In this work the development of just such a nanoparticle system has been undertaken using
silica doped with the a fluorescent ruthenium ′complex (Tris(2,2 -bipyridyl)
dichlororuthenium(II)hexahydrate)-Ru(II)(bypy)3). Using modifications of a simple, robust
synthesis procedure developed for this work it has been possible to produce strongly
fluorescent near monodisperse silica nanoparticles with sizes which can be controlled from
less than 10nm to above 140nm. The use of silica as the basis of these particles provides a
chemically stable matrix for the fluorophore which shows little/no leaching and limited
photobleaching. Furthermore, the surface chemistry of silica is well suited to controlled
functionalization to produce a wide variety of different surface chemistries and/or form the
core a more complex core shell systems. The principle factors controlling the synthesis have
been explored and relevant physico-chemical properties studied in both the as–synthesized
state and after chemically functionalization. These particles have potential for use in variety
of biological marker applications and have already been successfully applied in tracing the
intercellur fate of nanoparticles.
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4.1.4 Nanoparticles and their behaviour in biological fluid
Usawadee Sakulkhu1, Géraldine Coullerez1, Heinrich Hofmann1, Alke Fink2
1
Powder Technology Laboratory, École Polytechnique Fédérale de Lausanne, Switzerland
2
Department of Chemistry, University of Fribourg, Fribourg, Switzerland
Email: [email protected]
In a biological fluid, proteins associate with nanoparticles, the amount and presentation of the
proteins on the surface of the particles leads to an in vivo response. Proteins compete for the
nanoparticle ‘‘surface,’’ leading to a protein ‘‘corona’’ that largely defines the biological
identity of the particle [1]. This will be important for assessing nanoparticle toxicity (i.e.
translocation into cells and interference with viability and cellular function), advancing
nanoparticles for imaging, drug delivery, and therapeutic applications (i.e. targeting specific
cells, organs, or tumours), and for designing multifunctional nanoparticles (i.e., are there
dimensional limits to designing nanoparticles that can target and kill diseased cells?). Thus,
knowledge of protein adsorption on nanoparticles is important for understanding the nature of
the particle surface seen by the functional machinery of cells. The goals of these projects are
develop approaches to study nanoparticles behaviour in biological environment based on
Superparamagnetic iron oxide nanoparticles (SPIONs) core nanoparticles as well as
synthesis different surface of inorganic core-shell SPIONs which can be further modified by
functionalization. The main advantages of SPIONs core is in term of their magnetic
properties which allow for easy separation. Determining of protein compositions on different
surface of SPIONs core nanoparticles would facilitate the understanding of cell internalization
mechanism of the particles, transport pathways, interaction partners as well as cellular and
molecular function. In addition, these particles will be used for protein separation which will
further used for drug delivery, diagnostic, sensor, drug targeting and biomedical applications
in the future.
In this work, we use our developed technology “a magnetic reactor” for protein separation [2]
to minimize the contamination of proteins in each washing and elution step which normally
used a traditional centrifugation method. Our preliminary results showed that nanoparticle
surface strongly influence the adsorption of serum proteins on nanoparticle surface and
nanoparticle behaviour in biological environment. By using 12%SDS-PAGE 1D, same
surface nanoparticles, for instance, silica nanoparticles and silica/SPIONs shell/core
nanoparticles showed the same pattern of adsorbed proteins after incubation with DMEM
supplemented with 10% fetal bovin serum while different surface nanoparticles (e.g. polymer,
silica, gold, titania coated SPIONs) shared the same main adsorbed proteins; however, with
different pattern of adsorbed proteins.
[1] Dawson K.A, et al. 2006. Surface induced changes in protein adsorption and implications
for cell-surface response. Biomaterials 27: 3096-3108.
[2] Benedikt Steitz, et al. 2007. Fixed Bed Reactor for Solid-Phase Surface Derivatization of
Superparamagnetic Nanoparticles. Bioconjugate Chem. 18 (5): 1684–1690.
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4.2 Poster presentations
4.2.1 Bivalve immunocytes as a model for studying NP toxicity
Laura Canesi1, Caterina Ciacci2, Barbara Canonico2, Michele Betti2, Giulio Pojana3, Gabriella
Gallo1
1
University of Genoa, Dept. of Biology, Italy
2
University of Urbino, DISUAN, Italy
3
University of Venice, Dept. of Environmental Sciences, Italy
Email: [email protected]
The aquatic environment represents the ultimate sink for manufactured nanomaterials and
products. In this light, the possible impact of NPs on the aquatic biota is a growing area of
investigation.
Bivalve molluscs are abundant in different aquatic environments, from marine to freshwater,
and therefore they represent a relevant group of test organisms for investigating the effects
of NPs. Due to their filtering apparatus, the ciliated gill epithelium, there is short distance for
contaminants and particles to pass from the water to the blood. Moreover, bivalve cells have
highly developed processes for the cellular internalisation of nano- and microscale particles,
endocytosis and phagocytosis, that are integral to key physiological functions such as
intracellular digestion and cellular immunity. Bivalve blood cells, the hemocytes, resembling
the monocyte/macrophage lineage, are responsible for cell-mediated immunity through
phagocytosis and various cytotoxic reactions, such as the release of lysosomal enzymes and
antimicrobial peptides, the respiratory burst and nitric oxide production.
The marine bivalve (Mytilus spp.) is worldwide utilized in biomonitoring of marine coastal
areas. Several studies in M. galloprovincialis showed that the utilization of a battery of
immunotoxicity tests in mussels allows for the rapid and sensitive evaluation of the effects of
different emerging contaminants, including NPs. Data obtained on the effects and
mechanisms of action of different NPs (from carbon based NPs to n-oxides) on mussel
hemocytes are here summarized. The results demonstrate that bivalve immunocytes
represent a sensitive target for NP toxicity. NP suspensions can induce inflammatory and
pre-apoptotic processes like those observed in mammalian cells [1-2]. The effects of NPs
were confirmed in vivo, indicating that uptake of NP agglomerates through the digestive
system and subsequent transfer to the hemolymph and circulating hemocytes may occur [3].
Overall, the utilization of immunotoxicity tests in mussel hemocytes represents an useful
model that could provide rapid information when screening the potential impact of different
NPs in the aquatic environment.
[1] Canesi, L, Ciacci, C, Betti, M, Fabbri, R, Canonico, B, Fantinati, A, Marcomini, A, Pojana,
G., 2008. Immunotoxicity of carbon black nanoparticles to blue mussel hemocytes. Environ.
Int., 34:1114-1119.
[2] Canesi, L, Ciacci, C, Vallotto, D, Gallo, G, Marcomini, A, Pojana, G., 2010. In vitro effects
of suspensions of selected nanoparticles (C60 fullerene, TiO2, SiO2) on Mytilus hemocytes.
96:151-158.
[3] Canesi, L, Ciacci, C, Vallotto, D, Gallo, G, Marcomini, A, Pojana, G, 2010. Biomarkers in
Mytilus galloprovincialis exposed to suspensions of selected nanoparticles (Nano carbon
black, C60 fullerene, Nano-TiO2, Nano-SiO2) Aquat. Toxicol., 100: 168-77.
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4.2.2 Effect of Dissolved Copper and NanoCopper on Histopathology and
Haematopoietic Organs of Rainbow Trout (Oncorhynchus mykiss)
Genan Al-Bairuty, Benjamin J Shaw, and Richard D. Handy
School of Biomedical & Biological Sciences, University of Plymouth, UK
Email: [email protected]
It is unclear whether copper nanoparticles are more toxic than traditional forms of dissolved
copper. This study aimed to describe the toxicity and the pathologies in gill, liver, spleen, gut
and of juvenile rainbow trout (about 37 g fish) exposed in triplicate to either a control (no
added Cu), 20 or 100 µg/L of either dissolved Cu (as CuSO4.5H2O) or nano-Cu (99.9 %
purity; 50 nm nominal particle size) in semi-static exposure regime. Fish were sampled at
day 0, 4, and 10 for histology and spleen prints. The results showed that exposure to 100
µg/L of dissolved Cu caused 85 % mortality (treatment subsequently terminated) compared
to 14 % mortality of the same concentration of nano-Cu by day 4. At day 10, mortality was 4,
17, 10 and 19 % in control, low dissolved Cu, and low and high nano-Cu treatments
respectively. The cause of mortality may have been related to acute gill injury. All treatment
showed injuries that include hyperplasia and necrosis in gill; lipidosis and pyknotic nuclei in
liver; swelling of goblet cells and lifting of the epithelium in intestine; as well as lipidosis and
necrosis in the spleen. The spleen also showed a decrease in the proportion of red pulp,
while the proportion of white pulp increased for all treatment (statistically significant, ANOVA,
P < 0.05). The spleen prints showed changes in erythrocytes morphology and the
proportions of haematopoietic cell. Overall, the injuries observed were greater with Cu than
nano-Cu.
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4.2.3 Electrochemical Modelling of Nanoparticle Toxicity
Alexander Vakourov1, Ann Zhang1, Rik Brydson2, Iraida Loinaz3, Nerea Ormategui3, Andrew
Nelson1
1
School of Chemistry, University of Leeds, Leeds, UK
SPEME, Faculty of Engineering,University of Leeds, Leeds, UK
3
New Materials Department, CIDETEC, Donostia-San Sebastian, Spain
Email: [email protected]
2
This talk describes the interaction of SiO2,ZnO and organic polymeric nanoparticles with the
chip-supported phospholipid membranes [1] of the ENNSATOX nanosensor. SiO2
dispersions of particle size 14 to 150 nm were tested and were found to be stable within the
time of the experiment. The interaction of SiO2 particle dispersions with dioleoyl lecithin
(DOPC) membranes were inversely related to the particle size and characterised by an
interference with the electrically-induced phase transitions (figure 1). Impedance
measurements of the SiO2-DOPC interaction confirmed this finding. Uniquely novel
experiments using scanning electron microscopy (SEM) showed that SiO2 nanoparticles of
all size ranges adsorbed on the DOPC surface (figure 2). It can be concluded from these
results that the relationship of SiO2 activity on the DOPC membrane with particle size is due
to the geometrical proximity of the SiO2 surface to the DOPC polar groups. Similar
experiments were carried out with ZnO nanoparticle dispersions from different sources. Only
the ZnO dispersions with added dispersant were found to be stable and these dispersions
interacted strongly with the DOPC membrane. In comparison with the inorganic
nanoparticles, experiments were also carried out investigating the interaction organic
polymers and organic polymeric nanoparticles with DOPC membranes. The polymers
showed considerably stronger DOPC membrane activity. The rates of interaction of the
inorganic nanoparticles, the organic polymers and the organic polymeric nanoparticles with
the DOPC membrane are compared.
The biological relevance of the ENNSATOX nanosensor has been tested by intercalibrating
the results from the sensor with those obtained from the interaction of the nanoparticles with
biological organisms of increasing levels of complexity.
[1] Z. Coldrick, et al 2009, Electrochim.Acta 54,4944-4962.
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4.2.4 Establishment of a High Content Analysis (HCA) platform to assess
nano-toxicology and explore nanoparticle-induced cell death
Sergio Anguissola1, Fengjuan Wang1, Sonia Ramirez1, Anna Salvati1, Peter O’Brien2, Iseult
Lynch1, Kenneth Dawson1
1
Centre for BioNano interactions, School of Chemical Biology, University College Dublin,
Dublin, Ireland
2
College of Life Sciences, School of Agriculture, Food,Science & Veterinary Medicine,
Veterinary Science Centre, Dublin, Ireland
Email: [email protected]
Biosafety of nanomaterials is a relevant issue both for the industry and medical field as their
use ranges from computer industry, orthopaedics and medical applications as diagnostics
tools, therapeutic agents and drug delivery vehicles; it is therefore of primary importance to
assess, understand and manage their toxicity. Amino-modified polystyrene nanoparticles
(PS-NH2 NPs) can easily be modified to carry chemicals and proteins, therefore they are an
interesting model for drug delivery; Cerium Oxide (CeO2) NPs are used in ceramics, to
produce photosensitive glass, and in catalytic converters in automotive applications.
Preliminary results from IANH did not reveal toxic effects of CeO2 NPs, while our research
has revealed that PS-NH2 NPs cause apoptotic cell death by inducing cytosolic calcium
increase, lysosomal damage, mitochondrial membrane depolarization and activation of the
caspase cascade in astrocytoma cells. These two nanoparticles were chosen to establish an
HCA platform to assess NPs toxicity and to further characterize apoptosis induced by several
classes of nanomaterials. The cell lines chosen were HepG2 as an in vitro model for liver
toxicity and 1321N1 cells which are of interest as a model for targeted nanoparticles drug
delivery across the blood brain barrier.
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4.2.5 SiO2 nanoparticle trafficking across in vitro human blood-brain barrier
Dong Ye1, Michelle Nic Raghnaill1, Meredith Brown1,2, Tiago Santos1, Iseult Lynch1, Kenneth
A. Dawson1
1
School of Chemistry and Chemical Biology, University College Dublin, Dublin, Ireland
2
Veterinary Science Centre, University College Dublin, Dublin, Ireland
Email: [email protected]
Drug delivery to the brain is severely restricted by the formation of tight junctions between
adjacent brain capillary endothelial cells which result in an exceptionally low permeability and
low drug penetration. The aim of this work is to establish and validate a working in vitro blood
brain barrier (BBB) model system and to use this to screen a range of nanoparticles in order
to determine their ability to pass through the BBB. Here, we develop an in vitro BBB model
for this purpose using immortalized human endothelial cells (hCMEC/D3) [1] [2] grown on a
transwell membrane which allows formation of tight junctions and cellular polarization. We
used FITC-dextran 4 kDa as a permeable marker for detecting barrier integrity and ApoEAlexa Fluor 647 [3] as a positive control for intercellular transcytosis, and confirmed the
monolayer formation and barrier integrity using Transmission Electron Microscopy. Having
successfully validated the integrity of the BBB model, we proceeded to investigate the
trafficking of SiO2 nanoparticles of 50 nm, 100 nm and 200 nm diameters across the cell
monolayers at 4 °C and 37 °C (when the cells are depleted of energy and under normal cell
culture conditions, respectively). In parallel, nanoparticle uptake by this cell line was
quantified using flow cytometry. Additionally, nanoparticle localisation with regard to
endocytosis and transcytosis was illustrated through confocal microscopy and electron
microscopy.
Using a transwell system, apical to basolateral fluxes of the 3 sizes of SiO2 nanoparticles
across the BBB were found to depend mainly on the size of the nanoparticles, but also on
the environmental conditions (temperature). Results showed that 50 nm SiO2 nanoparticles
obtained a much higher permeability across the BBB than the two larger nanoparticles, as
expected. The fluxes of SiO2 nanoparticles at 4 °C were significantly lower than those at 37
°C, indicating that the particles cross the BBB via energy-dependent processes. In addition,
SiO2 nanoparticles were actively taken up by endocytosis into hCMEC/D3 cells, and some
SiO2 nanoparticels were found to bypass cellular lysosomes and were able to cross the BBB
monolayer by transcytosis. Significant additional work is needed to confirm the transport
mechanism, and to quantify the numbers of particles undergoing trancytosis.
[1] Birk Poller, et al. The human brain endothelial cell line hCMEC/D3 as a human bloodbrain barrier model for drug transport studies. Journal of Neurochemistry. 107, 1358-1368
(2008)
[2] B. B. Weksler, et al. Blood-brain barrier-specific properties of a human adult brain
endothelial cell line. The FASEB Journal. 19, 1872-1874 (2005)
[3] K. Michaelis, M. M. Hoffmann, S. Dreis, E. Herbert, R. N. Alyautdin, M. Michaelis, J.
Kreyter, and K. Langer. The Journal of Pharmacology and Experimental Therapeutics. 317,
1246-1253 (2006)
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4.2.6 Uptake of TiO2 nanoparticles across the isolated perfused intestine of
rainbow trout (Oncorhynchus mykiss)
Aliaa Al-Jubory1, Richard. D. Handy1
1
School of Biomedical and Biological Sciences, University of Plymouth, Devon, UK
Email: [email protected]
In vivo studies have raised concerns that titanium dioxide nanoparticles (TiO2 NPs) may be
taken up across the gut. The isolated, perfused intestine of rainbow trout (Oncorhynchus
mykiss) was used to determine the uptake rate of TiO2 NPs across fish gut. Differences
between bulk and nanoscale were tested. Experiments also examined the effect of CO2
buffering on TiO2 NP uptake by altering the CO2 content of perfusates. Luminal exposure to 1
mg l-1 TiO2 NP for 4 h gassed with 95% O2:5% CO2 showed a time-dependent accumulation
of NPs in the perfusate with a maximum initial uptake rate (mean ± S.E.M., n = 6-7) of
4.21±1.07, 2.03±0.81, and 0.38±0.26 nmol g-1 h-1 for TiO2 NPs, bulk powder, and no-added
Ti controls, respectively (statistically significant differences on all treatments, Kruskall-Wallis,
P <0.05). Notably, there was at least a 10 fold increase in TiO2 NP uptake to 69.65±61.48
nmol g-1 h-1 (mean ± S.E.M., n = 3), when the CO2 content of the perfusate gas was reduced
to 99.5% O2:0.5% CO2 (statistically significant, t-test, P <0.05). Titanium levels in the
intestine at the end of the experiments (5 % CO2) were 0.05±0.01, 0.19±0.07, and 0.01±0.01
µmol g-1 for TiO2 NPs, bulk powder, and no-added Ti control, respectively (mean ± S.E.M., n
= 6-7); while the titanium levels with low CO2 were 0.09±0.04 in TiO2 NP and 0.01±0.003 in
control group (mean ± S.E.M., n = 3). The data demonstrates that TiO2 NPs are taken up
across the intestine at a much faster rate than the bulk powder, and the uptake mechanism is
affected by gas mixture used.
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Symposiums
5 Stakeholder Session
5.1.1 What benefits might nanomedicine offer?
Yasemin J. Erden
Centre for Bioethics & Emerging Technologies, St Mary's University College, Twickenham,
United Kingdom
Email: [email protected]
Nanomedicine has the potential to fundamentally alter the ways in which we develop,
distribute and dispense medicines. This includes, for instance, the cost of producing
medicines, or how we ingest or inject treatment (e.g. vaccines). With change there inevitably
arise ethical issues to consider, whether for current or future developments in nanomedicine.
Particular focus in this presentation is given to the question of benefit, specifically through
raising questions about who holds an interest in the development of nanomedicines (such as,
for example, vaccines), and who will stand to benefit from them, or their production (industry,
patients, etc.). This generates related questions about benefit and impact in relation to
economic/political divisions between nations that are already wealthy, and those that are
developing wealth; and whether, for example, the patent system and commercial
confidentiality will be a hindrance for disseminating the full benefits of nanomedicine. To see
how these issues might play out, we will consider some pertinent issues arising from
previous medical breakthroughs, for example, in the distribution of clean water supplies
within developing countries.
As well as the direct impact of nanotechnology, there are also sociological and cultural
impacts to consider, including, for instance, public perceptions of nanomedicines in relation
to perceived and actual associated risks and benefits. One approach to this issue is of
course to seek consensus within stakeholder dialogue (between pharmaceutical companies,
governments, medical professionals, NGOs and patient groups), but the manner in which this
occurs is also central (whether occurring pre- or post planning, testing, production, or
distribution of nanomedicines). Questions about benefit therefore permeate the structure of
such dialogue (e.g. who benefits when dialogue does or doesn't occur?).
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5.1.2 Involving stakeholders in setting research priorities - Reflections from
consumers
Chiara Giovannini
ANEC, Brussels, Belgium
Consumers expect the products they buy and use to be safe, irrespective of their origin or
composition. Despite a serious lack of knowledge about the safety of nanomaterials and
nanotechnologies, consumer products containing nanomaterials and nanotechnologies are
present in the European market as illustrated our inventory of October 2010 1.
In light of the concerns raised by nanotechnologies and nanomaterials, in particular
regarding their safety, environmental and health aspects, much more needs to be done to
reassure citizens and consumers that a fair balance is achieved between economic benefits
and health, safety and environmental benefits.
ANEC calls for public funding for research on health, safety and environmental implications
(HSE) and ethical, legal and social implications (ELSI) to be increased. So far, the majority of
research resources have been allocated to innovation and commercial developments 2.
Prioritisation of areas for research funding would be an important field with which the public
could be engaged.
ANEC also calls for scientists’ capacity to communicate independent and balanced
information on the benefits and risks associated with the use of nanotechnology, in a
transparent manner, to be promoted. Specific actions shall be taken to improve the present
governance related to nanotechnologies, to guarantee full transparency and to ensure public
engagement and effective dialogue with citizens. Past experience has shown that citizens
including consumers are willing to know about nanotechnology and should be given the
power and means to take decisions and react in case of damage 3.
Potential product liability issues relating to nanotechnology, including in the pharmaceutical
sector, need to be carefully considered. Nanotechnology is a new field and not all potential
safety issues have been identified at this stage. ANEC calls for the precautionary principle to
be applied as the full extent of the risks posed by products containing nanomaterials is
unknown.
Because of the increasing amount of information from different sources, consumers are
struggling to get reliable sources of non-promotional information about health, medicines and
treatments. This is particularly true for products claiming to contain nanomaterials sold over
the Internet, including products which claim to have specific health effects such as food
supplements and medicines. All claims which are made about health, safety and/or
environmental aspects of products containing nanomaterials should be scientifically
substantiated and supported by publicly available information of the methods used to
substantiate the claim. Claims made for the efficacy of a product should be backed up by
rigorous evidence.
ANEC’s presentation will explain consumer expectations with regards to nanotechnologies
highlight the potential uncertainties and propose consumer relevant research priorities.
1
http://www.anec.eu/attachments/ANEC-PT-2010-Nano-017.xls
EU 2004-2009 Action Plan on nanosciences and nanotechnologies
3
E.g. Which? Consumer panel in the UK, VZBV Consumer survey in Germany, Publifocus undertaken in
Switzerland
2
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6 Symposiums
6.1 Immunosafety Task Force Kickoff
6.1.1 The need for concerted action toward immunosafety of nanomaterials
Albert Duschl
University of Salzburg, Salzburg, Austria
Nanomaterials are in a size range which overlaps that of viruses and are thus large enough
to be recognized by immune mechanisms as non-self. Indeed, it has been shown that
nanosized materials, including engineered ones, can bind to innate immune receptors and
stimulate immune activation. It has thus become apparent that immunosafety is an aspect
which needs attention in the nano-field.
The challenge of this task is daunting. Obvious problems include the large number of
materials to be tested, significant batch-to-batch variation, contamination with biological and
non-biological agents, storage and aging issues, complex endpoints which evolve over time,
and a multitude of available immunological tests in vitro and in vivo. The complexity of
immunity makes it urgent to set up communication platforms where the people working in this
field can exchange methodical experiences, theories and results, and to work together
towards a consensus on relevant methods, protocols and endpoints which would allow
intelligent testing for the immunosafety of nanomaterials. For immunologists, nanomaterials
are also an interesting research issue in themselves, since investigating e.g. the interactions
between particulate materials and innate immune mechanisms will allow a better
understanding of immunity.
The small but enthusiastic community of researchers in nanoimmunosafety needs a suitable
platform for exchange. The proposed immunosafety task force will provide such a platform. It
should be fully integrated into existing activities on networking in nanosafety, like the
NanoSafety Cluster, NanoImpactNet and NanoFutures. Many of the problems facing
immunologists are, after all, shared by a wider community. Questions like whether cells
remain viable or whether a specific nanomaterial aggregates in biological systems are of
general interest. A task force in immunosafety should, therefore, at least initially set up
opportunites like satellite meetings or sessions dedicated to this topic in the context of larger
meetings. The symposium on Immunosafety during the NanoImpactNet meeting 2011 is a
model of what could be organized in the near future. It is also feasible to organize workshops
on even more specific topics, methods or systems in immunosafety in the future, depending
on the needs of the community.
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6.1.2 Immunosafety of nanomedicines: an introduction
Bengt Fadeel
Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet,
Stockholm, Sweden
The immune system is our primary defence system against foreign intrusion including
microorganisms as well as particles. Understanding the interactions of engineered
nanomaterials with the immune system is a key challenge facing toxicologists and
immunologists. The recognition or non-recognition of engineered nanomaterials by immunecompetent cells may determine not only the toxic potential of such materials but also their
biodistribution. This has important ramifications for the biomedical application of engineered
nanomaterials. According to current guidelines for immunotoxicity studies for human
pharmaceuticals, data from standard toxicity studies should be evaluated for signs of
immunotoxic potential, including haematological changes, alterations in immune system
organ weights and/or histology (gross pathology), changes in serum globulins that occur with
or without a plausible explanation, increased evidence of infections, and increased
occurrence of tumours, indicative of immunosuppression in the absence of other plausible
causes. Will such animal studies be sufficient to reveal adverse effects of engineered
nanomaterials? Are standard assays for testing of human pharmaceuticals applicable to the
assessment of nanomedicines? Needless to say, particular attention should be paid to novel,
adverse properties arising as a consequence of the nano-scale size. For instance,
nanoparticles may escape immune surveillance and translocate to distal sites following entry
into the human body; will in vitro model systems suffice to capture such size-dependent
behaviour of nanomedicines? From a regulatory perspective: how should one define a
nanomedicine? Are multifunctional theragnostic particles medicines or devices? Many
challenges remain to be addressed.
[1] Fadeel B et al. Nanomedicine: reshaping clinical practice. J Intern Med. 2010
Jan;267(1):2-8.
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6.2 Nanoparticles in paints
6.2.1 Risks of nanoparticle handling and sanding nanoparticle-containing
paints
Koponen I K and Jensen K A
National Research Centre for Working Environment (NRCWE), Copenhagen, Denmark
Email: [email protected]
Increasing use of engineered nanoparticles (ENP) in different industrial applications has
raised a new potential health risk to the workers as well as to the consumers. Therefore, this
study investigates exposure levels from powder release in the paint production sites and the
particle size distributions of sanding dust released from paints produced with and without
ENPs.
Dust exposure during powder handling was measured during paint productions in two
companies using a TSI Fast Mobility Particle Sizer (FMPS; 6-540 nm) and a GRIMM
Dustmonitor Model 1.109 (0.28-30µm) for the aerosol size and number distribution next to
the mixing station. Background measurements were done using a CPC Model 2022 (TSI)
and GRIMM Dustmonitor Model 1.109.
Exposure risk during sanding dried paints was analyzed in an experimental set-up using a
commercial hand-held orbital sander. Sanding dust was led to a 0.03 m3 plastic chamber and
collected by an electrostatic precipitater. The particle size distribution was measured in the
chamber using an APS Model 3321 (Aerosol Particle Sizer, TSI Inc., 0.542 to 19.81 µm
(aerodynamic diameter)) and the FMPS mentioned above. The APS and FMPS data were
exported at a 10 seconds time resolution, which was sufficient to observe the relatively rapid
changes in the aerosol spectra.
Field measurement data shows that we are able to connect handling of powders to elevated
particle number concentrations. The maximum total particle concentration at the mixing
station reached a value of 4e5 cm-3, which lasted usually from few seconds to couple of
minutes. The dust particle number sizes were typically from 100 nm and up to 500 nm and 3
modes could be observed.
Dust emissions from sanding painted plates were found to consist of five size modes; three
modes under one µm and two modes around one and two µm. We observed that the sander
was the only source of particles smaller than 50 nm and they dominated the number
concentration spectra. Volume and surface area spectra were dominated by the 1 and 2 µm
modes. The number concentrations in the different size modes varied considerably in
between the studied products. Generally, there was no clear trend that ENP-doped paints
would produce more nanoparticles and therefore create a greater risk to the exposure of
nanoparticles during sanding surfaces covered with ENP-doped paints as compared to
sanding conventional paints. However, free pigment and nanoparticles fillers were observed
in some samples demonstrating their potential release.
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6.2.2 Inflammatory and genotoxic effects of nanoparticles and dust generated
from nanoparticle-containing paints and lacquers
Anne Thoustrup Saber1, Keld Alstrup Jensen1, Ismo Koponen1, Nicklas Raun Jacobsen1,
Renie Birkedal1, Lone Mikkelsen2, Peter Møller2, Steffen Loft2, Ulla Vogel1,3, Håkan Wallin1,2
1
National Research Centre for the Working Environment, Copenhagen, Denmark
Department of Environmental and Occupational Health, University of Copenhagen,
Copenhagen, Denmark
3
Institute for Science, Systems, and Models, University of Roskilde, Roskilde, Denmark
Email: [email protected]
2
Nanotechnology has potential applications in many processes and products. Therefore there
is a growing need to establish knowledge about the health risks in relation to exposure to
nanoparticles. The paint- and lacquer industry already use nanoparticles in substantial
amounts in their products and the number of applications will be growing in the near future.
The aim of NanoKem project is to investigate if the pure technical nanoparticles and sanding
dust generated from nanoparticle-doped paint cause inflammation or DNA damage,
associated with carcinogenicity. Nanoparticles (8 materials), nanoparticle-containing dust
and dust from reference paints and lacquers without nanoparticles (14 materials), were
tested in vitro and in vivo. Initially, the cytotoxicity of the materials was screened in vitro by
incubating FE1-MutaTMMouse lung epithelial cells, at concentrations between 50 and 800
µg/ml. The materials were little toxic in the in vitro screening why we decided that it was
justified to instill them in mice. Twenty four hours after a single intratracheal instillation of 54
µg particles in mice, the level of inflammation was assessed by measuring mRNA expression
of cytokines in lung tissue and bronchoalveolar lavage cell composition. DNA damage was
determined by the Comet assay in lung lining cells. One titanium dioxide nanomaterial and
the corresponding paint with and without titanium dioxide were tested for dose-responses at
different times. The mice were treated with a single intratracheal instillation with 18, 54 and
162 µg for the nanoparticles and 54, 162 and 486 for the paint dusts. DNA damage and
inflammation was evaluated 1, 3 and 28 days after intratracheal instillation. We will present
data on inflammation and DNA damage from in vivo experiments.
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6.2.3 Cardiovascular health effects of paint dust with
nanoparticles compared to the primary nanoparticles
and
without
Lone Mikkelsen1, Keld Alstrup Jensen2, Ismo Koponen2, Majid Sheykhzade3, Anne T. Saber2,
Ulla Vogel2,4, Håkan Wallin1,2, Steffen Loft1, and Peter Møller1
1
Department of Public Health, University of Copenhagen, Copenhagen, Denmark
National Research Centre for the Working Environment, Copenhagen, Denmark
3
Department of Pharmacology and Pharmacotherapy, University of Copenhagen,
Copenhagen, Denmark
4
Institute for Science, Systems, and Models, University of Roskilde, Roskilde, Denmark
Email: [email protected]
2
A number of studies on ambient air particles as well as a few studies on engineered
nanoparticles (NPs) have indicated associations between exposure and risk of
cardiovascular events. Inhalation of NPs may elicit pulmonary inflammation followed by an
increased release of cytokines, which might further affect the function of endothelial cells and
development of atherosclerosis. It is the small size of NPs that provide unique properties and
also causes concern about possible toxicological effects. Paints and lacquers are examples
of products containing particulate matter, where NPs can be added to provide special
properties such as resistance to bleaching by sunlight. The production, use, and removal of
paints give rise to different exposure situations where NPs may occur as primary nanosized
particles or embedded in a matrix with larger particles. It can also be speculated that NPs are
released and potentially inhaled by sanding of surfaces with NP-containing paint.
The aim of this project was to investigate the association between exposure to primary NPs,
and grinded paint dust samples, and vascular function in cultured human umbilical vein
endothelial cells (HUVECs) and dyslipidemic apolipoprotein E (ApoE-/-) knockout mice that
are susceptible to the development of atherosclerosis.
HUVECs were exposed to a panel of 22 different samples of primary NPs and dust samples
from grinded paint with and without NPs. The primary NPs included TiO2 and carbon black,
together with samples of fillers and binders, which were also investigated in order to evaluate
the effect of other particles than pigments in paints. Primary TiO2 and carbon black
(Flammruss 101) particles increased the production of reactive oxygen species (ROS) and
expression of cell adhesion molecules VCAM-1 and ICAM-1 in HUVECs, whereas paint dust
with and without NPs had substantially lower effects on mass basis. Samples of binders and
fillers in paint also increased the ROS production and expression of cell adhesion molecules
in HUVECs. The magnitude of expression of cell adhesion molecules was not dependent on
the particle size of the suspension determined by dynamic light scattering.
The effect on vasomotor function and progression of atherosclerosis was assessed in
atherosclerosis-prone ApoE-/- mice exposed to three different types of primary TiO2 particles
by intratracheal instillation. The mice were exposed to two instillations of 0.5 mg/kg
bodyweight of fine (21 m2/g), photocatalytic (17.8 m2/g) or nanosized (107.7 m2/g) of TiO2 at
26 and 2 hours before sacrifice. There were no effects on endothelium-dependent or
endothelium-independent vasodilation in aorta segments mounted in a myograph. However,
intratracheal instillation of nanosized TiO2 (0.5 mg/kg bodyweight) once a week for four
weeks was associated with a modest increase in the plaque area in the aorta.
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6.2.4 Developmental and reproductive toxicity of nanoparticles
Karin S. Hougaard1,3, Petra Jackson1, Keld A. Jensen1, Anne T. Saber1, Renie K Birkedal1,
Anni Vibenholt1, Jens J Sloth4, Katrin Löschner4, Erik H Larsen4, Anne-Mette Z Boisen1,
4
,Håkan Wallin1,3, Ulla Vogel1,2
1
National Research Centre for the Working Environment, Copenhagen, Denmark
2
Institute for Science, Systems, and Models, University of Roskilde, Denmark
3
Dep. Environmental and Occupational Health, University of Copenhagen, Denmark
4
National Food Institute, Technical University of Denmark, Mørkhøj, Denmark
E-mail: [email protected]
Engineering nanoparticles provides new properties to material and might change
toxicological properties. As yet work on developmental and reproductive toxicity of
nanomaterials is sparse [1], even if such testing is integrated into nanomaterials research
strategy of e.g. the US EPA and is recommended by the US NIOSH. We investigated
developmental and reproductive toxicity of two types of nanoparticles, titanium dioxide (TiO2)
and entangled multiwalled carbon nanotubes (MWCNT).
TiO2: Pregnant mice inhaled 40 mg TiO2/m3, 1 h/day on gestational days 8-18 (UV-titan
L181, Kemira: rutile coated with Al, Si, Zr and polyalcohol; 17 nm). RESULTS: In exposed
adult mice, 38 mg Ti/kg was detected in the lungs on day 5 after exposure and 33 mg Ti/kg
on days 26. No Ti was detected in milk. Lung inflammation was evident in exposed females
as judged by cell counts in bronchoalveolar lavage fluid on both time points. Body weights
were similar in control and exposed dams as were litter size, sex ratio, implantations and
offspring body weights. At 11 weeks, offspring were tested for learning and memory, and
data indicated similar cognitive ability in control and exposed offspring. In the open field test,
exposed offspring tended to avoid the central zone of the field, and in the acoustic startle
test, exposed female offspring displayed stronger prepulse inhibition [2].
MWCNT: Study 1: Time-mated mice were instilled intratracheally with either 67 µg MWCNT7 (Mitsui, Japan) or NM-400 (Nanocyl, Belgium), four times during gestation (gestational
days 8, 11, 15 and 18). Controls received vehicle (Millipore water with 2% mouse serum).
Endpoints included maternal lung inflammation and gestational and litter parameters. Followup study: Naive female mice were instilled once with 67 µg NM-400 on the day before
mating, and time-to-first-litter and litter parameters were recorded. In study 1, lung
inflammation was evident and gestational weight gain reduced in exposed females.
Relatively fewer time-mated females gave birth in females exposed to MWCNT, compared to
controls. In the follow-up study, delivery of litter was significantly delayed in exposed
compared to sham instilled females. In both studies litter size and birth weights compared
across groups.
Inhaled TiO2 induced lung inflammation in adult mice, and persisted in tissue. Gestationally
exposed offspring displayed moderate neurobehavioral alterations. For MWCNT, lung
exposure caused lung inflammation in adult mice and affected weight gain during gestation.
The relatively lower pregnancy rate and delayed delivery of first litter in exposed females
suggest that MWCNTs interfere with establishment of pregnancy, possibly due to increased
inflammatory status in the adult females. Both studies indicate that exposure to nanoparticles
might interfere with reproductive processes.
[1] Hougaard, KS et al. 2011. Developmental toxicity of engineered nanoparticles. In: Gupta,
R (ed.) Reproductive and Developmental Toxicology. Academic Press, Amsterdam, 2011.
[2] Hougaard, KS et al. 2010. Effects of prenatal exposure to surface coated nanosized
titanium dioxide. A study in mice. Part Fibre Toxicology 7: 16.
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6.2.5 Emission of Nanoparticles from Painted Surfaces
Ralf Kaegi1
1
Eawag, Swiss Federal Institute for Aquatic Science and Technology
Email: [email protected]
Nanoparticles (NP) are already used in many consumer products. Besides their beneficial
effects to mankind, the uncontrolled release NP into surface waters is of concern as the
impact on the aquatic environment is still unclear.
Results from material flow analysis suggest that the majority of NP in consumer products will
be released into sewer systems and thus reach wastewater treatment plants (WWTP).
Recent studies indicate that NP will be removed from the wastewater stream with a high
efficiency.
However, apart from these point sources, NP can also be released from so-called diffuse
sources, such as outdoor paints. Compared to the NP that take the route via the WWTP,
there is only a limited retention potential for NP released from diffuse sources. The rather fast
surface runoff under heavy rainfall conditions may transport NP released from outdoor paints
without significant retention mechanisms, which inevitably leads to a discharge of NP into
surface waters.
Amongst others, embedding nano-silica, nano-silver, nano-TiO2, and nano-ZnO into paints
have been described in the literature to enhance certain properties of the paints, such as
increasing bactericidal properties, reducing the organic content in the paints, increasing anti
pollution (destroying atmospheric pollutants such as NOx) and self cleaning properties.
Long term stability and weathering tests of NP-containing paints have been performed with a
focus in the degradation of paints containing photoactive (nano)-particles [1]. Scanning
electron microscopy (SEM) analysis revealed the formation of pits in the paints located
around the photo-active particles (both nano- and pigmentary grade). It is suggested that
these pits formed as a consequence of oxidation of the polymer at the particles surface.
Therefore, it can be anticipated that also the NP embedded in the paints are released to
some extent from the paints during weathering. This hypothesis has recently been confirmed
in an experimental study where the runoff from outdoor facades has been collected and
analyzed using transmission electron microscopy (TEM) [2].
The fate and transport behaviour of the NP released from painted surfaces will strongly
depend on whether they are released as freely dispersed, individual NP, as NP-aggregates
(several NP aggregated to a larger entity) or as composite colloids (NP attached to other,
naturally occurring colloids such as clays or humic substances). This will also significantly
affect the bioavailability and (eco)-toxicology of the NP. In order to assess the importance of
NP released from painted surfaces, mass balance considerations have to be complemented
by studies addressing the release pattern of the NP.
[1] Allen, N.S., et al., Interrelationship of spectroscopic properties with the thermal and
photochemical behaviour of titanium dioxide pigments in metallocene polyethylene and alkyd
based paint films: micron versus nanoparticles. Polymer Degradation and Stability, 2002.
76(2): p. 305-319.
[2] Kaegi, R., et al., Synthetic TiO2 nanoparticle emission from exterior facades into the
aquatic environment. Environmental Pollution, 2008. 156(2): p. 233-239.
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6.2.6
Coatings and Nanoparticles – Activities of the German Paint Industry on
Workers’ Safety and Consumer Protection in the field of Smart Coatings
Michael Bross
German Paint and Printing Ink Association (VdL), Frankfurt, Germany
Email: [email protected]
Smart Coatings are newly developed coatings with significantly improved properties or
formerly unknown new functionalities, designed by applying the scientific principles of
nanotechnology or using the results of materials science. Some smart coatings contain
intentionally manufactured nanoparticles. The coatings industry in Germany has been
confronted with some basic questions, all centring around the possible exposure of
consumers or workers to nanoparticles. Special concern has been expressed with respect to
private end consumers using products coated with smart coatings.
Rather than discussing potential risks of smart coatings VdL intended to clarify whether
nanoparticles will be released from the coatings matrix under various conditions [1]. A project
was carried out aimed at examining the mechanism of release of nanoparticles from the
coatings matrix. The project started with the simulation of an every-day-use situation,
proceeded to sanding processes and is currently studying release scenarios of deteriorated
coatings films.
The stress simulation of a typical domestic setting (cleaning, walking, polishing etc.) resulted
in no significant particle concentration in the ambient air [2]. During the simulation of the
sanding process a considerable generation of nanoparticles was measured. Further
investigation of the swarf material showed that the generated nanoparticles are rather made
up from the matrix material. The nanoparticulate additives remain embedded in the abrasive
wear [3].
Workers’ safety is subject of comprehensive regulation in Germany. Apart from legal
provisions and professional association requirements VdL issued a special guidance on the
handling of nanomaterial in the coatings production. Recommendations for the selection of
raw materials, appropriate exhaust technologies and personal protection equipment are laid
down in the guidance document [4].
[1] Rommert, A., et.al. 2010: Kleine Teilchen in der Luft? Farbe und Lack, 116 (12), 25 – 29
[2] Vorbau M., et.al. 2009: Method for the characterization of the abrasion induced
nanoparticle release into air from surface coatings. Journal of Aerosol Science, 40 (3), 209 –
217
[3] Göhler, D. et al 2010: Characterization of the nanoparticles release from surface coatings
by the simulation of a sanding process. Ann. Occup. Hyg., 54 (6), 615 – 624
[4] VdL-Guidance for the Handling of Nano-Objects at the Workplace, June 2010
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Other
7 Other
7.1 Poster presentations
7.1.1 Harmonization of Measurement Strategies for the Assessment of
Exposure to Manufactured Nano object; Report of a workshop
Derk Brouwer1, Wouter Fransman1, Rianda Gerritsen-Ebben1, Markus Berges2, Erik
Tielemans1
1
TNO Quality of Life, Zeist, The Netherlands
2
DGUV-IFA, Sankt Augustin, Germany
Email: [email protected]
The number of workplace air measurement studies focused on the assessment of exposure
to manufactured nano objects has increased substantially, the last few years. However, due
to the large variation of exposure situations with respect to the life cycle of nanomaterials and
nanoproducts, actual exposure data will remain scarce in the near future. Therefore, it is
acknowledged that data that will be generated should enable future use for either exposure
scenario building, exposure modelling, or meta-analysis in view of risk assessment or
epidemiology. Harmonization of data collection, data analysis and reporting, and data
storage are key conditions for such intended uses.
TNO and IFA under the PEROSH umbrella, in collaboration with University of Massachusetts
-Lowell and aligned with NIOSH and NanoImpactNet initiatives, organized and hosted
(December 2010) the First International Scientific Workshop on Harmonization of Strategies
to Measure and Analyze Exposure to (Manufactured) Nano objects in Workplace Air. The
objectives of the workshop were to discuss state-of-the-art approaches and reach consensus
on:
o
measurement strategies
o
analyzing and evaluating exposure data
o
needs and conditions for a Nano Exposure and Contextual Information
Database
Key players in the area of occupational (nano) exposure assessment from Europe, US,
Japan and Korea were invited to the workshop and discussed three position papers, i.e. on
actual measurement strategy issues, on data analysis, interpretation and reporting, and on
database structure and core information. The position papers describe the state of the art
and the challenges that are faced, and give guidance/recommendations to Regulatory
Authorities, Standardization Bodies, Researchers and OSH practitioners. The process of
harmonization is considered to be continued both by a follow-up workshop and through
OECD WPNM. A summary of the discussions and the main conclusions will be presented.
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7.1.2 Microvascular Distribution and Effects of Surface-modified Quantum
Dots in Postischemic Tissues
Markus Rehberg, Camila Ferreira-Leite, Fritz Krombach
Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians-Universität München,
Munich, Germany
Email: [email protected]
Background/Objective: In two recent in vivo studies performed in healthy mice, we reported
that the capillary endothelium considerably contributes to blood clearance and tissue
deposition of anionic quantum dots (QDs) [1] and that leukocyte recruitment is modulated in
the presence of QDs [2]. We found that the surface chemistry of QDs strongly affects their
localization in postcapillary venules and their potential to modify steps of leukocyte
recruitment [2]. However, interactions of QDs with microvessels and their impact on
inflammatory reactions under pathophysiological conditions are still largely unknown.
Therefore we designed this study to investigate the impact of surface modification (i) on
microvascular localisation of QDs and (ii) on the effects of QDs on the steps of leukocyte
recruitment during ischemia-reperfusion (I/R).
Methods and Results: For all experiments, QDs (emission 655 nm, size 20-30 nm) with
carboxyl (carboxyl-QD) or amine (amine-QD) surface coating were injected intra-arterially (3
pmol/g body weight) into anesthetized male C57BL/6 mice (n = 6 per group; control mice
received vehicle). The QDs were applied at the onset of reperfusion, 30 min after induction of
ischemia by clamping all supplying vessels of the cremaster muscle, or after sham operation.
As assessed by in vivo fluorescence microscopy, both types of QDs tested were found to be
associated with the endothelium of postcapillary venules in the postischemic cremasteric
tissue. Interestingly, amine-QD fluorescence intensity values in regions of interests along the
vessel walls were approximately 2 fold higher than carboxyl-QD fluorescence intensities at
15 min and 3- to 4-fold higher at 60 min after application. The carboxyl-QD fluorescence
intensities at the wall of post-ischemic vessels were comparable to carboxyl-QD as well as
amine-QD intensity values obtained in sham-operated animals. Using in vivo
transillumination microscopy on the mouse cremaster muscle, I/R-elicited firm adherence of
leukocytes was found to be significantly increased upon application of amine- as well as of
carboxyl-QDs. I/R-elicited leukocyte transmigration, however, was significantly enhanced
only upon application of amine-QDs.
Summary and Conclusions: Taken together, these in vivo findings show that in the
postischemic tissue amine-modified QDs (i) are strongly associated with the vessel wall, and
(ii) amplify I/R-elicited leukocyte transmigration. In conclusion, these data clearly indicates
that the surface chemistry of QDs strongly affects their interactions with microvessels in
postischemic tissue and that QDs are able to modulate the inflammatory response after I/R.
Thus, this study adds valuable information for future biomedical applications of
nanomaterials.
[1] Praetner, M et al. 2010. The contribution of the capillary endothelium to blood clearance
and tissue deposition of anionic quantum dots in vivo. Biomaterials 26: 6692-700
[2] Rehberg, M et al. 2010. Quantum dots modulate leukocyte adhesion and transmigration
depending on their surface modification. Nano Letters 9: 3656-64
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7.1.3 NanoRiskCat••• ••
Nanomaterials
94B
–
A
Conceptual
Decision
Support
Tool
for
Steffen Foss Hansen, Anders Baun, Keld Alstrup Jensen
U
U
1
2
DTU Environment, Technical University of Denmark
National Research Centre for the Working Environment, Copenhagen, Denmark
Email: [email protected]
U
U
A number of different preliminary hazard and exposure assessment methodologies and
approaches have been proposed in the literature for risk assessment of nanomaterials and
products thereof. Most of these methods address occupational risk assessment. Only a few
methods are useful for assessing the risk for professional end-users, consumers and the
environment.
To address this need, we developed a generic framework (NanoRiskCat••• ••) that can be
used by companies and risk assessors to categorize nanomaterials considering existing
environmental, health and safety information and known possible uncertainties about the
exposure risks and hazard of these materials.
U
U
U
U
In its simplest form, the final evaluation outcome for a specific nanomaterial in a given
application will be communicated in the form of a short title (e.g. TiO2 in sunscreen or MeO in
ship paint) describing the use of the nanomaterial and a color code where the first three
colored bullets (••• ••; always refer to potential exposure of professional end-users,
consumers and the environment in that sequence and the last two colored bullets always
refer to the hazard potential for humans and the environment.
The colours assigned to the exposure and hazard potential are green (•), yellow (•), red (•)
and grey (•) corresponding to none, possible, expected and unknown, respectively.
Taking outset in the REACH use categories [1] the exposure potential was evaluated based
on 1) the location of the nanomaterial and 2) a judgment of the potential of nanomaterial
exposure based on the description and explanation of each process, category, etc.
The hazard potential for humans is evaluated based on whether the nanomaterial in question
is known as a compound to have low solubility in water (biodurable); fulfil the fiber paradigm;
be regulated harder than nuisance materials, to have CMR-properties or other adverse
effects?
The environmental hazard potential is based on whether the nanomaterial in question is
known to be: readily dispersed, persistent, bioaccumulative, and/or has been reported to be
hazardous to environmental species.
The proposed approach was validated through the completion of a number of case studies
such as e.g. and C60 used in lubricants, CNT used in baseball bat and TiO2 in sunscreens
and badminton rackets.
[1] ECHA 2010. Guidance on information requirements and chemical safety assessment
Chapter
R.12:
Use
descriptor
system.
Available:
http://guidance.echa.europa.eu/docs/guidance_document/information_requirements_r12_en.
pdf (Accessed: 25-10-2010)
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7.1.4 Effect of nanoparticle morphology on the detection efficiency of
condensation particle counters (CPCs)
Markus Pesch1, Stephan Rennecke2, Alfred Weber2, Matthias Richter2, Lothar Keck2
1
GRIMM Aerosol Technik GmbH & Co. KG, Ainring, Germany
2
Institute of Particle Technology, Clausthal, Germany
Email: [email protected]
The exposure to inhaled nanoparticles is a major concern for the health impact of
nanomaterials. Condensation particle counters (CPCs) are the common instrument for
measuring the concentration to airborne nanoparticles, and, in combination with a Differential
Mobility Analyzer (DMA), also for the measurement of nanoparticle size distributions. A well
known limitation of such instruments, particularly for water-based counters, is the effect of
the particle composition on the detection efficiency as a function of particle size [1]. Less
frequently studied was however the effect of particle morphology on the detection efficiency.
The efficiency of the GRIMM CPCs model 5.403, 5.414, and 5.416 was measured following
the ISO 27891 standard for Ag particle of two different morphologies. Spherical Ag and NaCl
nanoparticles were generated by sublimation and condensation of bulk material.
Agglomerated Ag particles were produced with a spark generator and a partial sintering was
achieved with a downstream quartz furnace. Tungsten oxide nanoparticles were also
investigated. Monodisperse size fractions were selected using a DMA and an aerosol
electrometer (GRIMM 5.705) served as a reference for the efficiency measurements.
Figure1. Counting efficiency of the CPCs model 5.403 and 5.416 for different particle composition and
morphology
Figure 1 shows that the counting efficiency is significantly higher for the Ag agglomerates
than for the spherical Ag particles. The 50% detection efficiency for Ag agglomerates was
measured at 4.1 nm for the CPC 5.414 (4.5 nm for 5.403), the corresponding values for the
spherical particles were 5.0 nm and 6.2 nm [2].
[1] Hering, S. V. et al. 2005. A Laminar-Flow, Water-Based Condensation Particle
Counter (WCPC). Aerosol Science and Technology, 39:659–672
[2] Rennecke, A. and Weber, A. 2010. Characterisation of the efficiency of condensation
particle counters. Report of the Institute of Particle Technology, TU Clausthal
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7.1.5 In vitro evaluation of silver nanoparticles of different sizes in assays for
cytotoxicity, inflammation and developmental toxicity
Margriet Park1, H. van Loveren1,2, A.M. Neigh3 L.J.J. de la Fonteyne1, J.P. Vermeulen1, E.R.
Gremmer1 and W.H. de Jong1
1
RIVM, Bilthoven, The Netherlands.
Maastricht University, Maastricht, The Netherlands.
3
NanoComposix, San Diego, USA
Email: [email protected]
2
Silver nanoparticles are currently listed as the most commonly used nanomaterials in
consumer products, and their antibacterial properties are of interest for medical applications.
In light of the anticipated increased human exposure to silver nanoparticles, evaluation of
their potential adverse effects is necessary. We have studied well-characterized silver
nanoparticles of 20, 80 and 113 nm in vitro, using assays for cytotoxicity, inflammation and
developmental toxicity.
Silver nanoparticles were cytotoxic to both fibroblast and macrophage cell lines, and induced
a variety of cytokines. Furthermore, silver nanoparticles of all sizes inhibited the
differentiation of embryonic stem cells into cardiomyocytes, but only at cytotoxic
concentrations. At equal mass concentrations, the 20 nm nanoparticles appeared to be most
toxic for all toxicity endpoints considered, indicating that effects were determined by particle
numbers or surface area rather than mass. The toxic potential of the silver nanoparticles was
lower than that of silver ions, as determined from studies using silver nitrate.
0.8
0.6
0.4
0.2
0.0
Differentiated EBs (fraction of solvent control)
1.0
Our results demonstrate that safety evaluation of silver nanoparticles may need to be
considered separately from other forms of silver. In addition, health based exposure limits for
silver nanoparticles should not be expressed in mass concentration units, but rather in
particle number or surface area based units.
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
Log concentration (µg/ml)
Figure1. Silver nanoparticles and silver nitrate inhibited D3 stem cell differentiation.
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7.1.6 Assessing the toxicological impact of
nanoparticles for risk assessment purposes
a
panel
of
engineered
Ali Kermanizadeh1, Vicki Stone1, Birgit Gaiser1, Gary R Hutchison2
1
Heriot Watt University, Edinburgh, UK
Edinburgh Napier University, Edinburgh, UK
Email: [email protected]
2
Nanotechnology has become a global industry. It has been estimated that the economical
impact of nanoparticles (NPs) and nanotechnology in terms of industrial, medical and
consumer products is currently 292 billion US dollars and predicted to bypass two and half
trillion dollars by 2015 (1). As with any new technology there are a number of potential risks to
consumers and workers exposed to these particles. It is paramount that these risks are
assessed and managed promptly as the failure to do so could have hazardous
consequences for human and environmental health (2).
It is now known that following exposure via a number of routes (inhalation, instillation, dermal
or ingestion) some NPs can translocate to secondary tissues and can be potentially toxic in
these target organs. One organ identified as a site for accumulating blood borne particles is
the liver. Of particular interest are the hepatocytes due to their abundance and their
importance in the normal liver function.
The initial phase of this study has focused on C3A cells (human liver cell line derived from a
hepatoblastoma). The impact of the ENPRA panel of particles consisting of five titanium
dioxide NPs, two zinc oxide, two multi walled carbon nanotubes and a silver particle were
observed on hepatocyte toxicity and function.
We observed that the silver particles elicit the greatest levels of toxicity followed by the ZnO
NPs. It was also discovered that LC50 was not reached at the presence of the other ENPRA
nanomaterials after a 24 hour period of exposure. It was deduced that C3A cells produce
significantly increased levels of IL8 following exposure to the ENPRA nanomaterials, with
these levels peaking around the LC50. Meanwhile it was found that there was no significant
increase or decrease in the levels of TNF-α, IL6 or CRP secreted from these cells after
ENPRA nanomaterials exposure. Experiments were conducted to ascertain the potential
mechanism driving inflammation in C3A cells post NPs exposure. Intracellular ROS levels
were assessed and shown to increase, following exposure of the C3A cells, in a similar
pattern to the equivalent levels of IL8 produced.
We found that there was no significant increase or decrease in the levels of urea produced
by the C3A cells in the presence of any of the nanomaterials compared to the control.
Furthermore it seems that there was no significant increase or decrease in the levels of
albumin produced with the exception of the two ZnO NPs where there was a significant
reduction in the levels of albumin produced at LC50 concentrations.
[1] Hood, E., 2004. Nanotechnology: look as we leap. Environmental Health Perspectives.
112, 740-749
[2] Maynard, AD., Aitkin, RJ., Butz, T., Colvin, V., Donaldson, K., Oberdorster, G., Philbert,
MA., Ryan, J., Seaton, A., Stone, V., Tinkle, S., Tran, L., Walker, NJ., Warheit, DB., 2006.
Safe handling of nanotechnology. Nature. 444, 267-269
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7.1.7 Critical analysis of frameworks and approaches to assess the
environmental risks of nanomaterials
Khara D. Grieger1, Igor Linkov2, Steffen Foss Hansen1, Anders Baun1
1
Technical University of Denmark, Kgs. Lyngby, Denmark
Environmental Laboratory, U.S. Army Corps of Engineers, Brookline, USA
Email: [email protected]
2
Scientists, organizations, governments, and policy-makers are currently involved in
reviewing, adapting, and formulating risk assessment frameworks and strategies to
understand and assess the potential environmental risks of engineered nanomaterials (NM).
It is becoming increasingly apparent that approaches which are aimed at ultimately fulfilling
standard, quantitative environmental risk assessment for NM is likely to be not only
extremely challenging but also resource- and time-consuming. In response, a number of
alternative or complimentary frameworks and approaches to standard (environmental) risk
assessment have been subsequently proposed specifically for NM. However, further
information regarding the potential strengths and weaknesses of these strategies is currently
lacking.
This analysis aims to evaluate different environmental risk analysis or assessment
frameworks and approaches which have been developed or proposed by large organizations
or regulatory bodies for NM. These frameworks and approaches were evaluated and
assessed based on a select number of criteria which have been previously proposed as
important parameters for inclusion in successful risk assessment frameworks for NM: flexible
for a variety of NM, suitable for multiple decision contexts, incorporate uncertainty analysis,
include life cycle perspectives, iterative or adaptive, enable more timely decision making,
transparent, integrate various stakeholder perspectives, integrate precaution, and include
qualitative or quantitative data.
Among other results we find that most of the investigated frameworks and approaches are i)
flexible for multiple NM, ii) suitable for multiple decision contexts, iii) include life cycle
perspectives, iv) transparent, v) include precautionary aspects, and vi) able to include
qualitative and quantitative data. We also find that many of the frameworks and approaches
may be adapted for iterative or adaptive elements and timely decision making if needed,
although these criteria were not inherently embedded in many of the strategies based on
their current format. Furthermore, most frameworks and approaches are mainly applicable to
occupational settings with minor applications for the environment, and many (if not most) of
them have not been thoroughly tested on a wide range of NM or nano-applications. Given
these results, we recommend the use of a multi-faceted approach to assess the
environmental risks of NM, in which different frameworks may be used and combined for the
particular question considered. We also recommend further testing of these different
frameworks and approaches on concrete, real-world NM applications which are specifically
relevant for environmental risk contexts.
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7.1.8 NANOGENOTOX: European Joint action on «Safety evaluation of
manufactured nanomaterials by characterisation of their potential
genotoxic hazard»
N. Thieriet1, A. Cadène1 and F. Etore1
1
Anses, Maisons-Alfort, France
On behalf of NANOGENOTOX partners*
Email: [email protected]
Nanotechnology is a highly strategic economic sector revealing enormous potential benefits
for many societal and environmental domains. Products containing manufactured
nanomaterials (MNs) are already mass-produced. However, the lack of scientific knowledge
and absence of evidence of health and safety hazards of nanotechnology products make
regulation very difficult.
Within this context, NANOGENOTOX is a new European Joint Action (JA) on the safety of
MNs, complementary to the OECD sponsorship programme for testing MNs, involving 16
institutions from 11 member states. Its general objective brings added value by
complementing Member States’ policies and contributing to improving citizens’ health and
security. Starting in March 2010 for a 3-year period, its major outcomes are expected to be
to:
(i) increase health information and knowledge about the human and environmental safety of
MNs by generating relevant and reliable data sets for some selected MNs by
1- distinguishing specific hazards regarding the physical and chemical parameters of
MNs
2- establishing a correlation between in vivo and in vitro genotoxicological data and
completing information on MN bioaccumulation by identifying target organs.
(ii) promote a robust reliable methodology for testing genotoxicity of MNs by exchanging best
practices through a round robin test. The MNs to be tested are SiO2, TiO2 and carbon
nanotubes (CNT). The JA will provide quick, reliable and economical tests to assess
potential genotoxicity of MNs with alert signals useful for society and industries.
This presentation arises from the project NANOGENOTOX which has received funding from
the European Union, in the framework of the Health Programme.
ISS (Istituto Superiore di Sanita, Italy), CLMC-BAS (Central Laboratory of Mineralogy and
Crystallography Bulgarian Academy of Sciences, Bulgaria), IMB-BAS (Roumen Tsanev
Institute of Molecular Biology Bulgarian -Academy of Sciences, Bulgaria), FIOSH (Finnish
Institute of Occupational Health, Finland), NRCWE (the National Research Centre for the
Working Environment, Denmark), BfR (Bundesinstitut fûr Risikobewertung – Federal Institute
for Risk Assessment, Germany), NIOM (the Nofer Institute of Occupational Medicine,
Poland), IPL (Institut Pasteur de Lille, France), UAB (Universitat Autonoma de Barcelona,
Spain), IPH (Institute of Public Health, Belgium), INRS (Institut National de Recherche et de
Sécurité pour la prevention des accidents du travail et des maladies professionnelles,
France), VAR (centrum voor onderzoek in diergeneeskunde en agrochemie – centre
d’études et de recherches veterinaires et agrochimiques, Belgium), INSA (Instituto Nacional
de Saude DR. Ricardo Jorge, Portugal), CEA (Commissariat à l’Energie Atomique, France),
RIVM (Rijkinstituut voor Volksgezondheid en Milieu – National Institute for Public Health and
the Environment, Netherlands)
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7.1.9 Development of a control banding tool adapted to nanomaterials
M. Riediker1, C. Ostiguy2, J. Triolet3, P. Troisfontaines4, D. Vernez1, G. Bourdel5, N. Thieriet5, A.
Cadène5 and I. Daguet5
1
IST, Lausanne, Switzerland
2
IRSST, Montréal, Canada
3
INRS, Paris, France
4
WIV-ISP, Brussels, Belgium
5
Anses, Maisons-Alfort, France
Email: [email protected]
Control banding (CB) is an occupational risk management approach where hazard and
exposure of a substance are ranked and combined to bands of similar risk with associated
standardized control measures. CB may be useful for control of nanomaterials’ risks but a
way to rank hazards and exposures is needed. We propose an approach that starts with few
fundamental physico-chemical properties of the nanomaterials. It takes into account already
existing hazard and exposure data and allows for an easy integration of the many new data
that are expected to be generated over the coming years.
The proposed CB approach consists of three steps:
1) Plan: Analyse hazard and exposure information, attribute control bands and define an
action plan.
2) Implement: Set up the control measures and start the routines as defined in the action
plan.
3) Check and correct: regularly monitor workplaces, review knowledge and control
measures. Correct the control bands or action plan when needed.
The planning step (Fig.1) is central to this approach. First, product and exposure information is
identified, based on which hazard and emission potential bands are defined. If the hazard is
estimated as “very high” or if it cannot be estimated, a full hazard assessment is needed. The
combination of hazard and emission potential band leads to a possible control banding strategy.
The practical and financial feasibility of this strategy is then evaluated. If it is feasible, an action
plan will be defined. Otherwise, a full risk assessment is needed in lieu of the CB-approach.
To full risk
assessment
To full hazard
assessment
From Risk/Review management
Product and exposure informations
Hazard band
Emission potential
Possible CB
strategy ?
Action plan definition
To implementation step
Figure1. The planning step: estimates for hazard and emission potential are combined to define a possible CBstrategy, resulting in an action plan.
The control bands are defined by combining hazard and emission potential bands. The
corresponding control strategies range from room ventilation (CB1) to full containment with
an addition expert advice (CB5). This work was funded by Anses.
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7.1.10 Nanotubes of imogolite do not activate macrophages and modestly
perturb the barrier properties of airway epithelial cells in vitro
Ovidio Bussolati1, Bianca Maria Rotoli1, Pier Paolo Zanello1, Barbara Bonelli2, Cristina
Zanzottera2, Edoardo Garrone2, Ivana Fenoglio3, Bice Fubini3, Mara Ghiazza3, Enrico
Bergamaschi1
1
Dept. Cl. Medicine, Nephrology and Health Sciences & Dept. Experimental Medicine,
University of Parma, Italy
2
DISMIC, Politecnico di Torino, Italy and INSTM Unit of Torino Politecnico, Italy
3
Interdepart. Centre “G. Scansetti” and Dept. of Chemistry, University of Torino, Italy
Email: [email protected]
Since airways represent the first barrier for inhaled particles, the effects of nanomaterials on
the cells of Lung Blood Barrier (LBB) should be investigated. Previous findings showed that
MWCNT impair airway barrier function and are toxic to macrophage lines [1]. Here we
investigate the effects of nanotubes of imogolite (INT), a hydrated alumino-silicate with the
formula (OH)3Al2O3SiOH, previously proposed by some of us as a possible negative control
for HARN [2].
INT - i.d. 1 nm, BET 394 m2 g-1, total and microporous volume of 0.27 and 0.11 cm3 g-1,
respectively - were synthesized via sol-gel procedure and found organized into fibres at
FESEM [3]. As in vitro models of LBB cells, we used two murine macrophage cell lines
(Raw264.7 and MH-S) and the human airway epithelial cells Calu-3. Cell viability was
assessed with resazurin. RT-PCR was used to study the expression of NOS2 and ARG1,
markers of, respectively, macrophage classical or alternative activations, and concentration
of nitrites in the culture medium was measured as an indicator of NO production.. Epithelial
barrier integrity was evaluated from the trans-epithelial electrical resistance (TEER). At the
same doses, INT caused much smaller effects than MWCNT on macrophage viability, while
no significant damage was observed up to 40 µg/cm2 of monolayer for exposure times up to
24h. The incubation of macrophages with INT at doses as high as 120 µg/cm2 for 72h did not
alter either NOS2 or ARG1 expression nor increased NO production. In Calu-3 monolayers
exposed to INT (120 µg/cm2 for 7d) only modest TEER changes were recorded (< 20%).
As a whole, in spite of their fibrous nature, INT appear not markedly toxic for in vitro models
of LBB cells and could represent a low-toxicity reference for in vitro toxicological studies on
HARN, should further tests confirm their inertness.
Supported by MIUR-PRIN Grant No. 2007498XRF
[1] Bianca Maria Rotoli et al., 2008. Non-functionalized multi-walled carbon nanotubes alter
the paracellular permeability of human airway epithelial cells. Toxicol. Lett. 178:95-102.
[2] Bice Fubini et al., 2010. Physico-chemical features of engineered nanoparticles relevant
to their toxicity. Nanotoxicology, (DOI: 10.3109/17435390.2010.509519)
[3] Ilaria Bottero et al. 2010. Synthesis and characterization of hybrid organic/inorganic
nanotubes of the imogolite type and their behaviour towards methane adsorption. Phys.
Chem. Chem. Phys. (DOI: 10.1039/C0CP00438C)
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7.1.11 Nanotoxicology at European Center for the Sustainable Impact of
Nanotechnology-ECSIN and its research approach-an overview
Enrico Sabbioni1, Iolanda Olivato1
1
ECSIN Laboratory, Veneto Nanotech S.C.p.A., Rovigo, Italy
Email: [email protected]
When nano-containing products entered into the market the first nanotoxicological studies
suggested a potential hazard of such innovative materials. However, since at that time the
exposure was low the real health risks were seen as “far and mostly theorised”. Currently the
situation has dramatically changed, being number/amounts of nanomaterials on the market
drastically increased. A 2009 inventory shows the number of consumer products containing
nanomaterials at over 1,000 products, and has grown by nearly 379% since March 2006.
Thus new facilities have been developed to produce nanoparticles (nps) at levels of some
hundredths tons/year. Then, the exposure of general population, consumers and
occupational workers to nanomaterials is moving from a “mostly theorised phase” to a reality.
Unfortunately, the nanotoxicological data for a scientific-based nanoregulation are still
limited, showing how fast the market of nanomaterials is growing while the regulation of such
nanoproducts is alarmingly slow. Actually, the environmental and human health impact of
nanomaterials is largely unknown. These reflections stress the urgency for a much faster
development of nanotoxicology to generate reliable data for risk assessment before the
introduction of nanomaterials into the market, and becoming ubiquitous in every aspect of
life. Unfortunately, till now there was the perception that nanotoxicology would not be an
essential part for a sustainable development of nanotechnology, but rather a “break on
innovation”. Moreover, the slow progress of nanotoxicology is partly due to the marked
multidisciplinary character of such kind of studies that requires the use of specialized
structures, a combination of chemical, physical, biological, toxicological competences and
sophisticated expensive apparatus: this apparatus of competences in a same lab is rare. In
this context, ECSIN is a unit of Veneto Nanotech, the Italian Hi-Tech Cluster of
Nanotechnology applied to materials. ECSIN deals with environmental and health impact of
nanomaterials supporting a scientific-based nanoregulation and highlinting the concept that
nanomaterials are neither "nano-angels" nor "nano-demons". ECSIN aims:(i) creating
conditions that encourage innovation (ii) applying the most recent and advanced research
techniques to the characterization of the impact of nanomaterials on human health,
environment and society (iii) providing scientific and technical support to a policy of quality
where standards should be an essential tool in order to promote more rapid, safe and
effective development of nanomaterials, facilitating risk analysis and generate data useful for
regulatory bodies (iv) providing nanotechnology enterprises, public bodies and investors a
method to assess and reduce the risks connected with production and use of nanomaterials.
In this Conference we present on overview of ECSIN that is organized in two technology
platforms: in vivo and in vitro analysis of the effects of nps and nanostructured materials on
biological system, and nps monitoring in environmental matrices as well
econanotoxicological analysis. The scientific objectives and the current status of ECSIN
activities presented will refer to: integrating research themes to a unified research strategy
for risk assessment; conceptualization and guidelines that inspire the development of the
nanotoxicology; a view of the current/planned research projects, carried out in a
national/international context.
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7.1.12 The basic requirement for comparable results between laboratories from
in vitro tests remains a significant challenge
Matthias Roesslein1, John T. Elliott2, Marc Salit2, Cordula Hirsch1, Peter Wick1, Harald F.
Krug1
1
Empa, Laboratory Materials-Biology Interactions, St. Gallen, Switzerland
2
NIST Biochemical Science Division, Gaithersburg MD, USA
Email: [email protected]
Nanomedical research and development efforts are progressing rapidly. The outcome of
these efforts will soon be the focus of pharmaceutical industry and their regulating bodies,
such as the US food and drug administration (FDA). Therefore an international
harmonization of experimental protocols to assess the effects of nanoparticles in vitro and
subsequently also in vivo is urgently needed. To achieve this goal, a program of
interlaboratory comparisons was started under the auspices of the international alliance for
the NanoEHS harmonization (IANH) [1]. A first series of interlaboratory comparisons
revealed tremendously different outcomes for most in vitro assays under investigation. To
give a typical example figure 1 summarizes schematically the anonymized results of a
cytotoxicological test. The obtained pattern of results reveals over-dispersion, outliners and
sometimes huge variation within laboratories prevented the determination of the “true value”.
This lack of comparable results between laboratories hampers any direct stepwise approach
to improve test protocols.
Figure 1: Schematic display of interlaboratory comparison employing in vitro cell assay to determine the effect of
engineered nanoparticle. It shows a group of three laboratories (A) with mutually consistent results, but also overdispersion together with outlining values and sometimes huge variation within laboratories. A possible location of
a “true value” is given as dashed line
We undertook a considerable effort to resolve this stalemate. We present selected findings of
the before mentioned interlaboratory comparisons. Furthermore we elaborate on the use of
experimental design concepts, which were developed to determine the major sources of
variability within and between laboratories. Hence, the goal to generate comparable results
all over the world from in vitro test systems is advanced.
[1] www.nanoehsalliance.org
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7.1.13 Bioavailability of silver nanoparticles orally administered to rats
Meike van der Zande, Ruud Peters, Elly Wijma, Hans Bouwmeester
RIKILT – Institute of Food Safety, AE Wageningen, The Netherlands
Email: [email protected]
The physical and chemical properties of nanomaterials are known to be completely different
from their larger counterparts. This makes them attractive for exploitation in a wide range of
products. Amongst these nanomaterials are silver nanoparticles (AgNPs), which exert an
antibacterial effect. AgNPs are already used in the pharmaceutical and food industry and in
several consumer products [1]. As a consequence, consumers are likely being exposed to
these AgNPs. The extraordinary properties, that make NPs so attractive from a technical
point of view, also make them unpredictable and potentially harmful. It is unknown whether
natural barriers are protective. Moreover, the large reactivity of AgNPs adds up to their
hazardous potential [2].
At present, little is known about the kinetics and bioavailability of orally exposed AgNPs.
Therefore, we performed a pilot study to examine the potential of AgNPs to cross the
intestinal wall, enter the bloodstream and distribute to other organs after oral exposure in
rats. Rats (n=4) were exposed for three consecutive days and killed at the fourth day. AgNPs
sized <20 nm were administered in a dose of 500 mg/kg bw via different oral routes: 1) oral
gavage in water, 2) oral gavage in custard, 3) oral gavage in water after 6 h fasting and 4) via
suspending in drinking water. AgNPs sized 50-60 nm were administered at the same dose
via oral gavage in water. As controls, rats obtained either 50 mg/kg bw AgNO3 in water or
were left untreated. Blood and organs were examined for NP concentrations by single
particle ICPMS. This newly developed analytical method enables determination of the
presence and concentration of AgNPs, the particle size and the ionic Ag concentrations in
biological tissues. Preliminary results show the presence of NPs in blood and liver for the <20
nm AgNPs and AgNO3 groups. The measured NP concentration seems to be independent of
the type of oral exposure, or of the presence of a food matrix. No NPs were detected in the
blood or liver for the 50-60 nm NPs and untreated groups.
These initial results indicate that the <20 nm AgNPs are bioavailable. The <20 nm AgNPs
most probably translocate as nanoparticles through the intestines to the liver. Furthermore,
AgNPs appear to be formed from ions in the AgNO3 group. Possibly, these NPs consist of
AgCl particles, which assembled in the acidic environment of the stomach. The findings of
this pilot study will be validated in a 28-day oral exposure experiment in rats. The single
particle ICPMS method has proven to be a very sensitive tool for the detection of
nanoparticulate matter in biological tissues.
[1] Hagens WI, et al. 2007. What do we (need to) know about the kinetic properties of
nanoparticles in the body? Regulatory Toxicology and Pharmacology 217-229
[2] Bouwmeester H, et al. 2010. Minimal analytical characterisation of engineered
nanomaterials needed for hazard assessment in biological matrices. Nanotoxicology 52-62
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7.1.14 IANH assessment of Nano-particle Cytotoxicity
Daithi Garry, Sergio Anguissola, Sonia Ramirez, Iseult Lynch, K. A. Dawson
Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University
College Dublin, Dublin, Ireland
Email address: [email protected]
The production and development of nanoparticles (NP) have had a positive technological
impact on modern life, but at what cost? Highlighting the potential toxicological side effects of
all NPs is necessary for the growth of nanotechnology. Informing the relevant population of
the potentially hazardous toxicological effects on humankind and the surrounding
environment is of sincere and indisputable importance in ensuring a better future. The
International Alliance of NanoEHS Harmonization (IANH) has developed a ‘round robin’
stress-test model for NP toxicity which exposes NPs which possess highly toxic tendencies
and therefore clear non hazardous particles of all preconceived negative notions. The IANH
has composed a collection of assays to test cell viability, assess cell death and examine the
production of reactive oxygen species in RAW 264.7 murine macrophages. Throughout
these rigorous assays, the RAW 264.7 cells have been exposed to positive amino modified
Polystyrene (PS-NH2) and Cerium Oxide (CeO2, Ceria) NPs under reproducible time
constraints and concentrations. Understanding the biological impacts of NPs on human
health and that of the environment is paramount in securing a safe, responsible, world-wide
implementation of nanotechnology.
Amino Modified Polystyrene
Cerium Oxide
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7.1.15 Cytotoxicity and genotoxicity induced in human and murine cell assays
by copper oxide nanoparticles
Lucia Migliore1, M.Rita Fabbrizi1, Sebastiano Di Bucchianico1, Valentina Mariani2, Jessica
Ponti2, Francois Rossi2, Eugenia Valsami-Jones3, Deborah Berahnu3, Paul Reip4, Enrico
Bergamaschi5
1
Dept. of Human and Environmental Sciences, Faculty of Medicine, University of Pisa, Italy
EU Commission- Joint Research Centre, Institute of Health and Consumer Protection, NBS
Unit, Ispra, Italy
3
Department of Mineralogy, Natural History Museum, London, UK
4
Intrinsiq Materials Ltd, Cody Technology Park, Ively Road, Farnborough, Hampshire, UK
5
Dept. of Clin. Medicine, Nephrology and Health Sciences, University of Parma, Italy
Email: [email protected]
2
Metal oxide nanoparticles (NPs) are already present in commercial products and their
applications are expected to increase in the future. Among these, copper oxide (CuO) NPs
are used as antimicrobial preparations, heat transfer fluids and semiconductors. Since CuO
can induce toxic effects in several cell lines, we used CuO NPs from a commercial source to
set up cytotoxicity and genotoxicity assays suitable for screening purposes of metal NP.
The induction of toxicity and genotoxicity following in vitro exposure to CuO NPs [mass
concentration from 0.1 to 103 µg/ml] was assessed in three cell lines: the human A549 lung
epithelial cells, the murine macrophage RAW 264.7 and the murine fibroblast Balb/3T3; to
mimic possible interaction with blood cells, we also used peripheral blood monocytes
(PBMC) from volunteers. Cytotoxicity was assessed by MTT and Colony Forming Efficiency
(CFE) assays.
MTT assay revealed a significant dose-effect relationship between the testing concentrations
and a decrease of cell viability in A549 and RAW 264.7 cells, both after 24h and 48h with.
The CFE assay on A549 cells - that are able to form colonies - confirmed a dose-effect
relationship at 24 h and 72 h in the same range of CuO NPs concentrations used for MTT,
with an IC50 of about 4 µg/ml at 24h. The comet assay carried out on A549, RAW 264.7 cells
and PBMC (2h and 24h treatment; dose range: 0.1 to 100 µg/ml) revealed that the primary
DNA damage increased in a dose-dependent manner, with different sensitivity exhibited by
the different cell type. The version of the Comet assay that allows to specifically detect the
induction of oxidative damage to DNA was applied and both oxidised purines and
pyrimidines showed significant increases in all the cell types studied. Moreover, the
frequency of micronuclei in binucleated RAW 264.7 and A549 cells and PBMC increased in a
dose-related manner (dose range: 0.1 to 100 µg/ml) with differences in sensitivity due to the
specific cell type.
Supported by the Research Project “NANORETOX” funded under 7th EU FWP (Ref. No.
214478)
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7.1.16 Forecasting Nano Law: The Small Matter of Big Risks
Ilise L Feitshans1
1
Geneva School of Diplomacy Chateau des Penthese, Geneva Switzerland
Email: [email protected]
In the next five to ten years, billions of dollars will be spent on research and development
funding for the application of nanotechnology, touching the economy globally, across almost
every industry: food processing for retail markets, cosmetics, paintings and coatings,
agriculture, equipment and packaging. Yet the risks associated with the application of such
technologies are much slower to be emerging than the many new vistas of prosperity and
efficiency that nanotechnology promises to humanity throughout the world. The spectre of
new economic frontiers with wider horizons for new products and the attendant commerce
from their trade has caused many opinion leaders in science, law and health policy to herald
nanotechnology as an unprecedented opportunity for human development and growth. At the
same time, the known dangers of many of the substances whose molecular structure are
changed using nanotechnology has caused alarm among scientists and policymakers who
fear that unfettered use of such new technologies can unleash a public health crisis. Law and
science have partnered together in the recent past, to solve major public health issues,
ranging from the asbestos threat to industry to averting the threat of nuclear holocaust.
Wise people will try to foresee inevitable but presently unknown nanotechnology risks. Then
they will try to address these anticipated risks with best practices, codes of conduct and
scientific principles to prevent harm that will reshape the rule of law. Thus, the question
arises:
What law, if any applies to protect the general public, nanotechnology workers and their
corporate social partners from both liability and preventable harms?
This paper will travel to a legal and health policy frontier where no one has gone before. It
will be prepared in less than a year from the start date, because it tracks the author’s
doctoral research in International Relations “Forecasting nano Law” at the Geneva School of
Diplomacy in Geneva Switzerland, where the author, (a lawyer with public health training
from the Johns Hopkins University), currently serves as Faculty. This paper will translate
scientific principles into a legal framework, but mindful that some of the finest minds in the
nanotechnology industry, economics and sciences are unaware of basic methods for
studying law. The paper will be therefore geared to the use of plain English, avoiding legal
jargon as often as possible, and defining the legal terms that are employed when absolutely
necessary. This paper represents a first attempt in law or health policy to answer this vital
question. This paper provides succinct but comprehensive overview of the legal landscape.
This paper will examine the role of the scientific precautionary principles under international
law and the law of several key countries that already use nanotechnology as an emerging
sector of their economy by asking whether existing laws are consistent with the
precautionary principles that reflect scientific consensus. The final section of this paper will
offer a short proposal for legislation to fill apparent gaps, with a preliminary assessment of
areas where there is existing law to provide the basis for cautious application of
nanotechnology across a wide variety of industries.
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7.1.17 NANODEVICE: Novel Concepts, Methods, and Technologies for the
Production of Portable, Easy-to-use Devices for the Measurement and
Analysis of Airborne Engineered Nanoparticles in Workplace Air
Sari Sirviö1, Kai Savolainen1
1
Finnish Institute of Occupational Health, Helsinki, Finland
Email: [email protected]
NANODEVICE is a research project funded by the European Commission in the context of
the 7th Framework Program. The duration is 48 months starting 1st of April 2009.
Due to their unique properties, engineered nanoparticles (ENP) are now used for a myriad of
novel applications with great economic and technological importance. However, some of
these properties, especially their surface reactivity, have raised health concerns, which have
prompted scientists, regulators, and industry to seek consensus protocols for the safe
production and use of the different forms of ENP.
There is currently a shortage of field-worthy, cost-effective ways - especially in real time - for
reliable assessment of exposure levels to ENP in workplace air. In addition to the problems
with the size distribution, a major uncertainty in the safety assessment of airborne ENP
arises from the lack of knowledge of their physical and chemical properties, and the levels of
exposure. A special challenge of ENP monitoring is to separate ubiquitous background
nanoparticles from different sources from the ENP.
NANODEVICE will provide new information on the physico-chemical properties of
engineered nanoparticles (ENP) and information about their toxicology. Also a novel
measuring device will be developed to assess the exposure to ENP´s from workplace air.
The purpose of the project is also to promote the safe use of ENP through guidance,
standards and education, implementing of safety objectives in ENP production and handling,
and promotion of safety related collaborations through an international nanosafety forum.
The main project goal is to develop innovative concepts and reliable methods for
characterizing ENP in workplace air with novel, portable and easy-to-use devices suitable for
workplaces. Additional research objectives are:
1) Identification of relevant physico-chemical properties and metrics of airborne ENP;
establishment of reference materials.
2) Exploring the association between physico-chemical and toxicological properties of ENP;
3) Analyzing industrial processes as a source of ENP in workplace air;
4) Developing methods for calibration and testing of the novel devices in real and simulated
exposure situations.
5) Dissemination of the research results to promote the safe use of ENP through guidance,
standards and education, implementing of safety objectives in ENP production and handling,
and promotion of safety related collaborations through an international nanosafety forum.
Grant Agreement no. CP-IP 211464-2
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7.1.18 Social and Ethical Aspects of Nanomedicine: Sharing Benefits and Risks
Geoffrey Hunt
Centre for Bioethics & Emerging Technologies, St Mary’s University College, London
Email: [email protected]
While many ethical and governance issues coalesce around issues of nanotoxicity, in the
emerging field of nanomedicine there is another set of pressing ethical issues: What are the
nanomedicine priorities? By what criteria and processes are priorities set and by whom?
Who will benefit from nanomedicine? Who will be the first human recipients of
nanomedicines under development and will their consent be informed? Here we have
questions about global inequality and justice, corporate social responsibility, the governance
of research and development, and how risks and benefits are shared internationally.
According to one study (Pirages, 2005, p46), of 1,233 drugs on the global market in 1975 1997, only 13 were applicable to the tropical conditions causing the most infectious disease
deaths. Certainly nanotechnology developments in general could have public health benefits
such as detection of pathogens and contaminants in air-soil-water, filtration and remediation
of water supplies, and indirect effects such as reducing export of waste. But what about the
direct impacts of nanomedicine, and its priorities. Here, among other things, I argue for a
model in which priority is given to major global disease conditions through the development
of nano-vaccines. Novel and more effective vaccines for diseases such as HIV, Dengue
Fever, Hepatitis B and Tuberculosis are now possible, and in some cases under
development. I give the examples of nanomedicine research on hepatitis and TB.
Chronic hepatitis B infects 400 million people globally, with about 1m deaths especially in
Africa, Asia and Latin America. While there are three effective vaccines available, these
require needles, refrigeration, and three return visits which is not always feasible in
developing countries. But a nano-emulsion is a new delivery method for an antigen already
used in existing hepatitis B vaccines to activate the body’s immune defences. A needle-less
method, apparently non-toxic with “strong, sustained immune responses in animal studies”,
is under development.
Declared a global emergency by the WHO in1993, the re-emerging threat of TB continues to
be exacerbated by multi-drug resistance. Now, treatments with improved sustained release
profiles and bio-availability can increase compliance through reduced drug requirements and
so minimise MDR-TB. Additionally, improved diagnostic tools are required to meet the needs
of the WHO’s expansion of the Directly Observed Treatment Short-course. In India, country
with the highest estimated number of TB cases, there is ongoing research into the role
nanotechnology can play. A nanotechnology-based TB diagnostic kit (Central Scientific
Instruments Organisation of India), currently in the clinical trials phase, does not require
skilled technicians for use, offers efficiency, portability, user-friendliness, availability for as
little as one US dollar.
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7.1.19 Cellular effects of nanosilver in human macrophages: Uptake, oxidative
stress responses, lipid alterations and functional impairment
Andrea Haase1, Jutta Tentschert1, Philipp Graf2, Alexandre Mantion3, Felix Draude4, Harald
Jungnickel1, Johanna Plendl5, Heinrich F. Arlinghaus4, Andreas F. Thuenemann3, and
Andreas Luch1
1
German Federal Institute for Risk Assessment, Department of Product Safety, Berlin,
Germany
2
University of Basel, Department of Chemistry, Base, Switzerland
3
BAM - Federal Institute for Materials Research and Testing, Berlin, Germany
4
University of Münster, Institute of Physics, Münster, Germany
5
Free University of Berlin, Department of Veterinary Medicine, Institute of Veterinary
Anatomy, Berlin, Germany
Email: [email protected]
Silver nanoparticles (SNP) are among the most commercialized nanoparticles. Since
macrophages represent a physiological relevant cell system, we performed in vitro studies in
human macrophages derived from THP-1 cell line. We studied SNP of different sizes (20 nm
to 40 nm) and different coatings (citrate, peptide). Apart from clear dose- and time-dependent
effects, the toxicity strongly relied on size and the type of coating, with citrate coated particles
being less toxic. Gold nanoparticles, which served as control, showed nearly no adverse
effects.
The uptake was studied with Confocal Raman Spectroscopy as well as by TEM. SNP could
be detected as aggregates in the cytoplasm but also as individual particles throughout whole
cells, also in nucleus and lysosomes. Here we used a novel time-of-flight secondary ion
mass spectrometry (TOF-SIMS) and Laser postionization secondary neutral mass
spectrometry (Laser-SNMS) approach to visualize intracellular SNP and to study cellular
effects in parallel. We could detect significant changes in lipid composition of the outer
cellular membrane leaflet, which were indicative for oxidative stress and alterations in
membrane fluidity. We further supported this finding by different biochemical methods. We
could detect a time- and dose-dependent induction of heme oxygenase 1 and protein
carbonylation, both of which are established markers for oxidative stress. Each process
follows distinct kinetics, supporting a hierarchical model of oxdiative stress. We analyzed the
carbonylated proteins on 2D gels, thereby enabling visualization and separation of a complex
pattern of modified proteins. Importantly, with the 2D gel approach we were able to clearly
distinguish between the effects of different nanoparticles as they induce different
carbonylation patterns. In addition we detected functional alterations of the macrophages.
Even very low concentrations reduced the phagocytic activity considerably. Both endpoints,
impaired phagocytosis and lipid alterations could be linked to the particle-mediated
generation of oxidative stress. Some of these effects could be reversed depending on the
time and dose of nanoparticles used during treatment of cells. Thus we were able to
determine a “point of no return” that was found nicely fitting to that what was asserted as
overload dose for macrophages based on in vivo studies.
We conclude that SNP exert adverse effects in human macrophages also at subcytotoxic
concentrations via oxidative stress, leading to changes in membrane lipid composition and to
cell’s functional impairment. While all SNP were effectively taken up by macrophages,
different SNP induce distinguishable effects.
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7.1.20 Polystyrene: A potential standard for developing In Vitro cellular tracking
methods for nanotoxicology
Jennifer Dorney1, Franck Bonnier2, Alan Casey1, Gordon Chambers1, Hugh Byrne1
1
Nanolab, Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland
2
RESC, Focas Research Institute, Dublin Institute of Technology, Dublin, Ireland
Email: [email protected]
Nanotoxicology has emerged as a discipline of a result of the revolution of nanotechnology.
While nanotoxicology is in its infancy, there is a lack of toxicological data for nanoparticles,
naturally occurring or commercially produced. The need for information regarding cellular
uptake mechanisms associated with nanoparticle uptake, as well as internalisation and
accumulation of nanoparticles once penetrating cell membranes, is imperative. This study
focus’s on the internalisation studies of surface modified polystyrene nanoparticles. An in
vitro lung model consisting of A549 (ATCC No: CRL185) a carcinogenic lung epithelial cell
line, was employed to investigate the biocompatibility of nano scaled polystyrene particles in
pulmonary systems. Bulk polystyrene particles (above 3 µm) were employed as positive
control and biological effects were compared to that of 40nm carboxylated surface modified
nanopolystyrene, 40nm aminated surface modified nanopolystyrene and 50nm neutral
nanopolystyrene. Prior to cellular studies, a full particle size characterisation was carried out
using dynamic light scattering, atomic force microscopy, zeta potential and vibrational and
electronic spectroscopy. The cytotoxic effects of nano scale 40nm carboxylated, 40nm
aminated and 50nm neutral nanopolystyrene were then evaluated using five cytotoxic
endpoints namely the Neutral Red, Alamar Blue, Comassie Blue, MTT and Clonogenic
assays, with bulk polystyrene employed as a control. Cellular internalisation of the
fluorescently labelled particles was monitored with the aid of fluorescent confocal
microscopy. Raman spectroscopy and was employed as a novel technique for the
verification of nanoparticles internalised within live cells. Infra Red vibrational spectroscopy
was also employed to observe the internalisation of nanopolystyrene particles within cells.
Nanoparticle internalisation and accumulation within cells organelles such as lysosomes,
mitochondria and endoplasmic reticulum was monitored as a function of nanoparticle surface
charge and verified with the aid of commercially available transfection labelling kits.
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7.1.21 Protective effect of biosynthesized AgNPs from Melia azedarach against
Dalton’s Ascites Lymphoma
Achiraman S1, Sukirtha R1, Kamalakkannan S2, Sumedha N.C1, Gayathri R1, Jacob
Joe Antony1
1
Department of Environmental Biotechnology, Bharathidasan University,
Tiruchirappalli, India
2
University of Lausanne, Institute of Physiology, Lausanne, Switzerland
Email: [email protected]
Versatility of Silver nanoparticles renders them promising applications in various fields of
medicine including oncology. Plants harbour enumerable treasure of metabolites and confers
faster rate of synthesis through greener and safer biomimetic method. In the present study
the biosynthesized AgNPs from aqueous extract of M. azedarach were studied for their anti
tumour activity against Dalton’s ascites lymphoma (DAL) mice model. Animals were grouped
as normal, induced, four experimental groups (n=6). AgNPs and aqueous extract of M.
azedarach were administered intraperitoneally in the experimental groups. At the end of the
10 days treatment, mice were harvested and the cardiac blood was studied for
haematological variations. The vital organ liver was studied for their antioxidant defence
mechanism. The haematological profile regressed the increased total white blood cells
(WBC) count to normal level in the higher dose of AgNPs treated group than the aqueous
treated and induced group. The decreased red blood cells (RBC) and hemoglobin (Hb) count
relapsed in the AgNPs treated group, when compared to the aqueous treated and induced
group. The antioxidants levels of super oxide dismutase (SOD), catalase (CAT) and
glutathione peroxidase (GPx) also showed the coherent results as of the haematological
profile. Hence, we concluded the biosynthesized AgNPs from aqueous extract of M.
azedarach showed a protective effect on haematological and antioxidant system in DAL
induced mice model which reflects its potent antitumor activity.
Key words: Melia azedarach (M. azedarach), White blood cells (WBC), Red blood cells
(RBC) and Haemoglobin (Hb), Glutathione peroxidase (GPx).
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7.1.22 Ag and TiO2 Nanoparticles: Effects on Model Aquatic Organisms
A. Georgantzopoulou1, M. Dusinska2, M. Kruszewski3, Y.L. Balachandran4,
J.N. Audinot1, L. Hoffmann1, A.C. Gutleb1
1
Centre de Recherche Public - Gabriel Lippmann, Belvaux, Luxembourg
2
Norwegian Institute for Air Research, Kjeller, Norway
3
Institute of Nuclear Chemistry and Technology, Warszawa, Poland
4
Bharathiar University, Coimbatore, India
Email: [email protected]
The widespread application and use of nanoparticles (NPs) in numerous products will
unavoidably lead to their release in aquatic systems. However, there is a lack of knowledge
on the effects of NPs on living systems.
The present study aims to assess the effects of TiO2 (20 nm), and Ag (20, 27 and 200 nm)
NPs on Daphnia magna, the freshwater algae Desmodesmus subspicatus and validation of
the use of the marine bacterium Vibrio fischeri bioluminescence as a potential pre-screening
assay (mMicrotox-assay).
Characterised NPs and dispersion protocols were provided by the FP7 project NanoTEST,
the Polish-Norwegian Research Fund project NorPol and by the Bharathiar University, India.
Bulky TiO2 and Ag (200 nm) and AgNO3 were used for comparison. The organisms were
exposed to increasing concentrations of NPs or AgNO3. The inhibition of D. magna mobility,
the inhibition of D. subspicatus growth and inhibition of V. fischeri bioluminescence was
evaluated after 48, 72 hours and 30 minutes, respectively.
Both bulky and nano-TiO2 caused an increase in luminescence in the mMicrotox-assay in a
concentration dependent manner starting at concentrations higher than 20 and 200 mg/L for
nano- and bulky TiO2, respectively. TiO2 NPs had no effect on D. magna mobility and D.
subspicatus growth despite the high levels tested.
Size dependent effects were observed concerning Ag NPs in all organisms studied with D.
magna being the most sensitive organism (Table 1). Ag 20 nm and Ag 200 nm inhibited V.
fischeri bioluminescence by 12% and 15%, respectively at the highest tested concentration
of 10 mg/L and therefore no IC50 could be calculated.
These findings support the increasing concern on the potential harm of Ag NPs on aquatic
organisms and for the environment.
Table 1. IC50 for Ag NPs of different sizes as well as AgNO3.
D. magna
D. subspicatus
V. fischeri
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IC50 (mg/L)
Ag 20 nm Ag 27 nm
0.14
0.02
1.5
0.3
51
Ag 200 nm
0.4
5
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0.05
0.02
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7.1.23 Low-dose Single Wall Carbon Nanotubes affect embryonic development:
an in vitro and in vivo study
Antonio Pietroiusti1, Micol Massimiani1, Andrea Magrini1, Antonio Bergamaschi2, Luisa
Campagnolo1
1
Tor Vergata University, Rome, Italy
Tor Vergata University, Rome, Italy
[email protected]
2
The possible toxicity of engineered nanomaterial (ENM) has prompted numerous in vitro and
in vivo studies. However, limited data are available on ENM embryotoxicity in Mammals, and
none on carbon nanotubes (CNT), which are among the most promising ENM for industrial
and biomedical applications. Purpose of the present study was to investigate embryotoxicity
of three samples of single wall CNTs (SWCNTs), having different amounts of acidic
oxygenated functionalities on the surface, by intravenous administration to pregnant mice. In
parallel, SWCNT embryotoxicity was measured by the Embryonic Stem Cell Test (EST), an
in vitro assay used to predict embryotoxic effect of soluble chemical compounds. The in vivo
and in vitro experiments gave fully comparable results, clearly showing for the first time that,
by inducing developmental retard and severe embryo malformations, SWCNTs may
represent a risk for pregnancy, and that such effect can be accurately predicted in vitro by
the EST.
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7.1.24 Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro
technology
Sarah Schneider1, David Schürch1, Marianne Geiser1
1
Institute of Anatomy, University of Bern, Switzerland
Email: [email protected]
Medical literature regularly reports on accidental poisoning in children after accidental
ingestion and subsequent aspiration of lamp oils. These products contain paraffin oil or
petroleum distillates (mixtures of hydrocarbons). Animal experiments have shown that the
toxicity of lamp oils depends on the respective amount of hydrocarbons and on their physicochemical properties such as volatility, viscosity, surface tension and c-chain length. The aim
of this project is to assess and compare toxic effects of hydrocarbons with different c-chain
lengths and of commercial lamp oils on the constituents of the inner lung surface. Thus, we
will explore whether the data obtained from animal experiments are reproducible in an in vitro
model and further elucidate the underlying mechanisms. The studies are performed in view
of the “Globally Harmonized System” (GHS) to be introduced in Europe until 2015 and which
provides new regulations concerning the classification and labeling of hydrocarbons.
Furthermore, the acquired data may prepare the ground for future investigations of
nanomaterials containing organic compounds. In the first project phase, we exposed lung
epithelial cells to alkanes of various chain lengths (C6, C10, C16) at different, though realistic
doses. First experiments were performed with proliferating cells, i.e. the bronchial lung
epithelial cell line BEAS-2B [1]. Cells were cultured to 80% confluence on microporous filter
inserts. Then, the apical cell culture medium was removed to a minimum to establish
air-liquid interface (ALI) condition [2] and the substance was added to the cells for 1 hour.
Cellular responses were assessed at 1 hour and 24 hours after exposure to the alkane.
Biological endpoint measurements include cell viability, cytotoxicity, i.e., release of lactate
dehydrogenase (LDH) as well as release of inflammatory mediators such as interleukin-6
(IL-6), IL-8 and tumor necrosis factor alpha (TNF-α). First results demonstrate that the
toxicity correlates with the dose and is inversely proportional to the chain length. Direct
application of hydrocarbons on cells that lack the apical liquid lining layer increases toxic
effects. Hexane induces an immediate, strong toxicity at 1 hour, whereas with decane a
strong toxicity was observed at 24 hours. To assess effects of hydrocarbons on the
surfactant film, we use the Captive Bubble Surfactometer (CBS), one of the most effective
and well established devices for assessing pulmonary lung surfactant in vitro [3]. So far, we
tested hexane at 2 doses with CUROSURF®, a natural surfactant, prepared from porcine
lungs and therapeutically used to treat Respiratory Distress Syndrome (RDS) or hyaline
membrane disease in newborns. Particular attention has been paid to the rate and extent of
film formation (surface tension vs. time) and the dynamic film behavior during compression
and expansion (surface tension vs. area). The first results demonstrate that hexane - at the
dosages applied - does not influence the rate and extent of film formation, but affects the film
dynamics, i.e. its compression and expansion, in a dose dependent manner. The first results
confirm data from earlier animal experiments and give new insights into the mechanisms
underlying the adverse health effects observed. [Support: BAG]
[1] Savi, M et al. 2008 A novel exposure system for the efficient and controlled deposition of
aerosol particles onto cell cultures. Environ Sci Techn 42: 5667-5674
[2] Geiser, M et al. 2007. In vitro replica of the inner surface of the lungs for the study of
particle-cell interactions. Altex 24:82-84
[3] Schürch, S et al. 1992 Surface properties of rat pulmonary surfactant studied with a
captive bubble method: adsorption, hysteresis, stability. Biochim Biophys Acta 1103:127-136
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7.1.25 Ingested metal nanoparticles pass through intestine epithelia and enter
immune cells and gonads of the sea urchin Paracentrotus lividus
Carla Falugi1, Maria Grazia Aluigi1, Antonietta Gatti2, Alberto Fabrizi2,
Annalisa Pinsino3 and Valeria Matranga3
1
Dipartimento di Biologia, Università di Genova, Italy
Laboratorio dei Biomateriali, Dipartimento di Chirurgie Specialistiche, Università di Modena
e Reggio Emilia, Italy
3
Consiglio Nazionale delle Ricerche, Istituto di Biomedicina e Immunologia Molecolare
“Alberto Monroy”, Palermo, Italy
Email: [email protected]
2
Few data are available for what the effects of nanoparticles exposure in marine organisms
are concerned. In the last decades, the sea urchin has been used for innovative toxicity
tests, aimed at studying the hazard of different contaminants in laboratory conditions. In
order to test the distribution and effects of different metallic nanoparticles (NPs) in
Paracentrotus lividus adult tissues, specimens were exposed in triplicate to 4 kinds of NPs
(SiO2, CeO2, SnO2 and Fe3O4), by feeding the adults with algae containing NPs at
concentration of 10-5 particles/litre. We studied: a) the putative NPs passage to the
circulatory system and uptake by immune cells (coelomocytes); b) the presence of NPs in
gonads and effects on gametes morphology and functionality; c) effects on cellular stress
and pro-apoptotic proteins in coelomocytes. The presence and chemical characterization of
NPs present in tissues were detected by means of FEG- ESEM (Field Emission GunEnvironmental Scanning Electron Microscope) coupled with EDS (Energy Dispersive X-Ray
Spectroscopy). Gonads morphological features were analyzed by histology followed by light
microscopy. The expression of stress proteins was evaluated by WB and immunocytochemistry. Observation showed that NPs were present inside coelomocytes 5 days after
their forced ingestion, indicating the NPs ability to cross the intestine barrier, and to be
phagocitosized by macrophage-like coelomocytes. Defects were detected in the morphology
of gonads structure, mainly at the expenses of the testis cells, suggesting that NPs pass
through the coelomic wall, since gonads are found retroperitoneally in sea urchins as in all
deuterostoms. Effects on the expression of stress proteins as biomarkers of metal NPs
exposure were found according to their chemical nature in whole coelomocytes populations.
Results obtained in this preliminary study indicate that NPs are taken up by the digestive
system, transferred to the coelomic fluid and then uptaken by coelomocytes and/or gonads.
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7.1.26 Critical exposure to ultrafine particles during highway maintenance work
Reto Meier1, Wayne Cascio2, Michael Riediker1
1
Institute for Work and Health, Lausanne, Switzerland
2
East Carolina University, Greenville NC, USA
Email: [email protected]
Traffic-related emissions are associated with increased cardiovascular and pulmonary
morbidity and mortality. Highway maintenance workers spend up to eight hours per day
exposed to traffic emissions. The aims of our current project are to provide a better
understanding of the workers’ exposure to traffic stressors, particularly inhaled particles and
noise, and to assess their cardiovascular, pulmonary, and pro-inflammatory health effects.
We have a particular interest in the exposure to ultrafine particles as they have been
associated with increased pro-inflammatory and pro-thrombotic biomarkers, as well as
altered heart rhythm. These associations differ for particles from different sources such as
combustion, brake and road surface wear [1]. To quantify the workers’ exposure we use a
panel study design with repeated measurements to observe 50 road maintenance workers
over 5 non-consecutive working days. Measurements are ongoing.
Preliminary data shows that exposure to ultrafine particles is highly variable depending on
work site, work activity and work shift. Mean daily particle counts range from 20’000 to
200’000 particles per cm3. Transient peaks averaged over 15 minutes can reach more than
1’000’000 particles per cm3. This broad gradient of exposures offers an excellent opportunity
to establish dose-dependent effects of the particles generated and re-suspended on the
roadway.
[1] Riediker et al. 2004. Cardiovascular effects in patrol officers are associated with fine
particulate matter from brake wear and engine emissions. Particle and Fibre Toxicology
2004, 1:2
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7.1.27 Development of polysaccharide-based nanocarriers for drug delivery
applications
M. Borgogna, B. Bellich, and A .Cesàro
Department of Life Science, University of Trieste, Trieste (Italy)
Email: [email protected]; bbellich@units.it
Polysaccharide-based matrices play a fundamental role in nature, providing support and
protection to biological materials. Hence, the intrinsic biocompatibility and the capability to
create 3D-networks make such materials appealing candidates for technological
applications. Nanostructures (nanoparticles, nanocapsules, nanogels) based on
polysaccharidic matrices represent versatile carriers for drug targeting and delivery systems,
and are characterized by the possibility to tailor the polymer-based structure, controlling the
resulting properties. Such control can be achieved by engineering the polysaccharide
molecules and gels [1]. Moreover, polysaccharides and other biopolymers are widely
exploited in nanotechnology and nanomedicine fields also for the preparation of hybrid
systems which increase the biocompatibility and the stabilization of nanostructures of
inorganic origin (such as magnetic nanoparticles, carbon nanotubes and quantum dot
nanocolloids). Such polysaccharidic matrices are designed for imaging and therapeutic
applications, and have an embedding and targeting function for very complex and structured
systems. They contain several entities, also of very different nature, such as drugs, functional
polymers and inorganic nanostructures. It is mandatory the possibility to control and tune the
functional properties of such systems, in terms of stability, loading and release properties [2,
3]. In this work two functionally different polysaccharides (alginate and chitosan) have been
selected for a pilot study for the preparation of multicomponent systems. Alginates are a
family of polysaccharides of algal or bacterial origin. Their application ranges from food
additives to pharmaceutical formulations or cell immobilization matrices. Alginate based
nano-carriers loaded with model and therapeutic protein have been prepared and
characterized in terms of drug loading and release. The influence of molecular and physicochemical parameters (such as polymer composition, molecular weight and gelling conditions)
has been evaluated. Chitosan is obtained from the deacetylation of the chitin, a structural
component of the exoskeleton of crustaceans and insects. Its mucoadhesive capability
allows the immobilization of the carrier on specific sites for targeted release and optimal drug
delivery. The mucoadhesive behaviour can be also enhanced or tuned by functionalizing the
polymer backbone with signal molecules able to interact with the mucus structure. A study on
chitosan-based nanoparticles prepared by ionotropic gelation has been carried out. The
effect of several parameters on the final performances has been determined. Loading and
release profile of model protein have been determined in correlation with polymer features:
degree of deacetylation and substitution, interaction with other polymer and formation of
polyelectrolyte complexes (PEC).
[1] Liu, Z et al. 2008. Polysaccharides-based nanoparticles as drug delivery systems.
Advanced Drug Delivery Reviews 60: 1650-1662
[2] Moros, M et al. 2010. Engineering biofunctional magnetic nanoparticles for
biotechnological applications.
Nanoscale 2: 1746-1755
[3] Farokhzad, O C et al. 2009. Impact of nanotechnology on drug delivery.
ACS Nano 3 (1): 16-20.
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7.1.28 NanoImpactNet’s Stakeholder Engagement
Michael Riediker1, Nathalie Boschung1, Darren Hart1 and the NanoImpactNet Team2
1
Institute for Work and Health, University of Lausanne and Geneva, Lausanne, Switzerland
2
NanoImpactNet: 24 partner institutes and >300 associated members
Email: info@nanoimpactnet.eu
NanoImpactNet (NIN) is the European network on the health and environmental impact of
manufactured nanomaterials (MNMs). Functioning primarily as a coordination action and a
multidisciplinary platform for exchanging research ideas, NIN shares outputs with
stakeholders from academia, industry, professional associations, legislators, regulators, and
civil society across Europe and beyond. NIN identifies stakeholders' interests and needs to
improve this communication.
NIN has organised a series of workshops to discuss who needs or wants to know what, and
how this can be facilitated, with the ultimate goal of a healthy future in a world with MNMs.
Scientific cooperation and dialogue between researchers and other stakeholders were the
staring points for these three meetings on:
1.
2.
3.
Defining knowledge gaps in current research on MNM characterisation for use in life
cycle assessments, as well as identifying MNM behaviour in the environment.
‘How to make industrial data available’ - strategies for sharing potentially sensitive
proprietary (or negative) data and for allowing the comparison of protocols.
‘How to inform the public about nano-enhanced food contact materials’ – a sensitive and
potentially contentious debate will ensue if legislation fails to encourage communication.
All stakeholders agree that much more scientific data must be generated and shared, notably
on: potential toxic and safety hazards of MNMs throughout their lifecycles; fate and
persistence of MNMs in humans, animals and the environment and thus risks associated
with MNM exposure, for which researchers and workers are in the front line. Also highlighted
was the need for: nomenclature, standards, methodologies, benchmarks and protocols;
development of best practice guidelines; voluntary schemes on responsibility; and databases
of MNMs, research topics and themes.
Broadly speaking, NIN’s stakeholder sessions have shown: that regulatory agencies are
confident in Europe’s monitoring, control, expertise and legislation, whether for chemicals,
pharmaceuticals or food; that industries using or producing MNMs are positive that they have
the know-how to deal with MNMs because they see them as chemical, pharmaceutical or
biological problems - they do not wish to take undue risks with MNMs; and that consumers
will probably embrace nanotechnologies which improve their lives, as long as communication
on risks is transparent and from trustworthy sources.
Our workshops have shown that NIN researchers and other stakeholders share very similar
knowledge needs, and that open communication and free movement of knowledge are
wanted by and will benefit all parties. We encourage all organisations with a stake in the
possible health and environmental impacts that nanotechnologies may have to be active
members of NIN, to ensure safe and responsible development, production, use and disposal
of MNMs.
Funding: NanoImpactNet is a Coordination Action funded by the European Commission's 7th
Framework Programme (GA218539).
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7.1.29 Median lethal dose of titanium dioxide and oleic acid coated magnetite
nanoparticles after single intravenous injection to adult rats
Katarina Volkovova1, Milan Beno1, Mária Dusinska1,2
1
Slovak Medical University,Bratislava, Slovakia
Norwegian Institute for Air Research, Oslo, Norway
Email: katarina.volkovova@szu.sk
2
Median lethal dose (LD50) of a toxic substance is the dose required to kill half the members
of a tested population after a specified test duration. It is frequently used as a general
indicator of a substance´s acute toxicity. The objective of this study was to define the LD50
for TiO2 and oleic acid coated Fe3O4 nanoparticles after single i.v. injection to adult rats.
Results of this study can then be used as basic information to calculate the doses for further
in vivo experiments.
Female outbred Wistar rats (age 8 weeks, weight 205.5 ± 8.5 g) from Prague were used for
the experiment. I.v. injection was performed under xylazin anaesthetization. The study was
performed according to OECD guidelines 425 [1]: The main test consists of a single ordered
dose progression in which animals are dosed, one at a time, at a minimum of 48-hour
intervals. The first animal receives a dose a step below the level of the best estimate of the
LD50. If the animal survives, the dose for the next animal is increased by 3.2 times the
original dose; if it dies, the dose for the next animal is decreased by a similar dose
progression. Each animal has to be observed carefully for up to 48 hours before making a
decision on whether and how much to dose the next animal. A combination of stopping
criteria is used to keep the number of animals low. Dosing is stopped when one of these
criteria is satisfied at which time an estimate of the LD50 and a confidence interval are
calculated using the method of maximum likelihood for the test based on the status of all the
animals at termination (software AOT425 statpgm). We used TiO2 nanoparticles for the
study, obtained from Joint Research Center (Ispra, Italy). TiO2 was suspended in
physiological solution containing 10 volume % of rat serum (Sigma) and sonicated (Dynatech
Artek 300) for 15 min. in a tube with diameter of 9 mm, at 150 W. The size distribution was
bimodal, with peaks at 84 (±8) and 213 (±15) nm. pH was 7.5. Coated Fe3O4 nanoparticles in
concentration of 7 volume %, stabilised with oleic acid were obtained from Plasmachem
(Berlin). After heating to 38°C, the planned volume was pipetted and diluted with
physiological solution. The sample was homogenised by shaking. The size distribution for
coated Fe3O4 nanoparticles was also bimodal, with peaks at 31 (±4) and 122 (±3) nm. pH
was 6.0. Analyses of size distribution and pH of both types of nanoparticles were done in
University of Venice (by equipment NICOMP 370).
After single intravenous injection to adult rats the LD50 for titanium dioxide nanoparticles was
established to be 59.22 mg/kg with confidential interval from 55 to 70 mg/kg. For oleic acid
coated magnetite nanoparticles the LD was 36.42 mg/kg mg/kg with confidential interval (0 20 000 mg/kg). The experiment was finished by the software, when at dose 44 mg/kg one
animal survived and two died; and at dose 35 mg/kg one animal survived and one died.
Experimental modelling showed that continuing the study with additional animals would not
improve the statistics significantly.
[1] OECD (2000) Guidance Document on Acute Oral Toxicity. Environmental Health and
Safety Monograph Series on Testing and Assessment No 24.
This study was supported by NanoTest, EC contract No HEALTH-2007-201335.
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7.1.30 Novel Hydrophilic Ce(III)-Doped Maghemite (γ-Fe2O3) Nanoparticles Preliminary Toxicity Studies in Relation to the Nanoparticle Aggregation
Level
Jean-Paul Lellouche
Department of Chemistry, Nanomaterial Research Center, Institute of Nanotechnology &
Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel
Email: lellouj@biu.ac.il
Iron oxide-based nanoparticles (NPs) and/or nanocomposites have found quite diverse and
numerous magnetism-driven (bio)applications (cell separation, drug/gene-delivery(magnetic
drug targeting/gene therapy), non-invasive magnetic resonance imaging (MRI) of tissues,
and fluid hyperthermia for cancer therapy. Our recent work in the field led to the discovery
and successful implementation of a novel method/concept for the aggregation control of
hydrophilic magnetically responsive maghemite (γ-Fe2O3) NPs.
Quite remarkably and in contrast to any process described till now, this novel method does
not make use of any passivating organic species such as surface-interacting polymers or
ligands. Indeed, we demonstrated that the high-power ultrasound-assisted Ce3+ cation
doping of the surface of Massart pre-formed 10/15 nm-sized maghemite NPs strongly
modified their surface charge to highly positive values. This Ce3+ cation-doping process
enabled (i) a full charge-control of particle aggregation due to charge repulsive effects, as
well as (ii) their water-compatibility for biological applications. Due to its potential important
use in nanomedicine, this novel nanosized magnetic support was tested for cell toxicity at 24
and 48h using three different types of cell lines, i.e., HeLa, HEK 293, and MEF 3T3 (MTT
assay). Importantly, the MTT cytotoxicity test was validated regarding the potential
problematic bias introduced by NP aggregation during incubation. Cell incubation of Ce3+doped maghemite NPs in a complex physiological media such as a Fetal Calf Serumsupplemented Dulbecco Minimum Essential Medium resulted in a more or less pronounced
NP aggregation/NP sedimentation as expected from any nanosized particulate material. It
afforded NP aggregates in a 90-120 nm size range that remained below the critical 200 nm
values that would have precluded cell uptake. This aggregation phenomenon has been
tracked using Dynamic Light Scattering (DLS) & ζ potential measurements. Ce3+-doped
maghemite NPs have been found highly biocompatible for further in vivo uses (MRI,
drug/gene delivery) at concentrations as high as 1.0 g/L. Such combined cell toxicity-NP
aggregation studies will constitute the screening basis of innovative nanocarriers to be
developed for the soon-to-start FP VIIth large-scale collaborative European project SaveMe1.
In addition, Ce3+-doped maghemite NPs also have been incubated with T. brucei parasites
for cell penetration.2 TEM with compositional EDAX analysis demonstrated a preferred NP
incorporation into trypanosome acidocalcisomes (NP tropism) without any observable toxicity
(cell morphology modifications).
(1) NMP.2010.4.0-1: Development of nanotechnology-based systems for detection,
diagnosis and therapy for cancer
(2) These studies have been conducted in collaboration with Prof. Shulamit Michaeli and
Dror Eliaz (MSc), Faculty of Life Sciences, Bar-Ilan University
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7.1.31 Development of Novel Nanotechnology Based Diagnostic Systems for
Rheumatoid Arthritis and Osteoarthritis (NanoDiaRA)
Presented on behalf of the NanoDiaRA Consortium by
M. Hofmann-Amtenbrink, Scientific Coordination of NanoDiaRA
Although effective therapy of rheumatoid arthritis (RA) has improved considerably in recent
years, there is still no disease modifying treatment for osteoarthritis (OA). For treatments to
be effective it is considered extremely important to detect and treat these diseases early and
then be able to monitor treatment efficacy in days and months rather than years.
The project NanoDiaRA - Development of novel nanotechnology based diagnostic systems
for Rheumatoid Arthritis and Osteoarthritis-, funded by the European Commission under the
FP 7 Programme, aims to develop nanotechnology-based diagnostic tools for easy and early
detection of disease onset, progression and responses to therapy.
The poster provides an overview of the technology whereby modified superparamagnetic
nanoparticles are functionalized among others with special biomarkers to be used for in vitro
diagnostics, such as bioassays of biomarkers in body fluids, or as contrast agents in vivo in
combination with magnetic resonance imaging (MRI). The clinical partners strongly support
the project with different patient cohorts and MR imaging.
In parallel to these research activities the project aims to establish new standards that
address the social, ethical and legal aspects of the use of nanoparticles for medical
purposes. Young investigators involved in this study are learning about the complex
mutidisciplinary nanomedical approach that combines the natural and social sciences with
materials science and engineering. Fifteen partners from industry and academia are involved
in this large scale project, and their involvement will be described.
Project funded by FP7 under the Project No: NMP4-LA-2009-228929
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7.1.32 Biological responses induced in bronchial epithelial cells by carbon
black and titanium dioxide nanoparticles: similar outcomes but distinct
molecular pathways
Salik Hussain1, Stéphanie Val1, Armelle Baeza1, Karine Andreau1, Francelyne Marano1,
Sonja Boland1
1
Laboratory of Functional and Adaptative Biology (BFA), unit of Réponses Moléculaires et
Cellulaires aux Xénobiotiques (RMCX), University Paris Diderot-Paris7, Paris, France
Email: boland@univ-paris-diderot.fr
Although there is rapid transfer of technical research to consumer products in the sector of
nanotechnology the understanding of occupational and environmental health effects of the
nanoparticles (NPs) is still a neglected field.
We studied pro-inflammatory, oxidative and cytotoxic effects induced by well characterized
carbon black (CB) and titanium dioxide (TiO2) NPs of different sizes in human bronchial
epithelial cells (primary cells and 16HBE14o- cell line).
Both types of NPs induce pro-inflammatory (dose dependent mRNA expression and
secretion of GM-CSF, IL-6, TNF-α) and oxidative stress responses (mRNA expression of
HO-1 and O2•− production). These responses are size dependent and are highly correlated
with surface area. Moreover, the NP induced pro-inflammatory response is oxidative stress
dependent. These effects need to be carefully studied due to the adsorption of cytokines on
NPs. Utilization of surfactants (tween20/NP40) and serum albumin increases the recovery of
cytokines but modifies the biological responses to NPs.
We demonstrated that both CB and TiO2 induce cytotoxicity in bronchial epithelial cells in a
size and dose dependent manner through apoptosis induction. Cells exhibit decrease in cell
size, peripheral chromatin condensation, caspase activation and DNA fragmentation. A
decrease in mitochondrial membrane potential, activation of Bax and release of cytochrome
c from mitochondria were only observed in case of CB NPs whereas lysosomal membrane
destabilization, lipid peroxidation and release of cathepsin B was only observed for TiO2 NPs.
Furthermore, reactive oxygen species (ROS) production was observed after exposure to CB
and TiO2 but H2O2 was only implicated in apoptosis induction by CB NPs. Thus, although the
final outcome might be the same (apoptosis), the molecular pathways activated by NPs differ
depending upon the chemical nature of the NPs. CB NPs induce apoptosis by a ROS
dependent mitochondrial pathway whereas TiO2 NPs undergo cell death through lipid
peroxidation and lysosomal membrane destabilization.
In conclusion, our results demonstrate the significance of surface area and reactivity in
cellular responses and emphasize the need of studying molecular pathways induced by NPs
rather than only monitoring the final outcome after exposure.
Supported by the National grant ANR 0599-5 SET024-01; FP7 N° 201335 (NanoTest); FP7
N°228789 (ENPRA)
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7.1.33 Toxicology of iron oxide nanoparticles: impact of the size and surface
modifications.
P.Hugounenq1, R. Bazzi1, S. Boland2, A. Baeza2, V. Cabuil1
1
Laboratoire de Physicochimie des Electrolytes, Colloïdes et Sciences Analytiques, Paris,
France
2
Laboratoire des Réponses moléculaires et Cellulaires aux Xénobiotiques, Paris, France
Email: Pierre.Hugounenq@upmc.fr
The possible danger of nanoparticles is currently in the core of a public debate, and the
potential risk that nanoparticles could induce on the people and the environment is a key
society challenge. A regulation of type REACH is for the moment extremely difficult to apply
to nanoparticles, since the relation between exposure, dose and toxicity is almost impossible
to establish.
In this study, we focus on one type of nanoparticles, with varying size and grafted molecules,
to determine the relation between surface characteristics and toxicity. Maghemite
nanoparticles have been synthesized via the coprecipitation method or a polyol process,
leading to controlled size ranging from 6nm to 12nm. Various grafting on these iron oxide
nanoparticles have been carried out to tune their surface charge and stability in cell culture
medium. Grafting molecules such as citrates, dimercaptosuccinic acid (DMSA), dopamine
and 3,4-Dihydroxyhydrocinnamic acid (dopamine-like molecule with a terminal carboxylic
group) have been used. Since aggregation state potentially has a significant influence on the
toxicity, dynamic light scattering (DLS) measurements have been made to control the size of
nanoparticles or aggregates in cell culture medium at different incubation times. The surface
charge of the nanoparticles was estimated via zeta potential measurements in water.
We tested the cytotoxicity of these nanoparticles on A549 cells (adenocarcimonic human
alveolar basal epithelial cells) using WST-1 assays. Incubation time of cells with various
concentrations of nanoparticles has been set to 24 hours for this study. Results of these tests
show a potentially strong influence of surface modification on the cytotoxicity of iron oxide
nanoparticles of the same size (see fig.1). Nanoparticles with a negative surface charge tend
to be less toxic than the ones positively charged. This may be due to the interactions
between the coating and the membrane lipids of the cells.
Aggregation state of the nanoparticles has also an influence on their toxicity. Aggregated
particles tend to be more toxic than well dispersed ones. Aggregated nanoparticles tend to
settle in the culture well, leading to an increase of nanoparticle-cells contact that could
explain their higher toxicity.
100
viability %
80
60
40
20
0
a
b
c
d
e
Fig 2. WST assay value after 24 hours incubation with nanoparticles coated by different molecules: a. non grafted
nanoparticles, b. grafted with citrates, c. grafted with DMSA, d. grafted with 3,4-Dihydroxyhydrocinnamic acid, e.
Grafted with dopamine. The concentration of nanoparticles in the cells medium was set to 100µg/cm² in iron.
Control viability was set to 100%.
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7.1.34 In vitro Assessment of the Cellular Toxicity of Nanotubes
Lenke Horváth1,2, Arnaud Magrez1, Beat Schwaller2, László Forró1
1
Laboratory of Physics of Complex Matter, Ecole Polytechnique Fédérale de Lausanne,
Lausanne, Switzerland
2
Department of Medicine, Unit of Anatomy, University of Fribourg, Fribourg, Switzerland
Email: lenke.horvath@epfl.ch
The major contribution of nanotechnology to our life is the controlled synthesis of a large
variety of nanofilaments (nanowires and nanotubes) which could be the basis of future
devices. Although the expectations are large concerning the improvement of our everyday
life thanks to nanostructures (sensors, vectors for therapies, photovoltaic devices, fast
integrated circuits etc.), there is a growing fear related to their possible health hazards,
strongly reminiscent to those of asbestos. [1] Similar to carbon nanotubes (CNTs), boron
nitride nanotubes (BNNTs) as well as TiO2 nanotubes are among the most promising tubular
nanomaterials. Due to their structure, their size and their exceptional properties, nanotubes
possess a large number of advantages in terms of miniaturization and performances over
existing devices. These lead the commercial production of nanotubes to be scaled up and
non voluntary human exposure is very likely to increase dramatically.
Here, the in vitro toxicity of nanotubes made of carbon, [2] TiO2, [3] or BN [4] assessed by cell
proliferation and modification of their metabolism and their morphology, demonstrates
already at low concentrations an acute toxic action of nanotubes for all cell types studied
(including kidney and lung cells). The level of toxicity and the prominent morphological
alterations in the cell populations withstanding nanotube exposure are cell type dependent.
The presence of structural defects or putatively toxic functional groups on the nanotubes is
shown to enhance their toxicity. Finally, straight nanotubes with limited entanglement and
transversing easily the cell membrane exhibit higher toxic action. Our results point the
cellular accumulation to determine the toxic action of nanotubes as well as the sensitivity of
the cells tested.
[1] Jaurand, MC et al. 2009. Mesothelioma: Do asbestos and carbon nanotubes pose the
same health risk? Particle and Fibre Toxicology 6:16
[2] Magrez, A et al. 2006. Cellular Toxicity of Carbon-Based Nanomaterials. Nano Letters
6:1121-1125
[3] Magrez, A et al. 2009. Cellular Toxicity of TiO2-Based Nanofilaments. ACS Nano 3:227480
[4] Horváth, L et al. 2011. In vitro Investigation of the Cellular Toxicity of Boron Nitride
Nanotubes ACS Nano Submitted
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7.1.35 An in vitro integrated ultrasensitive approach to biocompatibility
analysis of silver nanowires
Jennifer Conroy1*, Navin K. Verma1,2*, Shane Tormey1, Philip Lyons2, Jonathan Coleman2,
Mary O’Sullivan1, Hardy Kornfeld3, Dermot Kelleher1, and Yuri Volkov1,2
1
Institute of Molecular Medicine, Trinity College Dublin, Ireland
Centre for Research on Adaptive Nanostructures & Nanodevices, Trinity College Dublin,
Ireland
3
University of Massachusetts Medical School, Worcester, Massachusetts
2
*
The global attraction of exploiting an array of nanomaterials and their engineered forms
including silver nanowires/nanorods due to their unique physicochemical properties has
generated numerous industrial and biomedical applications. The possibility of human
exposure during manufacture, use, and disposal of these nonmaterials has lead to major
concerns regarding their potential impact on human health. While an increasing number of
literature is reporting toxicity, interactions, biodistribution and bioactivity of nanoscale or
ultrafine particles in biological systems, there remains considerable uncertainty regarding the
approaches by which they are evaluated. This signifies the need for a series of standardized
in vitro tests and protocols to screen, understand, predict and manage potential toxicity of
these unique particles in biological systems for risk assessment. Here, utilizing real-time
impedance sensing technique in combination with an automated image acquisition and
analysis system, we have developed an ultrasentitive, high-content toxicity detection assay
platform suitable for high throughput screening of nanomaterials. Suitability of this screening
method was validated for silver nanowires of various lengths (3-6 µm) using four different
cultured human cell lines including epithelial cells A549, endothelial cells HUVEC, gastric
cells AGS, and phagocytic cells THP1 exposed to a range of concentrations (0.1-5 µg/ml) for
up to 96h. We observed a low level cytotoxic response that was dependent on cell types,
nanowire lengths, doses and incubation times. We demonstrate an in vitro, automated,
simple, sensitive and high throughput screening perspective for the biocompatibility
assessment of silver nanowires that could be applied for various nanomaterials.
A549 epithelial cells
48h
72h
Minimum
24h
Cell viability
0.2 µg/ml
1 µg/ml
2 µg/ml
5 µg/ml
Control
IPA
AgNW 1
AgNW 2
AgNW 3
AgNW 4
AgNW 5
AgNW 6
IPA
AgNW 1
AgNW 2
AgNW 3
AgNW 4
AgNW 5
AgNW 6
IPA
AgNW 1
AgNW 2
AgNW 3
AgNW 4
AgNW 5
AgNW 6
IPA
AgNW 1
AgNW 2
AgNW 3
AgNW 4
AgNW 5
AgNW 6
IPA
AgNW 1
AgNW 2
AgNW 3
AgNW 4
AgNW 5
AgNW 6
NiNW
Maximum
B
0.1 µg/ml
A
4h
Figure 1: (A) Confocal and bright field images illustrating internalisation of silver nanowires in a phagocytic cell
(B) A heatmap showing cytotoxicity of various length silver nanowires (Largest ~6 µm AgNW6 to the smallest
~3µm AgNW1) in a human epithelial cell line A549.
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7.1.36 Magnetic carbon nanotubes: a new tool for shepherding mesenchymal
stem cells by magnetic fields
Orazio Vittorio1,2, Vittoria Raffa1, Paola Quaranta2, Niccola Funel2, Daniela Campani2, Serena
Pelliccioni2, Biancamaria Longoni2, Franco Mosca2, Andrea Pietrabissa3 and Alfred
Cuschieri1
1
Medical Science lab, Scuola Superiore Sant’Anna, Pisa, Italy
Department of Oncology, Transplantation and Advanced Technologies in Medicine,
University of Pisa, Italy.
3
General Surgery, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Italy
Email: v.raffa@sssup.it
2
Here we investigated the interaction between magnetic carbon nanotubes (CNTs) and
mesenchymal stem cells (MSCs) and their ability to guide these cells injected in vivo by
using an external magnetic field [1]. MSCs were cultured in a CNT-containing medium. We
confirmed that CNTs and magnetic field do not alter cell viability, proliferation rate, cell
phenotype, cytoskeletal conformation and their ability to differentiate. We demonstrated that
MSCs labelled with CNTs can move towards the magnetic source in vitro. Finally we
investigated in vivo the possibility to guide MSCs labelled with CNTs into a target organ
(liver). One million of magnetized cells were injected in the hepatic portal vein of rats. We
demonstrated that the application of a proper magnetic field changes the MSC
biodistribution. Specifically, the histochemical studies revealed an increment of CNT labelled
MSC accumulation in the liver of the animals exposed to the magnetic field, with no evidence
of either inflammation or neoplastic proliferation.
This could pay the way for the development of new strategies for manipulation/guidance of
MSCs in regenerative medicine and cell transplantation.
Figure 1: MSC biodistribution. Mean and S.E.M. values of positive cells for Perls staining in each type of organ.
[1] Vittorio, A et al. in press. Nanomedicine UK
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7.1.37 CNT-mediated wireless cell permeabilisation: drug and gene uptake
Vittoria Raffa1, Lisa Gherardini2, Orazio Vittorio1, Giuseppe Bardi2, Afshin Ziaei3, Tommaso
Pizzorusso2, Cristina Riggio1, Stephanos Nitodas4, Theodoros Karachalios4, Khuloud AlJamal5, Mario Costa2, and Alfred Cuschieri1
1
Medical Science Lab, Scuola Superiore Sant’Anna, Pisa, Italy
2
Neuroscience Institute, CNR, Pisa, Italy
3
Thales Research & Technology France, Palaiseau cedex, France
4
Nanothinx S.A. Rio-Patras, Greece
5
The School of Pharmacy, University of London, London, United Kingdom
Email: v.raffa@sssup.it
The remit of nanomedicine is the utilisation of biocompatible nanomaterials with tailored
properties designed for targeted delivery and controlled release of drugs or to induce cell
stimulation in-vivo. The exploitation of chemical and physical properties of carbon nanotubes
(CNTs) constitutes one option to achieve this therapeutic goal.
CNTs can be engineered and integrated into biological systems as sensors [1], scaffolds [2],
or employed for intracellular delivery of therapeutics [3]. Here we describe for the first time by a theoretical and experimental approach - the use of the “antenna” properties of multiwalled carbon nanotubes (MWCNTs) for wireless cell permeabilisation by microwave energy
in-vitro and in-vivo.
We performed preliminary experiments in living eukaryotic NIH-3T3 cell line. We tested the
uptake of doxorubicin, a cytotoxic agent. The experimental data on doxorubicin uptake by
NIH 3T3 cells clearly indicated that the application of the EMF enhances drug uptake, and
the presence of the CNTs amplifies considerably the effect of the EMF. More importantly, we
observed that the presence of CNTs induces a strong increase of nuclear localization of the
drug which is a crucial consideration for the cytotoxic action of drugs like doxorubicin which
interact directly with the nuclear DNA.
Figure1. Doxorubicin is fluorescent and can be easily monitored via fluorescent microscopy (red field). a) cells
exposed to CNT+EMF and b) cells exposed only to EMF: in a) doxorubicin accumulation is preferentially nuclear
as opposite to b) where the drug concentration was found to be uniform in the cell.
These observations lead to in-vivo experiments on mice. Plasmid DNA alone or conjugated
with CNTs, was injected stereotactically into the primary motor cortex of mice. In view of
future possible applications in human CNS disease (e.g. cerebrovascular stroke), we used
Bcl-2, an anti-apoptotic gene. The data obtained in-vivo confirmed that CNTs possess
antenna-like properties that can be used to transfer genetic material in cells when exposed to
EMF in the microwave range.
This wireless application has the potential for CNT based electro- stimulation therapies and
targeted intracellular drug delivery.
[1] Chun, A L Nature Nanotechnology doi:10.1038/nnano.2009.291
[2] Edwards, S L et al. 2009. Expert Review of Medical Devices 6: 499-505.
[3] Lacerda, L et al. 2007. Nano Today 2: 38-43.
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7.1.38 Quantification of risk assessment in nanosafety: Determination of “runoff” effect of metallic nanoparticles in simulated body fluids
Maros Halama1, Andrea Fedorkova2, Dasa Halamova2, Vladimir Zelenak2
1
Technical University of Kosice, Kosice, Slovakia
2
P.J. Safarik University, Kosice, Slovakia
Email: maros.halama@tuke.sk
The corrosion properties of superparamagnetic Fe nanoparticles (SPIONs) made by our own
patent [1], commercially available ZnO NPs, surface treated NPs by 2%
carboxymethylcellulose (CMC) as carrier liquid and of Fe NPs doped mesoporous materials
were studied by using ultra-fast linear voltammetry which allow doing estimation of
degradation properties under conditions of simulated bioenvironments, esp. physiological
solution and simulated body fluids (SBFs) via measurements of polarization curves of NPs
attached on the surface of carbon paste electrode (CPE).
The aim of study is to assess life-time of pure and surface treated nanoparticles and thus
indirectly assess contribution of the part of drug delivery system on interaction with
application environment. This technique could achieve fruitful information for risk assessment
of NPs before their wide application such as potentially drug delivery carriers, as additives in
cosmetic industry etc. Developing new technique in this strong interdisciplinary topic
(nanotechnology, chemistry & corrosion and medicine) could lead to achieve more precise
information related to degradation of NPs, their life-time and interaction with chosen
environment. This is important step to guarantee more safety in nanotechnology and thus
prevent public against “black swan”.
Fig. 1 Degradation as final stage of NPs in biomedical and environmental application
Supported by the Scientific Grant Agenture of Ministry for Education, Science, Research and
Sport of Slovak Republic (VEGA, Grant No. 1/0324/10).
[1] Milkovic, O.- Halama, M. – Niznik, Š. – Longauer, S.: patent SK n0 286895
Vestník ÚPV B. Bystrica (2009)
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Author Index
8 Author Index
Achiraman, 23, 101
Ahuja, 9, 12
Aitken, 14
Alam, 9
Al-Bairuty, 66
Ali, 9, 12
Ali-Boucetta, 44
Al-Jamal, 44, 117
Al-Jubory, 70
Aluigi, 105
Andreau, 112
Angeloni, 51
Anguissola, 68, 94
Arlinghaus, 99
Audinot, 102
Baboota, 9, 12
Baeza, 17, 112, 113
Balachandran, 102
Baltensperger, 29
Barancokova, 38
Bardi, 117
Baun, 83, 87
Bazzi, 113
Bellich, 107
Beno, 109
Berahnu, 95
Bergamaschi, 90, 95, 103
Berges, 81
Betti, 65
Bhattacharya, 30, 57
Bianco, 44
Bilanicova, 26, 48
Bilaničová, 21
Birkedal, 76, 78
Boisen, 78
Boland, 16, 17, 112, 113
Bonelli, 90
Bonnier, 100
Boraschi, 28
Borgogna, 107
Borot, 16, 17
Boschung, 108
Bourdel, 89
Bouwmeester, 18, 54, 93
Boyd, 62
Brandenberger, 47
Brennan-Fournet, 62
Bross, 80
Brouwer, 14, 20, 81
Brown, 45, 69
Brydson, 67
Bussolati, 90
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Byrne, 10, 30, 44, 46, 49,
57, 58, 100
Byrnes, 62
Cabuil, 113
Cadène, 88, 89
Calzolai, 8
Campagnolo, 103
Campani, 116
Canesi, 65
Canonico, 65
Capitani, 35
Carella, 49
Carey, 57
Carreira, 33
Cartwright, 33
Casado, 58
Casals, 27, 28
Cascio, 106
Casey, 11, 13, 57, 100
Catalán, 15, 43, 50
Ceccone, 49, 63
Cesàro, 107
Chambers, 11, 13, 57, 100
Chapuis-Bernasconi, 40
Cho, 56
Choi, 56
Christensen, 14
Ciacci, 65
Clark, 14
Clift, 47
Coleman, 115
Conroy, 115
Costa, 117
Coullerez, 64
Cristofori, 21
Cronholm, 32, 53
Cunningham, 62
Cuschieri, 116, 117
Daguet, 89
Damme, 59
Davoren, 10
Dawson, 46, 68, 69, 94
De Boever, 27
de Jong, 7, 85
de la Fonteyne, 85
Deslarzes, 22
Di Bucchianico, 95
Dommen, 29
Donaldson, 44
Dorney, 100
Draude, 99
Drlickova, 48
- 119 / 121 -
Duffin, 44
Duschl, 28, 41, 73
Dusinska, 26, 38, 48, 102,
109
Elihn, 32
Elliott, 92
Erden, 71
Erdinger, 59
Etore, 88
Evans, 31
Fabbrizi, 95
Fabricius, 24
Fabrizi, 105
Fadeel, 25, 42, 74
Falck, 15, 50
Falugi, 105
Favre, 51
Fedorkova, 119
Feitshans, 96
Fenoglio, 49, 90
Ferguson, 33
Ferreira-Leite, 82
Fink, 64
Fjellsbø, 48
Flahaut, 36
Fleming, 62
Forró, 114
Foss Hansen, 83, 87
Franchini, 8
Fransman, 81
Fubini, 49, 90
Funel, 116
Gabsch, 59
Gaiser, 34, 86
Galibert, 36
Gallo, 65
Garcia, 10
Garrone, 90
Garry, 94
Gartiser, 59
Gasser, 39
Gatti, 35, 105
Gayathri, 101
Geertsma, 7
Gehr, 39, 52
Geiser, 29, 104
Georgantzopoulou, 102
Gerritsen, 14
Gerritsen-Ebben, 81
Gherardini, 117
Ghiazza, 90
Giazzon, 51
3rd NanoImpactNet Conference
The European Network on the Health and Environmental Impact of Nanomaterials
Gilliland, 8, 55, 63
Giovannini, 72
Giudetti, 55, 63
Gkanis, 20
Glaus, 24
Gonzalez, 41
Graf, 99
Grass, 52
Gremmer, 85
Grieger, 87
Guadagnini, 16, 17
Gubbins, 45
Günther, 24
Gutleb, 102
Haase, 99
Halama, 119
Halamoda Kenzaoui, 40
Halamova, 119
Handy, 66, 70
Hannukainen, 43
Hart, 108
Hellack, 59
Henry, 33
Herzog, 47
Hirsch, 92
Hoffmann, 102
Hofmann, 64
Hofmann-Amtenbrink, 111
Horváth, 114
Hougaard, 78
Housiadas, 20
Hugounenq, 113
Hungerbühler, 24
Hunt, 98
Hussain, 16, 112
Hutchison, 86
Italiani, 28
Jackson, 78
Jacob, 23, 101
Jacobsen, 76
Järventaus, 15, 43, 50
Jensen, 75, 76, 78, 83
Joshi, 62
Juillerat-Jeanneret, 40
Jungnickel, 99
Kaegi, 79
Kalberer, 29
Kamalakkannan, 23, 101
Karachalios, 117
Karlsson, 32, 53
Kazimirova, 38
Keck, 84
Kelleher, 115
Kermanizadeh, 86
Klingeler, 36
Koivisto, 15
Kontturi, 43
Koponen, 75, 76
Kornfeld, 115
Abstract Book
Kostarelos, 44
Krombach, 82
Krug, 39, 92
Kruszewski, 102
Krystek, 7
Kuhlbusch, 59
Kumar, 12
Künzi, 29
Kuricova, 26
Lankveld, 7
Larsen, 78
Ledwith, 62
Lellouche, 110
Li, 44
Liley, 51
Limbach, 52
Lindberg, 15, 50
Linkov, 87
Liskova, 26
Loft, 76
Loinaz, 67
Longoni, 116
Löschner, 78
Luch, 99
Lukanov, 36
Lynch, 45, 46, 68, 69, 94
Lyng, 10, 46, 58
Lyons, 115
Macken, 58
MacNee, 44
Maes, 59
Magdolenova, 48
Magrez, 114
Magrini, 103
Manohar, 7
Mantion, 99
Marano, 16, 17, 112
Marcomini, 21, 48
Mariani, 95
Marucco, 49
Massimiani, 103
Mather, 44
Matranga, 105
Matthey, 51
Megson, 41
Meier, 106
Mertes, 29
Micheletti, 14
Midander, 32, 53
Migliore, 95
Mikkelsen, 76
Mintova, 30
Möller, 19, 32, 53
Møller, 76
Monpoli, 45
,
Montanari , 35
Moreau, 16
Mosca, 116
Mukherjee, 10, 11
- 120 / 121 -
Murphy, 44
Naha, 46
Naydenova, 30
Neigh, 85
Nelissen, 27, 28
Nelson, 67
Neubauerova, 26
Nickel, 59
Nitodas, 117
Norppa, 15, 43, 50
Nunes, 44
Nykäsenoja, 15
Ó Claonadh, 11, 13
O’Brien, 68
O’Neill, 49
O’Sullivan, 115
Olivato, 91
Oomen, 7
Oostingh, 28, 41
Ormategui, 67
Ostiguy, 89
Park, 85
Paul, 33
Pelliccioni, 116
Pesch, 84
Peters, 18, 54, 93
Pietrabissa, 116
Pietroiusti, 103
Pilou, 20
Pinsino, 105
Pizzorusso, 117
Plendl, 99
Pojana, 21, 26, 48, 65
Poland, 44
Ponti, 95
Prato, 44
Prina-Mello, 44
Puntes, 27, 28, 41
Pylkkänen, 15
Quaranta, 116
Raemy, 52
Raffa, 116, 117
Raghnaill, 69
Ramirez, 68, 94
Rayavarapu, 7
Rehberg, 82
Reip, 95
Rennecke, 84
Richter, 84
Riediker, 22, 89, 106, 108
Riggio, 117
Rinna, 48
Rodhe, 19
Roesslein, 92
Rossi, 8, 15, 49, 55, 63, 95
Rothen-Rutishauser, 39, 47,
52
Rotoli, 90
Sabbioni, 91
3rd NanoImpactNet Conference
The European Network on the Health and Environmental Impact of Nanomaterials
Saber, 76, 78
Sakulkhu, 64
Salit, 92
Salvati, 68
Santos, 69
Saunders, 33
Sauvain, 22
Savolainen, 15, 43, 50, 97
Schmid, 14
Schneider, 29, 104
Schürch, 104
Schwaller, 114
Schwieso, 33
Shaw, 66
Siivola, 50
Sirviö, 97
Siva, 23
Sloth, 78
Soula, 36
Stark, 52
Stintz, 59
Stöhr, 41
Stone, 34, 45, 86
Storti, 22
Suhonen, 15, 50
Sukirtha, 23, 101
Sumedha, 101
Abstract Book
Tentschert, 99
Tenuta, 46
Tetley, 31
Thielecke, 28
Thieriet, 88, 89
Thorley, 31
Thuenemann, 99
Tielemans, 81
Tîlmaciu, 36
Tormey, 115
Triolet, 89
Troisfontaines, 89
Tulinska, 26
Turci, 49
Uboldi, 55, 63
Uzu, 20
Vakourov, 67
Val, 112
Valsami-Jones, 95
van der Zande, 54, 93
van Leeuwen, 7
van Loveren, 85
van Tongeren, 14, 20
Vanhala, 43
Verharen, 7
Verma, 115
Vermeulen, 85
- 121 / 121 -
Vernez, 89
Verstraelen, 27
Vibenholt, 78
Vippola, 15, 50
Vittorio, 116, 117
Vogel, 76, 78
Volkov, 115
Volkovova, 26, 109
von Goetz, 24
Vranic, 16, 17
Walczak, 54
Wallin, 76, 78
Wallinder, 32, 53
Wang, 68
Weber, 84
Weigel, 18
Wick, 39, 92
Wijma, 54, 93
Witasp, 42
Witters, 27
Wsolova, 38
Ye, 69
Zanello, 90
Zanzottera, 90
Zelenak, 119
Zhang, 67
Ziaei, 117
3rd NanoImpactNet Conference
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