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FP7 ERA-NET on
Nanosafety
Safe
Implementation of Innovative
Nanoscience
and Nanotechnology
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Toxicity in vivo
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Lead: Harvard University
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Objectives:
1.
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To evaluate the influence of surface modifications of nano-Ag
and -CeO2 in different aerosol formulations on their toxicological
potential using a mouse PCLS system. |
2.
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To evaluate the influence of surface modifications of nano-Ag
and -CeO2 in different aerosol formulations on their pulmonary toxic
and inflammatory effects in intact mouse animal model. |
3.
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To determine whether surface modifications of aerosolized
nano-CeO2 influence cerium lung clearance and extrapulmonary
biokinetics in a mouse model. |
4.
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To conduct a correlation study between in vitro and in vivo toxicity profiles |
5.
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To assist WP M with recommendations for modeling. |
Methodology:
1.
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Toxicological evaluations of surface-modified nano-Ag and -CeO2
will be performed ex vivo using living mice PCLS-based assays. The PCLS
will be prepared from 8 weeks old C57Bl/6 mice. The collapsed lung
lobes will be infused with 1.5ml of 1.5% agarose in Hanks’ balanced
salt solution (HBSS) through the inserted catheter, followed by 0.5ml
of air to clear the airways of agarose. Once the agarose solidified,
the lungs will be removed from chest cavity and immersed and kept in
cold HBSS. The left lobe will be sectioned into 250µm thick slices
using a vibratome. The lung slices will be incubated in Dulbecco's
Modification of Eagle's Medium/Ham's F-12 50/50 Mix (Corning) at 37°C
overnight before each experiment or freezing for later testing. Lung
slices will be cultured in 96-well plates. The first stage of this aim
will make use of test materials collected on filters during
aerosolization of various formulations (provided from WP E). The test
materials will be added to each well, and at selected timepoints,
several assays will be performed. The data obtained will guide the
experimental design of exposure of PCLS to aerosols in real time. This
next stage will be performed at TU Dresden (WP E). Additionally,
exposed PCLS will also be analyzed. |
2.
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Assessment of in vivo pulmonary effects of surface-modified
nano-Ag and -CeO2 in C57Bl/6 mice using bronchoalveolar lavage and
analyses. This experiment will determine whether and how surface
modifications of nano-Ag and -CeO2 in different aerosol formulations
may modify nano-Ag and and -CeO2 -induced lung toxicity and
inflammation. Groups of mice will be instilled intratracheally with
increasing doses of nano-CeO2 or Ag suspension provided by WP E
(Dresden). At selected timepoints later, mice will be euthanized and
the lungs will be lavaged multiple times. The cells from all washes
will be separated from the supernatant by centrifugation. Total cell
count and hemoglobin measurements will be made from the cell pellets.
Biochemical markers of lung injury (lactate dehydrogenase,
myeloperoxidase, glucosaminidase and albumin) as well as inflammatory
cytokines will be measured. |
3.
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Biokinetic studies will be performed using nano-CeO2 contained
in collected aerosols provided by WP E (Partner 3). Collected nano-CeO2
from the test rig developed in WP E will be used for evaluation of the
cerium biokinetics. Samples from WP E group will be neutron activated
at Massachusetts Institute of Technology (MIT) Nuclear Reactor
Laboratory (Cambridge, MA, USA). MA) with a thermal neutron flux of 5 x
1013 n/cm2/s for 24 hours. The activation process will generate the
radioisotope 141Ce, useful for biokinetic studies. Mice will be used to
determine if different MNM suspension preparations at BfR (described in
WP PC) and aerosolized/characterized at Dresden (WP E) alter the
pharmacokinetics of cerium after pulmonary exposure (intratracheal
instillation). |
4.
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The toxicity profiles obtained within this WP will be correlated with those gained in WP T-a of in vitro testing. |
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This project is part
of the SIINN ERA-NET and is funded under the ERA-NET scheme
of the Seventh Framework Programme of the European Commission,
Research
Directorate - General, Grant Agreement No. 265799
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