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RESEARCH
PROJECT IN NANOTOXICOLOGY |
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Manufactured nanomaterials and nanoparticles represent an emerging technological sector with more and more applications. At the present time, the manufactured nanoparticules are mainly involved in industrial processes (silicon wafers polishing, catalysts), but the prospects of applications for the general public are becoming more and more numerous and tangible (microelectronics, coatings, textiles, sport goods, cosmetics, pharmaceutical applications, food industry applications…)
Despite the risk of a spectacular increase of the professional and public exposure to nanomaterials, studies on the implications of the physico-chemical properties of those materials for human toxicology are only at their outset.
The production of new materials at the nanoscale raises several questions over the interaction of these materials with the living world. Indeed, at this scale, the size matters as well as the chemical properties when it comes to describe the properties of the material and its interactions with living cells. On the one hand, the nanoparticules and the nanoscaled materials show a huge surface area / mass ratio (from some dozen to hundreds of m2 / g of material), conferring specific properties to them. On the other hand, the small size of the nanoparticules facilitates their uptake by cells and transcytosis across the epithelial or the endothelial barrier into the blood and lymph circulation where they can reach sensitive organs.
The aim of this project is to contribute to the evaluation of the potential harmful effects of the nanoparticules on human health and, with an information from the in vitro mechanism, to optimize, adapt and/or replace the animal studies by alternative methods in order to respond to regulatory, politic, financial and ethical demands.
Since a few years
our laboratory of biochemical toxicology actively takes part in programs of
development and validation of alternative methods proposed by international
instances in order to reduce the use of laboratory animals in the toxicological
tests set by the 86/609/EU (A.R. 86/1839) guidance and in order to limit animal
suffering. Several in vitro techniques involving diverse cell lines
as an alternative to in vivo assays (kidney toxicity, LD50, general
toxicity, ocular irritation, dermal irritation) were adapted and developed.
It is our interest to refine the experimental conditions of these assays in
order to use them for the assessment of the acute or delayed toxicity of nanoparticules.
For this project we will test the in vitro techniques developed as
alternatives to in vivo testing, we will collect the data on cytotoxicity
and cellular responses after a short and/or a long exposure time to various
nanoparticules and nanomaterials available on the market and susceptible to
interact with living organisms in a form where their nanostructure is biologically
accessible.
Tested Nanoparticles :
The words nanomaterials and nanoparticles cover numerous materials and products with highly different physicochemical properties, but a common point for all these products and materials is that they have at least one dimension of about 100 nm or less (or 0.1 µm, or 100 millionth of a millimeter)
This project will evaluate nanoparticles manufactured at the industrial level and susceptible to come in contact with the human organism.
We present here a list of the materials that will be tested for our project and a non-exhaustive description of their actual or possible applications:
• Nanocrystalline materials (metal oxides and silica nanopowders) :
Titanium dioxide (IV) [TiO2] : applications: UV absorbent: sunscreens, transparent protection films, polymers and textiles; conducting films, photocatalytic surfaces (self-cleaning coatings), roofing, tiles, paint, glass (…)
Copper oxide (II) [CuO] : applications: anti-microbial preservative for food products or wood, textiles, microelectronics, batteries (…)
Aluminum oxide (II) [Al2O3] : applications: improvements of coatings, paper, ink toners, light bulbs and tubes, associated to silica: improvements for powders or liquid preparations (…)
Zinc oxide [ZnO] : applications: UV protection: coatings, textiles and cosmetics; bactericide and fungicide (biostatic), paints, coatings, textiles and care products (adhesive plaster); catalyst (…)
Iron oxide (III)
[Fe2O3] applications: medical applications: NMR for tumors,
as magnetic nanoparticles to target and destroy specific cancer cells, as
vector for some anticancer substances; others: hard drives (data storage),
coatings, plastics, textiles, microelectronics, catalyst (…)
Silica [SiO2] applications: abrasive for silicon wafers polishing,
toothpaste; coatings, joints, cosmetics, films, paint, adhesives…; additive
to enhance the properties of powders or liquid preparations (toners, paints
or with other nanopowders), dielectrics coatings, photovoltaic devices (…)
• Carbon derivatives: nanotubes and fullerenes :
Carbon nanotubes [CNT], applications: they are the most rigid and solid fibers known so far and they also possess unique electrical properties. The mechanical properties make them useful in the automotive and aerospace industry; these are also used to manufacture lightweight and abrasion resistant textiles, lightweight sport goods (skis, bikes…). Their electrical properties (conductor or semi-conductor according to their configuration) are also used in multiple applications: flat screens, scanning probes microscopes, electronics, plastics and coatings with conducting properties, batteries, fuel cells (…)
We will test single wall carbon nanotubes (swCNT), naked or functionalized (with hydrophilic PEG groups or soluble in organic solvents with octadecylamine groups)
Fullerene [C60, C70] (Buckminsterfullerene), applications: enhancement of electrical and/or optical properties of polymers, semi-conductors (…); functionalized to show biological compatible properties (medical and pharmaceutical applications)
We will test
naked fullerenes and functionalized fullerenes (with carboxylic or butyl-carboxylate
groups)
Other materials to be tested :
- composites
and vectors for drug delivery (ea dendrimers)
- silver [Ag] nanopowder: one of the most abundant nanopowder, used as antimicrobial
agent in textiles and other consumers applications
- quantum dots: biotechnology and medical imaging
Cell types used :
Two different cell lines will be used to evaluate the in vitro nanoparticles cytotoxicity assays:
• Hepatocytes (HepG2)
The HepG2 hepatocytes are commonly used in the in vitro pharmaco-toxicity assays. The liver is the main site for biotransformation and defense against the xenobiotics. It is already shown that nanoparticles (PEG coated quantum dots, polystyrene beads, metallo-fullerene) can translocate from the circulatory system to the liver.
• Kidney cells (HK2)
The kidney is the main filtration and elimination organ for toxics present in the blood circulatory system; it is already shown that the kidney excretes some nanoparticles.
In the long term we would like to tests other cell types to validate the results obtained with HepG2 and HK2: primary hepatocytes, Kupffer cells, HEK293, MDKK or LLC-PK1, reticulocytes.
In vitro assays – cell mechanisms tested
• Cell viability, cytotoxicity :
-
CBQCA total protein cytotoxicity test
This assay determines the GI50 (the concentration inducing 50% of growth inhibition) The CBQCA assay assess the survival/viability and is based on the capacity of the cells to incorporate and bind a fluorescent dye (CBQCA) to the protein amines. A toxic chemical, regardless of its site of action, will interfere with this process and results in a reduction of the growth rate as reflected by cell number. This decrease of cell number is highlighted by the decrease of fluorescence of the amine bound CBQCA.
-
NRU delayed cytotoxicity test
The cells are
treated for 24 hours with the tested nanoparticles, then the culture is maintained
for 5 more days in normal growth medium. The cytotoxicity is assessed from
the uptake of neutral red and the results quantified by the NI50del (the concentration
of nanoparticles required to reduce the neutral red uptake [NRU] by 50%) The
NI50del is compared to the NI50 obtained immediately after the 24 hours exposure.
In some cases, for some toxic substances, the acute human toxicity is best
predicted with the delayed cytotoxicity test rather than with the acute cytotoxicity
test.
We will also evaluate the ability of nanoparticles to elicit apoptosis by looking at the activation of caspase-3 with fluorescent substrates.
• The oxidative stress
Oxidative stress is a state of redox disequilibrium in the cell in which ROS (reactive oxygen species) production overwhelms the antioxidant defense capacity of the cell, causing irreversible damages to the cell machinery. Giving their catalytic properties, several nanoparticles are expected to induce oxidative stress. The ROS production can be evaluated with fluorescent molecules as dichlorofluoroscein diacetate (H2-DCFDA), which become fluorescent upon cleavage by intracellular esterase in oxidative conditions.
• Pro-inflammatory response
The measurement
of the pro-inflammatory response of the cell is another way to assess the
stress the cell is undergoing in contact with nanoparticles and the damages
nanoparticles may cause to a living organism. In vivo, unbalanced
pro-inflammatory response may lead to more damage than the event at the origin
of the response. The capacity of nanoparticles to elicit a pro-inflammatory
response will be tested through the secretion or the non-secretion of interleukine
(IL-8) by the cells in contact with nanoparticles in vitro.