New insight into artifactual phenomena during in vitro toxicity assessment of engineered nanoparticles: Study of TNF-α adsorption on alumina oxide nanoparticle |
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Authors: | Mélanie Pailleux Delphine Boudard Jérémie Pourchez Valérie Forest Philippe Grosseau Michèle Cottier |
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Affiliation: | 1. LINA, EA 4624, F-42023 Saint-Etienne, France;2. Ecole Nationale Supérieure des Mines de Saint-Etienne, SPIN-EMSE, CNRS:UMR5307, LGF, F-42023 Saint-Etienne, France;3. Université de Lyon, F-42023 Saint-Etienne, France;4. Université Jean Monnet, Faculté de Médecine, F-42023 Saint-Etienne, France;5. CHU de Saint-Etienne, F-42055 Saint-Etienne, France;6. Ecole Nationale Supérieure des Mines de Saint-Etienne, CIS-EMSE, LINA EA4624, F-42023 Saint-Etienne, France |
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Abstract: | Biomolecules can be adsorbed on nanoparticles (NPs) and degraded during in vitro toxicity assays. These artifactual phenomena could lead to misinterpretation of biological activity, such as false-negative results. To avoid possible underestimation of cytokine release after contact between NP and cells, we propose a methodology to account for these artifactual phenomena and lead to accurate measurements. We focused on the pro-inflammatory cytokine tumor necrosis factor TNF-α. We studied well-characterized boehmite engineered NP [aluminum oxide hydroxide, AlO(OH)]. The rate of TNF-α degradation and its adsorption (on boehmite and on the walls of wells) were determined in cell-free conditions by adding a known TNF-α concentration (1500 pg/ml) under various experimental conditions. After a 24-h incubation, we quantified that 7 wt.% of the initial TNF-α was degraded over time, 6 wt.% adsorbed on the walls of 96-well plates, and 13 wt.% adsorbed on the boehmite surface. Finally, boehmite NP were incubated with murine macrophages (RAW 264.7 cell line). The release of TNF-α was assessed for boehmite NP and the experimental data were corrected considering the artifactual phenomena, which accounted for about 20–30% of the total. |
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