New insight into artifactual phenomena during in vitro toxicity assessment of engineered nanoparticles: Study of TNF-α adsorption on alumina oxide nanoparticle

► TNF-α degradation in cell-free conditions. ► TNF-α adsorption on the walls of the wells in cell-free conditions. ► TNF-α adsorption on boehmite in cell-free conditions. ► Adsorption isotherms of TNF-α. 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 (1500pg/ml) under various experimental conditions. After a 24-h incubation, we quantified that 7wt.% of the initial TNF-α was degraded over time, 6wt.% adsorbed on the walls of 96-well plates, and 13wt.% 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.