Standard Cellular Testing Conditions Generate an Exaggerated Nanoparticle Cytotoxicity Profile.

Cellular internalization of nanoparticles (NPs) is key to many biomedical applications and serves as a model to investigate the potential toxicity of NPs on entire organisms. Large discrepancies between in vitro and in vivo nanotoxicity data however exist, suggesting that cellular systems may not be optimal for predictive in vivo toxicology. Here, we use validated multiparametric high-content imaging protocols to evaluate the impact of common cell culture conditions on NP cytotoxicity studies. The data show that high NP to cell ratios, typical for cellular studies, stress the cells by high endocytosis levels that overstimulate mitochondria, resulting in oxidative stress-mediated mitochondrial damage, which induces autophagy. Using proliferation-restricted models, we show that lowering endocytosis levels overcomes most toxicity while resulting in higher final cellular NP numbers. The data suggest that many common NP cytotoxicity mechanisms may partially be an artifact caused by overstimulated endocytosis.