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We used well-characterized and positively charged nanoparticles (NP+) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP+ physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP+ surface charge was significantly reduced, proportionally to NP+/serum and NP+/BSA ratios, while NP+ size was not modified. Microscopy studies showed high endocytosis of NP+ in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP+. No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP+ genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP+ might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.