Assessing the Cytotoxicity of TiO 2− x Nanoparticles with a Different Ti 3+ (Ti 2+ )/Ti 4+ Ratio

Titanium dioxide (TiO 2 ) nanoparticles are promising biomedical agents characterized by good biocompatibility. In this study, we explored the cytotoxicity of TiO 2− x nanoparticles with a different Ti 3+ (Ti 2+ )/Ti 4+ ratio and analyzed the efficiency of eryptosis indices as a tool in nanotoxicology. Two types of TiO 2− x nanoparticles (NPs) were synthesized by the hydrolysis of titanium alkoxide varying the nitric acid content in the hydrolysis mixture. Transmission electron microscopy (TEM) images show that 1-TiO 2− x and 2-TiO 2− x NPs are 5 nm in size, whereas X-ray photoelectron spectroscopy (XPS) reveals different Ti 3+ (Ti 2+ )/Ti 4+ ratios in the crystal lattices of synthesized NPs. 1-TiO 2− x nanoparticles contained 54% Ti 4+ , 38% Ti 3+ , and 8% Ti 2+ , while the relative amount of Ti 4+ and Ti 3+ in the crystal lattice of 2-TiO 2− x nanoparticles was 63% and 37%, respectively. Cell viability and cell motility induced by TiO 2− x nanoparticles were investigated on primary fibroblast cultures. Eryptosis modulation by the nanoparticles along with cell death mechanisms was studied on rat erythrocytes. We report that both TiO 2− x nanoparticles do not decrease the viability of fibroblasts simultaneously stimulating cell migration. Data from in vitro studies on erythrocytes indicate that TiO 2− x nanoparticles trigger eryptosis via ROS- (1-TiO 2− x ) and Ca 2+ -mediated mechanisms (both TiO 2− x nanoparticles) suggesting that evaluation of eryptosis parameters is a more sensitive nanotoxicological approach for TiO 2− x nanoparticles than cultured fibroblast assays. TiO 2− x nanoparticles are characterized by low toxicity against fibroblasts, but they induce eryptosis, which is shown to be a promising tool for nanotoxicity screening. The Ti 3+ (Ti 2+ )/Ti 4+ ratio at least partly determines the cytotoxicity mechanisms for TiO 2− x nanoparticles.