Assessing the Cytotoxicity of TiO2-x Nanoparticles with a Different Ti3+(Ti2+)/Ti4+ Ratio.

Titanium dioxide (TiO2) nanoparticles are promising biomedical agents characterized by good biocompatibility. In this study, we explored the cytotoxicity of TiO2-x nanoparticles with a different Ti3+(Ti2+)/Ti4+ ratio and analyzed the efficiency of eryptosis indices as a tool in nanotoxicology. Two types of TiO2-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-TiO2-x and 2-TiO2-x NPs are 5 nm in size, whereas X-ray photoelectron spectroscopy (XPS) reveals different Ti3+ (Ti2+)/Ti4+ ratios in the crystal lattices of synthesized NPs. 1-TiO2-x nanoparticles contained 54% Ti4+, 38% Ti3+, and 8% Ti2+, while the relative amount of Ti4+ and Ti3+ in the crystal lattice of 2-TiO2-x nanoparticles was 63% and 37%, respectively. Cell viability and cell motility induced by TiO2-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 TiO2-x nanoparticles do not decrease the viability of fibroblasts simultaneously stimulating cell migration. Data from in vitro studies on erythrocytes indicate that TiO2-x nanoparticles trigger eryptosis via ROS- (1-TiO2-x) and Ca2+-mediated mechanisms (both TiO2-x nanoparticles) suggesting that evaluation of eryptosis parameters is a more sensitive nanotoxicological approach for TiO2-x nanoparticles than cultured fibroblast assays. TiO2-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 Ti3+ (Ti2+)/Ti4+ ratio at least partly determines the cytotoxicity mechanisms for TiO2-x nanoparticles.