In Vitro Safety Assessment of In-House Synthesized Titanium Dioxide Nanoparticles: Impact of Washing and Temperature...

Titanium dioxide nanoparticles (TiO₂ NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO₂ NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO₂ NPs synthesized via the Stöber method under different washing and temperature conditions. TiO₂ NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO₂ NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO₂ NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO₂ NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO_2, indicating that toxicity is partially driven via non-oxidative pathways. TiO₂ NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions.