Keywords: 
• anatase titanium dioxide nanoparticles /
• rutile titanium dioxide nanoparticles /
• human hepatocellular carcinoma QGY cells /
• cytotoxicity /
• sedimentation rate

Abstract: Rutile TiO₂-NPs (TNPs-R) and anatase TiO₂-NPs (TNPs-A), both of which are nanoscale titanium dioxide (TiO₂ nanoparticles, TNPs), have been widely used in food, cosmetics, sunscreens and other products. However, some studies have confirmed that TNPs can accumulate in tissues through different exposure routes such as inhalation, injection, and skin penetration, thereby leading to the development of chronic diseases. In addition, mixtures of varied ratios of TNPs-R and TNPs-A are often used in the manufacturing process to optimize material properties. However, differences in the toxic effects of TNPs-R and TNPs-A on human hepatocytes after exposure to environmental changes are unclear. In this study, TNPs-R and TNPs-A with a particle size of 25 nm were selected, and the particles were formulated into 80 μg·mL^-1 after being treated individually or mixedly, as well as treated with natural light or UV light, and the sedimentation rate of TNPs of each treatment group was evaluated by the UV-absorption ratio A_e/A₀ method after a standing period of 3-12 h. The results were summarized as follows. The TNPs-R∶TNPs-A=1∶9 group, which had the highest sedimentation rate in the mixed treatment, was selected to treat human hepatocytes QGY for 48 h with the TNPs-R or TNPs-A groups alone, and the efficiency of particles entering the cells was assessed by flow cytometry lateral light scattering angle (SSC) ratio SSC_e/SSC₀, and cell necrosis was analyzed by Giemsa staining. It was found that the TNPs-R sedimentation rate was significantly lower than that of TNPs-A when water was used as the solvent (P<0.001), and the sedimentation rate was significantly higher in the TNPs-R∶TNPs-A = 1∶9 group than in the mixed groups with other ratios (P<0.05) when mixed treatments were used. When serum-free medium was used as the solvent, the TNPs-A and TNPs-R∶TNPs-A=1∶9 groups had significantly higher sedimentation rates than the TNPs-R alone group (P<0.001). After being exposed to UV light, the sedimentation rates of all groups of TNPs were significantly higher than those of the natural-light treated TNPs-R group (P<0.01). Compared with the control group, QGY cells showed an increasing trend of intracellular SSC_e/SSC₀ after 48 h exposure to TNPs-R, TNPs-R∶TNPs-A=1∶9 and TNPs-A, and all three exposure groups significantly induced necrosis in QGY cells (P<0.01). TNPs-R∶TNPs-A=1∶9 induced a rate of cell necrosis significantly higher than that of TNPs-R (P<0.001). In conclusion, the physicochemical properties of TNPs-R and TNPs-A mixtures are altered with the elevated fraction of TNPs-A and UV exposure conditions, leading to increased sedimentation rates, which in turn induce higher toxicity to human hepatocytes. This study provides scientific references for the safety evaluation of TNPs with different crystalline forms.