Both P25TiO₂NPs (with or without vitamin B2) were not found beyond the epidermis in 99% of the analyzed TEM images (Fig. 8). This is coherent with previous findings showing that nanoparticles greater than 50 nm can not penetrate the skin, even in vivo models with movement, stretching, and friction [54]. However, in one of the zones, a few nanoparticles were observed inside a hair follicle. This could be due to the follicle exposure after the localized rupture of this physical barrier when rats were shaved in order to clean the area for cream topical administration. This finding suggests that nanoparticle-based sunscreen should not be applied on recently shaved or harmed skin, in order to avoid nanoparticle skin penetration.

The production of Chinese anatase titanium dioxide involves a series of complex chemical processes, including hydrolysis and calcination of titanium precursors. These processes result in the formation of nanoscale particles of anatase titanium dioxide, which exhibit enhanced properties such as increased surface area and improved reactivity. The size and morphology of these nanoparticles can be controlled during the synthesis process, allowing for the production of tailored materials with specific properties for different applications.

The production of ROS was studied on white blood cells as a model to screen the effect on eukaryotic cells after being exposed to samples and solar simulated irradiation (according to the level of penetration under the skin). For that purpose, the leukocytes were separated from anticoagulated fresh blood using the Ficoll-Hypaque reactive in a well-known technique [33]. Then, 50 μL of suspensions of P25TiO₂NPs (0.2 mg/mL and 0.02 mg/mL), vitaminB2@P25TiO₂NPs (0.2 mg/mL and 0.02 mg/mL) and vitamin B2 (0.2 mg/mL and 0.02 mg/mL) were prepared and mixed with 50 μL of white blood cells suspension. A solution of 3% H₂O₂ was used as positive control and PBS as negative control. Then, the samples were irradiated using the LED panel for 3 and 6 h to simulate the light penetration into the skin. Also, a set of samples was kept in the dark as control. Finally, the ROS were detected through the colorimetric assay employing the nitroblue tetrazolium salt (NBT salt) and the absorbance at 650 nm was measured. The experiment was reproduced twice; the standard deviation was calculated and p-value < 0.05 were considered significant.