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.
The aim of this work was to examine particularly the Degussa P25 titanium dioxide nanoparticles (P25TiO2NPs) because they are among the most employed ones in cosmetics. In fact, all kinds of titanium dioxide nanoparticles (TiO2NPs) have gained widespread commercialization over recent decades. This white pigment (TiO2NPs) is used in a broad range of applications, including food, personal care products (toothpaste, lotions, sunscreens, face creams), drugs, plastics, ceramics, and paints. The original source is abundant in Earth as a chemically inert amphoteric oxide, which is thermally stable, corrosion-resistant, and water-insoluble. This oxide is found in three different forms: rutile (the most stable and substantial form), brookite (rhombohedral), and anatase (tetragonal as rutile), of these, both rutile and anatase are of significant commercial importance in a wide range of applications [3]. Additionally, the nano-sized oxide exhibits interesting physical properties, one of them is the ability to act as semiconducting material under UV exposure. In fact, TiO2NPs are the most well-known and useful photocatalytic material, because of their relatively low price and photo-stability [4]. Although, this photoactivity could also cause undesired molecular damage in biological tissues and needs to be urgently assessed, due to their worldwide use. However, not all nanosized titanium dioxide have the same behavior. In 2007, Rampaul A and Parkin I questioned: “whether the anatase/rutile crystal form of titanium dioxide with an organosilane or dimethicone coat, a common titania type identified in sunscreens, is appropriate to use in sunscreen lotions” [5]. They also suggested that with further study, other types of functionalized titanium dioxide could potentially be safer alternatives. Later, Damiani found that the anatase form of TiO2NPs was the more photoactive one, and stated that it should be avoided for sunscreen formulations, in agreement with Barker and Branch (2008) [6,7].