Description:

Hemolysis was studied on 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 500 μL of anticoagulated blood (donated by Laboratorio de Hemoderivados, UNC) in a rate of 1/10. A solution of NaCl 10% was used as the positive control and PBS as the negative control. Then, the samples were irradiated using the LED described above 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 samples were centrifuged and the absorbance at 540 nm was measured in the supernatants. The experiment was reproduced twice; the standard deviation was calculated and p-value < 0.05 were considered significant.

This constant high rate of ROS production leads rapidly to extreme macromolecular oxidation, here it is observed in the AOPP and MDA detected after 3 h in samples treated with bare P25TiO₂NPs (Fig. 6, Fig. 7). Macromolecular oxidation includes, among others, both protein and lipid oxidation. The ROS causes protein oxidation by direct reaction or indirect reactions with secondary by-products of oxidative stress. Protein fragmentation or cross-linkages could be produced after the oxidation of amino acid side chains and protein backbones. These and later dityrosine-containing protein products formed during excessive production of oxidants are known as advanced oxidation protein products (AOPP). They absorb at 340 nm and are used to estimate the damage to structural cell amino acids. Lipid oxidation is detected by the conjugation of oxidized polyunsaturated lipids with thiobarbituric acid, forming a molecule that absorbs light at 532 nm. Polyunsaturated lipids are oxidized as a result of a free-radical-mediated chain of reactions. The most exposed targets are usually membrane lipids. The macromolecular damage could represent a deadly danger if it is too extensive, and this might be the case. Moreover, it could be observed that cellular damage continues further and becomes irrevocable after 6 h and MDA could not be detected. This may be due to the fact that the lipids were completely degraded and cells were no longer viable. Lipids from the cell membrane are the most prone to oxidation. In fact, lipid peroxidation biomarkers are used to screen the oxidative body balance [51]. At the same time, AOPP values are up to 30 times higher for bare nanoparticles in comparison to the functionalized ones.

Infrared analysis showed that the characteristics bands for the bare nanoparticles are still exhibited in the vitamins@P25TiO₂NPs spectra, such as a wide peak in 450–1028 cm⁻¹ related to the stretching vibration of Ti-O-Ti and other peaks in 1630 cm⁻¹ and 3400 cm⁻¹, which represent the surface OH groups stretching. The IR spectrum of vitaminB2@P25TiO₂NPs showed signs of binding between compounds. The OH bending peak (1634 cm⁻¹) corresponding to bare nanoparticles disappeared, and the NH₂ bending band characteristic of vitamin B2 appeared (1650 cm⁻¹). The IR spectrum of vitaminC@P25TiO₂NPs also showed signs of successful functionalization. Bands at 1075 cm⁻¹; 1120 cm⁻¹; 1141 cm⁻¹ were observed, which are originated by CO-C vibrations present in the vitamin C. The intense band at 1672 cm⁻¹ is attributed to the C = O stretching in the lactone ring while the peak at 1026 cm⁻¹ is ascribed to the stretching vibration Ti-O-C. Wide bands at 3880–3600 cm⁻¹ are related to stretching vibration OH groups, but those disappear in the modified nanoparticles spectrum. These observations confirm the interactions between the P25TiO₂NPs and the vitamins [35].