Influência da biocorona proteica formada em soro fetal bovino nas propriedades físico-químicas e nos efeitos citogenotóxicos de nanopartículas de óxido de zinco in vitro

Abstract

Zinc oxide nanoparticles (ZnO NPs) are used in the cosmetic, pharmaceutical, food, textile, electronics, and agricultural sectors because of their physicochemical properties. However, they may interact with biological systems and induce cytotoxic and genotoxic effects. In biological fluids, proteins adsorb onto the NP surface and form a protein corona that can modify their biological identity. This study evaluated protein corona formation following the incubation of ZnO NPs with fetal bovine serum (FBS) and its influence on in vitro cytotoxicity and genotoxicity in Chinese hamster ovary (CHO) cells. The NPs were evaluated at concentrations of 0, 1, 10, and 100 µg/mL, with or without prior incubation with FBS. The dispersions were characterized by dynamic light scattering, Fourier-transform infrared spectroscopy, Raman spectroscopy, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). Cellular metabolic activity was assessed using the MTT and Alamar Blue assays, together with morphological analysis by inverted light microscopy. DNA damage was investigated using the comet assay. Data were analyzed using the Kruskal-Wallis test followed by Dunn’s multiple-comparison test, with a significance level of 5%. At 100 µg/mL, incubation with FBS reduced the mean hydrodynamic diameter of the NPs from 1,247 ± 250 to 123 ± 3 nm, while the Zeta potential ranged from −10.0 ± 5.1 to −13.4 ± 0.9 mV. SDS-PAGE demonstrated protein association with the NPs, with predominant bands between 25 and 35 kDa. The FTIR spectra showed bands between 456 and 683 cm⁻¹, consistent with Zn–O bond vibrations, whereas Raman signals between 1,075 and 2,337 cm⁻¹ were associated with organic components in the samples incubated with FBS. In the MTT assay, after 24 h, metabolic activity was reduced at 10 and 100 µg/mL, both with and without prior incubation with FBS, compared with the control (p < 0.05). At 1 µg/mL, NPs incubated with FBS resulted in higher metabolic activity than NPs without prior incubation with FBS (p < 0.05) and did not differ from the control (p > 0.05). After 48 h, only the concentration of 100 µg/mL reduced metabolic activity, regardless of prior incubation with FBS (p < 0.05). In the Alamar Blue assay, after 24 h, NPs without FBS reduced metabolic activity at 10 and 100 µg/mL, whereas NPs incubated with FBS reduced metabolic activity only at 100 µg/mL (p < 0.05). At 10 µg/mL, NPs incubated with FBS resulted in higher metabolic activity than NPs without FBS (p < 0.05) and did not differ from the control (p > 0.05). After 48 h, only the concentration of 100 µg/mL reduced metabolic activity, regardless of prior incubation with FBS (p < 0.05). In the comet assay, the percentage of DNA in the tail did not differ among the groups (p > 0.05). However, the Olive tail moment increased in cells exposed to 1 and 100 µg/mL of NPs incubated with FBS compared with both the control and the corresponding concentrations without FBS (p < 0.05). The results demonstrate that the adsorption of FBS proteins modified the physicochemical properties and biological effects of ZnO NPs. Therefore, protein corona formation should be considered when characterizing nanomaterial dispersions and interpreting in vitro nanotoxicology assays.