Effect of titanium dioxide nanoparticle aggregation on mouse myoblast cellular cytotoxicity and nitric oxide synthesis.

Abstract

ABSTRACT Introduction: The emerging interest of engineered titanium dioxide nanoparticles (TiO2 NPs) in medical, agricultural, industrial and manufacturing sectors have raised health questions worldwide. Therefore, the objective was to assess the effect of physiochemical properties of titanium dioxide nanoparticles (TiO2 NPs) on the cellular cytotoxicity, proliferation and physiological properties. Methods: TiO2 NPs were suspended in varying concentrations of bovine serum albumin (BSA γ-globulin) and characterised using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) for the determination of particle size, aggregation state, and zeta potential. The effect of TiO2 physiochemical properties on cellular cytotoxicity and proliferation was assessed in vitro on mouse myoblast (C2C12) cells using the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] and BrdU assay respectively. Nitric oxide (NO), a major signalling molecule was measured using a biochemical test. in vitro. Results: There was an increase in size, distribution, surface charge and reduced aggregation in BSA stabilised TiO2 NPs in comparison to non-stabilised TiO2 NPs. Increased cytotoxicity of cells treated with monodispersed TiO2 NPs compared to cells treated with aggregated TiO2 NPs (p<0.001) was observed. A significant decrease in cell viability in cells treated with BSA (0.5, 0.8 and 1.0 mg/ml) stabilised TiO2 NPs (40, 120, 240, 320 and 400 mg/ml) in a dose-dependent manner in contrast to cells treated with BSA (0.3 and 1.5 mg/ml) stabilised TiO2 NPs (40, 120, 240, 320 and 400 mg/ml) dose dependent manner was observed (p<0.05). However, there was a greater decrease in cell viability in BSA (0.8 mg/ml) stabilised TiO2 NPs (40, 120, 240, 320 and 400 mg/ml) compared to other BSA concentration (p<0.05). In addition, there was a significant difference in DNA proliferation of the control and treated cells. A significant difference in DNA damage was observed in cells treated with BSA compared to non-treated cells, especially at BSA concentrations of 0.8 and 1.5 mg/ml. A significant difference in DNA damage in cells treated with TiO2 NPs in combination with BSA (0.8 and 1.5 mg/ml) was obtained. There was greater difference in DNA damage of cells exposed to TiO2 NPs in combination with 0.8 mg/ml compared to TiO2 NPs in combination with 1.5 mg/ml. More interestingly there was a significant difference between the levels of nitric oxide (NO) in 40 and 400 mg/ml TiO2 NPs treated cells in comparison to cells treated with BSA (0.3-1.5 mg/ml) stabilised TiO2 NPs (40 and 400 mg/ml) (p<0.05). There was a significance difference in the levels of NO between cells treated with 40 mg/ml TiO2 NPs vs (0.3, 0.5, 0.8 and 1.0 mg/ml) BSA stabilised TiO2 NPs (40 mg/ml) (p<0.05). However, there was greater significant difference between 400 mg/ml TiO2 treated cells vs BSA (0.5, 0.8 and 1.0 mg/ml) stabilised 400 mg/ml TiO2 NPs (400 mg/ml) (p<0.05). Discussion/Conclusion: The use of BSA as a nanoparticle stabiliser impacted upon physiochemical properties for the determination of in vitro cytotoxicity. These findings indicate that particle size needs to be taken into consideration when assessing nanoparticle toxicity. The results also indicate that less aggregated TiO2 NPs are more toxic than more aggregated TiO2 NPs and have a potential to inhibit cellular signalling mechanisms such as NO signalling and cellular proliferation.