Pristine (C60) and hydroxylated [C60(OH)24] fullerene phototoxicity towards HaCaT keratinocytes: type I vs type II mechanisms.

The increasing use of fullerene nanomaterials has prompted widespread concern over their biological effects. Herein, we have studied the phototoxicity of gamma-cyclodextrin bicapped pristine C 60 [(gamma-CyD) 2/C 60] and its water-soluble derivative C 60(OH) 24 toward human keratinocytes. Our results demonstrated that irradiation of (gamma-CyD) 2/C 60 or C 60(OH) 24 in D 2O generated singlet oxygen with quantum yields of 0.76 and 0.08, respectively. Irradiation (>400 nm) of C 60(OH) 24 generated superoxide as detected by the EPR spin trapping technique; superoxide generation was enhanced by addition of the electron donor nicotinamide adenine dinucleotide (reduced) (NADH). During the irradiation of (gamma-CyD) 2/C 60, superoxide was generated only in the presence of NADH. Cell viability measurements demonstrated that (gamma-CyD) 2/C 60 was about 60 times more phototoxic to human keratinocytes than C 60(OH) 24. UVA irradiation of human keratinocytes in the presence of (gamma-CyD) 2/C 60 resulted in a significant rise in intracellular protein-derived peroxides, suggesting a type II mechanism for phototoxicity. UVA irradiation of human keratinocytes in the presence of C 60(OH) 24 produced diffuse intracellular fluorescence when the hydrogen peroxide probe Peroxyfluor-1 was present, suggesting a type I mechanism. Our results clearly show that the phototoxicity induced by (gamma-CyD) 2/C 60 is mainly mediated by singlet oxygen with a minor contribution from superoxide, while C 60(OH) 24 phototoxicity is mainly due to superoxide.