Mechanistic insight into the photodynamic effect mediated by porphyrin-fullerene C60 dyads in solution and in Staphylococcus aureus cells
The photodynamic action mechanism sensitized by a non-charged porphyrin-fullerene C60 dyad (TCP-C60) and its tetracationic analogue (TCP-C604+) was investigated in solution and in Staphylococcus aureus cells. The ability of both dyads to form a photoinduced charge-separated state was evidenced by the reduction of methyl viologen in N,N-dimethylformamide (DMF). Moreover, the formation of superoxide anion radicals induced by these dyads was detected by the reduction of nitro blue tetrazolium. Also, photosensitized decomposition of L-tryptophan (Trp) was investigated in the presence of reactive oxygen species (ROS) scavengers. The addition of β-carotene and sodium azide had a slight effect on reaction rate. However, photooxidation of Trp mediated by TCP-C60 was negligible in the presence of D-mannitol, while no protection was found using TCP-C604+. In a polar medium, these dyads mainly act by a contribution of type I pathway with low generation of singlet molecular oxygen, O2(1Δg). In S. aureus cell suspensions, an aerobic atmosphere was required for the photokilling of this bacterium. The photocytotoxicity induced by TCP-C60 was increased in D2O with respect to water, while a small effect was found using TCP-C604+. Furthermore, photoinactivation of microbial cells was negligible in the presence of sodium azide. The addition of D-mannitol did not affect the photoinactivation induced by TCP-C60. In contrast, S. aureus cells were protected by D-mannitol when TCP-C604+ was used as a photosensitizer. Also, generation of O2(1Δg) in the S. aureus cells was higher for TCP-C60 than TCP-C604+. Therefore, TCP-C60 appears to act in microbial cells mainly through the mediation of O2(1Δg). Although, a contribution of the type I mechanism was found for cell death induced by TCP-C604+. Therefore, these dyads with high capacity to produce photoinduced charge-separated state represent interesting photosensitizers to inactivate microorganisms by type I or type II mechanisms. In particular, TCP-C60 may be located in a non-polar microenvironment in the cells favoring a type II pathway, while a contribution of the type I mechanism was produced using the cationic TCP-C604+.