Mechanism of photodynamic action induced by a cationic phthalocyanine on Candida albicans cells

DI PALMA, M. ALBANA; OCHOA, A. LAURA, ÁLVAREZ, M. GABRIELA, MILANESIO, M. ELISA, DURANTINI, EDGARDO N.

Córdoba

Encuentro; XI ELAFOT, XI Encuentro Latinoamericano de Fotoquímica y Fotobiología,; 2012

Fungal diseases represent a critical problem to health and they are one of the main causes of morbidity and mortality worldwide. Candida albicans is the most common species associated with candidiasis and is the most frequently recovered species from hospitalized patients. The search for new therapeutic approaches is stimulated by the fact that standard drug treatments are prolonged and the appearance of drug resistant strains is more frequent in high risk groups. The photodynamic inactivation (PDI) has been proposed for the elimination of microorganisms. In this treatment, the photosensitizer is accumulated in microbial cells, which in the presence of light are inactivated by the formation of cytotoxic species. Basically, the photosensitizer excited state can react with molecules from its direct environment by electron or hydrogen transfer, leading to the production of radicals, or the photosensitizer can transfer its energy to oxygen, generating the highly reactive singlet molecular oxygen, O2(1Dg). In this work, the photodynamic mechanism of action induced by zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc+4) was studied in C. albicans. This phthalocyanine exhibited a high absorption coefficient in the visible region of the spectrum, characterized by the typical Soret (~374 nm) and Q-bands (~678 nm). Also, ZnPPc4+ emited two bands (~687 and 756 nm) with a fluorescence quantum yield of 0.22. The photodynamic activity of ZnPPc4+ indicated a high efficiency in the quantum yield of O2(1Dg) production (FD=0.59) in N,N-dimethylformamide. The C. albicans cultures treated with 10 µM of ZnPyPc4+ for 30 min showed a binding of ~4.80 nmol/106 cells. Cell survival produced a decrease of 5 log when treated with 10 µM ZnPyPc4+ and 30 min visible light irradiation. These results represent a value greater than 99.999% of cellular inactivation. Studies under anoxic conditions indicated that oxygen is necessary for the mechanism of action of photodynamic inactivation of this yeast. The photocytotoxicity induced by ZnPyPc4+ increased in D2O, which was used to enhance the lifetime of O2(1Dg). Furthermore, photoinactivation of C. albicans cells was negligible in the presence of 100 mM azide ion, a known quencher of O2(1Dg). In contrast, the addition of 100 mM mannitol produced a negligible effect on the cellular phototoxicity. This compound acts as scavenger of the superoxide anion radical and hydroxyl radical. Therefore, the results indicate that ZnPyPc4+ appears to act as photosensitizers mainly via the intermediacy of O2(1Dg). Thus, in the present in vitro experiments, the killing of C. albicans cells by ZnPyPc4+ and visible light irradiation seem to be mediated mainly by O2(1Dg). Although in a minor contribution, the participation of other active oxygen species could not be neglected particularly for C. albicans photoinactivated with ZnPyPc4+.