In vitro effect of photodynamic therapy on leishmania tropica

Leishmaniasis is a vectorborne disease transmitted by Phlebotomus species and the cuteonus form (CL) of disease is known as "Şark Çıbanı". Leishmania tropica is the most often agent of cutaneous leishmaniasis (CL) in Turkey. Photodynamic therapy (PDT) is a treatment which uses of a light-sensitive drug (a photosensitiser) in combination with light of a visible wavelength, to destroy target cells (canserous and non-cancerous) by generating mainly singlet oxygen in the presence of molecular oxygen. In this study, we investigated the effects of PDT using different photosensitizers, namely methylene blue (MB), toluidine blue (TB), alüminum ftalosiyanin chlorid (AIClPc) and pheporbide a (Pa) in L. tropica promastigotes. Parasites were incubated with increasing concentrations of TB, MB, AlClPc and Pa for one hour. After the media containing phosensitizers were removed, promastigotes were washed with PBS. Fresh PBS was added and parasites were illuminated with a white light source for 30 min. Following the irradiation of the cells, fresh growth medium was added. After incubation at 26°C for 18 h, the cell viability (analyzed using a hemocytometer and XTT colorimetric assay), morphological changes (staining with giemsa) and apoptotic assay (staining with DAPI and DNA fragmentation assay) of Leishmania tropica parasites were evaluated as a function of MB, TB, AlClPc and PPa concentration. The efficiency of PDT was investigated using irradiation+ phosensitizers, phosensitizers alone and in control group in all experiments. Parasite viability was significantly different (p<0,001 p<0,05 p<0,0001) between groups treated with MB,TB and PPA with or without irradiation. There were no statistically significant differences in cell viability between AlClPc trated L. tropica promastigotes with and without irradiation (p=0,07). Viability of L.tropica promastigotes was significantly lower than control groups in all PDT groups including all photosensitizers. Also, DAPI staning method, which is used to determine cellular changes in apoptotic cells, showed that MB, AlClPc and PA with irradiation affected on L.tropica promastigotes. Furthermore, DNA ladder pattern defining the apoptosis, was observed in irradiated MB,AlClPc and PA groups and not observed in TB with irradiation, AlClPc, TB, MB and PA without irradiation. The findings of this study promoted insight on the effects of PDT on Leishmania using the promastigote form of the parasite and has opened a new perspective for the alternative treatment of the parasites. The results also reveased that apoptosis induced cell death was observed in L.tropica promastigotes after the application of photosensitizers in combination with light irradiation