Cell death mechanisms in Leishmania amazonensis triggered by methylene blue-mediated antiparasitic photodynamic therapy

Antiparasitic photodynamic therapy (ApPDT) is an emerging approach to manage cutaneous leishmaniasis (CL) since no side effects, contraindications and parasite resistance have been reported. In addition, methylene blue (MB) is a suitable photosensitizer to mediate ApPDT on CL. In this study we aimed to look for the best parameters to eradicate Leishmania amazonensis and investigated the cell death pathways involved in MB-mediated ApPDT. MB uptake by parasites was determined using different MB concentrations (50, 100, 250 and 500 mu M) and incubation times (10, 30 and 60 min). L. amazonensis promastigotes were cultured and submitted to ApPDT using different concentrations of MB (50, 100 and 250 mu M) combined to a red LED emitting at 645 +/- 10 nm. The pre-irradiation time was 10 min. Two optical powers (100 mW and 250 mW) were tested and cells were exposed to 60 and 300 s of MB-mediated ApPDT delivering energies of 6, 15, 30 and 75 J and fluences of 21.2, 53.1, 106.2 and 265.4 J/cm(2), respectively. Following ApPDT, cells were prepared for flow cytometry and transmission electron microscopy to unravel the mechanisms of cell death. Our results showed the lowest MB concentration (50 mu M) and the lowest optical power (100 mW) promoted the highest percentage of cell decrease. ApPDT caused alterations on cell membrane permeability as well depolarization of mitochondrial membrane potential. We also observed ultrastructural changes of the parasites such as cell shrinkage, intense vacuolization of the cytoplasm, enlargement of mitochondrion-kinetoplast complex, and small blebs on parasite flagella and cell membrane after MB-mediated ApPDT. Taken together, our findings ratify that ApPDT parameters play a pivotal role in cell susceptibility and suggest that apoptosis is involved in parasite death regardless MB-mediated ApPDT protocol.