Effects of photodynamic inactivation mediated by Zn(II) porphyrin on promastigote and amastigote forms of Leishmania amazonensis

Abstract

Photodynamic inactivation (PDI) has been attracting attention as an innovative technology to treat topical diseases, such as cutaneous leishmaniasis (CL) and infections caused by multidrug-resistant microorganisms. Zn(II) mesotetrakis( N-n-hexylpyridinium-2-yl)porphyrin (ZnTnHex-2-PyP4+) is a lipophilic water-soluble Zn(II) porphyrin with improved photophysical properties, high chemical stability, and cationic/amphiphilic character that can enhance its interaction with cells. Thus, this study aimed to investigate the PDI effects mediated by ZnTnHex-2-PyP4+ on Leishmania amazonensis. Confocal fluorescence microscopy was explored to study the interaction of ZnTnHex-2- PyP4+ with promastigotes. The PDI action was analyzed by cell membrane integrity, mitochondrial membrane potential (ΔΨm), and cell morphology. Promastigotes were incubated with ZnTnHex-2-PyP4+ for 5 min at 0.62 and 1.25 μM and irradiated by a LED (410 nm) for 1 or 3 min (2.3 and 3.4 J/cm2, respectively). PDI on amastigotes and the cytotoxicity on macrophages were also analyzed (3.4 J/cm2). Fluorescence microscopy revealed that parasites efficiently uptake ZnTnHex-2-PyP4+ and displayed a punctate labeling pattern along with the cytoplasm. An intense ΔΨm depolarization was also observed, which in association with microscopy results, suggests that ZnTnHex-2- PyP4+ may accumulate in the mitochondrion, or other well-defined structures close to it. Moreover, ZnTnHex-2-PyP4+ at concentration as low as 0.62 μM led to the immediate inactivation of >95% of promastigotes, regardless of the light dose used. Loss of the fusiform shape and plasma membrane wrinkling were also observed. After a single treatment session in amastigotes, PDI led to a reduction of 70% in the infection index. No considerable toxicity was observed on mammalian cells. Thus, PDI of Leishmania parasites showed in vitro efficiency at a submicromolar concentration of ZnTnHex-2-PyP4+, with short pre-incubation and irradiation times. The results encourage further studies in CL pre-clinical assays and PDI of other microorganisms.