Photodynamic Sensitization of Leishmania amazonensis in both Extracellular and Intracellular Stages with Aluminum Phthalocyanine Chloride for Photolysis In Vitro

Abstract

Leishmania amazonensis, a causative agent of cutaneous leishmaniasis, is susceptible in vitro to lightmediated cytolysis in the presence of or after pretreatment with the photosensitizer aluminum phthalocyanine chloride. Cytolysis of both promastigotes and axenic amastigotes required less photosensitizer (e.g., one g · ml ؊1 ) and a lower light dose (e.g., 1.5 J · cm ؊2 ) than did the mammalian cells examined for comparison. Exposure of Leishmania cells to the photosensitizer alone had little effect on their viability, as judged from their motility, growth, and/or retention of green fluorescent proteins genetically engineered for episomal expression. Fluorimetric assays for cell-associated and released green fluorescence proteins proved to be even more sensitive for the evaluation of cell viability than microscopy for the evaluation of motility and/or integrity. Axenic amastigotes pretreated with the photosensitizer infected macrophages of the J774 line but were lysed intracellularly when the infected cells were exposed to light. Addition of the photosensitizer to the already infected cells produced no effect on their intracellular parasites. However, light irradiation lysed these macrophages and also those infected with parasites preincubated with the photosensitizer at a concentration of 5 g · ml ؊1 or higher. Photosensitized Leishmania cells are highly susceptible to cytolysis, apparently due to the generation of reactive oxidative species on light illumination, suggestive of inefficiency of their antioxidant mechanisms. Efficient delivery of photosensitizers to intracellular Leishmania is expected to increase their therapeutic potentials against leishmaniasis.

Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages

“…Leishmania amazonensis (LV78, clone 12-1) were cultured continuously as promastigotes in vitro at 25°C in HEPES-buffered Medium 199 (pH 7.4) containing 10% heat-inactivated fetal bovine serum [39]. Culture starting with an inoculum of 10 6 cells/ml reached a peak cell density of ~10 8 /ml in 4-5 days that was maintained throughout the stationary phase in the next several days.…”

“…DsRed was PCR amplified from pDsRed-monomer-C1 (Clontech) using a set of forward (5'-GGATCCATGGTGCGCTCCAAG-3') and reverse (5'-GGATCCTTATCTAGATCCGGTCG-3') primers with BamH1 sites (bold-faced) to facilitate its cloning into p6.5 [39]. The reverse primer sequence was designed such that original BamH1 site in the 3' end of the gene was obliterated without affecting the amino acid sequence of the product.…”

Topical and Intradermal Efficacy of Photodynamic Therapy with Methylene Blue and Light-Emitting Diode in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis

“…Several studies with PDT used LASER as a light source 7,14,17 and several compounds as photosensitizer substances. 8,[17][18][19] Although the LED unit is less potent than LASER, the price of LED is much lower. Furthermore, the array of the LED devices presents radiation per area higher than the LASER and the red LED emits light in the range of 655 nm wavelength, which penetrates deeper into the tissue and is relevant to the success of PDT.…”

Acumulación celular y el efecto de anti-Leishmania de protoporfirina IX exógena y endógena después del tratamiento fotodinámico

“…L. (L.) infantum promastigotes were more photosensitive to exogenous PpIX than L. (V.) panamensis promastigotes, demonstrating once again the interspecies intrinsic variation in drug sensitivity showed by some antileishmanial drugs, such as miltefosine, to which L. donovani (the main species involved in Old World leishmaniasis) was more sensitive than other Leishmania species. Leishmania parasites have been photosensitive to different exogenous photosensitizers like Al and Zn phthalocyanines, carboporphyrins, and benzophenoxazine (10,16,25,26).…”