Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment.

INTRODUCTION: Photodynamic therapy (PDT) using 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) constitutes an interesting alternative for cutaneous leishmaniasis treatment. OBJECTIVE: To evaluate the production of PpIXbased on the administration of ALA and MAL and the effect of ALA-PDTat cellular level on non-infected and infected THP-1 cells using Leishmania ( Viannia ) panamensis or Leishmania ( Leishmania ) infantum (syn Leishmania chagasi ) parasites. MATERIALS AND METHODS: Protoporphyrin IX (PpIX) production and mitochondrial colocalization were evaluated by confocal microscopy. Cell toxicities were evaluated after treatment with the compounds, followed by light irradiation (597-752 nm) at 2.5 J/cm 2 fluency using a colorimetric MTT assay for THP-1 cells and a standard microscopic analysis of parasites. RESULTS were expressed as compound concentration activity against 50% of cells or parasites (CC 50 or IC 50 ). RESULTS: ALA or MAL induced an endogenous PpIX with a red fluorescence localized mainly in the mitochondria inside human cells. ALA and MAL-PDT induced a similar range of toxicities on THP-1 cells (CC 50 0.16 ± 0.01 mM and 0.33 ± 0.019 mM, respectively) without any apparent inhibition of intracellular parasites in the infected cells as compared to untreated controls. Exogenous PpIX-PDT was toxic to THP-1 cells (CC 50 0.00032 ± 0.00002 mM), L. (L.) infantum (IC 50 0.003 ± 0.0001 mM) and L. (V.) panamensis (IC 50 0.024 ± 0.0001 mM) promastigotes. CONCLUSIONS: Despite the effectiveness of exogenous PpIX on promastigotes and the production of PpIX by human infected cells, treatment with ALA or MAL before irradiation was unable to completely destroy L. (L.) infantum or L. (V.) panamensis intracellular amastigotes.

Cell accumulation and antileishmanial effect of exogenous and endogenous protoporphyrin IX after photodynamic treatment / Acumulación celular y efecto anti- Leishmania de la protoporfirina IX exógena y endógena después del tratamiento fotodinámico

Introduction: Photodynamic therapy (PDT) using 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) constitutes an interesting alternative for cutaneous leishmaniasis treatment. Objective: To evaluate the production of PpIXbased on the administration of ALA and MAL and the effect of ALA-PDTat cellular level on non-infected and infected THP-1 cells using Leishmania ( Viannia ) panamensis or Leishmania ( Leishmania ) infantum (syn Leishmania chagasi ) parasites. Materials and methods: Protoporphyrin IX (PpIX) production and mitochondrial colocalization were evaluated by confocal microscopy. Cell toxicities were evaluated after treatment with the compounds, followed by light irradiation (597-752 nm) at 2.5 J/cm 2 fluency using a colorimetric MTT assay for THP-1 cells and a standard microscopic analysis of parasites. Results were expressed as compound concentration activity against 50% of cells or parasites (CC 50 or IC 50 ). Results: ALA or MAL induced an endogenous PpIX with a red fluorescence localized mainly in the mitochondria inside human cells. ALA and MAL-PDT induced a similar range of toxicities on THP-1 cells (CC 50 0.16±0.01mM and 0.33±0.019 mM, respectively) without any apparent inhibition of intracellular parasites in the infected cells as compared to untreated controls. Exogenous PpIX-PDT was toxic to THP-1 cells (CC 50 0.00032±0.00002 mM), L. (L.) infantum (IC 50 0.003±0.0001 mM) and L. (V.) panamensis (IC 50 0.024±0.0001 mM) promastigotes. Conclusions: Despite the effectiveness of exogenous PpIX on promastigotes and the production of PpIX by human infected cells, treatment with ALA or MAL before irradiation was unable to completely destroy L. (L.) infantum or L. (V.) panamensis intracellular amastigotes.

Introducción. El tratamiento fotodinámico con ácido 5-aminolevulínico como inductor de la protoporfirina IX (ALA-PpIX) constituye una alternativa interesante en el tratamiento de la leishmaniasis cutánea. Objetivo. Evaluar la producción de protoporfirina IX (PpIX) a partir de la administración de ALA o MAL y el efecto de la PDT con ALA a nivel celular en células THP-1 no infectadas e infectadas con Leishmania ( Viannia ) panamensis o Leishmania ( Leishmania ) infantum (syn. Leishmania chagasi ). Materiales y métodos. La producción de protoporfirina IX y su ‘colocalización´ mitocondrial se evaluaron mediante microscopía ‘confocal´. Se evaluó la toxicidad celular después del tratamiento con los compuestos y la aplicación de irradiación de luz (597-752 nm) en una fluencia de 2,5 J/cm 2 mediante el empleo de la prueba colorimétrica con metil-tiazol-tetrazolio (MTT) en las células, y de métodos microscópicos estándar en los parásitos. Los resultados se expresaron como la concentración del compuesto activo en el 50 % de las células o parásitos (CC 50 o CI 50 ). Resultados. El ácido aminolevulínico o el metil-5-aminolevulinato indujeron la protoporfirina IX endógena en células humanas, y se observó fluorescencia de color rojo en las mitocondrias. La actividad del ácido aminolevulínico y del metil-5-aminolevulinato utilizados con terapia fotodinámica fue similar en las células THP-1 (CC 50 0,16±0,01 mM y 0,33±0,019 mM, respectivamente) y, aparentemente, no inhibió los parásitos en las células infectadas, en comparación con los controles. El tratamiento exógeno con protoporfirina IX y terapia fotodinámica fue tóxico para las células THP-1 (CC 50 0,00032 ±0,00002 mM) y para los promastigotes de L. (L .) infantum (IC 50 0,003±0,0001 mM) y L. ( V .) panamensis (CI 50 0,024±0,0001 mM). Conclusiones. A pesar de la ‘fotoactividad´ del tratamiento con protoporfirina IX en promastigotes y de su producción después del tratamiento con ácido aminolevulínico y metil-5-aminolevulinato en las células infectadas con Leishmania , no se observó daño en los amastigotes presentes en las células de L. ( L .) infantum o L . ( V .) panamensis .

Photodynamic activity of aluminium (III) and zinc (II) phthalocyanines in Leishmania promastigotes.

INTRODUCTION: Photodynamic therapy is a two-step procedure, involving the use of photosensitizing agents followed by selective illumination of the target lesion with visible light. It produces highly reactive oxygen species and subsequent cellular damage. OBJECTIVE: This study was designed to determine whether Leishmania chagasi and L. panamensis promastigotes were sensitive to photodynamic therapy in vitro. MATERIAL AND METHODS: Leishmania promastigotes were treated with aluminium phthalocyanine chloride and zinc phthalocyanine photosensitizers before illumination with visible light at 670 nm. The parasite photoactivity was calculated by sigmoidal regression analysis. RESULTS: Leishmania chagasi promastigotes were highly photosensitive to aluminium phthalocyanine chloride treatment with effective inhibitory dose50 (ED50) concentration values of 0.0033, 0.0083 and 0.0093 microM upon exposure to 10.0, 5.0, and 2.5 J/cm2 light intensities respectively. By contrast, the activity of aluminium phthalocyanine chloride on L. panamensis was significantly lower (P < 0.01) with ED50 values of 0.17, 0.25, 0.34 microM at the same light intensities. Zinc phthalocyanine activity was significantly (P < 0.01) less active than aluminium phthalocyanine chloride on both strains of these two species and no differences in zinc phthalocyanine activity were found between them. A dose-response phototoxic effect with both phthalocyanines was observed. Parasite inhibition was not observed after aluminium phthalocyanine chloride or zinc phthalocyanine treatment in the dark. The reference drugs hexadecylphosphocholine and amphotericin B were not photoactive. CONCLUSION: Treatment of Leishmania promastigotes with aluminium phthalocyanine chloride and zinc phthalocyanine followed by illumination with visible light at 670 nm inhibited in vitro growth of promastigotes of L. chagasi and L. panamensis. Photodynamic therapy against Leishmania could be a promising strategy for leishmaniasis treatment.