Avaliação da atividade leishmanicida de porfirinas associadas à terapia fotodinâmica
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Data
2022-10-17
Autores
Sánchez Uría, Maricely
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
As leishmanioses são doenças negligenciadas que afetam milhões de pessoas em todo o mundo. Os fármacos disponíveis para o tratamento apresentam diversos efeitos colaterais e difícil via de administração, o que causa abandono do tratamento pelo paciente e surgimento de cepas resistentes. Nosso grupo de pesquisa atua na etapa pré-clínica de desenvolvimento de fármacos contra as leishmanioses, testando moléculas de origem natural e/ou sintética e investigando seus possíveis mecanismos de ação. Neste trabalho, foi avaliada a atividade leishmanicida (EC50) in vitro de oito porfirinas tetra-catiônicas (4-PtTPyP, 3-PtTPyP, 4-PdTPyP, 3-PdTPyP, 4-TPyP, 3-TPyP, 4-TMePyP, e 3-TMePyP) frente a L. amazonensis através de Terapia Fotodinâmica (TFD). Os compostos foram avaliados quanto à sua foto-citotoxicidade (CC50) em macrófagos murinos e seus Índices de seletividade (IS) foram calculados. As porfirinas 3-PtTPyP (EC50: 8,7 ± 0,4 nM; IS: 213), 3-PdTPyP (EC50: 15 ± 5 nM; IS: 85,2) e 3-TPyP (EC50: 25,5 ± 7,5 nM; IS: 61,9) utilizadas como fotossensibilizadores (FS) através de TFD mostraram-se as mais fototóxicas frente às formas promastigotas do parasita. Já na atividade anti-amastigota (EC50- AMA) foi constatado que em geral os FS: 3-TPyP (47,9 ± 4,4 nM; IS: 32), 3-PdTPyP (38,8 ± 11,5 nM; IS: 32,9) e 3-PtTPyP (27,6 ± 6,7nM; IS:67) são ótimos candidatos para serem futuramente testados in vivo. Devido a interação entre os FS e a luz, foi mensurada a produção de espécies reativas de oxigênio (EROs), encontrando-se que as porfirinas 4-PtTPyP, 3-PtTPyP e 3-PdTPyP induziram os níveis mais altos de EROs no parasita. Adicionalmente, ensaios de co-localização com as porfirinas foram realizados utilizando os marcadores MitoTracker Orange (marcador de mitocôndria) e LysoTracker Green (marcador de lisossomos ou pools acídicos) por microscopia confocal, encontrando-se co-localização das moléculas em ambas as organelas, mitocôndria e pools acídicos. Análises do tipo de morte celular potencialmente induzida pelos FSs foram realizadas através da marcação com brometo de etidio/laranja de acridina (BE/LA). As moléculas 3-PdTPyP, 3-PtTPyP e seus isômeros (4-PdTPyP, 4-PtTPyP) na presença de luz branca após 6 e 24 h de irradiação mostraram-se como os FSs com maior efeito leishmanicida. 3-PdTPyP e 3-PtTPyP induziram a morte do parasita principalmente por necrose (74,2% e 52,2% após 6h, respectivamente; e 79% e 78% após 24h, respectivamente). Enquanto que o isômero 4-PtTPyP induz a morte das promastigotas por like-apoptose (81%) após 6h de irradiação, e após 24 h direccionou os parasitas a morte por necrose (33,6%) e like-apoptose (28,2%), similar ao observado no isômero 4-PdTPyP em ambos os tempos (40% necrose/20%like-apoptose e 54,5%necrose/25,5%like-apoptose, após 6h e 24h de irradiação, respectivamente). Estes dados sugerem que as porfirinas tetra-catiônicas de platina e paládio aqui utilizadas têm um grande potencial para serem utilizadas no tratamento da leishmaniose cutânea por meio de terapia fotodinâmica. Palavras-chave: Leishmanioses, Terapia Fotodinâmica, Leishmania amazonensis, Porfirinas, mitocôndria, lisossomos.
Leishmaniasis are neglected diseases that affect millions of people worldwide. The drugs available for treatment have several side effects and a difficult route of administration, which causes the patient to abandon treatment and the emergence of resistant strains. Our research group works in the pre-clinical stage of drug development against leishmaniasis, testing molecules of natural and/or synthetic origin and investigating their possible mechanisms of action. In this work, the in vitro leishmanicidal activity (EC50) of eight tetracationic porphyrins (4-PtTPyP, 3-PtTPyP, 4-PdTPyP, 3-PdTPyP, 4-TPyP, 3-TPyP, 4-TMePyP, and 3 -TMePyP) against L. amazonensis through Photodynamic Therapy (PDT). Compounds were evaluated for their photocytotoxicity (CC50) on murine macrophages and their selectivity indices (IS) were calculated. The porphyrins 3-PtTPyP (EC50: 8.7 ± 0.4 nM; IS: 213), 3-PdTPyP (EC50: 15 ± 5 nM; IS: 85.2) and 3-TPyP (EC50: 25.5 ± 7.5 nM; IS: 61.9) used as photosensitizers (FS) through PDT proved to be the most phototoxic against the promastigote forms of the parasite. In the anti-amastigote activity (EC50-AMA) it was found that in general the FS: 3-TPyP (47.9 ± 4.4 nM; IS: 32), 3-PdTPyP (38.8 ± 11.5 nM; IS: 32.9) and 3-PtTPyP (27.6 ± 6.7nM; IS: 67) are excellent candidates to be tested in vivo in the future. Due to the interaction between FS and light, the production of reactive oxygen species (ROS) was measured, and the porphyrins 4-PtTPyP, 3-PtTPyP and 3-PdTPyP induced the highest levels of ROS in the parasite. Additionally, co-localization assays with porphyrins were performed using the markers MitoTracker Orange (mitochondria marker) and LysoTracker Green (lysosomes or acidic pools marker) by confocal microscopy, finding co-localization of the molecules in both organelles, mitochondria and acidic pools. Analyzes of the type of cell death potentially induced by FSs were performed by labeling with ethidium bromide/acridine orange (BE/LA). The molecules 3-PdTPyP, 3-PtTPyP and their isomers (4-PdTPyP, 4-PtTPyP) in the presence of white light after 6 and 24 h of irradiation showed to be the FSs with the greatest leishmanicidal effect. 3-PdTPyP and 3-PtTPyP induced parasite death mainly by necrosis (74.2% and 52.2% after 6h, respectively; and 79% and 78% after 24h, respectively). While the 4-PtTPyP isomer induced death of promastigotes by like-apoptosis (81%) after 6h of irradiation, and after 24h it directed the parasites to death by necrosis (33.6%) and like-apoptosis (28.2 %), similar to that observed in the 4-PdTPyP isomer at both times (40% necrosis/20%like-apoptosis and 54.5%necrosis/25.5%like-apoptosis, after 6h and 24h of irradiation, respectively). These data suggest that the tetracationic platinum and palladium porphyrins used here have great potential to be used in the treatment of cutaneous leishmaniasis by means of photodynamic therapy. Key-words: Leishmaniasis. Leishmania amazonensis. Porphyrins. photodynamic therapy
Leishmaniasis are neglected diseases that affect millions of people worldwide. The drugs available for treatment have several side effects and a difficult route of administration, which causes the patient to abandon treatment and the emergence of resistant strains. Our research group works in the pre-clinical stage of drug development against leishmaniasis, testing molecules of natural and/or synthetic origin and investigating their possible mechanisms of action. In this work, the in vitro leishmanicidal activity (EC50) of eight tetracationic porphyrins (4-PtTPyP, 3-PtTPyP, 4-PdTPyP, 3-PdTPyP, 4-TPyP, 3-TPyP, 4-TMePyP, and 3 -TMePyP) against L. amazonensis through Photodynamic Therapy (PDT). Compounds were evaluated for their photocytotoxicity (CC50) on murine macrophages and their selectivity indices (IS) were calculated. The porphyrins 3-PtTPyP (EC50: 8.7 ± 0.4 nM; IS: 213), 3-PdTPyP (EC50: 15 ± 5 nM; IS: 85.2) and 3-TPyP (EC50: 25.5 ± 7.5 nM; IS: 61.9) used as photosensitizers (FS) through PDT proved to be the most phototoxic against the promastigote forms of the parasite. In the anti-amastigote activity (EC50-AMA) it was found that in general the FS: 3-TPyP (47.9 ± 4.4 nM; IS: 32), 3-PdTPyP (38.8 ± 11.5 nM; IS: 32.9) and 3-PtTPyP (27.6 ± 6.7nM; IS: 67) are excellent candidates to be tested in vivo in the future. Due to the interaction between FS and light, the production of reactive oxygen species (ROS) was measured, and the porphyrins 4-PtTPyP, 3-PtTPyP and 3-PdTPyP induced the highest levels of ROS in the parasite. Additionally, co-localization assays with porphyrins were performed using the markers MitoTracker Orange (mitochondria marker) and LysoTracker Green (lysosomes or acidic pools marker) by confocal microscopy, finding co-localization of the molecules in both organelles, mitochondria and acidic pools. Analyzes of the type of cell death potentially induced by FSs were performed by labeling with ethidium bromide/acridine orange (BE/LA). The molecules 3-PdTPyP, 3-PtTPyP and their isomers (4-PdTPyP, 4-PtTPyP) in the presence of white light after 6 and 24 h of irradiation showed to be the FSs with the greatest leishmanicidal effect. 3-PdTPyP and 3-PtTPyP induced parasite death mainly by necrosis (74.2% and 52.2% after 6h, respectively; and 79% and 78% after 24h, respectively). While the 4-PtTPyP isomer induced death of promastigotes by like-apoptosis (81%) after 6h of irradiation, and after 24h it directed the parasites to death by necrosis (33.6%) and like-apoptosis (28.2 %), similar to that observed in the 4-PdTPyP isomer at both times (40% necrosis/20%like-apoptosis and 54.5%necrosis/25.5%like-apoptosis, after 6h and 24h of irradiation, respectively). These data suggest that the tetracationic platinum and palladium porphyrins used here have great potential to be used in the treatment of cutaneous leishmaniasis by means of photodynamic therapy. Key-words: Leishmaniasis. Leishmania amazonensis. Porphyrins. photodynamic therapy
Descrição
Palavras-chave
Leishmaniose, Porfirinas, Fototerapia, Doenças negligenciadas, Celulas e organelas