Fotossensibilização de sistemas miméticos de membrana celular induzida pelo fenotiazínico azul de toluidina
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Data
2020-02-17
Autores
Almeida Junior, Alexandre Mendes
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Universidade Estadual Paulista (Unesp)
Resumo
O aumento alarmante da resistência bacteriana aos antibióticos exige novas estratégias para inativação microbiana, como a terapia fotodinâmica (TFD), cuja atividade depende dos danos fotodinâmicos induzidos à membrana do microrganismo. Neste trabalho, os mecanismos de adsorção do fotossensibilizador azul de toluidina-O (ATO) em modelos simplificados de membrana bacteriana baseados em filmes de Langmuir de 1,2-dioleoil-sn-glicero-3-fosfoetanolamina (DOPE) e 1,2-dioleoil-sn-glicero-3-fosfo(1'-rac-glicerol) (DOPG) foram correlacionados com os efeitos da oxidação lipídica fotoinduzida. A escolha destes fosfolipídios ocorreu por análise da composição lipídica das membranas celulares de bactérias Gram-negativas, onde há maior abundância de fosfatidiletanolaminas e fosfatidilglicerol. Monocamadas de Langmuir de 1,2-dioleoil-sn-glicero-3-fosfocolina (DOPC) foram utilizadas como referência as membranas de células mamíferas. As moléculas de ATO foram diluídas em água ultrapura e concentradas em soluções distintas de 10-4 e 10-5 mol/L. As isotermas de pressão superficial indicaram que o ATO expande as monocamadas de DOPC, DOPE e DOPG e as tornam mais estáveis sobre a superfície da subfase devido às interações eletrostáticas com os grupos carregados negativamente nos fosfolipídios, com uma adsorção mais forte no DOPG, que possui carga superficial líquida. Os espectros de espectroscopia vibracional de absorção e reflexão no infravermelho com polarização modulada (PM-IRRAS) corroboram essa hipótese ao apontar que os grupos fosfato aniônicos são os mais afetados ao interagir com o ATO catiônico. A irradiação tornou as monocamadas de DOPC e DOPE associadas ao ATO menos instáveis como resultado da reação de oxigênios singletos (1O2) com as insaturações das cadeias e a formação de hidroperóxidos. Por outro lado, a estabilidade reduzida na monocamada de DOPG irradiada em solução de ATO sugere a clivagem das cadeias alifáticas. A natureza aniônica do DOPG permitiu uma penetração do ATO na região das cadeias, favorecendo as reações dependentes de contato entre o estado tripleto excitado do ATO e as insaturações lipídicas ou grupos hidroperóxidos, que é a chave para as reações de clivagem e permeabilização da membrana.
The alarming increase in bacterial resistance to antibiotics demand new strategies for microbial inactivation, which include photodynamic therapy (PDT) whose activity relies on the photoreaction damage to the microorganism membrane. In this work, the binding mechanisms of the photosensitizer toluidine blue-O (TBO) on simplified models of bacterial membrane with Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) were correlated to the effects of the photoinduced lipid oxidation. These phospholipids were chosen analyzing the lipid composition of the cell membranes of Gram-negative bacteria, which has greater abundance of phosphatidylethanolamines and phosphatidylglycerol. Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were also used as a reference of mammalian membranes. The TBO molecules were diluted in ultrapure water and concentrated in different solutions of 10-4 and 10-5 mol/L. The surface pressure isotherms indicated that TBO expands DOPC, DOPE and DOPG monolayers and makes than more stable on the subphase surface owing to electrostatic interactions with the negatively charged groups in the phospholipids, with a stronger adsorption on DOPG, which has a net surface charge. Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) spectra corroborate this hypothesis by pointing out that anionic phosphate groups are the most affected when interacting with cationic TBO. Light irradiation made the TBO-containing DOPC and DOPE monolayers less unstable as a result of the singlet oxygen (1O2) reaction with the chain unsaturation and hydroperoxide formation. In contrast, the decreased stability of the irradiated TBO-containing DOPG monolayers suggests the cleavage of carbon chains. The anionic nature of DOPG allowed a deeper penetration of TBO into the chain region, favoring contact-dependent reactions between the excited triplet state of TBO and lipid unsaturations or/and hydroperoxide groups, which is the key for the cleavage reactions and further membrane permeabilization.
The alarming increase in bacterial resistance to antibiotics demand new strategies for microbial inactivation, which include photodynamic therapy (PDT) whose activity relies on the photoreaction damage to the microorganism membrane. In this work, the binding mechanisms of the photosensitizer toluidine blue-O (TBO) on simplified models of bacterial membrane with Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (DOPG) were correlated to the effects of the photoinduced lipid oxidation. These phospholipids were chosen analyzing the lipid composition of the cell membranes of Gram-negative bacteria, which has greater abundance of phosphatidylethanolamines and phosphatidylglycerol. Langmuir monolayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were also used as a reference of mammalian membranes. The TBO molecules were diluted in ultrapure water and concentrated in different solutions of 10-4 and 10-5 mol/L. The surface pressure isotherms indicated that TBO expands DOPC, DOPE and DOPG monolayers and makes than more stable on the subphase surface owing to electrostatic interactions with the negatively charged groups in the phospholipids, with a stronger adsorption on DOPG, which has a net surface charge. Polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) spectra corroborate this hypothesis by pointing out that anionic phosphate groups are the most affected when interacting with cationic TBO. Light irradiation made the TBO-containing DOPC and DOPE monolayers less unstable as a result of the singlet oxygen (1O2) reaction with the chain unsaturation and hydroperoxide formation. In contrast, the decreased stability of the irradiated TBO-containing DOPG monolayers suggests the cleavage of carbon chains. The anionic nature of DOPG allowed a deeper penetration of TBO into the chain region, favoring contact-dependent reactions between the excited triplet state of TBO and lipid unsaturations or/and hydroperoxide groups, which is the key for the cleavage reactions and further membrane permeabilization.
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Palavras-chave
Terapia Fotodinâmica, Filmes de Langmuir, Azul de toluidina, Foto-oxidação, Membrana bacteriana