Avaliação do perfil lítico do micobacteriófago D29 livre e encapsulado em lipossoma frente Mycobacterium tuberculosis H37Rv em estado replicante e sua atividade intramacrofágica.
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Data
2018-10-22
Autores
Silva, Ana Paula Souza
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Universidade Estadual Paulista (Unesp)
Resumo
A Tuberculose é uma doença infecto contagiosa causada pelo agente etiológico Mycobacterium tuberculosis, sendo este a causa de 1,3 milhões de mortes ao redor do mundo no ano de 2018. O tratamento preconizado pela Organização Mundial da Saúde é eficaz para cepas sensíveis, porém os efeitos adversos dos fármacos levam muitos pacientes ao abandono da terapia e consequente surgimento de resistência micobacteriana. Dentro desse contexto e mediante o reduzido número de fármacos disponíveis para o tratamento se faz necessário o desenvolvimento de novas alternativas terapêuticas. Bacteriófagos, vírus que infectam bactérias, têm sido sugeridos como importantes agentes terapêuticos no combate a bactérias multirresistentes, os quais podem ser encapsulados em lipossoma objetivando a proteção contra a degradação pelo sistema imune. O objetivo deste trabalho foi avaliar o perfil do micobacteriófago D29 livre e encapsulado em lipossoma frente a cepa padrão de M. tuberculosis (H37Rv). Os lipossomas apresentaram tamanho, PDI e valores de potencial zeta pelo DLS (Dynamic Light Scaterring). De acordo com imagens obtidas por microscopia de transmissão, foram classificados com vesículas unilamelares gigantes com uma eficiência de encapsulação fágica de 9,4 % ± 0,023. Para garantir mais de 50 % em viabilidade celular, com a linhagem MRC-5 e J774A.1, respectivamente o volume máximo de 80% e 60% das amostras (tampão de fago, micobacteriófago D29, micobacteriófago D29 encapsulado em lipossoma e lipossoma vazio) foi utilizado nos ensaios. Além disso, com imagens por microscopia de fluorescência com a linhagem MRC-5, foi possível sugerir que o lipossoma contendo fagos atingiu o interior das células. A inibição do crescimento micobacteriano foi determinada por um ensaio de microdiluição e observou-se que o MOI de 0,001 para o fago D29 livre e encapsulado foi capaz de inibir 90% do crescimento micobacteriano. Avaliando a atividade intramacrofágica, em maiores concentrações, os micobacteriófagos D29 encapsulados em lipossoma apresentaram uma maior ação intramacrofágica em relação aos micobacteriófagos livres. Os resultados sugerem que os lipossomas possibilitam a proteção dos micobacteriófagos D29 para o interior dos macrófagos contribuindo com a eliminação de micobactérias no interior dos macrófagos.
Tuberculosis is a contagious infectious disease caused by the etiological agent Mycobacterium tuberculosis and the cause of 1.3 million of deaths around the world in the 2018. The treatment endorsed by the World Health Organization is effective for sensitive strains, but the adverse drug effects may lead patients to abandon therapy and consequently the emergence of mycobacterial resistance. Within that context and by the low number of drugs available for treating tuberculosis, bacteriophages, viruses that infect bacteria, have been remembered as important therapeutic agents to combat drug resistance which can be encapsulated in liposome aiming to protect against degradation by the immune system. The objective of this work was to evaluate the profile of the micobacteriophage D29 free and encapsulated in liposome in front of the standard strain of M. Tuberculosis (H37RV). Lipossomes presented size, IPD and Zeta Potential values by DLS (Dynamic Light scaterring). According to images obtained by transmission microscopy, they were classified with giant unilamelular vesicles with and efficiency of phagic encapsulation of 9.4% ± 0.023. To guarantee more than 50% in cellular viability, with the lineage MRC-5 and J774A. 1, respectively the concentration of 80% and 60% of the samples (phage buffer, micobacteriófago D29, micobacteriófago D29 encapsulated in liposome and liposome empty) was used in the tests. Furthermore, with fluorescence microscopy imaging with the MRC-5 lineage, it was possible to suggest that the liposomal containing phage reached the inside of the cells. The inhibition of mycobacteria growth was determined by a microdilution assay and it was observed that the MOI of 0.001 for D29 phage free and encapsulated in liposome was able to inhibit 90% of mycobacteria growth. Evaluating the intra macrophages activity, in higher concentrations, encapsulated phages presented a greater intra macrophages action in relation to free phage. The results suggest that liposomes allow the protection of mycobacteriophages D29 for the inside of macrophages, contributing to the elimination of mycobacteria within the macrophages.
Tuberculosis is a contagious infectious disease caused by the etiological agent Mycobacterium tuberculosis and the cause of 1.3 million of deaths around the world in the 2018. The treatment endorsed by the World Health Organization is effective for sensitive strains, but the adverse drug effects may lead patients to abandon therapy and consequently the emergence of mycobacterial resistance. Within that context and by the low number of drugs available for treating tuberculosis, bacteriophages, viruses that infect bacteria, have been remembered as important therapeutic agents to combat drug resistance which can be encapsulated in liposome aiming to protect against degradation by the immune system. The objective of this work was to evaluate the profile of the micobacteriophage D29 free and encapsulated in liposome in front of the standard strain of M. Tuberculosis (H37RV). Lipossomes presented size, IPD and Zeta Potential values by DLS (Dynamic Light scaterring). According to images obtained by transmission microscopy, they were classified with giant unilamelular vesicles with and efficiency of phagic encapsulation of 9.4% ± 0.023. To guarantee more than 50% in cellular viability, with the lineage MRC-5 and J774A. 1, respectively the concentration of 80% and 60% of the samples (phage buffer, micobacteriófago D29, micobacteriófago D29 encapsulated in liposome and liposome empty) was used in the tests. Furthermore, with fluorescence microscopy imaging with the MRC-5 lineage, it was possible to suggest that the liposomal containing phage reached the inside of the cells. The inhibition of mycobacteria growth was determined by a microdilution assay and it was observed that the MOI of 0.001 for D29 phage free and encapsulated in liposome was able to inhibit 90% of mycobacteria growth. Evaluating the intra macrophages activity, in higher concentrations, encapsulated phages presented a greater intra macrophages action in relation to free phage. The results suggest that liposomes allow the protection of mycobacteriophages D29 for the inside of macrophages, contributing to the elimination of mycobacteria within the macrophages.
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Palavras-chave
Resistência micobacteriana, Micobacteriófago D29, Lipossomas, Mycobacterial Resistance, Mycobacteriophage D29