Estudos de caracterização e aplicação de lipossomas associados à peptídeos antifúngicos derivados da histatina-5 visando o combate de Candida albicans
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Data
2022-08-19
Autores
Oliveira, Jessica Ellen
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Universidade Estadual Paulista (Unesp)
Resumo
Os lipossomas PEGuilados apresentam capacidade de duração in vivo capaz de realizar a liberação prolongada de moléculas, esta ação, pode ser um fator capaz inibir o crescimento de microrganismos patogênicos e a formação de biofilme. Assim, a proposta deste trabalho consiste no desenvolvimento de formulações PEGuiladas associada ao derivado de Histatina-5, 0WHistatina-5, para tratamento de candidíase oral e inibição de biofilmes orais formadas por Candida albicans. Para isso, foi aplicado o método de síntese de peptídeo em fase sólida, utilizada a cromatografia de alta eficiência e caracterização em espectrometria de massas com ionização por eletrospray modo positivo (LC/ESI-MS+) para obtenção dos derivados de 0WHistatina-5 e 1WHistatina-5, com obtenção dos peptídeos foram realizadas as análises antimicrobianas de concentração inibitória mínima (CIM), no qual 0WHistatina-5 demonstrou atividade inibitória a 1,03 μg/mL, sendo o peptídeo escolhido para as demais análises. Para os sistemas lipossomais foi proposto o método de evaporação em fase reversa, o qual, permitiu a associação aos derivados peptídicos em até 88,65% de eficiência para o peptídeo 0Whistatina-5. Visando a caracterização morfológica, os lipossomas foram analisados por técnica de espalhamento de luz dinâmico com estabilidade a 4ºC por 42 dias, tendo como desestruturação em pH 4,5 sendo um fator de seletividade. As análises de variação de temperatura de 0ºC a 100ºC por calorímetro diferencial de varredura e dicroísmo circular evidenciaram a interação entre o sistema lipossomal PEGuilado e o peptídeo 0Whistatina-5. Para curva de crescimento e cinética de morte, ensaio de morte celular, viabilidade celular por resazurina (AlamarBlue®) e microscopia confocal com aplicação de calcofluor-white, Concavalina A e isocianato de fluoresceína foi utilizada a cepa ATCC 10231 resistente ao fluconazol e produtora de biofilme orais. Tais, técnicas permitiram inferir que a formulação composta por fosfolipídio e polietilenoglicol (PP) gerou a liberação prolongada do peptídeo antifúngico 0Whistatina-5 após 12 horas com inibição parcial do microrganismo, assim como as células presente no biofilme. Portanto, o método de evaporação reversa demonstrou-se eficiente para associação de macromoléculas em sistemas lipossomais de segunda geração, como os lipossomas PEGuilados gerando efeito prolongado do peptídeo 0WHistatina-5.
PEGylated liposomes have the ability to last in vivo capable of carrying out the prolonged release of molecules and this action may be a factor capable of inhibiting the growth of pathogenic microorganisms and the formation of biofilms. Thus, the purpose of this work is the development of PEGylated formulations associated with the Histatin-5 derivative, 0WHistatin-5, for the treatment of oral candidiasis and inhibition of oral biofilms formed by Candida albicans. For this, the solid phase peptide synthesis method was applied, using high performance chromatography and characterization in positive mode electrospray ionization mass spectrometry (LC/ESI-MS+) to obtain the 0WHistatin-5 and 1WHistatin-5 derivatives to obtain the peptides, antimicrobial analyses of minimal inhibitory concentration (MIC) were performed, in which 0WHistatin-5 showed inhibitory activity at 1.03 μg/mL being the peptide, 0WHistatin-5 chosen for the other analyses. For liposomal systems, the reversed-phase evaporation method was proposed, which allowed the association with peptide derivatives in up to 88.65% efficiency for the 0Whistatin-5 peptide. Aiming at morphological characterization, the liposomes were analysed by dynamic light scattering technique with stability at 4ºC for 42 days, with destructuring at pH 4.5 being a selectivity factor. The analysis of temperature variation from 0ºC to 100ºC by differential scanning calorimeter and circular dichroism showed the interaction between the PEGylated liposomal system and the 0Whistatin-5 peptide. For the growth curve and death kinetics, cell death assay, cell viability by resazurin (AlamarBlue®) and confocal microscopy with application of calcofluor-white, Concavalin A and fluorescein isocyanate, fluconazole-resistant strain ATCC 10231 was used oral biofilm. Such techniques allowed us to infer that the formulation composed of phospholipid and polyethylene glycol (PP) generated the prolonged release of the antifungal peptide 0Whistatin-5 after 12 hours with partial inhibition of the microorganism, as well as the cells present in the biofilm. Therefore, the reverse evaporation method proved to be efficient for the association of macromolecules in second-generation liposomal systems, such as PEGylated liposomes, generating a prolonged effect of the 0WHistatin-5 peptide.
PEGylated liposomes have the ability to last in vivo capable of carrying out the prolonged release of molecules and this action may be a factor capable of inhibiting the growth of pathogenic microorganisms and the formation of biofilms. Thus, the purpose of this work is the development of PEGylated formulations associated with the Histatin-5 derivative, 0WHistatin-5, for the treatment of oral candidiasis and inhibition of oral biofilms formed by Candida albicans. For this, the solid phase peptide synthesis method was applied, using high performance chromatography and characterization in positive mode electrospray ionization mass spectrometry (LC/ESI-MS+) to obtain the 0WHistatin-5 and 1WHistatin-5 derivatives to obtain the peptides, antimicrobial analyses of minimal inhibitory concentration (MIC) were performed, in which 0WHistatin-5 showed inhibitory activity at 1.03 μg/mL being the peptide, 0WHistatin-5 chosen for the other analyses. For liposomal systems, the reversed-phase evaporation method was proposed, which allowed the association with peptide derivatives in up to 88.65% efficiency for the 0Whistatin-5 peptide. Aiming at morphological characterization, the liposomes were analysed by dynamic light scattering technique with stability at 4ºC for 42 days, with destructuring at pH 4.5 being a selectivity factor. The analysis of temperature variation from 0ºC to 100ºC by differential scanning calorimeter and circular dichroism showed the interaction between the PEGylated liposomal system and the 0Whistatin-5 peptide. For the growth curve and death kinetics, cell death assay, cell viability by resazurin (AlamarBlue®) and confocal microscopy with application of calcofluor-white, Concavalin A and fluorescein isocyanate, fluconazole-resistant strain ATCC 10231 was used oral biofilm. Such techniques allowed us to infer that the formulation composed of phospholipid and polyethylene glycol (PP) generated the prolonged release of the antifungal peptide 0Whistatin-5 after 12 hours with partial inhibition of the microorganism, as well as the cells present in the biofilm. Therefore, the reverse evaporation method proved to be efficient for the association of macromolecules in second-generation liposomal systems, such as PEGylated liposomes, generating a prolonged effect of the 0WHistatin-5 peptide.
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Palavras-chave
Lipossomas, Peptídeos antimicrobianos catiônicos, Candidíase, Candida albicans, Biofilmes