Derivados anfifílicos de quitosana como agentes antimicrobianos: estudo da atividade e mecanismo de ação
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Data
2017-05-05
Autores
Dias, Amanda Manchini [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho descreve a síntese, caracterização e o estudo do mecanismo da atividade antimicrobiana de derivados anfifílicos de quitosana contra o fungo Aspergillus flavus. As quitosanas utilizadas nas sínteses foram obtidas pela desacetilação heterogênea de quitosana comercial. O derivado hidrofílico foi obtido pela reação de quitosana com cloreto de 2-cloro-N,N-dietilaminoetil (DEAE). O grau de substituição (GS) de grupos DEAE inseridos na cadeia polimérica foi determinado utilizando-se as técnicas de Ressonância Magnética Nuclear de Hidrogênio (RMN¹H) e Espectroscopia de absorção na região do Infravermelho (FTIV), obtendo-se 40% de substituição (QD 40% DEAE). Em seguida, a quitosana-DEAE foi degradada com nitrito de sódio para obtenção de polímeros com diferentes massas moleculares (Mw). As massas moleculares foram determinadas por Cromatografia de Permeação em Gel (GPC), e os valores obtidos foram 8 - 25 - 60 - 116 e 136 kDa respectivamente. Na sequência, as quitosanas de diferentes Mw foram alquiladas com dodecilaldeído seguido pela redução com borohidreto de sódio, obtendo-se derivados com ~20% de conteúdo hidrofóbico caracterizados por RMN¹H e FTIV. O comportamento em solução dos derivados anfifílicos foi estudado utilizando Espectroscopia de Fluorescência, e os resultados mostraram que em baixas concentrações os derivados de quitosana se auto-associam formando domínios hidrofóbicos. A agregação ocorre a partir da concentração de agregação crítica (CAC) que depende da massa molecular e do grau de substituição dos grupos hidrofóbicos presentes na estrutura polimérica. Os resultados dos ensaios microbiológicos mostram uma maior inibição do crescimento fúngico para os derivados de menor massa molecular (25 e 8 kDa) que pode estar relacionada a uma melhor interação entre o polímero e a membrana e/ou parede celular do A. flavus. Para investigar o mecanismo de interação da macromolécula com o fungo, realizou-se o estudo da interação dos diferentes polímeros anfifílicos com membranas modelo preparadas com os fosfolipídios L-α-fosfatidilcolina e L-α-fosfatidil-DL-glicerol, utilizando-se medidas de Espalhamento de Luz Dinâmico (DLS) e Potencial Zeta. Os resultados obtidos permitiram postular um modelo baseado na força de interação polímero-lipossoma, que depende principalmente da massa molecular e da carga superficial da vesícula. Para as vesículas zwiteriônicas a interação com a bicamada depende mais criticamente da massa molecular, visto que nos derivados anfifílicos de 8 e 25 kDa a interação polímero-lipossoma levou a formação de vesículas estabilizadas, ou seja revestidas por uma camada de quitosana que se deposita na superfície vesicular de forma gradativa, sem a formação de agregados. Para os derivados de maiores Mw (136, 116 e 60 kDa) as interações são mais intensas provocando a formação de agregados de vesículas em concentrações muito baixas (> 0,1 g L-1), porém o aumento abrupto do potencial zeta leva a desagregação das partículas originando lipossomas de ~300 nm de diâmetro. As imagens obtidas por Microscopia Eletrônica de Transmissão mostraram que o polímero atua na desestruturação da parede celular, pela associação com os polissacarídeos e proteínas da parede. Correlacionando-se as imagens obtidas pela microscopia confocal com as imagens de microscopia eletrônica de transmissão, pode se afirmar que a maior interação observada entre o polímero e os conídios se deve pela presença de maior concentração de proteínas hidrofóbicas na superfície dos mesmos, potencializando as interações entre o polímero e os constituintes da parede celular.
This study describes the synthesis, characterization and the study of mechanism and antimicrobial activity of chitosan derivatives against the fungus Aspergillus flavus. Chitosan was obtained by the heterogeneous deacetylation of commercial chitosan. The hydrophilic derivative was obtained by reaction of chitosan with 2-chloro-N, N-diethylaminoethyl (DEAE) chloride. The degree of substitution (GS) of DEAE groups was determined using the techniques of Nuclear Magnetic Resonance of Hydrogen (NMR¹H) and Absorption Spectroscopy in the Infrared region (FTIV), obtaining 40% of substitution. Afterwards, samples of the obtained derivative were degraded with sodium nitrite to obtain polymers with different molecular weights (Mw). Molecular weights were determined by Gel Permeation Chromatography (GPC), and the obtained values were 8 - 25 - 60 - 116 and 136 kDa respectively. Subsequently, chitosans with different molecular masses were alkylated with dodecyl aldehyde followed by reduction with sodium borohydride to provide ~ 20% hydrophobic content that was characterized by NMR¹H. The solution behavior of the amphiphilic derivatives was studied using Fluorescence Spectroscopy, and the results showed that at low concentrations, chitosan derivatives form self-assembled particles with hydrophobic domains. Derivatives self-aggregate at the critical aggregation concentrations (CAC), which were shown to be dependent on the molecular mass and degree of substitution by hydrophobic groups present in the polymeric structure. The results of the microbiological assays showed a greater inhibition for derivatives having low molecular weights which may be related to a better interaction between the polymer chain and the cell wall and cell membrane of the A. flavus, which can also be associated to the smaller size of the chain polymer of such derivatives. To investigate the mechanism of interaction of the macromolecule with the fungus, the study of the different amphiphilic polymers with model membranes prepared with the phospholipids L-alpha-phosphatidylcholine and L-alpha-phosphatidyl-DL-glycerol was carried out using measurements of Dynamic Light Scattering (DLS) and zeta potential. The results allowed to postulate an interaction model based on the strength of interaction, which depends mainly of the molecular weight and the surface charge of the vesicle. For the zwitterionic vesicles the interaction with the surface depends more critically on the molecular mass, for the amphiphiles of low Mw (Mw 25 and 8 kDa) the interaction leads to the formation of aggregates of vesicles. For the derivatives with higher Mw the interactions are stronger and the abrupt increase of the zeta potential leads to the disintegration of the aggregates. The images obtained by Transmission Electron Microscopy showed that the polymer acts in the disassembly of the cell wall by the association with its constituents. By the correlation of the images obtained by the confocal microscopy, it can be affirmed that the greater interaction observed with the conidia is due to the presence of a greater concentration of hydrophobic proteins in their surface, strengthening the interactions between the polymer and the constituents of the cell wall.
This study describes the synthesis, characterization and the study of mechanism and antimicrobial activity of chitosan derivatives against the fungus Aspergillus flavus. Chitosan was obtained by the heterogeneous deacetylation of commercial chitosan. The hydrophilic derivative was obtained by reaction of chitosan with 2-chloro-N, N-diethylaminoethyl (DEAE) chloride. The degree of substitution (GS) of DEAE groups was determined using the techniques of Nuclear Magnetic Resonance of Hydrogen (NMR¹H) and Absorption Spectroscopy in the Infrared region (FTIV), obtaining 40% of substitution. Afterwards, samples of the obtained derivative were degraded with sodium nitrite to obtain polymers with different molecular weights (Mw). Molecular weights were determined by Gel Permeation Chromatography (GPC), and the obtained values were 8 - 25 - 60 - 116 and 136 kDa respectively. Subsequently, chitosans with different molecular masses were alkylated with dodecyl aldehyde followed by reduction with sodium borohydride to provide ~ 20% hydrophobic content that was characterized by NMR¹H. The solution behavior of the amphiphilic derivatives was studied using Fluorescence Spectroscopy, and the results showed that at low concentrations, chitosan derivatives form self-assembled particles with hydrophobic domains. Derivatives self-aggregate at the critical aggregation concentrations (CAC), which were shown to be dependent on the molecular mass and degree of substitution by hydrophobic groups present in the polymeric structure. The results of the microbiological assays showed a greater inhibition for derivatives having low molecular weights which may be related to a better interaction between the polymer chain and the cell wall and cell membrane of the A. flavus, which can also be associated to the smaller size of the chain polymer of such derivatives. To investigate the mechanism of interaction of the macromolecule with the fungus, the study of the different amphiphilic polymers with model membranes prepared with the phospholipids L-alpha-phosphatidylcholine and L-alpha-phosphatidyl-DL-glycerol was carried out using measurements of Dynamic Light Scattering (DLS) and zeta potential. The results allowed to postulate an interaction model based on the strength of interaction, which depends mainly of the molecular weight and the surface charge of the vesicle. For the zwitterionic vesicles the interaction with the surface depends more critically on the molecular mass, for the amphiphiles of low Mw (Mw 25 and 8 kDa) the interaction leads to the formation of aggregates of vesicles. For the derivatives with higher Mw the interactions are stronger and the abrupt increase of the zeta potential leads to the disintegration of the aggregates. The images obtained by Transmission Electron Microscopy showed that the polymer acts in the disassembly of the cell wall by the association with its constituents. By the correlation of the images obtained by the confocal microscopy, it can be affirmed that the greater interaction observed with the conidia is due to the presence of a greater concentration of hydrophobic proteins in their surface, strengthening the interactions between the polymer and the constituents of the cell wall.
Descrição
Palavras-chave
Aspergillus flavus, Aflatoxinas, Atividade antifúngica, Quitosana, Modelos de membrana