Estudo do potencial antifúngico de compostos de coordenação de Ag (I) não incorporados e incorporados em sistema nanotecnológico de liberação de fármacos
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Data
2020-06-26
Autores
Fortunato, Giovanna Capaldi
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Universidade Estadual Paulista (Unesp)
Resumo
O aumento de linhagens resistentes aos medicamentos utilizados nos tratamentos clínicos, configuram a principal razão dos casos de falhas das terapias antifúngicas existentes. Neste sentido, novos compostos como os complexos de Ag(I) têm sido testadas contra Candida sp. A literatura tem demonstrado, que o uso de compostos de coordenação de Ag(I) frente as linhagens fúngicas são promissores no que se refere ao potencial antifúngico. Todavia, os complexos são insolúveis em água, necessitando de recursos nanotecnológicos para sua veiculação. Sistemas nanoestruturados baseados em lipídios tem sido empregado com o objetivo de promover a melhora da performance farmacológica dos mesmos. Este estudo propõe avaliar o potencial antifúngico de compostos de coordenação de Ag(I) incorporados e não incorporados em um sistema lipídico nanoestruturado, voltando-se ao controle de espécies de Candida. Foi desenvolvido um sistema de liberação de fármacos (nanoemulsão lipídica – NL) seguido da caracterização físico-química dos sistemas (dispersão de luz dinâmica, Microscopia Eletrônica de Transmissão e ensaios reológicos). A atividade antifúngica dos compostos,CCP1.[Ag(PhTSC∙HCl)2](NO3)∙H2O,CCP2.[Ag(HTSC∙HCl)2](NO3)∙H2O,,CCP3.[Ag(PhTSC∙HCl)2](Cl)∙H2O e CCP4.[Ag(HTSC∙HCl)2](Cl)∙H2O (não incorporado) frente as linhagens de C. albicans ATCC10231, C. glabrata ATCC2001, C. krusei, ATCC6258, C. parapsilosis ATCC22019 e C.tropicalis, ATCC750, foram avaliadas por determinação da concentração inibitória mínima (CIM) pelo método de microdiluição. Além disso foi realizado o ensaio de concentração fungicida mínima, que permitiu avaliar a capacidade de crescimento fúngico decorrido 48 horas de incubação a 37ºC. Os ensaios foram realizados por metodologias in vitro, a fim de elucidar o perfil antifúngico, dando destaque aos resultados referentes ao composto 1. [Ag(PhTSC∙HCl)2](NO3)∙H2O (CCP1), o qual, apresentou CIM de 3,9 μg/mL e CFM de 7,8 μg/mL para Candida albicans. Adicionalmente foi realizado e ensaio de biofilme em formação, e biofilmes maduros em ensaios de 48h e 72h, os quais obtiveram inibição de 100% a 2000μg/mL para o CCP1. Ainda foram realizados ensaios de sorbitol e ergosterol os quais sugerem que o CCP1 possa atuar na biossíntese de parede celular, bem como, na permeação de íons na membrana fúngica. O ensaio de mutagenicidade revelou que que não há índices mutagênicos significantes para o composto [Ag(PhTSC∙HCl)2](NO3)∙H2O carreado em nanoemulsão lipídica (CCPNL1). O ensaio tóxico agudo em modelo de G. mellonella revelou que em concentrações que variaram de 5 mg/kg a 100 mg/kg foi atóxica, visto que, a sobrevida larval foi de 100% nas condições avaliadas.
The increase in drug resistant strains used in clinical treatments is the main reason for the failure of existing antifungal therapies. In this sense, new molecules such as Ag(I) complexes have been tested against Candida sp. The literature has demonstrated that the use of Ag(I) coordination compounds against fungal strains are promising in terms of antifungal potential. However, the complexes are insoluble in water, requiring nanotechnological resources for their solubility. Nanostructured lipid systems have been employed to improve their pharmacological performance. This study proposes to evaluate the antifungal potential of unloaded and loaded Ag (I) coordination compounds into nanostructured lipid system, focusing on the control of Candida species. A drug release system (lipid nanoemulsion - LN) was developed and characterized by (dynamic light scattering, transmission electron microscopy, Zeta Potential, rheological assays), aiming at the incorporation of Ag (I) coordination compounds. CCP1. [Ag (PhTSC∙ HCl)2](NO3)∙ H2O, CCP2. [Ag (HTSC ∙ HCl) 2](NO3)∙H2O, CCP3. [Ag (PhTSC ∙ HCl) 2] (Cl)∙H2O and CCP4. [Ag (HTSC∙ HCl)2](Cl)∙H2O against C. albicans strains ATCC10231, C. glabrata ATCC2001, C. krusei, ATCC6258, C. parapsilosis ATCC22019 and C. tropicalis ATCC750, were evaluated by determination of the minimum inhibitory concentration (MIC) by the 96-well plate. In addition, the minimum fungicidal concentration test was performed, to evaluate the fungal growth capacity after 48 hours of incubation at 37º. The assays were performed by in vitro methodology in order to elucidate the desired antifungal profile, highlighting the results related to the compound CCP1. [Ag(PhTSC∙HCl)2](NO3)∙H2O, which presented MIC of 3.9 μg / mL and MFC of 7.8 μg/mL for C. albicans. It is noteworthy that all compounds tested against the five fungal strains show satisfactory results. Thus, the MIC and MCF assays showed the great potential of Ag (I) coordination compounds to inhibit the fungal, showing to be promising against the infections caused by Candida species. In addition, a biofilm assay in formation, (48h) and mature (72h) were perfomed, which obtained 100% inhibition at 2000μg / mL for coordinated silver compound (CSC1). Additionally, sorbitol and ergosterol assays were performed which suggest that CSC1 may act on cell wall biosynthesis, as well as ion permeation in the fungal membrane. The mutagenicity test (Ames) for loaded coordinated silver compound into lipid nanoemulsion (CSCLN1) showed a mutagenicity index lower 2.0 against all strains, demonstrating the absence of mutagenic activity in the absence and presence of metabolism. The acute toxic assay in the G. mellonella model revealed that at concentrations ranging from 5μg / mL to 100μg / mL it was non-toxic, since the larval survival was 100% under the conditions evaluated.
The increase in drug resistant strains used in clinical treatments is the main reason for the failure of existing antifungal therapies. In this sense, new molecules such as Ag(I) complexes have been tested against Candida sp. The literature has demonstrated that the use of Ag(I) coordination compounds against fungal strains are promising in terms of antifungal potential. However, the complexes are insoluble in water, requiring nanotechnological resources for their solubility. Nanostructured lipid systems have been employed to improve their pharmacological performance. This study proposes to evaluate the antifungal potential of unloaded and loaded Ag (I) coordination compounds into nanostructured lipid system, focusing on the control of Candida species. A drug release system (lipid nanoemulsion - LN) was developed and characterized by (dynamic light scattering, transmission electron microscopy, Zeta Potential, rheological assays), aiming at the incorporation of Ag (I) coordination compounds. CCP1. [Ag (PhTSC∙ HCl)2](NO3)∙ H2O, CCP2. [Ag (HTSC ∙ HCl) 2](NO3)∙H2O, CCP3. [Ag (PhTSC ∙ HCl) 2] (Cl)∙H2O and CCP4. [Ag (HTSC∙ HCl)2](Cl)∙H2O against C. albicans strains ATCC10231, C. glabrata ATCC2001, C. krusei, ATCC6258, C. parapsilosis ATCC22019 and C. tropicalis ATCC750, were evaluated by determination of the minimum inhibitory concentration (MIC) by the 96-well plate. In addition, the minimum fungicidal concentration test was performed, to evaluate the fungal growth capacity after 48 hours of incubation at 37º. The assays were performed by in vitro methodology in order to elucidate the desired antifungal profile, highlighting the results related to the compound CCP1. [Ag(PhTSC∙HCl)2](NO3)∙H2O, which presented MIC of 3.9 μg / mL and MFC of 7.8 μg/mL for C. albicans. It is noteworthy that all compounds tested against the five fungal strains show satisfactory results. Thus, the MIC and MCF assays showed the great potential of Ag (I) coordination compounds to inhibit the fungal, showing to be promising against the infections caused by Candida species. In addition, a biofilm assay in formation, (48h) and mature (72h) were perfomed, which obtained 100% inhibition at 2000μg / mL for coordinated silver compound (CSC1). Additionally, sorbitol and ergosterol assays were performed which suggest that CSC1 may act on cell wall biosynthesis, as well as ion permeation in the fungal membrane. The mutagenicity test (Ames) for loaded coordinated silver compound into lipid nanoemulsion (CSCLN1) showed a mutagenicity index lower 2.0 against all strains, demonstrating the absence of mutagenic activity in the absence and presence of metabolism. The acute toxic assay in the G. mellonella model revealed that at concentrations ranging from 5μg / mL to 100μg / mL it was non-toxic, since the larval survival was 100% under the conditions evaluated.
Descrição
Palavras-chave
Compostos de Ag (I);, Nanoemulsão lipídica, Concentração inibitória mínina (CIM), Ag (I) compounds, Lipid Nanoemulsion, Minimal Inhibitory Concentration (MIC)