Avaliação da atividade antifúngica e citotoxicidade de microcristais de alfa vanadato de prata (α-AgVO3) sintetizados em diferentes temperaturas
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Data
2017-06-30
Autores
Pimentel, Bruna Natália Alves da Silva [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Nos últimos anos, os microcristais de prata têm se tornado foco de estudos. Uma das propriedades evidenciadas destes materiais é a sua atividade antimicrobiana contra diferentes microrganismos, devido a presença da prata na sua composição. Neste estudo, investigou-se a atividade antifúngica de microcristais de alfa vanadato de prata (α-AgVO3) contra Candida albicans (ATCC 90028) e sua citotoxicidade sobre células do tipo queratinócitos orais normais espontaneamente imortalizados (NOK-si). Os microcristias de α-AgVO3 foram sintetizados pelo método da co-precipitação sob três diferentes temperaturas (10, 20 e 30ºC) e caracterizados através de difração de raios-x, microscopia eletrônica de varredura por emissão de campo e espectroscopia Raman. A atividade antifúngica foi avaliada a partir da microdiluição seriada dos microcristais (de acordo com o Clinical & Laboratorial Standards Institute - CLSI), onde foram determinadas as concentrações inibitória (CIM) e fungicida mínimas (CFM). Imagens de microscopia de fluorescência com os microcristais nas concentrações inibitória e fungicida mínimas foram obtidas a fim de confirmar os achados microbiológicos. A viabilidade celular de células NOK-si foi avaliada através do ensaio Alamar Blue, e imagens de microscopia eletrônica de varredura (MEV) de todos os grupos avaliados foram realizadas. Nos ensaios celulares foram utilizadas apenas quatro concentrações dos microcristais (CIM, CFM, CIM diluída 10 vezes e concentrada 10 vezes). Os resultados obtidos foram submetidos ao teste de normalidade Shapiro-Wilk, e então avaliados com o teste Kruskal-Wallis, seguidos do pós teste de Dunn (α = .05), no software GraphPad Prism 7. Os três microcristais apresentaram os mesmos valores de CIM e CFM (3,90 μg/mL e 15,62 μg/mL, respectivamente). Ao avaliar a microscopia de fluorescência foi possível confirmar os resultados obtidos com o ensaio microbiológico, onde na CIM notou-se uma redução do número de células em comparação ao controle, e na CFM notou-se inviabilidade de todas as células. Quando avaliados com relação a citotoxicidade, os três microcristais apresentaram-se biocompatíveis nos valores de CIM, CFM e CIM diluída 10 vezes (p <0,05). As imagens de MEV foram consistentes com os achados no ensaio de Alamar Blue, onde apenas observou-se morte celular na concentração CIM concentrada 10 vezes. Através da morfologia da morte celular, sugeriu-se que os microcristais avaliados causam morte celular por necrose. Os três microcristais de α-AgVO3 mostraram resultados promissores como agentes antifúngicos contra C. albicans e apresentaram biocompatibilidade com o tipo celular estudado, tornando-os interessante para posteriores estudos in vivo.
In recent years, silver microcrystals have become the focus of studies. One property of these materials is its antimicrobial activity against different microorganisms, which is due to the presence of silver in its composition. In this study the antifungal activity of alpha silver vanadate (α-AgVO3) microcrystals against Candida albicans (ATCC 90028) and cytotoxicity on normal oral keratinocytes spontaneously immortalized (NOK-si) cells were investigated. The microcrystals were synthesized by co-precipitation (CP) method under three different temperatures (10, 20 and 30°C) and were characterized by x-ray diffraction, scanning electronic microscopy field emission guns and Raman spectroscopy. The antifungal activity was evaluated from serial microdilution of microcrystals (according to Clinical & Laboratorial Standards Institute - CLSI), where minimum inhibitory (MIC) and fungicide (MFC) concentrations were determined. Fluorescence microscopy images were obtained in order to confirm microbiological findings. Cell viability of NOK-si cells was evaluated by Alamar Blue assay, and scanning electron microscopy (SEM) images of all groups were performed. In these tests, only four concentrations of microcrystals (MIC, MFC, MIC 10 times diluted and MIC 10 times concentrated) were used. The results were submitted to Shapiro-Wilk’s test, and then they were evaluated using the Kruskal-Wallis test, followed by Dunn's post-test (α = .05), in GraphPad Prism 7 software. All three microcrystals presented the same values of MIC and MFC (3.90 μg/mL and 15.62 μg/mL, respectively). When evaluated fluorescence microscopy it was possible to confirm the results obtained with the microbiological assay, where at the MIC noticed a reduction in cell number when compared with the control, and at the MFC noticed unviability of all cells. When evaluated for its cytotoxicity, the three microcrystals were biocompatible in the MIC, MFC and MIC 10 times diluted values (p <0.05). SEM images were consistent with the findings in the Alamar Blue assay, where cell death was observed only at MIC 10 times concentrated. Through the morphology analyses of cell death, it was observed that the microcrystals at MIC 10 times concentrated caused cell death by necrosis. The three α-AgVO3 microcrystals showed promising results as antifungal agents against C. albicans and showed biocompatibility with the studied cell type, making them interesting for later in vivo studies.
In recent years, silver microcrystals have become the focus of studies. One property of these materials is its antimicrobial activity against different microorganisms, which is due to the presence of silver in its composition. In this study the antifungal activity of alpha silver vanadate (α-AgVO3) microcrystals against Candida albicans (ATCC 90028) and cytotoxicity on normal oral keratinocytes spontaneously immortalized (NOK-si) cells were investigated. The microcrystals were synthesized by co-precipitation (CP) method under three different temperatures (10, 20 and 30°C) and were characterized by x-ray diffraction, scanning electronic microscopy field emission guns and Raman spectroscopy. The antifungal activity was evaluated from serial microdilution of microcrystals (according to Clinical & Laboratorial Standards Institute - CLSI), where minimum inhibitory (MIC) and fungicide (MFC) concentrations were determined. Fluorescence microscopy images were obtained in order to confirm microbiological findings. Cell viability of NOK-si cells was evaluated by Alamar Blue assay, and scanning electron microscopy (SEM) images of all groups were performed. In these tests, only four concentrations of microcrystals (MIC, MFC, MIC 10 times diluted and MIC 10 times concentrated) were used. The results were submitted to Shapiro-Wilk’s test, and then they were evaluated using the Kruskal-Wallis test, followed by Dunn's post-test (α = .05), in GraphPad Prism 7 software. All three microcrystals presented the same values of MIC and MFC (3.90 μg/mL and 15.62 μg/mL, respectively). When evaluated fluorescence microscopy it was possible to confirm the results obtained with the microbiological assay, where at the MIC noticed a reduction in cell number when compared with the control, and at the MFC noticed unviability of all cells. When evaluated for its cytotoxicity, the three microcrystals were biocompatible in the MIC, MFC and MIC 10 times diluted values (p <0.05). SEM images were consistent with the findings in the Alamar Blue assay, where cell death was observed only at MIC 10 times concentrated. Through the morphology analyses of cell death, it was observed that the microcrystals at MIC 10 times concentrated caused cell death by necrosis. The three α-AgVO3 microcrystals showed promising results as antifungal agents against C. albicans and showed biocompatibility with the studied cell type, making them interesting for later in vivo studies.
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Palavras-chave
Candida albicans, Antifúngicos, Testes de toxicidade, Queratinócitos, Antifungal agents, Toxicity tests, Keratinocytes