Microcristais de tungstato de prata, molibdato de prata e vanadato de prata: biocompatibilidade e atividade antifúngica sobre biofilme de Candida albicans
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Data
2022-02-10
Autores
Pimentel, Bruna Natália Alves da Silva [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A presente tese foi dividida em dois estudos que tiveram como objetivos (1) avaliar a citotoxicidade dos microcristais vanadato de prata (α-AgVO3; 3,9 µg/mL e 15,62 µg/mL), tungstato de prata (α-Ag2WO4; 7,81 µg/mL) e molibdato de prata (β-Ag2MoO4; 15,62 µg/mL) sobre células inflamatórias THP-1 e macrófago em monocamada, bem como quantificar a produção de superóxido, citocinas pró-inflamatórias e metaloproteinases de matriz (MMP); (2) avaliar a atividade antifúngica de α-AgVO3, α-Ag2WO4 e β-Ag2MoO4, sobre biofilme de Candida albicans cultivados sobre modelos de co-cultivo tridimensional (3D). A citotoxicidade e a resposta inflamatória de α-AgVO3, α-Ag2WO4 e β-Ag2MoO4 foram avaliadas sobre os modelos de co-cultivo 3D. No estudo 1, a viabilidade celular de THP-1 e macrófagos, após 24 horas de contato com α-AgVO3, α-Ag2WO4 e β-Ag2MoO4, foi determinada a partir do ensaio de alamarBlueTM. A produção da espécie reativa de oxigênio (superóxido) foi quantificada por fluorimetria e visualizada por microscopia de fluorescência confocal. Já a produção de citocinas e metaloproteinases de matriz foi quantificada por citometria de fluxo e ELISA, respectivamente. Os microcristais α-Ag2WO4, β-Ag2MoO4 e α-AgVO3, na concentração fungicida mínima (CFM: 15,62 µg/mL), promoveram pequena diminuição na viabilidade de THP-1 (77,3%, 69,29% e 73,96%, respectivamente). Já em macrófagos, apenas α-AgVO3 (CFM) foi capaz de diminuir a viabilidade celular (70,36%). Em ambas as células foram observadas produção de superóxido. Com relação às citocinas, houve aumento na produção de IL-8 por ambas as células, bem como aumento na produção de IL-1β por macrófagos. A MMP-8 não foi detectada no sobrenadante de THP-1 em nenhuma condição avaliada, enquanto a MMP-9 teve sua produção reduzida nas células THP-1 expostas a α-Ag2WO4 e β-Ag2MoO4. Já os sobrenadantes dos macrófagos apresentaram diminuição de MMP-8 e -9, independente do microcristal ao qual a célula foi exposta. No estudo 2, um modelo de co-cultivo 3D envolvendo três linhagens celulares foi desenvolvido e validado. Nestes modelos foi determinada a citotoxicidade, pelo ensaio colorimétrico MTT, de α-AgVO3, α-Ag2WO4 e β-Ag2MoO4, nas mesmas concentrações testadas no estudo 1. Foi avaliada também a ação dos microcristais sobre biofilmes de Candida albicans crescidos sobre os modelos 3D, simulando um processo infeccioso. Além disso, a modulação da resposta inflamatória foi verificada a partir da dosagem de citocinas pró-inflamatórias, na ausência e na presença dos microcristais, tanto nos modelos com infecção, quanto nos modelos sem infecção. O modelo de co-cultivo 3D se mostrou adequado para avaliar a biocompatibilidade de biomateriais bem como processos infecciosos. Entre os microcristais avaliados, apenas α-AgVO3 mostrou-se citotóxico, além de não ter sido efetivo contra o biofilme de C. albicans e não ter modulado a resposta inflamatória; α-Ag2WO4 apresentou atividade contra biofilme de C. albicans, com redução de 1,5 log10, semelhante à redução observada no controle com a droga padrão (fluconazol), e induziu a produção de IL-6, IL-8 e IL-1β; β-Ag2MoO4 não apresentou atividade contra o biofilme de C. albicans, pesar de ter sido biocompatível e capaz de diminuir a produção de TNFα nos modelos infectados. Com base nos resultados destes estudos, α-Ag2WO4 foi considerado um material promissor no tratamento de infecções por C. albicans.
This present thesis was divided into two studies that aimed to (1) evaluate the cytotoxicity of microcrystals silver vanadate (α-AgVO3; 3.9 μg/mL and 15.62 μg/mL), silver tungstate (α-Ag2WO4; 7.81 μg/mL) and silver molybdate (β-Ag2MoO4; 15.62 μg/mL) on THP-1 and macrophage inflammatory cells in monolayer model, as well as quantifying the production of superoxide, proinflammatory cytokines and matrix metalloproteinases (MMP); (2) evaluate the antifungal activity of α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 on Candida albicans biofilm cultivated on three-dimensional (3D) coculture models. The cytotoxicity and inflammatory response of α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 were also evaluated on 3D co-culture models. In study 1, the cell viability of THP-1 and macrophages, after being challenged with α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 for 24 hours, was determined using the alamarBlueTM assay. The production of reactive oxygen species (superoxide) was quantified by fluorimetry and visualized by confocal laser scanning microscopy. The production of cytokines and matrix metalloproteinases was quantified by flow cytometry and ELISA, respectively. The α-Ag2WO4, β-Ag2MoO4 and α-AgVO3 microcrystals, at the minimum fungicidal concentration (MFC: 15.62 μg/mL), promoted a small decrease in THP-1 viability (77.3%, 69.29% and 73 .96%, respectively). In macrophages, only α-AgVO3 (MFC) was able to decrease cell viability (70.36%). In both cells, superoxide production was observed. Regarding cytokines, there was an increase in IL-8 production by both cells, as well as an increase in IL-1β production by macrophages. MMP-8 was not detected in the THP-1 supernatant in any condition evaluated, whereas MMP-9 had its production reduced in THP-1 cells exposed to α-Ag2WO4 and β-Ag2MoO4. Macrophage supernatants showed a decrease in MMP-8 and -9, regardless of the microcrystals to which cells were exposed. In study 2, a 3D co-culture model involving three cell lines was developed and validated. In these models, the cytotoxicity of α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 was determined by MTT colorimetric assay, at the same concentrations tested in study 1. The antimicrobial activity of microcrystals on C. albicans biofilms grown on 3D models was also evaluated, simulating an infectious process. Furthermore, the modulation of the inflammatory response was assessed by measuring the production of pro-inflammatory cytokines, in the absence and presence of microcrystals, in both infected and noninfected models. The 3D co-culture model proved to be adequate to assess the biocompatibility of biomaterials as well as the infectious processes. Among the microcrystals evaluated, α-AgVO3 was cytotoxic, had no effect against C. albicans biofilm and did not modulate the inflammatory response; α-Ag2WO4 showed activity against C. albicans biofilm with a 1.5 log10 reduction, similar to the control with the standard drug (fluconazole). α-Ag2WO4 also induced the production of IL-6, IL-8 and IL-1β; β-Ag2MoO4 showed no activity against C. albicans biofilm, despite being biocompatible and able to reduce the production of TNFα in infected models. Based on the findings of these studies, α-Ag2WO4 appears to be a promising material in the treatment of C. albicans infections
This present thesis was divided into two studies that aimed to (1) evaluate the cytotoxicity of microcrystals silver vanadate (α-AgVO3; 3.9 μg/mL and 15.62 μg/mL), silver tungstate (α-Ag2WO4; 7.81 μg/mL) and silver molybdate (β-Ag2MoO4; 15.62 μg/mL) on THP-1 and macrophage inflammatory cells in monolayer model, as well as quantifying the production of superoxide, proinflammatory cytokines and matrix metalloproteinases (MMP); (2) evaluate the antifungal activity of α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 on Candida albicans biofilm cultivated on three-dimensional (3D) coculture models. The cytotoxicity and inflammatory response of α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 were also evaluated on 3D co-culture models. In study 1, the cell viability of THP-1 and macrophages, after being challenged with α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 for 24 hours, was determined using the alamarBlueTM assay. The production of reactive oxygen species (superoxide) was quantified by fluorimetry and visualized by confocal laser scanning microscopy. The production of cytokines and matrix metalloproteinases was quantified by flow cytometry and ELISA, respectively. The α-Ag2WO4, β-Ag2MoO4 and α-AgVO3 microcrystals, at the minimum fungicidal concentration (MFC: 15.62 μg/mL), promoted a small decrease in THP-1 viability (77.3%, 69.29% and 73 .96%, respectively). In macrophages, only α-AgVO3 (MFC) was able to decrease cell viability (70.36%). In both cells, superoxide production was observed. Regarding cytokines, there was an increase in IL-8 production by both cells, as well as an increase in IL-1β production by macrophages. MMP-8 was not detected in the THP-1 supernatant in any condition evaluated, whereas MMP-9 had its production reduced in THP-1 cells exposed to α-Ag2WO4 and β-Ag2MoO4. Macrophage supernatants showed a decrease in MMP-8 and -9, regardless of the microcrystals to which cells were exposed. In study 2, a 3D co-culture model involving three cell lines was developed and validated. In these models, the cytotoxicity of α-AgVO3, α-Ag2WO4 and β-Ag2MoO4 was determined by MTT colorimetric assay, at the same concentrations tested in study 1. The antimicrobial activity of microcrystals on C. albicans biofilms grown on 3D models was also evaluated, simulating an infectious process. Furthermore, the modulation of the inflammatory response was assessed by measuring the production of pro-inflammatory cytokines, in the absence and presence of microcrystals, in both infected and noninfected models. The 3D co-culture model proved to be adequate to assess the biocompatibility of biomaterials as well as the infectious processes. Among the microcrystals evaluated, α-AgVO3 was cytotoxic, had no effect against C. albicans biofilm and did not modulate the inflammatory response; α-Ag2WO4 showed activity against C. albicans biofilm with a 1.5 log10 reduction, similar to the control with the standard drug (fluconazole). α-Ag2WO4 also induced the production of IL-6, IL-8 and IL-1β; β-Ag2MoO4 showed no activity against C. albicans biofilm, despite being biocompatible and able to reduce the production of TNFα in infected models. Based on the findings of these studies, α-Ag2WO4 appears to be a promising material in the treatment of C. albicans infections
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Palavras-chave
Teste de materiais, Candida albicans, Prata, Citocinas, Metaloproteinases da matriz