Síntese e avaliação dos efeitos de um nanocarreador de miconazol sobre microrganismos orais
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Data
2020-01-16
Autores
Arias, Laís Salomão [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A presente tese teve como objetivo geral preparar, caracterizar e avaliar os efeitos antimicrobianos de um nanocarreador de miconazol (MCZ) à base de nanopartículas magnéticas de óxido de ferro (NPsMOF) funcionalizadas com quitosana (QS). Assim, dois subprojetos (SP1 e SP2) foram desenvolvidos e apresentaram os seguintes objetivos específicos: SP1) Preparar, caracterizar e avaliar os efeitos do nanocarreador NPsMOF-QS-MCZ sobre células planctônicas e biofilmes simples e misto de Candida albicans e Candida glabrata; SP2) Avaliar o efeito do nanocarreador na composição de três diferentes modelos de biofilmes polimicrobianos patogênicos orais (gengivite, prótese total e cárie dentária). A primeira etapa do SP1 consistiu em revestir as NPsMOF com QS e carregar este core-shell com MCZ, a fim de caracterizar este nanocarreador por métodos físico-químicos. As concentrações inibitórias mínimas (CIMs) do nanocarreador foram determinadas pelo método da microdiluição em caldo, usando o índice da concentração inibitória fracionária a fim de avaliar se houve interação sinergística entre os compostos. Ainda, biofilmes simples e mistos de Candida foram formados no fundo de placas de 24 ou 96 poços por 48 h e, em seguida, tratados por 24 h com NPsMOF-QS-MCZ carreando MCZ a 31,2 e 78 µg/ml, na presença e ausência de um campo magnético externo. Os biofilmes foram avaliados quantitativamente por biomassa total, atividade metabólica, contagem de unidades formadoras de colônias (UFCs) e composição da matriz extracelular. Os dados foram analisados por ANOVA a dois fatores, seguida pelo teste de Holm-Sidak (p<0,05). Ainda, a estrutura dos biofilmes foi avaliada qualitativamente por microscopia eletrônica de varredura e microscopia confocal. Os resultados do SP1 mostraram que o nanocarreador apresentou diâmetro menor que 50 nm e valores de CIM menores do que aqueles encontrados para MCZ sozinho, com efeito sinérgico sobre C. albicans. NPsMOF-QS-MCZ a 78 µg/ml foi mais eficaz que MCZ sozinho na redução de UFCs e atividade metabólica de biofilmes misto e simples de C. albicans. A biomassa total dos biofilmes e a matriz extracelular não foram afetadas pelo nanocarreador, e a aplicação de um campo magnético externo não melhorou seu efeito antibiofilme. As imagens de microscopia confirmam que tratamentos com o nanocarreador, principalmente na maior concentração, apresentaram maior atividade antibiofilme. Com relação ao SP2, as CIMs de NPsMOF-QS-MCZ foram determinadas para diferentes espécies microbianas, e todos os biofilmes polimicrobianos foram desenvolvidos por 5 dias e tratados por 24 h com NPsMOF-QS-MCZ a 64 µg/ml. Após o tratamento, os biofilmes foram avaliados quanto à biomassa total, atividade metabólica, contagem de UFCs e análise composicional por PCR quantitativo. Microscopia eletrônica de varredura foi usada para analisar a estrutura do biofilme. As diferenças entre os grupos foram determinadas por teste t não pareado (p<0,05). Os resultados do SP2 mostraram que NPsMOF-QS-MCZ foi mais eficaz que MCZ sozinho contra a maioria das células fúngicas e bacterianas testadas. Ainda, este nanocarreador foi capaz de reduzir a atividade metabólica, biomassa total e UFCs (p<0,05) dos biofilmes, além de alterar a sua ultraestrutura. Por fim, NPsMOF-QS-MCZ afetou a composição dos três biofilmes polimicrobianos avaliados, reduzindo principalmente os números de Streptococcus spp. e alterando a prevalência das espécies nos biofilmes. Em suma, os resultados dos SP1 e SP2 permitiram concluir que o nanocarreador melhorou o efeito antimicrobiano do MCZ, dependendo da espécie e parâmetro de biofilme avaliados. O nanocarreador também mostrou potencial para interferir nas interações sinergísticas entre células fúngicas e bacterianas dentro de biofilmes polimicrobianos.
The present thesis aimed to prepare, characterize and evaluate the antimicrobial effects of a miconazole (MCZ) nanocarrier based on iron oxide magnetic nanoparticles (IONPs) functionalized with chitosan (CS). Thus, two subprojects (SP1 and SP2) were developed and had the following specific objectives: SP1) To prepare, characterize and evaluate the effects of the IONPs-CS-MCZ nanocarrier on planktonic cells and singleand dual-species biofilms of Candida albicans and Candida glabrata; SP2) To evaluate the effect of IONPs-CS-MCZ on the composition of three different models of oral pathogenic biofilms (gingivitis, denture and dental caries). The first step of SP1 was to coat IONPs with CS and to load this core-shell association with MCZ, in order to characterize this nanocarrier by physicochemical methods. The minimum inhibitory concentrations (MICs) of the nanocarrier were determined by the microdilution method, using the fractional inhibitory concentration index in order to assess whether there was synergistic interaction between the compounds. In addition, single- and dual-species biofilms of Candida species were formed at the bottom of 24- or 96-well plates for 48 h and, in sequence, treated for 24 h with IONPs-CS-MCZ carrying MCZ at 31.2 and 78 µg/ml, in both the presence and absence of an external magnetic field. Biofilms were quantitatively evaluated by total biomass, metabolic activity, counting of colony forming units (CFUs) and extracellular matrix components. Data were analyzed by twoway ANOVA, followed by Holm-Sidak test (p <0.05). In addition, the structure of biofilms was qualitatively evaluated by scanning electron microscopy and confocal microscopy. The results from SP1 showed that IONPs-CS-MCZ presented diameter smaller than 50 nm, and MIC values lower than those found for MCZ alone, with synergistic effect on C. albicans. Moreover, 78 µg/ml IONPs-CS-MCZ was more effective than MCZ alone in reducing CFUs and metabolic activity of single biofilms of C. albicans and dual-species biofilms. Total biofilm biomass and extracellular matrix were not affected by the nanocarrier, and the application of an external magnetic field did not improve the nanocarrier effects. Microscopy images confirm that treatments with the nanocarrier, mainly in the highest concentration, exhibited greater antibiofilm activity. Regarding SP2, the MICs of IONPs-CS-MCZ were determined for different microbial species, and all polymicrobial biofilms were developed for 5 days and treated for 24 h with IONPs-CS-MCZ at 64 µg/ml. After treatment, the biofilms were evaluated for total biomass, metabolic activity, counting of CFUs and quantitative PCR analysis. Scanning electron microscopy was used to analyze the biofilm ultrastructure. Differences between groups were determined by unpaired t-test (p<0.05). Results from SP2 showed that IONPs-CS-MCZ was more effective than MCZ alone against most fungal and bacterial cells tested. Moreover, this nanocarrier was able to reduce the metabolic activity, total biomass and CFUs (P <0.05) of the biofilms, besides altering their ultrastructure. Finally, IONPs-CS-MCZ affected the composition of the three evaluated biofilms, mainly reducing the numbers of Streptococcus spp. and changing the prevalence of species in the biofilms. From the results obtained by SP1 and SP2, it was possible to conclude that the nanocarrier improved the antimicrobial effect of MCZ, depending on the species and biofilm parameter evaluated. Nanocarrier also showed potential to interfere in the synergistic interactions among fungal and bacterial cells within polymicrobial biofilms.
The present thesis aimed to prepare, characterize and evaluate the antimicrobial effects of a miconazole (MCZ) nanocarrier based on iron oxide magnetic nanoparticles (IONPs) functionalized with chitosan (CS). Thus, two subprojects (SP1 and SP2) were developed and had the following specific objectives: SP1) To prepare, characterize and evaluate the effects of the IONPs-CS-MCZ nanocarrier on planktonic cells and singleand dual-species biofilms of Candida albicans and Candida glabrata; SP2) To evaluate the effect of IONPs-CS-MCZ on the composition of three different models of oral pathogenic biofilms (gingivitis, denture and dental caries). The first step of SP1 was to coat IONPs with CS and to load this core-shell association with MCZ, in order to characterize this nanocarrier by physicochemical methods. The minimum inhibitory concentrations (MICs) of the nanocarrier were determined by the microdilution method, using the fractional inhibitory concentration index in order to assess whether there was synergistic interaction between the compounds. In addition, single- and dual-species biofilms of Candida species were formed at the bottom of 24- or 96-well plates for 48 h and, in sequence, treated for 24 h with IONPs-CS-MCZ carrying MCZ at 31.2 and 78 µg/ml, in both the presence and absence of an external magnetic field. Biofilms were quantitatively evaluated by total biomass, metabolic activity, counting of colony forming units (CFUs) and extracellular matrix components. Data were analyzed by twoway ANOVA, followed by Holm-Sidak test (p <0.05). In addition, the structure of biofilms was qualitatively evaluated by scanning electron microscopy and confocal microscopy. The results from SP1 showed that IONPs-CS-MCZ presented diameter smaller than 50 nm, and MIC values lower than those found for MCZ alone, with synergistic effect on C. albicans. Moreover, 78 µg/ml IONPs-CS-MCZ was more effective than MCZ alone in reducing CFUs and metabolic activity of single biofilms of C. albicans and dual-species biofilms. Total biofilm biomass and extracellular matrix were not affected by the nanocarrier, and the application of an external magnetic field did not improve the nanocarrier effects. Microscopy images confirm that treatments with the nanocarrier, mainly in the highest concentration, exhibited greater antibiofilm activity. Regarding SP2, the MICs of IONPs-CS-MCZ were determined for different microbial species, and all polymicrobial biofilms were developed for 5 days and treated for 24 h with IONPs-CS-MCZ at 64 µg/ml. After treatment, the biofilms were evaluated for total biomass, metabolic activity, counting of CFUs and quantitative PCR analysis. Scanning electron microscopy was used to analyze the biofilm ultrastructure. Differences between groups were determined by unpaired t-test (p<0.05). Results from SP2 showed that IONPs-CS-MCZ was more effective than MCZ alone against most fungal and bacterial cells tested. Moreover, this nanocarrier was able to reduce the metabolic activity, total biomass and CFUs (P <0.05) of the biofilms, besides altering their ultrastructure. Finally, IONPs-CS-MCZ affected the composition of the three evaluated biofilms, mainly reducing the numbers of Streptococcus spp. and changing the prevalence of species in the biofilms. From the results obtained by SP1 and SP2, it was possible to conclude that the nanocarrier improved the antimicrobial effect of MCZ, depending on the species and biofilm parameter evaluated. Nanocarrier also showed potential to interfere in the synergistic interactions among fungal and bacterial cells within polymicrobial biofilms.
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Palavras-chave
Biofilmes, Candida, Anti-infecciosos, Nanopartículas, Magnetita, Quitosana, Biofilms, Antimicrobials, Magnetite, Chitosan