Avaliação antifúngica e de citoqueratinas epiteliais após terapia fotodinâmica mediada por Curcumina nanoparticulada em modelo murino de candidose oral
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Data
2017-07-31
Autores
Sakima, Vinicius Tatsuyuji [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Este estudo avaliou os efeitos fotodinâmicos da curcumina (CUR) encapsulada em nanopartículas poliméricas (NP) em um modelo murino com infecção prolongada de candidose bucal. NP de CUR foram sintetizadas utilizando o polímero poli-ácido lático (PLA) e sulfato de dextrana (DEX). Brometo de cetiltrimetil-amônio foi também utilizado para NP catiônicas. Para caracterização dessas formulações, foram determinadas as propriedades fisicoquímicas (tamanho, polidispersão e potencial zeta), testes de eficiência de encapsulamento, espectro de absorção, fotoestabilidade e liberação. Um total de 140 camundongos fêmeas com 6 semanas de vida foram imunossuprimidos e inoculados com uma cepa padrão de Candida albicans (Ca) em uma concentração de 1x107 unidades formadores de colônias por mililitro (UFC/mL) na região dorsal da língua. Para a Terapia Fotodinâmica antimicrobiana (aPDT), uma das formulações sintetizadas ou CUR livre foi aplicada no dorso lingual dos animais que, após 20 min, foram irradiados com uma fonte de luz LED azul a 37,5 J/cm2 (grupo F+L+). Animais adicionais foram tratados somente com uma das formulações ou CUR livre sem luz (F+L-), Nistatina 1 vez ao dia (NIS1x), Nistatina 4 vezes ao dia (NIS4x) ou não receberam nenhum tratamento (controle). Em todos os grupos, os procedimentos foram realizados em 5 dias consecutivos. Após a última aplicação do tratamento, coletas da língua dos animais foram plaqueadas em meio de cultura específico (CFU/mL). Os animais foram eutanasiados 24 h e 7 dias após o último dia de tratamento, as línguas foram removidas e submetidas às análises histológica e expressão de citoqueratinas (CK) 13 e 14 por ensaio imunohistoquímico (IHQ). Os dados [log10(CFU/mL)] foram analisados por ANOVA/Welch e Games-Howell (α=5%). Após a síntese, as CUR-NP apresentaram propriedades nanomêtricas farmacotécnicas adequadas, com valores médios de tamanho, polidispersão e potencial zeta 225.38; 0.15 e -30.96 mV; 248.12; 0.21 e +35,84 mV respectivamente para as CUR-NP aniônica e catiônica. Foi observada uma eficiência de encapsulamento de 60.84% e 73.81% para as CUR-NP aniônicas e catiônicas. O espectro de absorção das CUR-NP foram semelhantes à CUR livre, com sinais mais elevados de absorbância. CUR livre apresentou uma menor fotodegradação após 20 min de iluminação (43,2 J/cm2). A liberação de CUR das NP aniônicas e catiônicas foi de 52% após 12 horas de estudo. No estudo in vivo, foi observado uma redução significativa de 1,19 log10 para o grupo F+L+ com CUR livre (p<0,001), 1,39 log10 para o grupo NIS4x (p<0,001) e uma maior redução de 2,59 log10 para o grupo NIS1x (p<0,001), quando comparados com o grupo controle. aPDT mediada por CUR-NP aniônica não apresentou efeito antifúngico e a CUR-NP catiônica apresentou efeito antifúngico com e sem luz. Na análise histológica foi observada presença de Ca no epitélio de todos os animais inoculados, porém os animais tratados com nistatina e F+L+ com CUR livre apresentaram menor quantidade de Ca para ambos os períodos de eutanásia. Na análise IHQ foi observada uma intensa marcação para CK 13 e 14 em todos os animais inoculados com exceção dos animais tratados com F+L+ com CUR livre, que apresentaram marcações similares do animal saudável. O encapsulamento da CUR em NP melhorou a solubilidade em água e a estabilidade da CUR, porém a CUR livre mostrou um melhor efeito fotodinâmico do que as CUR-NP.
The aims of this study were to synthetize and evaluate the antimicrobial photodynamic effects of curcumin (CUR) encapsulated in polymeric nanoparticles (NP) on a murine model with prolonged infection of oral candidosis. CUR-NP was synthesized using poly-lactic acid (PLA), and dextran sulfate (DEX). Cetyltrimethylammonium bromide was used for cationic NP. To characterize the formulations, the physicochemical properties (size, polydispersity and zeta potential) were determined and tests of encapsulation efficiency, absorption spectrum, photostability (photodegradation of CUR) and release were also performed. The effectiveness of CUR-NP-mediated aPDT was evaluated in a murine model of oral candidiasis. Female mice (N = 140) were immunosuppressed and inoculated with C. albicans (Ca) to induce oral candidosis. For aPDT, formulations or free CUR was applied on the dorsum of tongue and, after 20 min, illumination at 37.5 J/cm2 was performed (F+L+ group). Additional animals were treated only with formulations or free CUR without light (F+L-), nystatin once a day (NYS1x), nystatin four times a day (NYS4x) or received no treatment (control). In each group, procedures were performed for 5 consecutive days. After the last treatment application, microbial samples were plated in specific agar media (CFU/mL). The animals were euthanatized 24 hours and 7 days after the treatments for histological evaluation and expression of cytokeratins (CK) 13 and 14 by immunohistochemistry (IHC). Data [log10(CFU/mL)] were analyzed by ANOVA/Welch and Games-Howell (α = 5%). After synthesis CUR-NP had appropriate pharmacotechnical nanometric properties, mean values of size, polydispersity, and zeta potential were 225.38; 0.15 and -30.96 mV; 248.12; 0.21 and +35.84 mV, respectively for anionic and cationic CUR-NP. Encapsulation efficiency of 60.84% and 73.81% for anionic and cationic CUR-NP were observed. Peaks absorption for CUR-NP and CUR were close to UV-visible range, the absorption spectrum of CUR-NP were similar to free CUR, with higher absorbance signals. Free CUR showed a lower photodegradation after 20 min of illumination (43.2 J/cm2).The release of CUR from anionic and cationic NP was 52% after 12 hours of study. In vivo study, a significant mean reduction of 1.19 log10 for the F+L+ group (p<0.001), 1.39 log10 for the NIS4x group (p<0.001) and a greater reduction of 2.59 log10 for the NIS1x group (p<0.001) was observed when compared with the control group. aPDT mediated by anionic CUR-NP had no antimicrobial effect, and cationic CUR-NP showed antimicrobial effect even in light absence. In histological analysis a few amount of Ca in the epithelium was observed for the nystatin and aPDT with free CUR groups compared with the control group. In IHC analysis, for both CK, animals submitted to aPDT with free CUR showed a less intense expression similar to the healthy animals compared with all other groups. The synthesized formulation improved the water solubility and stability of CUR, but free CUR showed a better photodynamic effect than CUR-NP.
The aims of this study were to synthetize and evaluate the antimicrobial photodynamic effects of curcumin (CUR) encapsulated in polymeric nanoparticles (NP) on a murine model with prolonged infection of oral candidosis. CUR-NP was synthesized using poly-lactic acid (PLA), and dextran sulfate (DEX). Cetyltrimethylammonium bromide was used for cationic NP. To characterize the formulations, the physicochemical properties (size, polydispersity and zeta potential) were determined and tests of encapsulation efficiency, absorption spectrum, photostability (photodegradation of CUR) and release were also performed. The effectiveness of CUR-NP-mediated aPDT was evaluated in a murine model of oral candidiasis. Female mice (N = 140) were immunosuppressed and inoculated with C. albicans (Ca) to induce oral candidosis. For aPDT, formulations or free CUR was applied on the dorsum of tongue and, after 20 min, illumination at 37.5 J/cm2 was performed (F+L+ group). Additional animals were treated only with formulations or free CUR without light (F+L-), nystatin once a day (NYS1x), nystatin four times a day (NYS4x) or received no treatment (control). In each group, procedures were performed for 5 consecutive days. After the last treatment application, microbial samples were plated in specific agar media (CFU/mL). The animals were euthanatized 24 hours and 7 days after the treatments for histological evaluation and expression of cytokeratins (CK) 13 and 14 by immunohistochemistry (IHC). Data [log10(CFU/mL)] were analyzed by ANOVA/Welch and Games-Howell (α = 5%). After synthesis CUR-NP had appropriate pharmacotechnical nanometric properties, mean values of size, polydispersity, and zeta potential were 225.38; 0.15 and -30.96 mV; 248.12; 0.21 and +35.84 mV, respectively for anionic and cationic CUR-NP. Encapsulation efficiency of 60.84% and 73.81% for anionic and cationic CUR-NP were observed. Peaks absorption for CUR-NP and CUR were close to UV-visible range, the absorption spectrum of CUR-NP were similar to free CUR, with higher absorbance signals. Free CUR showed a lower photodegradation after 20 min of illumination (43.2 J/cm2).The release of CUR from anionic and cationic NP was 52% after 12 hours of study. In vivo study, a significant mean reduction of 1.19 log10 for the F+L+ group (p<0.001), 1.39 log10 for the NIS4x group (p<0.001) and a greater reduction of 2.59 log10 for the NIS1x group (p<0.001) was observed when compared with the control group. aPDT mediated by anionic CUR-NP had no antimicrobial effect, and cationic CUR-NP showed antimicrobial effect even in light absence. In histological analysis a few amount of Ca in the epithelium was observed for the nystatin and aPDT with free CUR groups compared with the control group. In IHC analysis, for both CK, animals submitted to aPDT with free CUR showed a less intense expression similar to the healthy animals compared with all other groups. The synthesized formulation improved the water solubility and stability of CUR, but free CUR showed a better photodynamic effect than CUR-NP.
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Palavras-chave
Nanopartículas, Queratinas, Camundongos, Candida albicans, Nanoparticles, Keratins, Mice