Desenvolvimento e caracterização de nanopartículas poliméricas contendo associação de Fenil-tio-ftalocianinica de Zinco e Anfotericina B para aplicação em Terapia Fotodinâmica Antimicrobiana
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Data
2019-12-04
Autores
Evangelista, Rodrigo Passos
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Universidade Estadual Paulista (Unesp)
Resumo
O avanço tecnológico cresce rapidamente no mundo, inclusive na área farmacêutica. Nesse contexto, sistemas nanoestruturados ganham progressivo destaque, porquanto carregam um enorme potencial para o aperfeiçoamento das práticas terapêuticas atuais. A administração de fármacos na forma livre pode apresentar problemas como baixa concentração no sítio ativo, inadequada distribuição pelo organismo, baixa biodisponibilidade, presença de efeitos adversos, entre outros. As nanocápsulas poliméricas são capazes de contornar tais vieses, possibilitando, inclusive, a diminuição da toxicidade com a diminuição de doses e aumento da estabilidade de fármacos em meios biológicos. Foram desenvolvidas nanocápsulas poliméricas contendo Anfotericina B (AMB) e Fenil-tio-ftalocianina de zinco (ZnS4Pc) na concentração teórica de 0,05 mg.mL-1 objetivando, com a associação entre os dois ativos, melhorar a atividade antifúngica conseguida pelo antifúngico isoladamente utilizando-se a Terapia Fotodinâmica Antimicrobiana. A avaliação das características físico-químicas das nanocápsulas mostraram diâmetro médio de partícula de 253,8 ± 17,3 nm, índice de polidispersidade médio de 0,36 ± 0,01 e potencial Zeta médio de – 31,03 ± 5,54 mV. Tais análises revelaram-se satisfatórias já que o diâmetro de partícula conseguido pelo método de deposição interfacial de polímero pré-formado utilizado na produção das nanocápsulas varia de 250 a 500 nm (MORA-HUERTAS et. al., 2010), enquanto que o potencial zeta considerado para uma formulação estável deve ser maior que 30 mV em módulo (RAVAL et al., 2019). Análises de espectroscopia de absorção e emissão realizadas na região do UV-Vis não encontraram alterações nas propriedades fotofísicas no estado estacionário de NC/ZnS4Pc e NC/ZnS4Pc + AMB em relação a ZnS4Pc livre. Os ativos nas NC foram quantificados utilizando-se uma curva analítica de correlação de absorção e concentração variando de 0,255 a 2,04 µg.mL-1 para a ZnS4Pc e 0,50 a 10,00 µg.mL-1 para AMB, de cuja análise obtivemos uma quantificação, para as NC com ativos isolados, de 43,8% para ZnS4Pc e 75,0% para AMB; Para as NC mistas, isto é, NC/ZnS4Pc + AMB, esses teores foram de 30,7% e 67,7%, respectivamente. No que diz respeito ao teor de encapsulação, para as nanocápsulas com ativos isolados, notou-se um alto valor de 89,6% para ZnS4Pc e que se manteve com 89,2% para as NC Mistas. Além disso, um estudo morfológico das formulações de NC foi realizado com Microscopia de Força Atômica denotando a presença de nanocápsulas de formato esférico e com distribuição homogênea. O ensaio de viabilidade celular com as células NIH/3T3 ATCC® CRL-1658™ não demonstrou citotoxidade para as mesmas na presença das NC mistas com fatores de diluição de até 15% (v/v). Para o ensaio de atividade fotodinâmica, foram utilizadas NC mistas diluídas de 1% e 5% (v/v) junto às leveduras Candida albicans ATCC® 90028 e, diferentemente do que ocorreu para as células NIH/3T3, as NC mistas apresentaram toxicidade fúngica tanto na ausência de luz quanto na presença da mesma com irradiação de 12 J/cm2 e 25 J/cm2. Todos os resultados foram tratados estatisticamente através do teste de análise de variância One-way ANOVA seguido do pós teste Tukey t-test para múltiplas comparações (p<0,05).
Technological advancement is growing rapidly in the world, including the pharmaceutical area. In this context, nanostructured systems gains progressive prominence, as they carry enormous potential for the improvement of current therapeutic practices. The drugs administration in the free-form may present problems such as low active site concentration, inadequate distribution throughout body, low bioavailability, presence of adverse effects, among others. Polymeric nanocapsules allowing the reduction of toxicity with decreasing doses and increasing the stability of drugs in biologic media. Polymeric nanocapsules containing Amphotericin B (AMB) and zinc phenyl-thio-phthalocyanine (ZnS4Pc) were developed at a 0.05 mg/mL theoretical concentration to improve antifungal activity with the association between the two actives over antifungal alone. The nanocapsules physical-chemical characteristics showed an average particle diameter of 253,8 ± 17,3 nm, an average polydispersity index of 0,36 ± 0,01 and negative Zeta potential -31,03 ± 5,54 mV. These analyzes proved satisfactory since the particle diameter achieved by the preformed polymer interfacial deposition method used in nanocapsule production ranges from 250 to 500 nm (MORA-HUERTAS et. al., 2010), while the Zeta potential considered for a stable formulation should be bigger than 30 mV in modulus (RAVAL et al., 2019). Absorption and emission spectroscopy analyzes performed in the UV-Vis region found no changes in the steady state photophysical properties of NC / ZnS4Pc and NC / ZnS4Pc + AMB in relation to free form ZnS4Pc. Assay quantification was performed using a concentration and absorption correlation analytical curve ranging from 0.255 to 2.04 µg.mL-1 for ZnS4Pc and 0.50 to 10.00 µg.mL-1 for AMB. For NC with isolated assets, we got 43.8% for ZnS4Pc and 75.0% for AMB; for the mixed NC (NC / ZnS4Pc + AMB) these contents were 30.7% and 67.7% respectively. Regarding the encapsulation content, for nanocapsules with isolated actives, a high value of 89.6% was observed for ZnS4Pc and remained with 89.2% for Mixed NC. In addition, a morphological study of NC formulations was performed with Atomic Force Microscopy denoting the presence of homogeneously distributed spherical-shaped nanocapsules. Cell viability assay with NIH/3T3 ATCC® CRL-1658 ™ cells showed no cytotoxicity to them in the presence of mixed NC with dilution factors of up to 15% (v/v). For the photodynamic activity assay, 1% and 5% (v/v) diluted mixed NC were used with Candida albicans ATCC® 90028TM yeasts and, unlike for NIH/3T3 cells, mixed NC showed fungal toxicity in both the absence of light and in its presence with irradiation of 12 J/cm2 and 25 J/cm2. All results were statistically treated using the One-way ANOVA analysis of variance test followed by the Tukey t-test post-test for multiple comparisons (p <0.05).
Technological advancement is growing rapidly in the world, including the pharmaceutical area. In this context, nanostructured systems gains progressive prominence, as they carry enormous potential for the improvement of current therapeutic practices. The drugs administration in the free-form may present problems such as low active site concentration, inadequate distribution throughout body, low bioavailability, presence of adverse effects, among others. Polymeric nanocapsules allowing the reduction of toxicity with decreasing doses and increasing the stability of drugs in biologic media. Polymeric nanocapsules containing Amphotericin B (AMB) and zinc phenyl-thio-phthalocyanine (ZnS4Pc) were developed at a 0.05 mg/mL theoretical concentration to improve antifungal activity with the association between the two actives over antifungal alone. The nanocapsules physical-chemical characteristics showed an average particle diameter of 253,8 ± 17,3 nm, an average polydispersity index of 0,36 ± 0,01 and negative Zeta potential -31,03 ± 5,54 mV. These analyzes proved satisfactory since the particle diameter achieved by the preformed polymer interfacial deposition method used in nanocapsule production ranges from 250 to 500 nm (MORA-HUERTAS et. al., 2010), while the Zeta potential considered for a stable formulation should be bigger than 30 mV in modulus (RAVAL et al., 2019). Absorption and emission spectroscopy analyzes performed in the UV-Vis region found no changes in the steady state photophysical properties of NC / ZnS4Pc and NC / ZnS4Pc + AMB in relation to free form ZnS4Pc. Assay quantification was performed using a concentration and absorption correlation analytical curve ranging from 0.255 to 2.04 µg.mL-1 for ZnS4Pc and 0.50 to 10.00 µg.mL-1 for AMB. For NC with isolated assets, we got 43.8% for ZnS4Pc and 75.0% for AMB; for the mixed NC (NC / ZnS4Pc + AMB) these contents were 30.7% and 67.7% respectively. Regarding the encapsulation content, for nanocapsules with isolated actives, a high value of 89.6% was observed for ZnS4Pc and remained with 89.2% for Mixed NC. In addition, a morphological study of NC formulations was performed with Atomic Force Microscopy denoting the presence of homogeneously distributed spherical-shaped nanocapsules. Cell viability assay with NIH/3T3 ATCC® CRL-1658 ™ cells showed no cytotoxicity to them in the presence of mixed NC with dilution factors of up to 15% (v/v). For the photodynamic activity assay, 1% and 5% (v/v) diluted mixed NC were used with Candida albicans ATCC® 90028TM yeasts and, unlike for NIH/3T3 cells, mixed NC showed fungal toxicity in both the absence of light and in its presence with irradiation of 12 J/cm2 and 25 J/cm2. All results were statistically treated using the One-way ANOVA analysis of variance test followed by the Tukey t-test post-test for multiple comparisons (p <0.05).
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Palavras-chave
Nanocápsulas, Fenil-tio-ftalocianina, Anfotericina B, Espectroscopia, Microscopia de Força Atômica, Citotoxicidade, Terapia Fotodinâmica Antimicrobiana, Nanocapsules, Phenyl-thio-phthalocyanine, Amphotericin B, Spectroscopy, Atomic Force Microscopy, Cytotoxicity, Antimicrobial Photodynamic Therapy