Síntese e caracterização de nanopartículas poliméricas para veiculação de lignanas bioativas
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2018-04-20
Autores
Lima, Regiane Godoy de
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Universidade Estadual Paulista (Unesp)
Resumo
Os produtos naturais são uma fonte inesgotável de compostos químicos com as mais variadas propriedades biológicas, entretanto, muitos deles têm baixa biodisponibilidade e/ou são degradados pelas condições fisiológicas, o que os torna de pouco interesse para uso como potenciais fármacos. Dentre os vários produtos naturais bioativos as lignanas apresentam um significativo potencial citotóxico contra várias linhagens de células tumorais, como a (-)-hinoquinina (HNK), (-)-cubebina (CB) e (-)-6,6’-dinitrohinoquinina (DN), determinado em outros trabalhos. Entretanto, essa lignanas possuem caráter hidrofóbico e podem ser degradadas em condições fisiológicas dependendo do pH. Desta forma, o presente trabalho teve por objetivo estudar a incorporação dessas lignanas em nanopartículas poliméricas (NNPs), como forma de melhorar a biodisponibilidade, perdas por degradação e os efeitos da incorporação sobre a atividade citotóxica desses compostos. Neste estudo a HNK e CB foram extraídas da Piper cubeba e a DN foi sintetizada a partir da reação de nitração da HNK. Essa lignanas foram submetidas a ensaios de citotoxicidade contra as linhagens tumorais Hep-2 e Si-Ha. As NNPs com e sem a lignana foram sintetizadas utilizando como matriz polimérica inicialmente o PLGA (Poli(ácido lático-co-ácido glicólico). As NNPs sintetizadas foram caracterizadas quanto ao seu tamanho, morfologia e estabilidade utilizando análises de MEV, DSC, MET, UV/Vis, FTIR, DLS e potencial zeta. A eficiência da incorporação foi determinada através de analises de UV/Vis. Nos ensaios de citotoxicidade todas as lignanas foram ativas, com destaque para a inibição de 98% da HNK sobre a linhagem Hep-2. As três lignanas foram incorporadas em NNPs de PLGA utilizando o método de nanoemulsão-evaporação com agitação mecânica e gotejamento, entretanto a estabilidade dessas NNPs e as porcentagens de incorporação indicaram que apenas as NNPs-PLGA-HNK apresentaram os parâmetros necessários para a utilização nos ensaios de citotoxicidade. Portanto, outros estudos foram realizados com as três lignanas visando a melhora nos métodos de obtenção e estabilidade com o uso de sonicador e diferente composição do PLGA (50:50) e PLGA-PEG. As análises físicas mostraram que as nanopartículas de PLGA (50:50) obtidas através da metodologia por sonicação foram mais estáveis com menores tamanhos e maior potencial zeta em valores absolutos, entretanto, a DN e a CB não foram encapsuladas de forma eficiente. Desta forma, somente a HNK prosseguiu no estudo. O uso do PLGA-PEG alterou de forma significante os parâmetros físicos e químicos das NNPs-PLGA-PEG-HNK em relação as de PLGA-HKN. Os testes de liberação mostraram melhores resultados com as NNPs-PLGA-HNK, principalmente em pH 7,4. O tempo de liberação total da HNK em todas as NNPs avaliadas foi em torno de 30 minutos o que sugere que as nanopartículas obtidas são nanocápsulas. A comparação dos resultados dos ensaios de citotoxicidade mostraram que o CI50 das NNPs de PLGA-HNK foi de 29,8 µM sobre as células tumorais Si-Ha, enquanto a HNK livre apresentou CI50 = 110 µM. Para as outras células tumorais mais agressivas as NNPs de PLGA(50:50)-HNK foram menos eficazes do que a HNK livre, com cerca de 50% de perda da atividade. Esses resultados indicam a efetividade das NPPs de PLGA(65:35)-HNK sobre as células da linhagem Si-Ha, e que o encapsulamento da HNK em NNPs de PLGA pode ser uma alternativa para diminuir a decomposição e aumentar a biodisponibilidade para a condução de em ensaios in vivo.
The natural products are inexhaustible source of chemical compounds with the most varied biological properties; however, many of them have low bioavailability and / or are degraded by physiological conditions, which makes them less interesting for use as potential drugs. Among the several bioactive natural products the lignans present a significant cytotoxic potential against several tumor cell lines, as (-)-hinokinin (HNK), (-)-cubebin (CB) and (-)-6,6’-dinitrohinokinin (DN), determined in previous works. However, these lignans have a hydrophobic character and can be degraded in physiological conditions depending on pH. Thus, the present work aimed to study the incorporation of these lignans into polymeric nanoparticles (PNPs), as a method to improve bioavailability, avoid losses by degradation and evaluate as this incorporation affects the its cytotoxic activity. In this study, the HNK and CB were extracted from Piper cubeba and the DN was synthesized from the nitration reaction of the HNK. These lignans were submitted to cytotoxicity assays against tumoral cells Hep-2 and Si-Ha. The empty and content lignans PLGA (Poly Lactic-co-Glycolic Acid, 65:35) PNPs were synthesized using the emulsion-evaporation methodology on mechanical agitation and dripping. The PNPs were investigated for their size, morphology and stability through SEM, DSC, TEM, UV/Vis, FTIR, DLS and zeta potential. Encapsulation efficiency incorporation of the lignan was determined by analysis of UV/Vis. In the cytotoxicity assays all lignans were active, with emphasis on inhibition of 98% da (-)-hinokinin about the Hep-2 tumoral line. The three lignans were incorporated into PLGA (65:35) NNPs, however the stability of these PNPs and the percentages of incorporation indicated that only the PLGA-HNK PNPs had the parameters required for use in the cytotoxicity assays. Therefore, other studies were performed with the three lignans aiming to improve the methods of obtaining and stability from sonicator and different composition in PLGA: (50:50) and PLGA-PEG. The physical analyzes showed that PLGA (50:50) nanoparticles obtained by sonication were more stable with smaller sizes and higher zeta potential in absolute values. However, even in this method, DN e CB were not efficiently encapsulated. In this way, only HNK continued the study. The use of PLGA-PEG altered the physical and chemical parameters of PLGA-PEG-HNK NNPs obtained when compared to PLGA-HNK NNPs. Release tests showed better results with NNPs-PLGA-HNK, especially at pH 7.4. The total release time of HNK in all NNPs evaluated was around 30 minutes, which suggests that the nanoparticles obtained are nanocapsules. Comparison of cytotoxicity assay results showed that PLGA-HNK PNPs IC50 was 29.8 μM on the Si-Ha tumor cells, while the free HNK showed IC50 = 110 μM. For the other more aggressive tumor cells PLGA (50:50)-HNK PNPs were less effective than free HNK with about 50% loss of activity. These results indicate the effectiveness of PLGA(65:35)-HNK NPPs on cells of the Si-Ha line, and that the encapsulation of HNK in PLGA NNPs may be an alternative to decrease decomposition and increase bioavailability for conducting biological assays in vivo.
The natural products are inexhaustible source of chemical compounds with the most varied biological properties; however, many of them have low bioavailability and / or are degraded by physiological conditions, which makes them less interesting for use as potential drugs. Among the several bioactive natural products the lignans present a significant cytotoxic potential against several tumor cell lines, as (-)-hinokinin (HNK), (-)-cubebin (CB) and (-)-6,6’-dinitrohinokinin (DN), determined in previous works. However, these lignans have a hydrophobic character and can be degraded in physiological conditions depending on pH. Thus, the present work aimed to study the incorporation of these lignans into polymeric nanoparticles (PNPs), as a method to improve bioavailability, avoid losses by degradation and evaluate as this incorporation affects the its cytotoxic activity. In this study, the HNK and CB were extracted from Piper cubeba and the DN was synthesized from the nitration reaction of the HNK. These lignans were submitted to cytotoxicity assays against tumoral cells Hep-2 and Si-Ha. The empty and content lignans PLGA (Poly Lactic-co-Glycolic Acid, 65:35) PNPs were synthesized using the emulsion-evaporation methodology on mechanical agitation and dripping. The PNPs were investigated for their size, morphology and stability through SEM, DSC, TEM, UV/Vis, FTIR, DLS and zeta potential. Encapsulation efficiency incorporation of the lignan was determined by analysis of UV/Vis. In the cytotoxicity assays all lignans were active, with emphasis on inhibition of 98% da (-)-hinokinin about the Hep-2 tumoral line. The three lignans were incorporated into PLGA (65:35) NNPs, however the stability of these PNPs and the percentages of incorporation indicated that only the PLGA-HNK PNPs had the parameters required for use in the cytotoxicity assays. Therefore, other studies were performed with the three lignans aiming to improve the methods of obtaining and stability from sonicator and different composition in PLGA: (50:50) and PLGA-PEG. The physical analyzes showed that PLGA (50:50) nanoparticles obtained by sonication were more stable with smaller sizes and higher zeta potential in absolute values. However, even in this method, DN e CB were not efficiently encapsulated. In this way, only HNK continued the study. The use of PLGA-PEG altered the physical and chemical parameters of PLGA-PEG-HNK NNPs obtained when compared to PLGA-HNK NNPs. Release tests showed better results with NNPs-PLGA-HNK, especially at pH 7.4. The total release time of HNK in all NNPs evaluated was around 30 minutes, which suggests that the nanoparticles obtained are nanocapsules. Comparison of cytotoxicity assay results showed that PLGA-HNK PNPs IC50 was 29.8 μM on the Si-Ha tumor cells, while the free HNK showed IC50 = 110 μM. For the other more aggressive tumor cells PLGA (50:50)-HNK PNPs were less effective than free HNK with about 50% loss of activity. These results indicate the effectiveness of PLGA(65:35)-HNK NPPs on cells of the Si-Ha line, and that the encapsulation of HNK in PLGA NNPs may be an alternative to decrease decomposition and increase bioavailability for conducting biological assays in vivo.
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Palavras-chave
Sistema de entrega de drogas, (-)-6,6’-dinitrohinoquinina, (-)-hinoquinina, (-)-cubebina, PLGA, PLGA-PEG, Materiais biocompatíveis, Drug delivery system, (-)-6,6’-dinitrohinokinin, (-)-hinokinin, (-)-cubebin, Biocompatible materials