Síntese, caracterização e estudos in vitro de derivados acetilados de quitosana como carreadores de RNA de interferência
Carregando...
Arquivos
Data
2020-08-13
Autores
Martins, Grazieli Olinda
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
A quitosana tem recebido muita atenção como carreador de siRNA devido à sua boa capacidade de complexação para posterior liberação no interior celular. No entanto, uma de suas limitações é a insolubilidade em pH neutro e a tendência de suas nanopartículas (NPs, quitosana + siRNA) agregarem em condições fisiológicas. Portanto, realizar modificações na quitosana, junto às caracterizações físico-químicas e biológicas são necessárias para superar e compreender estas limitações. No presente trabalho, quitosanas anfipáticas foram sintetizadas, variando-se o grau de acetilação (GA) em aproximadamente 20 e 30 %, o grau de substituição (GS) por dietilaminoetil (DEAE) entre 5 e 25 % e o GS com cadeias de polietilenoglicol (PEG) na faixa de 1,5. Os resultados mostraram que o ajuste destes parâmetros diminuiu as interações intermoleculares mediadas por ligações de hidrogênio para obtenção de nanopartículas com estabilidade coloidal e sérica: nanopartículas com tamanho de 150 nm, com baixo índice de polidispersão (0,15 - 0,2) e potencial Zeta positivo (entre ~ +4 e +12 mV). A resistência à agregação é fornecida por mudanças na superfície das nanopartículas e destaca a importância de uma automontagem mais organizada para fornecer estabilidade em condições fisiológicas. A peguilação dos vetores reduziu o tamanho das nanopartículas para 100 nm em pH 6,3, além de melhorar a estabilidade sérica do polímero com menor grau de acetilação em pH 7,4. Os polímeros na forma livre e suas respectivas nanopartículas com siRNA exibiram baixa toxicidade celular, eficiência da captação celular das nanopartículas, bem como a distribuição e a liberação em tempo real do siRNA-FAM no interior celular, que foram confirmados pelos marcadores fluorescentes. Os estudos de transfecção in vitro em macrófagos mostraram a influência do grau de acetilação na redução da expressão relativa de TNF- em até 63 % com as NPs dos derivados modificados com DEAE e PEG, com eficiência de transfecção superior à lipofectamina, que reduziu em 48 % a expressão de TNF. De forma análoga, estudos de transfecção em célula epitelial de carcinoma cervical (HeLa-GFP) também mostraram que os derivados modificados com DEAE e PEG formaram NPs que promoveram o knockdown de GFP. E, portanto, os resultados forneceram uma abordagem clara para superar a limitada estabilidade de nanopartículas de quitosana em condições fisiológicas.
Chitosan has received a lot of attention as a carrier of siRNA due to its good complexing capacity for subsequent release into the cell interior. However, one of its limitations is the insolubility in neutral pH and the tendency of its nanoparticles (NPs, chitosan + siRNA) to aggregate under physiological conditions. Therefore, to make changes in the chitosan, together with physicochemical and biological characterizations are necessary to overcome and understand these limitations. In the present work, amphipathic chitosans were synthesized, varying the degree of acetylation (GA) in approximately 20 and 30%, the degree of substitution (GS) by diethylaminoethyl (DEAE) between 5 and 25% and the GS with polyethylene glycol chains (PEG) in the range of 1, 5.The results showed that the adjustment of these parameters decreased the hydrogen bond mediated intermolecular interactions to obtain colloidal and serum stability nanoparticles: ~150 nm size nanoparticles, with low polydispersion index (0.15 - 0.2) and positive Zeta potential (between ~ +4 and +12 mV). Aggregation resistance is provided by changes in the surface of nanoparticles and highlights the importance of more organized self-assembly to provide stability under physiological conditions. Vector pegylation reduced nanoparticle size to ~100 nm at pH 6.3, and improved serum stability of the less acetylated polymer at pH 7.4. Polymers in free form and their respective nanoparticles with siRNA exhibited low cell toxicity, efficient cell uptake of the nanoparticles, as well as the real-time distribution and release of siRNA-FAM inside the cell, which were confirmed by fluorescent markers. In vitro transfection studies on macrophages showed the influence of the degree of acetylation on the reduction of the relative expression of TNF- by up to 63% with the NPs of derivatives modified with DEAE and PEG, with greater transfection efficiency than lipofectamine, which reduced by 48% TNF expression. Similarly, transfection studies on epithelial cell of cervical carcinoma (HeLa-GFP) also showed that derivatives modified with DEAE and PEG formed NPs that enabled GFP knockdown. And therefore, the results provided a clear approach to overcome the limited stability of chitosan nanoparticles under physiological conditions
Chitosan has received a lot of attention as a carrier of siRNA due to its good complexing capacity for subsequent release into the cell interior. However, one of its limitations is the insolubility in neutral pH and the tendency of its nanoparticles (NPs, chitosan + siRNA) to aggregate under physiological conditions. Therefore, to make changes in the chitosan, together with physicochemical and biological characterizations are necessary to overcome and understand these limitations. In the present work, amphipathic chitosans were synthesized, varying the degree of acetylation (GA) in approximately 20 and 30%, the degree of substitution (GS) by diethylaminoethyl (DEAE) between 5 and 25% and the GS with polyethylene glycol chains (PEG) in the range of 1, 5.The results showed that the adjustment of these parameters decreased the hydrogen bond mediated intermolecular interactions to obtain colloidal and serum stability nanoparticles: ~150 nm size nanoparticles, with low polydispersion index (0.15 - 0.2) and positive Zeta potential (between ~ +4 and +12 mV). Aggregation resistance is provided by changes in the surface of nanoparticles and highlights the importance of more organized self-assembly to provide stability under physiological conditions. Vector pegylation reduced nanoparticle size to ~100 nm at pH 6.3, and improved serum stability of the less acetylated polymer at pH 7.4. Polymers in free form and their respective nanoparticles with siRNA exhibited low cell toxicity, efficient cell uptake of the nanoparticles, as well as the real-time distribution and release of siRNA-FAM inside the cell, which were confirmed by fluorescent markers. In vitro transfection studies on macrophages showed the influence of the degree of acetylation on the reduction of the relative expression of TNF- by up to 63% with the NPs of derivatives modified with DEAE and PEG, with greater transfection efficiency than lipofectamine, which reduced by 48% TNF expression. Similarly, transfection studies on epithelial cell of cervical carcinoma (HeLa-GFP) also showed that derivatives modified with DEAE and PEG formed NPs that enabled GFP knockdown. And therefore, the results provided a clear approach to overcome the limited stability of chitosan nanoparticles under physiological conditions
Descrição
Palavras-chave
Policátions, Derivados de quitosana, Estabilidade fisiológica, N-acetilação, Transfecção in vitro, Polycations, Chitosan derivatives, Physiological stability, N-acetylation, In vitro transfection