Síntese, caracterização e transfecção in vitro mediada por nanopartículas de diisopropiletilamina e dietilaminoetil-quitosana: efeito da massa molar na liberação de RNA de interferência
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Data
2017-03-17
Autores
Souza, Ricchard Hallan Félix Viegas de [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A quitosana, polímero catiônico de origem natural, biocompatível, biodegradável e de baixa toxicidade vem sendo largamente estudada e utilizada no desenvolvimento de vetores não-virais para uso em terapia gênica. Nesse estudo, derivados de quitosana foram modificados com grupos hidrofílicos dietilaminoetil (DEAE) e diisopropiletilamina (DIPEA) tendo como objetivo melhorar a eficiência de transfecção e a liberação do material genético no ambiente intracelular. Poliplexos dos derivados de quitosana com diferentes massas molares foram investigados em ensaios de transfecção in vitro realizados com células HeLa, utilizando siRNA SSB como agente silenciador. Os resultados obtidos mostraram bons níveis de silenciamento e bloqueio de expressão do RNAm SSB, apresentando, em algumas situações, eficiência superior aos vetores não-virais Lipofetamina®, TransIT-siQUEST® e PEI. Fatores que podem afetar a eficiência de transfecção como pH, massa molar, grau de ionização, tamanho dos poliplexos, potencial zeta e razão N/P foram avaliados. Tanto as quitosanas desacetiladas quanto os seus derivados mostraram níveis de viabilidade celular superiores a 80% em ensaios de citotoxicidade realizados com MTS-PMS. Os resultados sugerem que os derivados de quitosana sintetizados e caracterizados nesse estudo possuem características favoráveis que os habilitam como potenciais vetores não-virais para serem usados em terapia gênica.
Chitosan, a natural biocompatible, biodegradable and low toxicity cationic polymer has been widely studied and used in the development of non-viral vectors for use in gene therapy. In this study, chitosan derivatives were modified with hydrophilic groups diethylaminoethyl (DEAE) and diisopropylethylamine (DIPEA) aiming to improve the transfection efficiency and the release of the genetic material in the intracellular environment. Poliplexes of chitosan derivatives with different molar masses were investigated in vitro transfection assays performed with HeLa cells using SSB siRNA as the silencing agent. The results showed significant knockdown of SSB mRNA expression for chitosan and its derivatives, presenting, in some situations, superior efficiency to the non-viral vectors Lipofetamina®, TransIT-siQUEST® and PEI. Factors that could affect transfection efficiency such as pH, molar mass, degree of ionization, nanoparticle size, zeta potential and N/P ratio were evaluated. Both deacetylated chitosan and its derivatives showed cell viability above 80% in MTS-PMS cytotoxicity assays. The results suggest that the chitosan derivatives synthesized and characterized in this study have favorable characteristics that enable them as potential non-viral vectors to be used in gene therapy.
Chitosan, a natural biocompatible, biodegradable and low toxicity cationic polymer has been widely studied and used in the development of non-viral vectors for use in gene therapy. In this study, chitosan derivatives were modified with hydrophilic groups diethylaminoethyl (DEAE) and diisopropylethylamine (DIPEA) aiming to improve the transfection efficiency and the release of the genetic material in the intracellular environment. Poliplexes of chitosan derivatives with different molar masses were investigated in vitro transfection assays performed with HeLa cells using SSB siRNA as the silencing agent. The results showed significant knockdown of SSB mRNA expression for chitosan and its derivatives, presenting, in some situations, superior efficiency to the non-viral vectors Lipofetamina®, TransIT-siQUEST® and PEI. Factors that could affect transfection efficiency such as pH, molar mass, degree of ionization, nanoparticle size, zeta potential and N/P ratio were evaluated. Both deacetylated chitosan and its derivatives showed cell viability above 80% in MTS-PMS cytotoxicity assays. The results suggest that the chitosan derivatives synthesized and characterized in this study have favorable characteristics that enable them as potential non-viral vectors to be used in gene therapy.
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Palavras-chave
Derivados de quitosana, siRNA, Massa molar, Transfecção in vitro, Chitosan derivatives, Molar mass, in vitro transfection