Síntese e caracterização de derivados de baixa massa molecular de diisopropiletilamina-quitosana: estudo da interação com RNA de interferência
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Data
2021-07-07
Autores
Jonas, Víctor Augusto dos Santos
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A terapia gênica tem o potencial de tratar doenças hereditárias para as quais há poucos tratamentos eficazes. As terapias baseadas em RNA interferente (siRNA) tem demonstrado grande potencial para silenciar proteínas alvo causadoras de doenças. Essa modalidade de terapia exige carreadores seguros e eficientes, capazes de transportar o material genético até o ambiente intracelular, um desafio que tem limitado suas aplicações terapêuticas. Nesse estudo, foram sintetizados e caracterizados policátions derivados de quitosana, para avaliação como potenciais carreadores para RNA de interferência. Derivados de quitosana de baixa massa molar e graus variados de acetilação (76,0 – 97,3%) foram modificados com grupos diisopropiletilamina (DIPEA) variando de 13,8 a 104,6 %. Os polímeros foram caracterizados por RMN 1H e as massas molares (10,5 – 16,4 kDa) determinadas por GPC. O grau de ionização dos derivados foi estudado em função do pH e os resultados mostraram que a densidade de cargas pode ser controlada variando-se o grau de substituição por DIPEA. O potencial de interação desses derivados com siRNA, e da formação de nanopartículas foi estuda por meio das técnicas de eletroforese, e espalhamento de luz. As nanopartículas apresentaram potencial Zeta positivo a partir da razão de carga 3,0 de amino/fosfato (N/P) para maioria dos polímeros testados. O estudo de eletroforese indicou que os derivados de quitosana interagiram fortemente com o siRNA e graus de substituição intermediários de 20 e 40 % se mostraram mais favoráveis para a formação de poliplexos nanométricos (~200 nm) em baixas razões de carga (N/P 10,0 e 20,0). A toxicidade dos polímeros e das nanopartículas foram testadas para maioria dos polímeros até 1,5 g/L, e a totalidade dos derivados estudados apresentou baixa toxicidade e atividade antioxidante significativa. A partir dos estudos conduzidos foram selecionados derivados que apresentam propriedades físico-químicas favoráveis para à aplicação como carreadores de RNA de interferência.
Gene therapy has the potential to treat hereditary diseases for which there are few effective treatments. Interfering RNA (siRNA)-based therapies have shown great potential to silence disease-causing target proteins. This modality of therapy requires safe and efficient carriers, capable of transporting genetic material to the intracellular environment, a challenge that has limited its therapeutic applications. In this study, polycations based on chitosan derivatives were synthesized and characterized for evaluation as potential carriers for interfering RNA. Chitosan derivatives with low molar mass and varying degrees of acetylation (76.0 – 97.3%) were modified with diisopropylethylamine (DIPEA) groups ranging from 13.8 to 104.6%. The polymers were characterized by 1H NMR and the molar masses (10.5 – 16.4 kDa) determined by gel permeation chromatography GPC. The degree of ionization of derivatives was studied as a function of pH and the results showed that the charge density can be controlled by varying the degree of substitution by DIPEA. The ability of these derivatives to interact with siRNA, and the formation of nanoparticles was studied by electrophoresis and light scattering techniques. The nanoparticles exhibited positive Zeta potentials at charges ratios (amino/phosphate (N/P) higher than 3.0 for most of the tested polymers. The electrophoresis study indicated that chitosan derivatives strongly interacted with siRNA and polymers having intermediate degrees of substitution of 20 and 40% were more favorable for the formation of nanometric polyplexes (~200 nm) at low charge ratios (N/P 10.0 and 20.0). The toxicity of polymers and nanoparticles were tested and for the majority of the polymers up to 1.5 g/L, and all polyplexes studied showed low toxicity and significant antioxidant activity. From the studies conducted, it was possible to select derivatives that have displayed favorable physicochemical properties for application as small interfering RNA carriers.
Gene therapy has the potential to treat hereditary diseases for which there are few effective treatments. Interfering RNA (siRNA)-based therapies have shown great potential to silence disease-causing target proteins. This modality of therapy requires safe and efficient carriers, capable of transporting genetic material to the intracellular environment, a challenge that has limited its therapeutic applications. In this study, polycations based on chitosan derivatives were synthesized and characterized for evaluation as potential carriers for interfering RNA. Chitosan derivatives with low molar mass and varying degrees of acetylation (76.0 – 97.3%) were modified with diisopropylethylamine (DIPEA) groups ranging from 13.8 to 104.6%. The polymers were characterized by 1H NMR and the molar masses (10.5 – 16.4 kDa) determined by gel permeation chromatography GPC. The degree of ionization of derivatives was studied as a function of pH and the results showed that the charge density can be controlled by varying the degree of substitution by DIPEA. The ability of these derivatives to interact with siRNA, and the formation of nanoparticles was studied by electrophoresis and light scattering techniques. The nanoparticles exhibited positive Zeta potentials at charges ratios (amino/phosphate (N/P) higher than 3.0 for most of the tested polymers. The electrophoresis study indicated that chitosan derivatives strongly interacted with siRNA and polymers having intermediate degrees of substitution of 20 and 40% were more favorable for the formation of nanometric polyplexes (~200 nm) at low charge ratios (N/P 10.0 and 20.0). The toxicity of polymers and nanoparticles were tested and for the majority of the polymers up to 1.5 g/L, and all polyplexes studied showed low toxicity and significant antioxidant activity. From the studies conducted, it was possible to select derivatives that have displayed favorable physicochemical properties for application as small interfering RNA carriers.
Descrição
Palavras-chave
Derivados de quitosana, siRNA, Vetor não viral, Transfecção in vitro, Terapia gênica, Chitosan, Non-viral vector, In vitro transfection, Gene therapy