Estudo físico-químico de nanopartículas de DNA com derivado de quitosana contendo grupos fosforilcolina
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Data
2009-10-09
Autores
Picola, Isadora Pfeifer Dalla [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Na presente dissertação, foram preparadas quitosanas com diferentes massas molares, 16 kDa, 18 kDa e 29 kDa, e com diferentes graus de substituição de fosforilcolina (PC). As quitosanas de baixa massa molecular foram obtidas pela degradação de quitosana desacetilada. Essas quitosanas foram purificadas com membranas de diálise de tamanho de exclusão apropriadas e caracterizadas por meio de titulação potenciométrica, H-RMN e Cromatografia de permeação em gel (GPC). O estudo físico-químico da interação das quitosanas com DNA foi realizado utilizando-se as técnicas de fluorescência, eletroforese, microscopia óptica, microscopia eletrônica de transmissão e espalhamento de luz dinâmico. As propriedades avaliadas foram a eficiência de interação, a estabilidade dos complexos, potencial zeta e tamanho de poliplexos. Os experimentos foram conduzidos variando-se a força iônica, pH do meio, massa molar de policátion e razão de cargas para quitosanas com diferentes conteúdos de grupos PC. Além das quitosanas preparadas durante o projeto, também se utilizou quitosanas de diferentes massas molares, de 5 e 150 kDa, para estudo dos poliplexos em diferentes forças iônicas. Os resultados mostraram que a eficiência de interação entre quitosana e DNA é reduzida com a presença do grupo PC. Nos estudos de DLS, verificou-se que, em baixos valores de pH, o método de coacervação permite a obtenção de nanopartículas de 150 a 300 nm. A força de interação, o tamanho e a estabilidade das nanopartículas dependem do pH do meio, da força iônica da solução, da massa molar e do conteúdo de fosforilcolina. Em pHs mais elevados, os poliplexos são mais estáveis quanto maior a massa molar ou quando há a presença de grupo fosforilcolina. O trabalho permitiu ampliar os estudos sobre os efeitos da força iônica, do pH, da massa molar e conteúdos de PC e como, tais parâmetros...
In this work chitosans with different molecular weights and their derivatives containing different amounts of phosphorylcholine (PC) were prepared. The low molecular weights chitosans were obtained by degradation of deacetylated chitosan. These chitosans were purified by dialysis membranes of appropriate sizes of exclusion and characterized by potentiometric titration, H-NMR and gel permeation chromatography (GPC) techniques. The physico-chemical study of the interaction between chitosan and DNA was performed using the ethidium bromide fluorescence assay, gel electrophoresis, optical microscopy, transmission electron microscopy and dynamic light scattering techniques. The experiments were carried out at varying experimental conditions as ionic strength, pH, and charge ratio with chitosans of different molecular weights and phosphorylcholine contents. The results showed that the efficiency of the interaction between chitosan and DNA was reduced with the incorporation of PC on the chitosan chain. The results showed that at low pH values the sizes of the nanoparticles obtained by the coacervation method varied from 150 to 300 nm. The strength of the interaction and the size of the nanoparticles were shown to be dependent of pH, ionic strength, chitosan molecular weight and of the phosphorylcholine contents. The study at high pH values showed that more stable nanoparticles were formed with chitosans having the higher molecular weights. The attaching of phosphorylcholine groups to the chitosan main chain allows obtaining more stable particles at high pH values. In this work we provide new insights on the effects of molecular weight, pH, ionic strength and PC contents on both the chitosan-DNA interaction and the stability of formed nanoparticles
In this work chitosans with different molecular weights and their derivatives containing different amounts of phosphorylcholine (PC) were prepared. The low molecular weights chitosans were obtained by degradation of deacetylated chitosan. These chitosans were purified by dialysis membranes of appropriate sizes of exclusion and characterized by potentiometric titration, H-NMR and gel permeation chromatography (GPC) techniques. The physico-chemical study of the interaction between chitosan and DNA was performed using the ethidium bromide fluorescence assay, gel electrophoresis, optical microscopy, transmission electron microscopy and dynamic light scattering techniques. The experiments were carried out at varying experimental conditions as ionic strength, pH, and charge ratio with chitosans of different molecular weights and phosphorylcholine contents. The results showed that the efficiency of the interaction between chitosan and DNA was reduced with the incorporation of PC on the chitosan chain. The results showed that at low pH values the sizes of the nanoparticles obtained by the coacervation method varied from 150 to 300 nm. The strength of the interaction and the size of the nanoparticles were shown to be dependent of pH, ionic strength, chitosan molecular weight and of the phosphorylcholine contents. The study at high pH values showed that more stable nanoparticles were formed with chitosans having the higher molecular weights. The attaching of phosphorylcholine groups to the chitosan main chain allows obtaining more stable particles at high pH values. In this work we provide new insights on the effects of molecular weight, pH, ionic strength and PC contents on both the chitosan-DNA interaction and the stability of formed nanoparticles
Descrição
Palavras-chave
Biofísica, Biologia molecular, Quitosana, Nanopartículas, Vetores genéticos, Terapia gênica, Chitosan, Gene therapy, Non-viral vector, nanoparticles, Phosphorylcholine
Como citar
PICOLA, Isadora Pfeifer Dalla. Estudo físico-químico de nanopartículas de DNA com derivado de quitosana contendo grupos fosforilcolina. 2009. 118 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, 2009.