Síntese, caracterização e estudo físico-químico de nanopartículas formadas pela interação de derivados hidrofílicos de quitosana com DNA
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Data
2011-12-07
Autores
Casé, Ana Helena [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A quitosana tem emergido na última década como um bom candidato para a entrega genética devido suas propriedades, como baixa toxicidade, biodegradabilidade e versatilidade, como vetor em administração intravenosa e oral. Além disso, possui a capacidade de condensar de forma eficiente com o DNA formando poliplexos resistentes, o que é importante para evitar a degradação por desoxirribonucleases ( DNAses). As nanopartículas resultantes têm recebido grande atenção por suas potenciais aplicações em terapia gênica não viral. Neste trabalho foi realizada a síntese, caracterização e estudo físico químico das nanopartículas formadas pela interação de derivados hidrofílicos de quitosana com DNA. As interações entre derivados de quitosana contendo os grupos fosforilcolina e os grupos poli(etileno glicol) com DNA foram investigadas com base nos efeitos da razão de cargas, pH e conteúdo dos grupos substituintes (PC e PEG) sobre o tamanho e estabilidade dos complexos. Nos estudos foram utilizadas as técnicas de fluorescência com o ensaio do brometo de etídio (EtBr), eletroforese em gel de agarose, espalhamento de luz dinâmico e microscopia de fluorescência. O tamanho e estabilidade coloidal das nanopartículas preparadas com quitosana desacetilada (CHD), quitosana-fosforilcolina (CH-PC) e quitosana-PEG (CH-PEG) mostraram-se fortemente dependentes do conteúdo dos grupos hidrofílicos, bem como, da razão de cargas e pH. A força de interação foi avaliada por meio de ensaio com EtBr. Para razões de carga (N/P) maiores que 5,0, nenhuma liberação do DNA foi observada das nanopartículas por eletroforese em gel. As nanopartículas formadas pela interação dos derivados com DNA (CH-PC-DNA e CH-PEG-DNA) em baixos valores de pH exibiram uma maior resistência à agregação quando...
Chitosan has been recognised in the past decade as a good candidate for gene delivery due to its properties as low toxicity, biodegradability and versatility as a carrier for intravenous and oral administration. Also, the ability to condense efficiently with DNA forming tight polyplexes is important to avoid the degradation by DNAses. The resulting nanoparticles have received great attention for their potential applications in nonviral gene therapy. In this work it was carried out the synthesis, characterization and physicochemical studies of nanoparticles formed by the interaction of hydrophilic derivatives of chitosan with DNA. The interactions between chitosan derivatives containing phosphorylcholine (CH-PC) and poly(ethylene glycol) (CH-PEG) moieties and calf thymus DNA (DNA) were investigated focusing on the effects of the charge ratio, the pH and phosphorylcholine content on the size and stability of the complexes using the ethidium bromide fluorescence assay, gel electrophoresis, dynamic light scattering and fluorescence microscopy. The size and colloidal stability of deacetylated chitosan (CH-DNA), CH-PC-DNA and CH-PEG-DNA complexes were strongly dependent upon phosphorylcholine (PC) and poly(ethylene glycol) contents, charge ratios and pH. The interaction strengths were evaluated by ethidium bromide fluorescence and, at N/P ratios higher than 5.0, no DNA release was observed in any synthesized CH-PC-DNA and CH-PEG-DNA polyplexes by gel electrophoresis. The CH-PC-DNA and CH-PEG-DNA polyplexes exhibited a higher resistance to aggregation compared to deacetylated chitosan (CHD) at neutral pH. At low pH values highly charged chitosan and its phosphorylcholine and PEG derivatives had strong binding affinity with DNA, whereas at higher pH values chitosan (CHD) formed... (Complete abstract click electronic access below)
Chitosan has been recognised in the past decade as a good candidate for gene delivery due to its properties as low toxicity, biodegradability and versatility as a carrier for intravenous and oral administration. Also, the ability to condense efficiently with DNA forming tight polyplexes is important to avoid the degradation by DNAses. The resulting nanoparticles have received great attention for their potential applications in nonviral gene therapy. In this work it was carried out the synthesis, characterization and physicochemical studies of nanoparticles formed by the interaction of hydrophilic derivatives of chitosan with DNA. The interactions between chitosan derivatives containing phosphorylcholine (CH-PC) and poly(ethylene glycol) (CH-PEG) moieties and calf thymus DNA (DNA) were investigated focusing on the effects of the charge ratio, the pH and phosphorylcholine content on the size and stability of the complexes using the ethidium bromide fluorescence assay, gel electrophoresis, dynamic light scattering and fluorescence microscopy. The size and colloidal stability of deacetylated chitosan (CH-DNA), CH-PC-DNA and CH-PEG-DNA complexes were strongly dependent upon phosphorylcholine (PC) and poly(ethylene glycol) contents, charge ratios and pH. The interaction strengths were evaluated by ethidium bromide fluorescence and, at N/P ratios higher than 5.0, no DNA release was observed in any synthesized CH-PC-DNA and CH-PEG-DNA polyplexes by gel electrophoresis. The CH-PC-DNA and CH-PEG-DNA polyplexes exhibited a higher resistance to aggregation compared to deacetylated chitosan (CHD) at neutral pH. At low pH values highly charged chitosan and its phosphorylcholine and PEG derivatives had strong binding affinity with DNA, whereas at higher pH values chitosan (CHD) formed... (Complete abstract click electronic access below)
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Biologia molecular, Biofísica, Nanopartículas
Como citar
CASÉ, Ana Helena. Síntese, caracterização e estudo físico-químico de nanopartículas formadas pela interação de derivados hidrofílicos de quitosana com DNA. 2011. 88 f. Tese (doutorado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, 2011.