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Previous issue date: 2007-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Broadly speaking, the concept of gene therapy involves the transfer of a genetic material into a cell, tissue, or organ in order to cure a disease or at least improve the clinical status of a patient. Making it simple, gene therapy consists in the insertion of functional genes into cells containing defective genes by substituting, complementing or inhibiting them. The achievement of a foreigner DNA expression into a population of cells requires its transfer to the target. Therefore, it is a key issue to create systems able to transfer and protect the DNA until it reaches the target, the vectors. The disadvantages related to the use of viral vectors have encouraged efforts to develop emulsions as non-viral vectors. In fact, they are easily produced, present controllable stability and enable transfection. The aim of this work was to develop an emulsion for gene therapy and evaluate its ability to compact nucleic acids by the development of a complex with the plasmid pIRES2-EGFP. The first step was to determine the Hydrophilic Lipophilic Balance (HLB) of the Captex? 355 (oily internal phase of the emulsion) through long and short term stability assays. Based on the results, emulsions composed of Captex? 355, Tween 20? and Span 60? with 10.7 HLB were produced by three different methods: phase inversion, spontaneous emulsification and sonication. The results showed that the lowest diameter and best stability of the emulsions were achieved by the sonication method. The cationic emulsions were made by adding DOTAP to the basic emulsion. Its association with pIRES2-EGFP was evaluated by electrophoresis. Several rates of emulsion and DNA were evaluated and the results showed that 100% of the complex was formed when the rate DOTAP/DNA(nmol/?g) was 130. In conclusion, the overall results show the ability of the proposed emulsion to compact pIRES2-EGFP, which is a requirement to a successful transfection. Therefore, such formulation may be considered a promising candidate for gene therapy / Terapia g?nica, em uma ampla defini??o, ? o tratamento de doen?as baseado na transfer?ncia de material gen?tico a uma c?lula, tecido ou ?rg?o com o intuito de curar ou melhorar o estado cl?nico do paciente. Em sua forma mais simples, a terapia g?nica consiste na inser??o de genes funcionais em c?lulas com genes defeituosos objetivando substituir, complementar ou inibir esses genes causadores de doen?as. Para que o DNA ex?geno seja expresso em uma popula??o celular faz-se necess?ria a sua transfer?ncia at? o local. Assim, ? necess?rio criar ve?culos, os vetores, que transportem e protejam o DNA at? que este chegue a uma popula??o celular alvo. Os obst?culos encontrados com a utiliza??o de vetores virais t?m proporcionado o interesse no desenvolvimento de emuls?es cati?nicas como vetores n?o-virais, por serem f?ceis de produzir, apresentarem estabilidade control?vel e facilitarem a transfec??o g?nica. O objetivo deste trabalho foi desenvolver um sistema emulsionado para terapia g?nica e avaliar sua capacidade de compacta??o de ?cidos nucl?icos atrav?s da sua associa??o com o plasm?deo pIRES2-EGFP. Primeiramente o EHLc do TCM utilizado, o Captex? 355, foi determinado atrav?s de ensaios de estabilidade acelerada e a longo termo. Com base nos resultados obtidos, emuls?es de EHL 10,7 compostas de Captex? 355, Tween 20? e Span 60? foram preparadas pelos m?todos de invers?o de fases, emulsifica??o espont?nea e sonica??o e elegeu-se o melhor m?todo para o preparo das emuls?es cati?nicas. As emuls?es de menor granulometria e maior estabilidade foram obtidas atrav?s do m?todo de sonica??o. As emuls?es cati?nicas foram preparadas acrescendo-se ? emuls?o base o DOTAP e a sua associa??o com o pIRES2-EGFP foi avaliada atrav?s da t?cnica de eletroforese em gel de agarose. V?rias propor??es de emuls?o e DNA foram testadas e os resultados demonstraram que houve forma??o de 100% dos complexos quando a propor??o DOTAP/DNA(nmol/?g) foi igual a 130. Em conclus?o, o conjunto dos resultados obtidos demonstra a capacidade da emuls?o proposta neste trabalho de compactar o DNA, requisito necess?rio para uma boa transfec??o, tornando a formula??o uma forte candidata ? utiliza??o em terapia g?nica
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/16784 |
| Date | 26 February 2007 |
| Creators | Ver?ssimo, Lourena Mafra |
| Contributors | CPF:38878240400, http://lattes.cnpq.br/6907806915889763, Magalh?es, Nereide Stella Santos, CPF:34939610497, Medeiros, S?lvia Regina Batistuzzo de, CPF:32398336468, http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781004Y8, Lima, Lucymara Fassarela Agnez, Egito, Eryvaldo S?crates Tabosa do |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular, UFRN, BR, Gen?tica e Biologia Molecular |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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