Nanoparticulas de adna quitosano evaluacion y desarrollo para su uso com vector de terapia genica
- Autores: Miguel Noel Centelles Llorente
- Directores de la Tesis: Juan Manuel Irache Garreta (dir. tes.), Cheng Qian (codir. tes.)
- Lectura: En la Universidad de Navarra ( España ) en 2006
- Idioma: español
- Tribunal Calificador de la Tesis: Ignacio Javier Melero Bermejo (presid.), José Ramón Isasi Allica (secret.), Alicia Rodríguez Gascón (voc.), Ramon Alemany Bonastre (voc.), Juan Ruiz Echeverria (voc.)
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- Resumen
Manopartículas DE adn-quitosano: evaluación y desarrollo para SU USO como vector DE terapia genica RESUMEN: Chitosan is a partially deacetylated polymer of N-acetyl glucosamine. it is usually prepared from chitin [(1 - 4) -2 -acetamido-2-deoxy-beta-D-glucan], the second most universally abundant biopolymer behind cellulose. The presence of amino groups on chitosan backbone confers a high positive charge density dependent on its deacetylation degree, and makes it behave as a polycation at acidic and neutral pH. Cationic polymers have the potential for dna complexation and it is recognised that they may be useful as non-viral vectors for gene delivery. Tne objective of this work was to evalúate and develop the DNA-chitosan nanoparticles as non-viral gene delivery systems. To this end, three commercially available chitosans, differing in their molecular weight (150kDa, 400kDa and 600kDa), were purchased and the chitosan with a molecular weight (mw) of 400 Kda and a degree of deacetylation (DD) of 85% was modified by acetylation with acetic anhydride to obtain a chitosan with a DD of 75%. All polysaccharides were used to prepare DNA-chitosan nanoparticles by charge interactions with pDNA (coacervation process). The influence of the chitosan-to-DNA ratio, molecular weight Cmw) of chitosan and size of the plasmid on the physico-chemical properties of the resulting nanoparticles was examined. Then, gene transfection efficacy and toxicity of the different DNA-chitosan nanoparticles was determined on 293T cells (FACS and bioluminescence assays).The in vivo studies were performed by two administration procedures in mice: the hydrodynamic and the intraduodenal injections. The transgene expression was followed using either the green fluorescent protein (GFP) or the luciferase (Luc) as repórter genes. Admimstered by the hydrodynamics-based procedure, the DNA-chitosan nanoparticles were accumulated in the liver, where the transgene was expressed for at least 105 days. No significant influence of mw was observed on the levels of transgene expression along the whole kinetic, but interestingly, the DD improved the initial burst effect, and accelerated the DNA release, facts that leaded to increase of the levels of transgene expression. The kinetics of transgene expression observed with the CCD camera were described and divided in 3 phases: (i) the initial burst release, (ii) the sustained release step and (iii) the decline phase (promotor inactivation and immunological processes). The calculation of the different kinetic parameters (Ke, tl/2 Ke, Cmax, AUC, and MET), characterizing the kinetics of transgene expression, enable us to propose a new methodology that would allow further comparisons between a wide variety of gene delivery systems. Finally, the intraduodenal administration of naked DNA permitted the gene transfer in a dose dependent manner quantifiable with the CCD camera within 3 days. Nevertheless, the same administration procedure of the different formulations did not improve the levels of transgen expression obtained with naked DNA. This fact could be explained by the rapid physiological turn-over of enterocytes and by the ability of chitosan nanoparticles to control the DNA release
