Paulina Alejandra MONTAÑO GONZÁLEZ PhD defense
Insights into the physicochemical parameters of DNA/Poly(ethylenimine) polyplexes governing stability and promoting an efficient gene delivery into cells
Vrijdag 13 december 2024, 14u00Verlopen
Organic Chemistry & Interfaces (COrInt) team
Abstract
The lack of understanding of the stability and dissociation mechanisms of nucleic acid-based polyplexes, allowing the release of DNA, is currently a major limitation in non-viral gene delivery. Although a great deal of progress has been made in this field, the efficacy of these synthetic systems remains much lower than that achieved by viral systems, with only 0.01 to 2% of initially loaded nucleic acids able to exert their biological function within the cell. Poly(ethylenimine) (PEI) is one of the most widely used polycations for gene delivery due to its proven high transfection efficiency, making it an interesting model of DNA complexing agent for this work. The research performed and stated in this PhD thesis aims to reduce the gap in the knowledge about PEI/DNA polyplexes stability and dissociation in presence of biological media and anionic biomacromolecules, as well as on the impact of PEI/DNA polyplexes physicochemical properties in intracellular trafficking and transfection efficiency. To achieve these goals, the complexation of a plasmid DNA (peGFP-C3) was conducted using four different PEI samples, having different molecular weights and structures (branched or linear), and at different charge ratios. The stability of PEI/peGFP-C3 polyplexes was studied in terms of particle size and plasmid DNA release at different environments of ionic strength, as well as in presence of biological macromolecules such as glycosaminoglycans (GAGs). It was found that heparin’s high charge density, compared to other GAGs and polyanions, as well as strong interactions between heparin and PEI, determined from thermodynamic studies, are the crucial parameters that drive the strength of complexation with PEI and subsequently facilitate the release of DNA from the polyplexes. Circular dichroism measurements revealed that the plasmid maintains its B-type conformation after complexation with PEI and after subsequent release in heparin. Furthermore, correlations between data of the expression of reporter protein transfection assays in HEK293T cells with both the molecular characteristics of PEI and the physicochemical characteristics of the polyplexes allowed revealing that the branched PEI with a molecular weight of 25 kg/mol promoted higher internalization and GFP expression on HEK293T cells. In the long term, the outcome of this work will be to propose guidelines to help design more effective, and less cytotoxic non-viral gene carriers with a great potential for new therapeutic applications.
Jury
- Christophe CHASSENIEUX, Professeur des Universités, IMMM, Université de Le Mans
- Gilles JOUCLA, Maître de Conférence, ENSTBB, Université de Bordeaux
- Adeline BOIRE, Chargée de Recherche, INRAE, Nantes
- Jean-Paul CHAPEL, Directeur de Recherche, CRPP, Université de Bordeaux
- Paul-Alain JAFFRÈS, Professeur des Universités, UBO, Université de Brest
- Sophie TOMASI, Professeure des Universités, ISCR, Université de Rennes
- Fabienne GAUFFRE, Directrice de Recherche, ISCR, Université de Rennes / Directrice de thèse
- Lourdes Mónica BRAVO ANAYA, Chargée de Recherche, ISCR, Université de Rennes / Co-encadrante de thèse
Guests
- Christophe SCHATZ, Maître de Conférence, LCPO, Université de Bordeaux
- Pascal LOYER, Directeur de Recherche, INSERM, Université de Rennes
Contacts
Fabienne Gauffre, fabienne.gauffre@univ-rennes.fr
Lourdes Mónica Bravo Anaya, lourdes-monica.anaya@univ-rennes.fr
