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Author
Navalón López, María
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Abstract
Gene therapy is currently emerging as a treatment for a wide range of acquired and genetic disorders, including the use of oligonucleotides as therapeutic agents. However, gene therapy from the beginning has the challenge to overcome: the successful delivery of the load in a target cell. To make this possible, a vehicle must be designed to protect the oligonucleotyped and deliver it to the specific tissue and intracellular compartment.
The first approaches were carried out using genetically modified viruses as vectors for their natural ability to infect and cross cellular barriers to delivering genetic material. However, viral vectors have some negative aspects such as cytotoxicity, immunogenicity, insertion mutagenesis, low carrying capacity, and high production costs. Therefore, these drawbacks triggered the development of non-viral delivery systems. The most widely used are based on polymeric systems, specifically, polyplexes, which consist of the combination of cationic polymers with negatively charged oligonucleotides to condense into colloidal nanostructures. This project proposes the use of polymeric nanoparticles, specifically poly (b-amino ester) s (pBAE) modified oligopeptides due to their high transfection efficiency, low toxicity, high ease of modification, and excellent biocompatibility and biodegradation. In addition, pBAEs can be designed to increase their buffering capacity and their ability to compact nucleic acids thus promoting cell internalization. In particular, alterations of this polymer are made in the terminal groups which are modified by the addition of cationic peptides such as lysine (K), arginine (R), and histidine (H). As a result of these modifications, the polymer will have an intensely positive charge, so it will make the nanoparticle very promiscuous as the cell membrane is anionic. Therefore, endocytosis will increase and nanoparticles will transfect cells without discrimination. In order to make a more specific treatment, these nanoparticles can be modified to decrease the positive surface charge and therefore reduce the efficiency of non-selective transfection. For this, a coating of an anionic polymer and what aspartic acid would be like can be added (D).
In this thesis the research has focused on OM-pBAE with aspartic acid, synthesizing combinations of RHD, KHD, and RKD nanoparticles together with the cationic polyplexes RH, KH, and RK, as proof of concept. With the help of advanced techniques, the traffic of the different polyplexes can be unmasked and, therefore, efficient delivery systems can be designed. Starting from the carrier and the stability of the nanoparticle and ending with the understanding of the intracellular release of genetic material. These primary studies are critical to the successful development of careers that release the charge into specific tissues.
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