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Title: Nucleic acid delivery : biocompatible yet efficient platforms
Author: Carlos, M. I.
ISNI:       0000 0004 5364 9962
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2015
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Gene therapy aims to act on the genetic cause of a pathology by gene inhibition or substitution. Gene delivery systems are necessary to deliver intact nucleic acids to the cells in order to achieve a therapeutic effect. Chitosan is a linear polysaccharide that displays properties such as biocompatibility and biodegradability. Despite the efforts to develop ch itosan-based vectors, the therapeutic effectiveness of ch itosan-base gene therapy still needs to be improved in order to achieve clinical significance. This work introduces a new chitosan-based polymer: N-(2-ethylami no)-6-O-g lycol chitosan (EAGC). The new polymer aims to overcome the disadvantages of chitosan for gene delivery, such as poor solubility at physiological pH and low buffer capacity, in order to enhance its transfection efficiency while retaining its main benefits of low toxicity and biocompatibility. Three batches of EAGC were synthesized with different degrees of ethylamino substitution: EAGC1 7, EAGC21 and EAGC30. The EAGC synthesis and the degree of substitution of monomers with the new ethylamino group were confirmed by Nuclear Magnetic Resonance. The agarose gel retardation assay revealed that all polymers had the ability to condense with DNA/siRNA at different polymer, DNA/siRNA mass ratios. EAGC30 was able to condense both nucleic acids at smaller polymer, DNA/siRNA mass ratios due to its higher charge density. The nanoparticles formed between the different polymers and DNA/siRNA presented sizes between 100 and 450nm with a positive charge of +40mV and spherical shape. The stability of the DNA/siRNA nanoparticles was tested in the presence of different biological challenges. All EAGC polymers were able to deliver the β-galactosidase plasmid to A431 cells in vitro. EAGC30 showed the best transfection capacity at lower polymer, DNA mass ratios. Differences in charge density of the polymers resulted in different gene activity. Nevertheless, all EAGC polymers were superior transfection agents to lipofectamine, particularly at high polymer, DNA mass ratios. In vitro down regulation of proteins was obtained for EAGC30, siRNA mass ratios 30 and 60. Delivery to the brain of a complex formed between siRNA and a chitosan derivative, with a nose to brain delivery method, will be presented for the first time. Fluorescent EAGC30, siRNA nanoparticles were visualized on the olfactory bulb tissue after nasal administration. The results of this study confirmed Ethylamino Glycol Chitosan as a good candidate for in vitro and in vivo gene delivery.
Supervisor: Uchegbu, I. F. ; Schatzlein, A. G. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available