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Title: Engineered nanoparticles : safe and efficient vectors for nucleic acid delivery
Author: Alamoudi, A. A.
ISNI:       0000 0004 7230 2155
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2016
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Therapeutics based on nucleic acids offer the possibility of specific and potent treatment of diseases. However, the core obstacle is the ability to deliver these nucleic acid drugs safely and efficiently. In our laboratories we have formulated the nucleic acids: plasmid DNA and short interfering RNA in a synthetic delivery system that facilitates cellular delivery. The delivery system (4PPA) was synthesised, characterised with overall 10% modification level and complexed electrostatically with β-galactosidase plasmid DNA and siRNA-ITCH, in phosphate buffer (pH = 6.0). The resulting complexes were sized, their zeta potential measured and imaged using scanning electron microscopy and transmission electron microscopy. The complexes were further characterised for binding stability and nucleic acid protection using gel electrophoresis. The in vitro transfection efficiency and cell cytotoxicity of the polyplexes were determined in MIA PaCa-2 and A-431 cells. Cellular uptake of a fluorescently labelled nucleic acid was also investigated using flow cytometry and confocal microscopy. Additionally, in vivo studies were conducted to obtain comprehensive data on the behaviour of polyplex formulations following an intranasal brain delivery. Polyplex nanoparticles were 150–350 nm in diameter (polydispersity < 0.5), with a zeta potential of 10–20 mV (n=3) and were spherical in shape. The polymer delivered nucleic acids to the cells. Efficient β-galactosidase up-regulation and Itchy E3 Ubiquitin protein ligase (ITCH) down-regulation were achieved in vitro. The new polymer was as efficient as Lipofectamine® 2000 and 3rd Generation poly(propylenimine) dendrimer in transfecting, but more than several hundred times less toxic. In vivo, 4PPA successfully delivered siRNA-ITCH to the CNS of the olfactory bulb and brain tissue and knocked-down ITCH expression with twice the efficiency of siRNA-ITCH alone. A novel, biocompatible delivery system that provides efficient gene and siRNA transferring vehicles was thus synthesised, achieving good in vitro and in vivo transfection/ down-regulation.
Supervisor: Uchegbu, I. F. ; Schatzlein, A. G. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available