Non-viral gene therapy : design and characterisation of novel non-viral vectors for improved cellular transfection

The promise of gene therapy, once hailed as the medical treatment of the future, has yet to be achieved. Problems related to the safety and efficacy of gene therapies have checked the enthusiasm once surrounding this field. The future of gene therapy relies on the development of safe and effective non-viral vectors for gene delivery.This doctoral thesis describes the development and evaluation of a novel nanoparticle system that demonstrates characteristics suitable for gene delivery purposes. The work presented in this thesis is divided into three main phases: (1) Development of a novel delivery system. The development of a novel carrier was undertaken with the goal of producing a system that is biocompatible, biodegradable and non-toxic. The alginate-chitosan polyelectrolyte system, chosen for development due to its desirable characteristics at the macro- and micro-scales, was successfully used to prepare nanoparticles of appropriate size for delivery purposes. (2) Evaluation of the delivery system as a gene carrier. Alginate-chitosan nanoparticles were shown to complex DNA and effectively protect it from degradation. The inclusion of alginate in the system was confirmed to reduce the strength of binding between chitosan and DNA, thereby facilitating its release intracellularly. Cell viability studies confirm the non-toxicity of the system, while in vitro studies confirm the ability of alginate-chitosan nanoparticles to mediate efficient transfection. (3) Investigation of the transfection process. The cell line-dependent transfection ability of these nanoparticles, as observed with many non-viral vectors, led to the investigation of the processes involved in successful transfection. The intracellular trafficking of the non-viral vectors to the endosomal-lysosomal pathway, determined to be critical for efficient transfection, was found to be directly dependent on the internalisation mechanisms of the complexes.The development and evaluation of the alginate-chitosan nanoparticle system confirms their suitability for gene delivery applications. The additional information provided by the thorough investigation of the cellular internalisation and intracellular trafficking pathways of the alginate-chitosan nanoparticles can be exploited to further develop the system to allow tailoring to improve transfection