Katas, Haliza; (2007) Development of nanocarriers for siRNA based on cationic polymers. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

A diverse range of viral and non-viral strategies has been developed more than a decade for a gene delivery such as plasmid DNA (pDNA) and oligodeoxynucleotides (ODN). Recently, the development has been extended to a newly discovered class of molecule, small interfering RNA (siRNA). The use of cationic and biodegradable polymeric particles has been widely utilised for delivery of these moieties. This study therefore, aimed to develop and investigate cationic polymers mainly chitosan and polyethylenimine (PEI) as well as their derivative particles; PLGA-PEI and -chitosan nanoparticles as siRNA carriers. Additionally, TAT-peptide has also been investigated as one of the delivery systems for siRNA. Certain process and formulation parameters have been extensively studied with regards of physical and biological properties of the above systems to obtain an optimal delivery system. Physical properties particularly particle size, surface charge as well as particle morphology have been found to be influenced by certain parameters such as homogenisation or stirring rate, molecular weight and concentration of polymers as well as other processes involved in obtaining final products (e.g. centrifugation, freeze-drying). In-vitro evaluations in cultured cells have revealed that the derivative PLGA-PEI nanoparticles are capable of transfecting mammalian cells with siRNA better than the parent compound, PEI. In addition to that, chitosan nanoparticles simply prepared by ionic gelation have been shown to be more competent as siRNA carriers compared to other types of chitosan-based nanoparticles investigated in this study, either chitosan- siRNA complexes or PLGA-chitosan nanoparticles. Although type and molecular weight of chitosan are important in delineating characteristics of particle size and surface charge (the two factors normally important in determining capability of particulate systems to transfect cells), it appears that the type and molecular weight of chitosan have not shown any obvious correlation with the level of the targeted gene knockdown by siRNA. TAT-peptide siRNA complexes were shown to be capable of successfully delivering siRNA into cells without the need of chemical conjugation and the effect could be enhanced by the addition of calcium into the particle suspensions before transfection. Further investigations using chitosan nanoparticles prepared by ionic gelation, have been used to deliver MAPK-14 siRNA in the macrophage cell line, J774A.1 have shown that the system has the ability to transfect cells and subsequently allow the delivered siRNA to knockdown the targeted endogenous gene, MAPK p38α with a sustained effect and a relatively low cytotoxicity. In conclusion, the ability of these polymers as a carrier for siRNA is highly dependent on the method of preparation and their physicochemical characteristics of each of these polymeric particles.

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