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Title: PMPC-PDPA polymersomes-mediated siRNA delivery
Author: Patikarnmonthon, Nisa
ISNI:       0000 0004 5348 8353
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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Polymersomes made from the amphiphilic diblock copolymers, PMPC-PDPA, are proposed to serve as a siRNA carrier with pH-responsive property that provides endosomal escape. The main purpose of this work is to investigate the ability of polymersomes to provide effective intracellular delivery of siRNA into HeLa cells. Encapsulation of siRNA into polymersomes was performed by pH-switch and electroporation method, both techniques enable siRNA encapsulation. No alteration of polymersomes size and morphology was observed in DLS and TEM. Purification of polymersome was conducted to ensure that no free siRNA or polymer remained. Intracellular delivery was examined by using fluorescence-labelled siRNA to track the internalisation. Flow cytometry and fluorescence microscope were used to study the cellular uptake of polymersomes and siRNA. siRNA is successfully delivered with the distribution of siRNA signal throughout the cell, with stronger signal compared with Lipofectamine. Kinetic uptake of siRNA suggests that siRNA can be effectively delivered to most cells within 20 hours. In addition, evidence of endosomal escape of siRNA delivered by polymersomes was observed. Silencing activity of siRNA was determined by qPCR and Western blot, mRNA and protein expression of Lamin A/C as a target gene were not significantly decreased. Cytotoxicity and other cellular response, including pro-inflammatory response and interferon response, were investigated. Polymersomes provide very low cytotoxicity and no pro-inflammatory response, unlike Lipofectamine. Moreover, the gene expression profile of interferon response indicates the possible apoptosis occurrence in Lipofectamine treated cells, but not in polymersomes treated cells. The information suggests two possible factors that influence the silencing activity of siRNA delivered by polymersomes; the incomplete characterisation of siRNA process and the cellular response from carriers.
Supervisor: Battaglia, Giuseppe Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available