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Title: Cellular gene delivery via polymeric biguanides
Author: Chivu, Alexandru
ISNI:       0000 0004 7661 0729
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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Controlled and efficient cell and tissue delivery of exogenous nucleic acids for the purposes of gene delivery remains an enormous technical challenge. Multiple cellular and pre-cellular barriers must be overcome for this to take effect and research into identification of potential candidates is ongoing. This project has explored the ability of widely used antiseptic poly(hexamethylene biguanide) (PHMB) to transport DNA into model mammalian cell lines HepG2, HEK293T and HeLa in vitro, under serum conditions. PHMB was shown to bind, neutralise and condense DNA at ratios above 3 (w/w), similar to the well established polymer polyethyleneimine (PEI), and form submicron particles with positive zeta potentials (> +26 mV). Particle internalisation was successful in all three cell lines in higher extent than for the PEI control. Despite this, gene expression following pEGFP-N1 and pLuciferase transfection was only significant in HEK293T, and only if the polyplex was assembled under basic conditions (pH 12). Two oligomeric biguanides - P(HMB-EEG) and P(HMB-EDSB) - were also synthesised, with the aim to improve the transfection of PHMB. Even though they also formed submicron, positively charged (+25 mV) polyplex particles above ratios 3 (w/w), their uptake properties were not as good as for PHMB. However, higher molecular weight analogues remain to be synthesised for proper comparison and assess the contribution of the biguanide moiety. The branched widely used polymers PAMAM and bPEI were also functionalised with terminal biguanide moieties. They also interacted with and neutralised DNA at ratios above 9 and 1 (w/w) respectively. PAMAM-biguanide hydroiodide presented solubility issues and due to time constraints it was not pursued, and its efficiency assessment is pending ion exchange. bPEI-biguanide promoted higher fluorescent oligodeoxynucleotide uptake compared to its nonfunctionalised (amine-terminated) counterpart, but it did not induce GFP or luciferase expression in any of the three cell lines. The current study showed that the presence of the biguanide functional group in a vector promotes uptakes of molecular cargoes it interacts with electrostatically and condenses into submicron particles.
Supervisor: Song, W. ; Davidson, B. ; Good, L. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available