Development of a cationic polymer-based siRNA delivery system for the regeneration of tendon injury

Liao, Xin (2019) Development of a cationic polymer-based siRNA delivery system for the regeneration of tendon injury. Doctoral thesis, University of East Anglia.

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Abstract

Tendon injury is one of the most common forms of musculoskeletal injuries that could occur in both human and equine patients. However, the conventional therapies have been unsuccessful when addressing the clinical need for the treatment of tendon injury. Therefore, the urgent need for more effective therapeutic strategies has set researchers along the route of exploring the causes underlying tendon injuries. In the past few decades, advances have been made in the understanding of tendon injury and its repair. Standing on the foundation of such discoveries, novel therapeutic approaches including gene therapy have been applied to the treatment of tendon injury.

It is established that tendon tissue repaired via the natural healing process after injury has a weakened mechanical property compared to the healthy tendon due to the scar tissue and adhesion formed during the repair process. Type III collagen and several cytokines including transforming growth factor-beta is reported to be closely associated with the formation of scar tissue. Consequently, it is hypothesised that through the suppression of the COL3α1 gene, which is responsible for the production of type III collagen, the formation of scar tissue could be reduced to restore the mechanical property in the repaired tendon.

RNA interference is a reliable way to suppress the expression of a target gene using specific siRNA. However, the use of siRNA has several limitations including extracellular degradation by enzymes in tissue fluid, intracellular degradation by lysosomes and difficulties penetrating the cell membrane. Therefore, an efficient and safe siRNA delivery system is required, and the cationic polymer-based delivery system is a strong candidate. Cationic polymer poly (dimethylaminoethyl acrylate) (PDMAEA) is a biodegradable synthetic polymer that showed great potential as a delivery vector. This prompted the development of a new siRNA delivery system based on four-armed PDMAEA. It is hypothesized that the cationic four-armed PDMAEA polymer can effectively bind to negatively charged siRNA to form a nano-sized polyplex. It is also hypothesized that a four-armed PDMAEA could be more efficient than counter-part linear polymers in terms of binding to siRNA and the cytotoxicity profile. Cationic polymer PDMAEA was selected as a delivery vector for its good buffering capability, siRNA condensation efficiency and biodegradable property.

The work presented in this thesis describes the synthesis of a library of PDMAEA polymers of different molecular weights and architectures via RAFT polymerisation. The synthesized polymers were characterized by 1H-NMR, 13C-NMR, GPC and potentiometric titration to confirm the desired molecular weight, architecture and pKa value. Firstly, a dsDNA with low base pair number was chosen as a model molecule to optimise the experimental conditions. Afterwards, the formation of the PDMAEA/dsDNA and PDMAEA/siRNA polyplexes were confirmed by agarose gel electrophoresis, and the solution properties, in particular the hydrodynamic diameter and zeta-potential, were confirmed using DLS. Later, the polymers and their polyplexes were tested on mouse 3T3 fibroblast cells and adult horse tenocytes to determine their cytotoxicity profiles. Finally, the optimised therapeutic siRNA-PDMAEA polyplex was applied to adult horse tenocytes stimulated with TGF-β1 to observe the transfection and silencing effect of the COL3α1 gene in induced in vitro conditions. The expression level of the COL3α1 gene was determined by qPCR and type III collagen protein expression was monitored by immunocytochemistry. The results showed that the developed cationic PDMAEA was effective in delivering therapeutic siRNA in a clinically relevant in vitro condition and achieved controlled suppression of type III collagen expression. The developed delivery system could potentially be used to reduce scar tissue formation in many other tissues.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Gillian Aldus
Date Deposited: 13 Jun 2019 12:49
Last Modified: 13 Jun 2019 12:49
URI: https://ueaeprints.uea.ac.uk/id/eprint/71386
DOI:

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