RNA interference (RNAi) is receiving increasing attention as a form of gene regulation able to temporarily silence gene expression through post transcriptional mechanisms. RNAi agents have shown promise in targeting a range of ailments from cancers to myocardial infarction. However, RNAi based therapeutics have not passed the clinical trial stage of development, in part due to lack of optimal delivery mechanisms. One means towards improving delivery is the development of localised and sustained delivery systems for the RNAi molecule. Such approaches are important as it has previously been found that systemic delivery often leads to off target effects and rapid clearance. The aim of this project has been to assess the utility of an enzymatically degradable polyethylene glycol (PEG) hydrogel as a localised delivery vehicle. Enzymatically degradable PEG hydrogels that rely on cellular invasion for their degradation might enable the release of entrapped RNAi agents to surrounding cells as well as transfection of invading cells. The cationic polymer poly(ethyleneimine) (PEI), was used as a tool to investigate the PEG hydrogel as a localised delivery vehicle. Thus, an initial objective of this study was the establishment and characterisation of the PEI/siRNA nanoparticle technology in our group. siRNA was found to be fully complexed at a PEI nitrogen to siRNA phosphate ratios of 5:1 and higher. A 10:1 ratio and higher were able to protect the siRNA from RNases present in serum for 7 days with up to 65% of RNA still intact. A 40:1 ratio was found to be cytotoxic. A 20:1 ratio was found to be the most effective at gene knock down and determined to be optimal. As there is a relatively limited volume available in the PEG hydrogel system used here for loading with PEI/siRNA nanoparticles, a study was conducted to assess the maximum concentration of siRNA at which effective nanoparticles could be formed. Somewhat unexpectedly it was found that at a concentration of 7.5 µM siRNA, nanoparticles showed a significant 40% reduction in transfection efficacy of cells cultured in 2D. This finding for PEI nanoparticles indicating a limitation in the dosage attainable in the PEG hydrogel system. The influence of PEG encapsulation for PEI/siRNA nanoparticles on RNase protection was assessed by exposing hydrogels with entrapped nanoparticles to serum RNases. The PEG hydrogel was found to significantly improve PEI based protection from RNase degradation in the initial 24 hours and this protection persisted for up to 5 days. PEI/siRNA nanoparticles were retained after encapsulation within PEG hydrogels for up to 7 days whilst siRNA alone was entirely released after 24 hours. It is possible that this is a limitation of the system. A 3D invasion assay was developed to more closely mimic the in vivo scenario where cells could invade a PEG hydrogel with entrapped nanoparticles but are not initially exposed to high concentrations of nanoparticles prior to hydrogel polymerisation. The assay involved the formation of dermal equivalents (cells entrapped within contracted collagen) that were encapsulated within a PEG hydrogel. Cells were observed invading the hydrogel within 1 hour of polymerising and continued to do so for up to 7 days. Invading cells were seen to take up the fluorescent siRNA although this uptake was scant and challenging to quantify. When commercial cell death siRNA sequence nanoparticles were encapsulated, a trend towards higher death levels was observed. In conclusion, PEI/siRNA nanoparticles and related RNAi based assays have been formally established in our laboratory and can be used in the future for other applications. A 3D cell invasion assay was developed which more closely mimics the in vivo scenario where hydrogels bearing siRNA are polymerised within tissue. The increased RNase protection though relatively slight is important for the use of hydrogels as localised delivery depots in an in vivo environment. Potential dose limitations of the PEG hydrogel system when using PEI nanoparticles and their apparent very tight encapsulation in the PEG hydrogels suggests the need for modifications of both nanoparticles and hydrogels to optimise efficacy. Future investigations into scaffold based localised siRNA delivery should be facilitated by the methodologies and assays established in this study.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/29621 |
| Date | 18 February 2019 |
| Creators | Doubell, Emma |
| Contributors | Davies, Neil H |
| Publisher | University of Cape Town, Faculty of Health Sciences, Department of Surgery |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc |
| Format | application/pdf |
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