Browsing by Author "Davies, Neil H"

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    Investigation into synthetic protease-sensitive hydrogels as siRNA nanoparticle delivery vehicles.
    (2018) Doubell, Emma; Davies, Neil H
    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.
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    Open Access
    Long-Term Left Ventricular Remodelling in Rat Model of Nonreperfused Myocardial Infarction: Sequential MR Imaging Using a 3T Clinical Scanner
    (2012) Saleh, Muhammad G; Sharp, Sarah-Kate; Alhamud, Alkathafi; Spottiswoode, Bruce S; van der Kouwe, André J W; Davies, Neil H; Franz, Thomas; Meintjes, Ernesta M
    Purpose. To evaluate whether 3T clinical MRI with a small-animal coil and gradient-echo (GE) sequence could be used to characterize long-term left ventricular remodelling (LVR) following nonreperfused myocardial infarction (MI) using semi-automatic segmentation software (SASS) in a rat model. Materials and Methods. 5 healthy rats were used to validate left ventricular mass (LVM) measured by MRI with postmortem values. 5 sham and 7 infarcted rats were scanned at 2 and 4 weeks after surgery to allow for functional and structural analysis of the heart. Measurements included ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), and LVM. Changes in different regions of the heart were quantified using wall thickness analyses. Results. LVM validation in healthy rats demonstrated high correlation between MR and postmortem values. Functional assessment at 4 weeks after MI revealed considerable reduction in EF, increases in ESV, EDV, and LVM, and contractile dysfunction in infarcted and noninfarcted regions. Conclusion. Clinical 3T MRI with a small animal coil and GE sequence generated images in a rat heart with adequate signal-to-noise ratio (SNR) for successful semiautomatic segmentation to accurately and rapidly evaluate long-term LVR after MI.
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    Stimulating angiogenesis into biomaterials through the delivery of growth factors
    (2007) Schmidt, Christian Alexander Peter; Davies, Neil H
    lschemic disease in form of ischemic heart disease (IHD), ischemic stroke and peripheral arterial disease (PAD) due to atherosclerosis represents a massive clinical and economic burden to healthcare and is currently the number one cause of death in the world. Treatment modalities for peripheral arterial disease include bypass surgery involving autologous vein or synthetic materials such as ePTFE. Long term patency of small diameter vascular grafts used for infra-inguinal reconstructions, however, is below 50 % 5 years after implantation. Therefore, novel vascular graft concepts and materials are needed. The concept of transmural in vivo endothelialisation of vascular grafts holds great promise for increasing long term patency. To achieve complete luminal endothelial cell coverage and optimal integration of the porous synthetic graft material into the host tissue, transmural ingrowth of tissue and vasa vasorum might have to be facilitated. Since VEGF1ss and PDGF-BB are growth factors known to stimulate and consolidate angiogenesis, this PhD thesis hypothesized, that neovascularisation of porous polyurethane (PU) can be increased by delivery of vascular endothelial growth factor (VEGF1ss) and platelet derived growth factor (PDGF-BB). To prove this hypothesis, subcutaneous implantation of PU discs was established as a valid, reproducible, relatively simple and quantifiable neovascularisation model. Three different ways of growth factor delivery were investigated. The gene encoding for human VEGF15s was cloned into the genome of adeno associated viruses (AAV), which served as a vector for gene transduction of autologous wound healing cells in vivo using the "Gene Activated Matrix" approach. Genetically modified matrix embedded AAV-VEGF155 was loaded into porous PU and transduced autologous ingrowing wound cells. In contrast to the excellent transduction efficiency in myocytes, AA V showed a poor tropism for wound healing cells. The second approach to increase neovascularisation into porous PU was the surface modification of PU by covalent attachment of nitrous acid degraded heparin. Neovascularisation into the biomaterial was increased by 77 % after 10 days of subcutaneous implantation. Since certain angiogenic growth factors show a high affinity for heparin, additional loading of heparin surface modified PU with VEGF165 increased neovascularisation even further up to 115 % at 10 days compared to control. Dual growth factor delivery of VEGF 165 and PDGF-BB not only initiated increased vascularisation of porous PU, but also created a stable vascular network 2 months after implantation. In contrast, PU loaded with VEGF165 alone showed regression of total vascular area of 61 % compared to vascular area at 10 days. Thirdly, to study the effects of controlled, prolonged growth factor delivery, a "Neovascularisation Construct" was developed which was implanted subcutaneously in rats. The construct consisted of an osmotic mini pump and a tube of porous PU lined with ePTFE, into which a defined amount of VEGF16s was pumped for 10 days. After implantation, granulation tissue was growing into the pores of the PU and neovascular area was increased up to 265 % compared to PBS control. Furthermore, using different growth factor concentration, a dose dependency was shown. In addition, this thesis investigated the functional perfusion of the micro vascular network growing into PU by four different vascular quantification techniques. lntravital perfusion with biotinylated lycopersicon esculentum followed by microscopical analysis, vascular corrosion casting quantified by scanning electron microscopy as well as the novel micro-CT analysis of silicone rubber perfused vessels were compared to conventional immunhistochemical analysis of endothelial cells by CD31. Interestingly, PBS perfused "Neovascularisation Constructs" showed a relatively poor perfusion; therefore CD31 immunohistochemistry "overestimated" functional neovascularisation 3 fold. All perfusion techniques indicated a strong effect of VEGF 165 delivery on vessel perfusion (10 to 20 fold increases of vascular area and volume compared to PBS control). Micro-CT scanning was shown to be an excellent tool to study micro vascular networks in a three-dimensional fashion across the whole length of the sample in a limited amount of time and to provide reliable and reproducible data on vessel density, vascular volume, and connectivity. Since resolution is still limited today to about 10 μm using a commercially available bench top scanner, this new technology still needs to be complemented by immunohistochemistry and perfusion studies such as lectin perfusion and corrosion casting. In summary, the induction of neovascularisation was achieved by heparin surface modification alone, which was even increased through additional delivery of growth factors into the biomaterial PU. The development of a stable micro vascular network at 2 months was achieved and the functionality was shown using four different, independent techniques including the novel micro-CT scanning of neovascularisation into biomaterials. Towards the development of an in vivo, spontaneously and transmurally endothelialising vascular graft with superior long-term patency further investigations are necessary. As an initial step, increased spontaneous neovascularisation of the possible graft material polyurethane was achieved. Future steps are clearly indicated to study the translation of increased neovascularisation of the biomaterial polyurethane towards increased endothelialisation in a vascular graft model.