An injectable collagen scaffold delivering exogenous microRNA as a therapy to modulate extracellular matrix remodelling

Abstract

Cardiovascular disease is the leading cause of death in the developed world and is responsible for approximately 36% of Irish mortality. Myocardial infarction (MI), which is literally the death of cardiac tissue due to lack of oxygenation, accounts for the majority of deaths associated with cardiovascular disease. This death of cardiac tissue leads to a loss of cardiac function as the damaged area becomes a non-contractile scar. Amelioration of this process is a main aim of regenerative cardiac strategies such as anti-fibrotic therapies. Thus, anti-fibrotic interfering RNA (RNAi) therapy with exogenous microRNA (miR)-29B was proposed as a method to modulate extracellular matrix (ECM) remodelling following MI. It was hypothesized that miR-29B scaffold delivery will efficiently modulate the ECM remodelling response and reduce maladaptive remodelling, such as aggressive deposition of collagen type I, after injury. The primary objective of this doctoral project was to develop a scaffold-based, controlled release gene therapy system. A co-polymer of a linear poly (dimethylamino) ethyl methacrylate (pDMAEMA) block and a hyperbranched poly (ethylene glycol) methyl ether acrylate (PEGMEA) based unit with poly (ethylene glycol) diacrylate (PEGDA) as the branching agent (pD-b-/PDA) was synthesised, with the purpose of complexing miRs, using deactivation enhanced atom transfer radical (De-ATRP) synthesis. Non-viral complexes of miR-29B and pD-b-/PDA were optimized for both monolayer and three-dimensional delivery from a crosslinked collagen-based scaffold in vitro. The release of these complexes from the scaffolds was assessed and their ability to silence collagen type I and collagen type III expression was evaluated. When cardiac fibroblasts were cultured with complex loaded scaffolds, relatively low levels of collagen type I and collagen type III mRNA expression were observed for up to two weeks of culture. When scaffolds loaded with miR-29B or miR-29B complexes were applied to a rat excisional wound model, reduced wound contraction, improved collagen type III/I ratios and a significantly higher MMP-8: TIMP-1 ratio were detected. From these investigations it was concluded miR-29B functionalization of the scaffold significantly increased the ratio of collagen type III/I and MMP-8/TIMP-1 ratios. Furthermore, these effects were not improved through the use of pD-b-/PDA, and were significantly influenced by the dose of miR-29B in the collagen scaffold (0.5 micro g versus 5 micro g). This is the first study to describe a biomaterial scaffold combined exogenous miRs capable of improving ECM remodelling following injury. There is significant potential for further development of this platform