Chronic wounds are a major financial and clinical burden causing the deaths of millions per year. Over expression of elastase is well documented as the main culprit that delays the normal wound repair process within chronic wounds. The aim of this thesis is to design a responsive chronic wound dressing based on the hydrogel polymer, PEGA (polyethylene glycol acrylamide) in the form of particles to mop-up excess elastase by exploiting polymer collapse in response to elastase hydrolytic activity within sample fluids mimicking the environment of chronic wounds. PEGA particles were functionalised with enzyme cleavable peptides (ECPs) containing charged residues. Upon cleavage the charge balance changes, causing polymer swelling and consequent elastase entrapment. The pH range of chronic wounds is reported in the range of 5.45 - 8.65. Due to its pI which is around 8.3, within this range elastase exist both in its cationic and anionic forms. To accommodate a hydrogel dressing that could selectively entrap excess elastase both in its cationic and anionic, oppositely charged ECPs were designed. In its cationic form, elastase was found to have a high preference of cleaving ECPs and penetrating into PEGA particles bearing negative charges. In contrast, in its anionic form the opposite effect was observed, wherein elastase preferred to cleave ECPs and penetrate PEGA particles bearing positive charges. The diffusion, accessibility and entrapment of elastase into functionalised PEGA particles was explored using various fluorescence microscopy techniques. Removal of the charged residue by elastase showed a reduction in particle swelling causing the pores of PEGA particles to become restricted. In this manner, cleaved PEGA particles prevented the accessibility of molecules with a molecular weight as low as 20 kDa into the cleaved PEGA particles. Since elastase has a molecular weight of 25.9 kDa the collapsing of the pores within PEGA particles entrapped elastase inside the interior of cleaved PEGA particles. In its cationic form (at pH 7.4) elastase was found to penetrate and become trapped more into both negative and positive PEGA particles compared to neutral particles. The negative particles were shown to trapped cationic elastase within 2 minutes compared to the positive particles. In contrast, the neutral particles failed to retain and encapsulate elastase as the fluorescence inside the neutral particles was found to decrease. Coinciding with these observations, after sample fluids containing elastase were treated with functionalised PEGA particles, the residual elastase activity in sample fluids was reduced more by the charged PEGA particles compared to neutral particles. The cell culture studies demonstrated that the elastase activity observed in human dermal fibroblasts (HDF) was also reduced more by the charged particles compared to the neutral particles. However, the positive particles were found to significantly reduced HDF-elastase activity compared to both the negative and neutral PEGA particles. Overall, this thesis exemplifies that on the basis of charge selective cleaving of ECPs coupled to PEGA particles can be exploited to selectively remove excess proteases such as elastase from sample fluids mimicking the environment of chronic wounds.

no / The resurgence of larval biotherapy as a debridement tool in wound management has been accompanied by several clinical reports highlighting concomitant tissue regeneration. Studies employing in vitro cell motility assays have found that purified excretory/secretory (ES) products from Greenbottle larvae (blowfly, Lucilia sericata) are motogenic for human dermal fibroblasts when used as a supplement in culture media. The objective of the present study was to determine whether ES delivered using a prototype hydrogel wound dressing induced similar motogenic effects on fibroblastic (3T3) and epithelial cells (HaCaTs) comprising a scratched-monolayer wound model. Quantitative analysis by MTT assay failed to detect significant mitogenic effects of ES on either cell type. Quantitative image analysis revealed that ES exposure markedly accelerated wound closure through a motogenic effect on both fibroblasts and keratinocytes. Quantitative histochemical analysis detected significantly higher phosphotyrosine (pTyr) expression in ES-exposed cell cultures than in controls; moreover immunocytochemistry revealed conspicuously raised levels of pTyr expression in cells located at the wound margin. By attenuation with a panel of enzyme inhibitors these effects were attributed to the protease components of ES. The present results suggest that controlled delivery of ES as a follow-up to maggot debridement therapy may be an effective therapeutic option for stimulation of tissue regeneration in wound management.