Chronic wounds have an exaggerated inflammatory response which disrupts the proteolytic microenvironment, increasing proteases, particularly matrix metalloproteinases (MMPs). This causes degradation of vital matrix components which prevent wound healing. It was therefore hypothesised that a dressing incorporating a peptide based MMP inhibitor may promote healing. Novel peptides designed to chelate the active site zinc, were successfully synthesised using Solid Phase Peptide Synthesis (SPPS) and the inhibitory potential of each evaluated against MMPs associated with chronic wounds (MMP-1, -2, -8 and -9). Results indicated that three peptides, namely A1, A2 and T2A inhibited MMPs in a reversible, competitive manner with Ki values in the sub-micromolar range. Furthermore, biocompatibility studies suggested that the compounds did not affect cell viability or migration. The lead compound, T2A, was then manipulated in order to bind it to a number of solid supports. The most successful of those analysed were in the form of resins designed for use in SPPS, namely NovaPEG and Spheritide. MMP inhibitor peptides were immobilised onto each via standard SPPS methods before optimisation of TFA promoted removal of the protecting trityl group. A further inhibitor was designed to increase the projection of the inhibitor from Spheritide resin (peptide T2A-PEG). Enzyme inhibition assays suggested both NovaPEG and Spheritide resins, with immobilised peptides successfully acted as broad spectrum MMP inhibitors with preference given to Spheritide bound T2A-PEG (S -T2A-PEG). The use of this combination in further studies was vindicated by promising cytotoxicity outcomes from indirect cell viability analysis. Finally, S-T2A-PEG significantly removed MMPs from biologically relevant human and equine chronic wound fluid. Development of a prototype dressing was accomplished by enclosing the immobilised compound within a sealed nylon 'bag' which enabled retention of the peptides' inhibitory ability. In conclusion, the advanced material and resulting prototype emerging from this project is a commercially important and novel wound care product.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:677454 |
| Date | January 2015 |
| Creators | Robb, Charlene Marie Alma |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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