Hydrogels have been increasingly used in the treatment of skin and soft tissue wounds in recent years due to their superb water holding and cell-growth promoting properties. When impregnated with antibiotics, they can also treat or prevent bacterial infections. Due to the global increase in antibiotic resistance, antibiotics are now becoming less effective and bacteriophage (viruses able to kill bacteria) offer a new alternative. Triggered release mechanisms also slow resistance development, as bacteria are not continually exposed to sub-lethal levels of therapeutic. Here, Staphylococcus aureus (S. aureus) was focussed on as it is the most common pathogen present in skin and soft tissue infections. The main aim of this work was to form biocompatible hydrogel systems which were able to give passive or triggered release of bacteriophage particles by S. aureus virulence factors. Micropatterned hydrogels were also investigated to assess the response in eukaryotic systems. This study found that Bacteriophage K was highly infective against S. aureus, with 94% strains (out of 86 strains) showing complete or intermediate sensitivity. Bacteriophage were immobilised into PVA and agarose hydrogel systems without a significant loss in concentration or efficacy. A photocrosslinkable polymer, HAMA (hyaluronic acid methacrylate) was also investigated which could be crosslinked in situ into a robust hydrogel; this hydrogel was sensitive to hyaluronidase (HAase), an enzyme secreted by the majority of S. aureus strains. On incubation with purified HAase and S. aureus supernatant, hydrogel degradation was recorded (Carbazole assay and SEM imaging) compared to buffer and HAase – negative S. aureus strains. When combined into a bilayered hydrogel system, 2% agarose/HAMA hydrogels gave triggered release of bacteriophage by pure HAase and HAase positive bacterial supernatant. However, in live culture triggered killing was not possible. HAMA could also be successfully printed, and NIH-3T3 fibroblasts showed directed aggregation but no adhesion to HAMA hydrogels. In general, hydrogel systems which gave triggered release of Bacteriophage K only in the presence of S. aureus HAase were successfully formed. However, the passive leakage of bacteriophage into the wider environment was seen in live culture. This could be remedied by direct coupling of the bacteriophage to the polymer network, and is a possible avenue for future work. This system was significant as it proved that S. aureus HAase can be used as a trigger for bacteriophage release from hydrogel systems. In eukaryotic cells, HAMA hydrogels did not promote cell growth, although some promise was seen in gelatin-based gels.
|Date of Award||29 Oct 2015|
|Supervisor||Toby Jenkins (Supervisor)|