Abstract
Purpose: The aim of this thesis was to investigate a suitable formulation for the topical delivery of growth factors to chronic wounds, and then to determine the concentrations reached within an animal wound model. A secondary aim was to determine if the chosen growth factor was present at levels able to stimulate the production of other cytokines, specifically IL-1β and MCP-1.
Methods: An in vitro testing apparatus was designed and made and the release of model actives [bromophenol blue (BPB) and horseradish peroxidase (HRP)] from gels and films of hydroxylpropylmethylcellulose (HPMC) (E4M CR, K4M CR and E10M CR) was determined. In this study, Fibroblast Growth Factor -2 (FGF-2) (0.3 μg) was incorporated into three formulations (solution, gel and dried gel film on Melolin™ backing) and together with a control formulation were administered to punch biopsy wounds in rats. The in vivo release was followed over three time periods (two, five and eight hours) and the amount of FGF-2 at various wound depths was quantified by ELISA. Two biological markers IL-1β and MCP-1 were quantified using ELISA. The FGF-2 was additionally tagged with a fluorescent dye so that visualisation of the penetration could be obtained via confocal microsopy.
Results: For the HPMC gels, the more viscous gel (E10M) provided a greater diffusional barrier and slowed the release of BPB (12 ± 3.5 μg/min compared with 16 ± 1.7 μg/min and 18 ± 1.4 μg/min for K4M and E4M respectively). However, when HPMC was formulated as a dried film a burst release was seen and release of BPB was slowest from the more rapidly hydrating K4M. With the larger model active HRP, there was a slower diffusion through the gel barrier formed upon film hydration, such that time of 100% release was up to 300 minutes compared to approximately 60 minutes for BPB. When the film was dried onto a supportive backing, the initial burst release was minimised as the film did not break apart on contact with the wound, and hence film integrity was maintained and release prolonged. The in vivo studies showed that, two hours after application, the highest concentration of FGF-2 was seen in the surface granulation tissue of rats that received the solution formulation (2280 ± 790 pg/g). The concentration decreased with increasing tissue depth but was significantly greater than the saline control in the surface granulation and subcutaneous fat layers (p<0.05). Tissue concentrations following application of the gel and film formulations were only marginally greater than control in the surface granulation layer. After eight hours, rats that received the solution retained elevated surface tissue concentrations (surface granulation and subcutaneous fat) of FGF-2. Repeated measures ANOVA using a general linear model showed statistically significant differences in the mean FGF-2 level with respect to formulation and length of time of application of the formulation (p<0.05). In terms of other cytokines, there was a release of both IL-1β and MCP-1 in all groups, immediately post-wounding, probably in response to cellular damage. After eight hours, the film formulation appeared to elevate IL-1β levels which may be useful to drive wound healing. Confocal microscopy images showed diffuse distribution of FGF-2 eight hours after application of the solution formulation after eight hours and that with the gel formulation FGF-2 initially aggregated at the wound surface.
Conclusion: In vitro experiments investigating the effect of hydration rate and viscosity of HPLC polymers allowed a formulation to be chosen for further in vivo study. Elevated FGF-2 could be measured in superficial wound tissues up to eight hours after application of a solution. However, application of a comparable amount of FGF-2 in HPMC gels or films did not produce appreciable elevations in FGF-2 in wound tissues, although confocal microscopy indicated the penetration of FGF-2 into the wound for up to eight hours.