Formulation development of highly stable collagenase-containing hydrogels for wound healing

Collagenases are enzymes that break down collagen and are used in wound healing and treating various disorders. Currently, collagenase is commercially available in only ointment and injectable forms and is sensitive to various environmental factors. In the present study, different hydrogel formulations of collagenase have been prepared at pH 6.5 using carboxymethylcellulose sodium and zinc acetate with and without humectants such as propylene glycol (PG) and glycerin (GL) in varying concentrations. The formulated gels were stored at room temperature (25±2°C, 60±5% RH) and refrigerator temperature (5±3°C) for six months to evaluate their physical and up to six years for chemical stability. The gels were subjected to various tests, including organoleptic studies, spreadability, moisture content, swelling index, swelling/de-swelling, syneresis, viscosity, gelation time, and weight variation. The purity and molecular weight of collagenase have been determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). At the same time, its activity during the storage period was evaluated by gelatin zymography. Casein zymography was also performed to detect any caseinase contamination in the formulations. The release of the enzyme from different gel formulations was assessed using the Franz diffusion apparatus and analyzed by gelatin zymography. The results showed some physical changes that were more prominent in gels stored at room temperature than those kept refrigerated. The difference in humectant concentration was also found to affect the stability of gels. PG was found to be a better humectant than GL, particularly in a concentration of 25%. The zymography results indicated that collagenase was stable in all formulations kept in the refrigerator. In contrast, its complete degradation was noted in the preparations stored at room temperature within a month. The data generated in this study will help the formulators to commercialize a relatively economical gel formulation of collagenase that is highly stable for up to six years at refrigerator temperature (5±3°C).