Purpose: Marginal stability of proteins under environmental stress conditions limit their clinical application. Our study indicates that combination of osmolytes can enhance the activity of the proteins at optimal concentrations and also preserve their wound healing properties under environmental stress conditions.
Methods: We selected trypsin as a model protein for its wound healing properties considering its current clinical use in alternative medicine. We evaluated its wound healing properties in the presence of 6 chemically different osmolytes and their combinations at different concentration levels. We tested the proteolytic activity of trypsin under elevated temperatures in the presence and absence of various concentration levels of osmolyte solutions. To study the activity of trypsin in-vitro, we cultured the cells in skin mimicking 3-D model comprising of epithelial cells, monocytes, and collagen trapped in a hydrogel. A deep wound was made to the 3-D skin mimicking scaffold using a biopsy punch. The wound was treated with various concentrations and combinations of osmolytes. The rate of monocyte migration and wound healing was observed by time-lapse imaging of the scaffold over 48 hours. The control group of wounded skin scaffold received treatment with PBS. The cytotoxicity of the added osmolytes was tested in-vitro by mobilizing the cells with accumax and almar blue assay. The formulations of trypsin and osmolytes were then exposed to elevated temperatures for 1 hour, and their proteolytic and wound healing activity was studied.
Results: Our studies indicated that counteracting osmolytes TMAO (0.5M) and urea (0.5M) can shield the trypsin at elevated temperatures and preserve the proteolytic and the wound healing activity after exposure to 85ᵒC. We also observed that activity of trypsin was enhanced by combining TMAO or trehalose with urea at optimum concentrations. We did not observe significant in-vitro toxicity after treatment with trypsin combined with optimized osmolyte combinations.
Conclusion: In conclusion, osmolytes can improve the wound healing activity of trypsin and can be used in topical formulations for enhancing the activity and stability of the protein formulation.
Luke Theogarajan– University of California Santa Barbara