Effect of freeze drying on stability, thermo-responsive characteristics, and in vivo wound healing of erythropoietin-loaded trimethyl chitosan/glycerophosphate hydrogel

Erythropoietin (EPO) was successfully incorporated into a bioadhesive thermosensitive hydrogel based on trimethyl chitosan (TMC)/beta-glycerophosphate (GP) for prevention and treatment of oral mucositis in cancerous patients. The aim of the present study was to evaluate the effect of freeze drying on thermo-responsive property of the hydrogel and structural stability of the loaded protein. The freeze-dried EPO-loaded hydrogel were characterized using various methods. Gelation property by rheological analysis, EPO aggregation in formulations by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), protein secondary structure by far ultraviolet-circular dichroism (CD), and the antigenic activity of EPO with ELISA techniques. The healing effects of the freeze-dried formulation was also investigated in Sprague-Dawley rats with chemotherapy-induced mucositis and compared with freshly prepared mixture. Finally, the retention time of the gel in the oral cavity was assessed in healthy volunteers. SDS-PAGE, CD, and ELISA confirmed the stability of conformational structure of loaded and released EPO. Severity of mucositis was markedly reduced in animals treated with freeze-dried EPO hydrogel; whereas the group received normal saline did not show any significant healing. EPO salvia level was decreased rapidly following EPO solution compared to the gel application. Approximately, 40% of EPO was maintained on the buccal areas in patients receiving the hydrogel system after 30 min. Therefore, the TMC/GP could preserve EPO stability after freeze drying and has the potential in the treatment of oral mucositis and other oral or subcutaneous wounds.