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

Vajihe Akbari , Mahboubeh Rezazadeh , Mohsen Minaiyan, Marjan Amirian, Azadeh Moghadas, Ardeshir Talebi


Erythropoietin (EPO) was successfully incorporated into a bioadhesive thermosensitive hydrogel based on trimethyl chitosan (TMC)/β-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.


Freeze-drying; Thermosensitive hydrogel; Erythropoietin; In vivo.

Full Text:



Spivak JL, Gascón P, Ludwig H. Anemia management in oncology and hematology. Oncologist. 2009;14(Suppl 1):143-156.

Sayan H, Ozacmak VH, Guven A, Aktas RG, Ozacmak ID. Erythropoietin stimulates wound healing and angiogenesis in mice. J Invest Surg. 2006;19(3):163-173.

Hamed S, Ullmann Y, Masoud M, Hellou E, Khamaysi Z, Teot L. Topical erythropoietin promotes wound repair in diabetic rats. J Invest Dermatol. 2010;130(1):287-294.

Galeano M, Altavilla D, Bitto A, Minutoli L, Calò M, Lo Cascio P, et al. Recombinant human erythropoietin improves angiogenesis and wound healing in experimental burn wounds. Crit Care Med. 2006;34(4):1139-1146.

Haroon ZA, Amin Kh, Jiang X, Arcasoy MO. A novel role for erythropoietin during fibrin-induced wound-healing response. Am J Pathol, 2003;163(3):993-1000.

Galeano M, Altavilla D, Cucinotta D, Russo GT, Calò M, Bitto A, et al. Recombinant human erythropoietin stimulates angiogenesis and wound healing in the genetically diabetic mouse.Diabetes. 2004;53(9):2509-2517.

Ferri C, Giuggioli D, Manfredi A, Quirici N, Scavullo C, Colaci M, et al. Recombinant human erythropoietin stimulates vasculogenesis and wound healing in a patient with systemic sclerosis complicated by severe skin ulcer. Clin Exp Dermatol. 2010;35(8):885-887.

Rezazadeh M, Jafari N, Akbari V, Amirian M, Tabbakhian M, Minaiyan M, et al. A mucoadhesive thermosensitive hydrogel containing erythropoietin as a potential treatment in oral mucositis: in vitro and in vivo studies. Drug Deliv Transl Res. 2018;8(5):1226-1237.

Herrstedt J. Prevention and management of mucositis in patients with cancer. Int J Antimicrob Agents. 2000;16(2):161-163.

Wang T, Hu Y, Leach MK, Zhang L, Yang W, Jiang L, et al. Erythropoietin-loaded oligochitosan nanoparticles for treatment of periventricular leukomalacia. Int J Pharm. 2012;422(1-2):462-471.

Naughton CA, Duppong LM, Forbes KD, Sehgal I. Stability of multidose, preserved formulation epoetin alfa in syringes for three and six weeks. Am J Health Syst Pharm. 2003;60(5):464-468.

Aksungur P, Sungur A, Unalc S, Iskit AB, Squier CA, Senel S. Chitosan delivery systems for the treatment of oral mucositis: in vitro and in vivo studies. J Control Release. 2004:98(2):269-279.

Raber-Durlacher JE, Elad S, Barasch A. Oral mucositis. Oral Oncol. 2010;46(6):452-456.

Saadeh CE. Chemotherapy- and radiotherapy-induced oral mucositis: review of preventive strategies and treatment. Pharmacotherapy. 2005;25(4):540-554.

Oztürk E, Demirbilek S, Köroğlu A, But A, Begeç ZO, Gülec M, et al. Propofol and erythropoietin antioxidant properties in rat brain injured tissue. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32(1);81-86.

Guneli E, Cavdar Z, Isleke H, Sarioglu S, Erbayraktar S, Kiray M, et al. Erythropoietin protects the intestine against ischemia/ reperfusion injury in rats. Mol Med. 2007;13(9-10):509-517.

Cetin H, Olgar ş, Oktem F, Ciris M, Uz E, Aslan C, et al. Novel evidence suggesting an anti-oxidant property for erythropoietin on vancomycin-induced nephrotoxicity in a rat model. Clin Exp Pharmacol Physiol. 2007:34(11):1181-1185.

Hamed S, Bennett CL, Demiot C, Ullmann Y, Teot L, Desmoulière A. Erythropoietin, a novel repurposed drug: an innovative treatment for wound healing in patients with diabetes mellitus. Wound Repair Regen. 2014;22(1):23-33.

Hosseinjani H, Hadjibabaie M, Gholami K, Javadi M, Radfar M, Jahangard-Rafsanjani Z, et al. The efficacy of erythropoietin mouthwash in prevention of oral mucositis in patients undergoing autologous hematopoietic SCT: a double-blind, randomized, placebo controlled trial. Hematol Oncol. 2017;35(1):106-112.

Watson C, Sharp JS. Conformational analysis of therapeutic proteins by hydroxyl radical protein footprinting. AAPS J. 2012;14(2):206-217.

Emami J, Hamishekar H, Najafabadi AR, Gilani K, Minaiyan M, Mahdavi H, et al. A novel approach to prepare insulin-loaded poly (lactic-co-glycolic acid) microcapsules and the protein stability study. J Pharm Sci. 2009;98(5):1712-1731.

Tejada G, Barrera MG, Piccirilli GN, Sortino M, Frattini A, Salomón CJ, et al. Development and evaluation of buccal films based on chitosan for the potential treatment of oral candidiasis. AAPS PharmSciTech. 2017;18(4):936-946.

Jayakumar R, Prabaharan M, Sudheesh KPT, Nair SV, Tamura H. Biomaterials based on chitin and chitosan in wound dressing applications. Biotechnol Adv. 2011;29(3):322-237.


  • There are currently no refbacks.

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License which allows users to read, copy, distribute and make derivative works for non-commercial purposes from the material, as long as the author of the original work is cited properly.