Selectively crosslinked hyaluronic acid hydrogels for sustained release formulation of erythropoietin

Abstract A novel sustained release formulation of erythropoietin (EPO) was developed using hyaluronic acid (HA) hydrogels. For the preparation of HA hydrogels, adipic acid dihydrazide grafted HA (HA‐ADH) was synthesized and analyzed with 1 H NMR. The degree of HA‐ADH modification was about 69%. EPO was in situ encapsulated into HA‐ADH hydrogels through a selective cross‐linking reaction of bis(sulfosuccinimidyl) suberate (BS 3 ) to hydrazide group (p K a = 3.0) of HA‐ADH rather than to amine group (p K a > 9) of EPO. The denaturation of EPO during HA‐ADH hydrogel synthesis was drastically reduced with decreasing pH from 7.4 to 4.8. The specific reactivity of BS 3 to hydrazide at pH = 4.8 might be due to its low p K a compared with that of amine. In vitro release of EPO in phosphate buffered saline at 37°C showed that EPO was released rapidly for 2 days and then slowly up to 4 days from HA‐ADH hydrogels. When the hydrogels were dried at 37°C for a day, however, longer release of EPO up to 3 weeks could be demonstrated. According to in vivo release test of EPO from HA‐ADH hydrogels in SD rats, elevated EPO concentration higher than 0.1 ng/mL could be maintained from 7 days up to 18 days depending on the preparation methods of HA‐ADH hydrogels. There was no adverse effect during and after HA‐ADH hydrogel implantation. © 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006