Scheme 3. Schematic of synthetic route for HA-SH. HA-SH was synthesized and characterized as described in Scheme 3. HA (MW = 80 kDa) was dissolved in DI water (5 mg/mL) and conjugated with three molar amounts of cystamine dihydrochloride at pH 4.8 overnight after activation of carboxyl group of HA with EDC and HOBT (3 equiv.) for 2 hours. The resulting product was purified by dialysis tubing (MWCO of 10,000 kDa) against DI water for 3 days. Then, the solution was treated with TCEP (5 equiv.) to cleave the disulfide linkage of cystamine component. After stirring for 2 hours, the pH of the solution was adjusted to 3.5 with HCl. Finally, HA-SH was precipitated in ethanol, re-dissolved in DI water, lyophilized and stored at -20 ºC.