Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.