Enzymatically Disulfide-Crosslinked Chitosan/Hyaluronic Acid Layer-by-Layer Self-Assembled Microcapsules for Redox-Responsive Controlled Release of Protein

Disulfide-crosslinked hollow polyelectrolyte microcapsules composed of thiolated chitosan (CS-SH) and hyaluronic acid (HA-SH) were prepared by combining the layer-by-layer (LBL) technique and horseradish peroxidase (HRP)-mediated oxidative cross-linking reaction in mild conditions. FITC-dextran-doped CaCO₃ microspheres were used as template core and removed after LBL depositing CS-SH and HA-SH on the surface. The disulfide-crosslinked (CS/HA) microcapsules were readily fabricated by HRP-mediated oxidative coupling of the thiol groups in CS/HA shell layer in the presence of HRP (10 units/mL) and Tyramine hydrochloride (Tyr, 35 mmol/L). The kinetics of enzymatic disulfide-crosslinking reaction was investigated through the real-time monitoring of the consumption of thiol groups by UV absorption spectra. It found that the formation of disulfide linkages by the enzymatic thiol oxidation reaction showed a gradual acceleration. The disulfide-crosslinked CS/HA hydrogel were rapidly formed in gelation time between approximately 17 and 30 min, which were dependent on the concentrations of HRP and Tyr. The disulfide linkages endowed the microcapsule-enhanced physical stability and low permeability under physiological conditions and redox-responsive degradability in reducing environments. The structural stability of disulfide-crosslinked (CS/HA) microcapsules was visualized by confocal laser scanning microscopy in phosphate-buffered saline containing 5.0 mmol/L dithiothreitol (DTT) to evaluate the redox-responsive disassembly process. Redox-responsive controlled release of encapsulated FITC-dextran from the disulfide-crosslinked (CS/HA) microcapsules were obtained. The release profiles of FITC-dextran could be manipulated by controlling the shell thickness and the concentration of DTT. The conformational stability analyses and more than 94% esterase activity of released bovine serum albumin (BSA) from (CS/HA) microcapsules conformed that the structural integrity and bioactivity were well preserved during the encapsulation and release process. The microcapsules exhibited excellent cytocompatibility for HEK 293 cells up to a concentration of 1.0 mg/mL. The microcapsules efficiently delivered loaded FITC-BSA into HeLa cells and released the protein in the reducing cytosol. This study proposed a novel approach for producing disulfide-crosslinked microcarriers for intracellular delivery and redox-responsive controlled release of protein.