Synthesis Of Enzyme-mediated Cross-linking Hyaluronic Acid Hydrogel And The Applications In Drug Controlled Release

In recent years, enzymatic preparation of hydrogels have attracted much attention. Enzymes as biological catalysts can be used to mediate hydrogel formation with the advantages of mild preparation conditions, unique chemo-selectivity and no need for toxic cross-linkers. Horseradish peroxidase(HRP) is an enzyme that efficiently catalyzes the radical coupling of phenol and aniline derivatives, with the aid of exogenous hydrogen peroxide(H2O2). HRP has been successfully used for the efficient preparation of hydrogel. Aqueous H2O2 is usually directly supplied into the reaction system, to activate HRP for the enzymatic hydrogelation of phenol-containing polymeric substrates. An excess of H2O2 can inactivate HRP, and may negatively affect the activity of the encapsulated drug. In this study, HRP-mediated preparation of redox-sensitive disulfide-cross-linked hydrogels and microgels through the gelation of thiol functional Hyaluronic acid with tyramine as the catalyst promoters and requiring no added H2O2. The composite gel was fabricated with the microgels dispersed throughout the HA hydrogels matrix, and systematically studied their controlled release properties.In the first part of this dissertation, the HA-SH and HA-Tyr hydrogels were prepared by HRP/tyramine and HRP/H2O2 reaction system respectively, and the catalytic efficiency of these two reaction systems were studied. Experimental results showed that in the HRP/tyramine reaction system, the rate of gelation could be controlled by altering the concentration of HRP and tyramine, the HA-SH hydrogels could be formed in 20 min without addition of H2O2, and had the same catalytic efficiency of the HRP/H2O2 reaction system. Hydrogel degradation experiments showed that the disulfide-cross-linked hydrogels could be degraded rapidly under reducing conditions, and the degradation rate could be controlled by changing the concentration of the reducing medium(L-cysteine). The hydrogel swelling experimental results showed that the equilibrium swelling ratio decreased with the increase of the concentration of HA-SH, and was independent of the concentration of HRP and Tyramine. Rheological analysis showed that HA-SH hydrogel had good stability and flexible network structure, the storage modulus increased from 64 Pa to 220 Pa when the concentration of HA-SH increased from 1% to 3%. The experimental results of FITC-Dextran drug release showed that the increase of HA-SH concentration and the molecular weight of the drug could effectively reduce the burst release effect, and prolong the drug release time. The controllable gelation and degradation properties of the hydrogel are likely to be useful for drug delivery carriers.In the second part of this dissertation, the HA-SH microgels were prepared by reversed phase emulsion processes, the HA-SH microgels were embedded in the HA-SH and HA-Tyr hydrogels matrix. The particle size distribution, microscopic morphology and structure of the microgels were studied by laser particle size analyzer and SEM. The microgels dispersion with a diameter of 10μm could be uniformly dispersed in the network structure of the macroscopic hydrogel. The rheological analysis showed that compared with HA hydrogel, the mechanical properties of composite hydrogels were significantly improved, the storage modulus of HA-SH and HA-Tyr hydrogel increased from 63 Pa, 38 Pa to 93 Pa, 48 Pa after 0.2 wt.% microgels were incorporated. Laser scanning confocal microscopy(CLSM) analysis showed that in situ loading was beneficial to the uniform distribution of the drug in the microgels. The results of drug release showed that the composite hydrogel could be used as a dual drug delivery system, and the sustained drug release property of the composite hydrogel system was significantly better than the performance of the single microgels.