Biopolymer Hydrogels Prepared By Enzymatic Crosslinking And Native Chemical Ligation

Natural polymer hydrogels have excellent biocompatibility and degradability, but usually suffer from a lack of mechanical strength. Chemical crosslinking could improve their mechanical property. But, the residual crosslinker often reduce the biocompatibility of the hydrogels severely limiting their applications in biomedical area. In order to overcome these shortcomings, a series of biohydrogels were prepared via enzymatic reaction and native chemical ligation reaction using hyaluronic acid (HA), gelatin, poly (glutamic acid) (PGA) and ε-polylysine (EPL) as raw materials. And we further improved the mechanical strength of the hydrogel by interpenetrating network technology. In this thesis, the morphology, degradability and biocompatibility of biohydrogels were also systematically investigated, as following described:In the first Chapter, hybrid polypeptide hydrogels were produced via native chemical ligation (NCL) reaction. Firstly, thiolactone grafted poly (glutamic acid) (PGA-HC) and cysteine grafted s-poly-lysine (EPL-C) precursors were synthesized. Then, NCL crosslinking of PGA-HC and EPL-C precursors was triggered at room temperature, resulting in hybrid polypeptide hydrogel with gelation time in 40-100 minutes. The mechanical strength could be changed between 40-60 KPa by adjusting precursor compositions. Furthermore, the morphology, equilibrium water content, degradation property and biocompatibility of the hybrid hydrogels were also characterized in detail. The results showed that the hybrid polypeptide hydrogels had well-interconnected pore structure, tunable equilibrium water content and good biocompatibility.In the Chapter Two, poly (glutamic acid) (PGA) hydrogels were designed and prepared using native chemical ligation (NCL) reaction. After mixing cysteine grafted PGA (PGA-C) precursor with PGA-HC precursor, the pure PGA hydrogels could be yielded under mild conditions through NCL reaction. The hydrogels had tunable gelation time in 3.5-8 hours and mechanical strength was in the range of 241.9-347.5 KPa. What’s more, the morphology, equilibrium water content, enzymatic degradation, swelling property and biocompatibility of hydrogels were characterized in detail. The results revealed that PGA hydrogels had tunable equilibrium water content and all the hydrogels with different compositions exhibited excellent biocompatibility.In the Chapter Three, in situ injectable poly (glutamic acid) hydrogels were prepared by enzymatic crosslinking reaction. Firstly, the precursor of poly (glutamic acid)-tyramine (PGA-Ty) was synthesized through PGA with tyramine (Ty). The PGA-Ty solution was enzymatically crosslinked with HRP in the presence of low concentration (10 mmol/L) H2O2 to provide gelation time within 10±2s at room temperature. Mechanical strength of PGA-Ty hydrogel was in the range of 3796.7-11568.6 Pa and equilibrium water content could be varied from 13.2-18.1. Porous scaffolds, whose pore size of internal structure was in the range of 10-100μm, could be obtained after PGA hydrogel be lyophilized. The data of MTT assay confirmed the injectable PGA-Ty hydrogels had no cytotoxicity.In the Chapter Four, bienzymatically crosslinked gelatin/hyaluronic acid interpenetrating network (IPN) hydrogels were studied and prepared. The mixture solution of tyramine grafted hyaluronic acid (HA-Ty) precursor and gelatin could form IPN hydrogel under the coexistence of horseradish peroxidase (HRP) and transglutaminase (mTG) in 37℃ water bath. Rheological tests confirmed that:one gelatin network was crosslinked by mTG and the other HA network was crosslinked by HRP. Both of the networks were intertwined and independent of each other. Mechanical strength of IPN hydrogel was much higher than that of single enzymatically crosslinked hydrogel, and was higher than that of semi-IPN hydrogel. The degradation behaviors of IPN hydrogels were controllable. Biological experiments revealed the IPN hydrogels had excellent biocompatibility and could also promote mouse fibroblasts (L929) spreading and proliferation well.In this thesis, utilizing mild crosslinking reactions, a series of biopolymer hydrogels with good biocompatibility, tunable mechanical strength, degradability and equilibrium water content were successfully prepared. They have great potential in tissue engineering scaffolds, wound repair material and drug or cell carrier.