Multienzyme Catalyzed Hydrogel Construction And Its Biomedical Application

In nature,biocatalysis plays a vital role in molecule synthesis,energy conversion,and immune defense.The development of ideal multienzyme carriers to achieve efficient biocatalytic functions and participate in the homeostasis regulation of the biological microenvironment has attracted much attention but is remaining a challenge in the biomedical material field.Inspired by the cellular structure and the respiratory chain enzyme metabolism,and based on biocompatible natural enzymes and natural polysaccharides,we developed cascade enzyme-catalyzed free radical polymerization and cascade enzyme-oxidized crosslinking methods,and then mildly prepared an enzyme-polymer hydrogel platform with multiple physical and chemical properties and adjustable mechanical behaviors.As a result,the enzymes were regularly immobilized in the resulting hydrogels and showed high-efficient cascade catalysis abilities.The biomimetic hydrogels with bioactive multienzyme are promising tissue filling materials,which will apply to regulate the redox balance in pathological microenvironments and treat inflammatory diseases.The details are as follows:（1）Using glucose oxidase/cytochrome c cascade reaction,a new hydrogelation method was developed by the enzyme-catalyzed free radical polymerization and crosslinking of the modified polysaccharide.Specifically,the tyrosine residues in cytochrome c were transformed to a protein radical Tyr·by the GOx/Cyt c cascade catalysis,which can initiate the polymerization of unsaturated vinyl monomers and prepare multienzyme-contained polymeric hydrogels with tunable mechanical properties and 3D printing capabilities.The synergy of the dynamic assembly of the multienzyme and polysaccharide and the multienzyme polymerization selection contribute to the self-immobilization of adjacent GOx/Cyt c in the compartmental structure of the hydrogel.Thereby,the cascade catalysis activities of the immobilized multienzyme were significantly enhanced,which was 33.9-fold more than that of the free enzymes.The superacitivity of immobilized multienzyme has two reasons.One is the intermediate transferring improvement caused by the hydrogel compartmental effect,and another is the cytochrome c activity increment originated from its ligand change of the active center after anchoring,which was verified by UV-vis spectra,low-temperature ESR spectra,and DFT calculation.In addition,both the crosslinking density of the hydrogel network and the cascade catalysis activity of the immobilized multienzyme can be adjusted by varying the ratio and concentration of the two enzymes.The hydrogels also show excellent recyclability and stability,which provides a new insight for the design and fabrication of multienzyme-contained hydrogel materials and their catalysis-related applications.（2）Combined galactose oxidase/catalase catalyzed oxidation reaction with dynamic crosslinking of polysaccharides,a new hydrogel integrated multienzyme platform was constructed.During the hydrogelation,the aldehyde group was formed from galactoside in guar catalyzed by galactose oxidase,and simultaneously oxygen was consumed with H₂O₂ production.Subsequently,the aldehyde groups of guar crosslinked with the amino groups of glycol chitosan by a Schiff-base reaction leading to the hydrogel formation.The H₂O₂ was instantly decomposed to nontoxic water and oxygen by catalase.This mild and efficient enzyme-catalyzed oxidation hydrogelation method can realize in vivo injection and in situ rapid molding without being affected by the body liquids.The porous network and the mechanical properties of the hydrogels were tunable by varying the concentrations of two polysaccharides to meet the requirements of on-demand fabrication.The resulting hydrogels are self-healing,3D printable,anti-swelling,and biodegradable due to the dynamic reversible Schiff-base crosslinking.Furthermore,with the hydrogel formation,the multienzyme was self-immobilized in the hydrogel network by covalent bond anchoring.It maintains the catalytic activities and specificity of the multienzyme,as well as endows it with good storage stability,enzymatic hydrolysis stability,and lyophilization stability.These multienzyme immobilized hydrogels formed by multienzyme-catalyzed oxidation provide a new strategy for soft tissue filling applications.（3）Based on the above enzyme-catalyzed hydrogelation investigation,immobilized multienzyme endows the hydrogels with the functions of biocatalytic regulation.The galactose oxidase in the hydrogels has a superoxide dismutase-mimic activity and can catalyze the disproportionation of overproduced superoxide anion（·O₂^-）into H₂O₂ and oxygen in an inflammatory microenvironment.Then,the H₂O₂ is decomposed by the catalase immobilized in the hydrogels.Meanwhile,the cascade reaction can inhibit a series of detrimental reactive species generation,such as hydroxyl radical（·OH）,singlet oxygen（¹O₂）,and peroxynitrite（ONOO^-）.The in vitro experiment results showed that this multienzyme-contained hydrogel has the ability to regulate the levels of reactive oxygen species（ROS）and reactive nitrogen species（RNS）in osteoarthritic synovial fluids.It also down-regulated the inflammatory cytokines of IL-6 and TNF-αin RAW264.7 cells with a LPS stimulus.At last,the therapy of the multienzyme-contained hydrogel was investigated by an osteoarthritis mouse model.After joint section,staining,and scoring,the results showed that,compared to the non-treated DMM group,the hydrogel treatment group had a better cartilage matrix and a lower osteophyte and synovitis degree,indicating the anti-inflammatory effect of the multienzyme-contained hydrogel.In addition,owing to the optimized biocatalytic abilities and the favorable cytocompatibility,other multienzyme-contained compartmental hydrogels prepared by the enzyme-catalyzed free radical polymerization method are promising biomaterials to applied in more biomedical fields such as bioimaging and biosensors in the future.