Regulating Hydrogel Structure Via Enzyme Initiation Technology To Construct Multilevel Functional Materials

Hydrogels are widely applied as intelligent soft actuators,biological scaffolds and drug delivery due to their adjustable mechanical properties and fine biocompatibility among other cumulative properties.Among them,3D printable hydrogels with preconfigurable structure and gradient hydrogels with continuous or semi-continuous gradient microstructure have been paid special attention to by researchers due to their rich functions.The regulation of microstructure of hydrogel is the key factor to realize the function of hydrogel.Enzyme-mediated polymerizations by peroxidases,oxidases,or their complexes have been widely applied for precise macromolecular synthesis and hydrogel moldings because of their physiological conditions and controllable efficiency.Currently,many biological enzymes such as horseradish peroxidase,glucose oxidase and laccase are used in the field of polymer preparation,but these enzymes are only used in homogeneous preparation systems.The rate of enzymatic polymerization is commonly higher than that of traditional polymerization due to the quicker enzymemediated radical generation.The initiation stage of enzymatic polymerization follows the Michaelis-Menten equation.The substrate diffusion and enzyme concentration can significantly affect the formation of free radicals and thus determine the polymer hydrogel microstructure,which has been confirmed by researchers and applied to the preparation of microgels.At present,there are few reports that multifunctional hydrogel devices can be obtained by regulating the structure of macro-hydrogel using biological enzyme catalysis system.Therefore,in this paper,the structure of macro-hydrogel is regulated by biological enzyme catalysis system to prepare new functional biomaterials.There are three main innovations in this paper: firstly,a polysaccharide-polymer composite hydrogel with good biocompatibility,adjustable mechanical strength and 3D printing was prepared by rapid crosslinking of polysaccharides and post-polymerization of monomers catalyzed by multiple enzymes;Secondly,a composite hydrogel with multiple continuous gradients was prepared for the first time by spatiotemporal regulation of substrates competitive reactions in multi-enzyme catalytic system;Thirdly,a conductive hydrogel with multiple continuous gradients was prepared by free radical growth induced via interfacial diffusion of initiator in a multi-enzyme catalyzed system.The specific contents are as follows:(1)We employed the HRP/GOx multi-enzyme system to initiate the immediate crosslinking of chondroitin sulfate grafted with tyrosine and the gradual polymerization of monomers to form the composite hydrogels.The detailed two-step gelation mechanism was confirmed by the Fluorescence spectroscopy,Electron paramagnetic resonance spectroscopy and Gel permeation chromatography,respectively.The final composite hydrogel combines the merits of enzymatic crosslinking hydrogels and polymerized hydrogels to achieve adjustable mechanical strength and facile printing performance.The multi-enzyme regulated polymer composite hydrogels are the promising precision devices for tissue repair,organoid materials,implant materials and other biomedical applications.(2)we utilized the spatiotemporal distribution of initiator acetylacetone to regulate the multi-enzyme polymerization and fabricate a chitosan-polymer hydrogel.The temporal priority order of acetylacetone was higher than phenol-modified chitosan by density functional theory calculation.The acetylacetone within the gelatin could gradually diffuse spatially into the chitosan hydrogel to fabricate the composite hydrogel with gradient network structure.The gradient hydrogel possessed a transferring topography from the two-dimensional pattern,which continuously decreased modulus along with acetylacetone diffusion was confirmed by atomic force microscope-based force mapping experiment.The water-retaining ability of various regions was confirmed by low-field NMR and TG analysis,which led to the spontaneous actuation of gradient hydrogel with maximum 1821 °/h curling speed and227 ° curling angle.Consequently,the promising gradient hydrogels could be applied as intelligent actuators and flexible robots.(3)We designed a conductive polymer hydrogel with continuous gradient structure through HRP/GOx multi-enzyme system biocatalysis and free radical polymerization mediated by interfacial diffusion of initiator acetylacetone.The second gradient was designed by doping Fe3+ to form-COO-/Fe3+ coordination bond and photodissociation to enhance the functionality of gradient hydrogel.The elastic modulus of gradient hydrogel gradually decreased from 143.2 KPa to 76.2 KPa along the direction of initiator diffusion.The conductivity of gradient hydrogel decreases from 101.2 m S/cm to 9.46 m S/cm along the direction of initiator diffusion by doping multi-walled carbon nanotubes and their sedimentation.At the same time,the conductivity gradient hydrogel can bend spontaneously because of its different water retention ability in different regions.In addition,the gradient hydrogels were bonded by aqueous carbon diimine activation(adhesion energy 162 J m-2),and the transition from simple monomer to complex hydrogels was realized.The conductive gradient hydrogel has the potential to be a flexible robot and wearable electronic device.