Enzyme-catalyzed Self-healing Hydrogel Preparation And Its Application For The Treatment Of Diabetic Chronic Wounds

Due to the complex microenvironment of wound tissue,diabetic chronic wounds often cause scar tissue hyperplasia and long-term non-healing,resulting in diabetic foot ulcers,diabetic retinopathy,cardiovascular diseases and other diseases,and even sepsis that threat the health of patients.Normal wound repair process can be mainly divided into the following four stages:coagulation stage,inflammatory stage,proliferation stage,and remodeling stage.The four stages occur in a specific order and at a specific time to ensure the effective healing of the wound.However,the hyperglycemic environment in diabetic chronic wounds tends to trigger persistent bacterial infection,inflammatory overreaction,redox imbalance and angiogenesis disorders,which in turn reduces the activity of biomacromolecules with important repair functions.This makes wound repair stagnate in the inflammatory stage and delays the transformation of the proliferation stage,which seriously hinders the wound healing process.Therefore,it is of great significance to prepare repair materials to address the multiple factors that make diabetic chronic wounds difficult to heal.In this thesis,a p H-responsive multifunctional self-healing hydrogel was successfully constructed by the reversible covalent crosslinking of a variety of natural enzymes for satisfying the treatment requirement towards the four stages of diabetic wound healing.A multifactorial synergistic treatment strategy can be realized by the combination of multi-enzyme cascade reaction and encapsulated drugs to regulate local blood glucose level,bacterial infection,inflammatory responses,redox balance,and cell migration.The detailed content is as follows:1.Enzyme-catalyzed self-healing hydrogel preparationA pH-responsive self-healing hydrogel was prepared by lysozyme（LY）,glucose oxidase（GOx）,and catalase（CAT）via the crosslinking between the amino groups on the surface of three enzymes and the aldehyde group of glutaraldehyde to form the dynamically reversible imine bonding.Firstly,the immobilized GOx catalyzes the production of H₂S and H₂O₂ from 1-thio-β-D-glucose and generates the gluconic acid to reduce the p H of the system,which leads to the cleavage of imine bonds and makes the hydrogel self-healable.Secondly,H₂O₂ will be removed by CAT to protect the imine bonds in the gel crosslinking process against oxidative damage.Therefore,both p H and redox microenvironment of the hydrogels can be adjusted by multi-enzyme cascade reaction to achieve reversible self-healing.Subsequently,the microstructure and physical properties of the hydrogel,including swelling ratio,air permeability,adhesion strength,and self-healing ability were characterized.Because the 3D porous network structure of the hydrogel changes under acidic conditions,the p H controlled-release curves of the drug rosuvastatin calcium and other different sized molecules were also determined.In addition,the excellent enzymatic properties of hydrogels,including GOx and CAT enzyme activities,cascade rate and H₂S yield,were evaluated in detail.2.Self-healing hydrogel for the treatment of diabetic chronic woundsIn order to systematically study in-vitro antibacterial,anti-inflammatory,antioxidant activities and biosafety of self-healing hydrogels,the inhibition rate of hydrogels against gram-positive and negative bacteria was first determined by spread plate method,zone of inhibition test and growth curve analysis.Secondly,DPPH radical scavenging and DCFH-DA probe for reactive oxygen species were used to explore the antioxidant performance of H₂S in cells.Based on lipopolysaccharide-induced macrophages,the in-vitro inflammatory model was established to detect the levels of TNF-αand IL-6 in macrophages by ELISA method,which confirmed their strong anti-inflammatory ability.Finally,the biosafety of hydrogels was identified by MTT cell viability and hemolysis experiments.Based on the trauma model of diabetic mice induced by streptozotocin,the healing effect of hydrogel was further studied,the wound healing and mouse weight change were monitored within 14 days after treatment,and the antibacterial and anti-inflammatory effects of hydrogel were evaluated in vivo by extracting bacteria from the tissue surface at different time periods for culture and ELISA detection.