Preparation Of Tissue Adhesive Hydrogel Loaded With Thymol And Application In Promoting Diabetic Wound Healing

Background:Diabetic wound（DW）is one of the most common and harmful complications of the many complications of diabetes.In diabetic wounds,the process of skin repair is disturbed by many factors,which hinder the normal healing of the wound and eventually develops into chronic wounds,with the diabetic foot ulcer（DFU）as the representative.As many as 25%of diabetics will have DFUs in their lifetime,of which the amputation rate is 7-20%,the mortality rate within 5 years is nearly 50%,and the annual cost of related treatment is more than 3 billion dollars.Diabetic wounds not only impose the economic burden of family and society and consume a lot of medical resources,but also make patients suffer great physical and mental pain.Therefore,it is urgent to develop new methods to speed up the healing of diabetic wounds and shorten their course.The wound dressings play an important role in the treatment and management of the wounds,which can not only cover the wound and protect it,but also induce tissue regeneration and accelerate the wound healing.Common wound dressings include fiber textile materials,polymer membranes,foam,sponges and hydrogels.Among them,hydrogel is a kind of moisturizing dressings composed of hydrophilic polymer materials,which has the soft and moist characteristics similar to extracellular matrix,and is widely concerned and studied in wound management;At the same time,it has a good application prospect in tissue engineering and drug delivery for its three-dimensional network structure.However,existing hydrogels are prone to swelling and rupture in humid and complex wound environments,and their unstable physical properties will damage their adhesion and mechanical properties.In addition,most hydrogels have limitations such as weak antibacterial activity,poor biocompatibility,and no antioxidant function,which makes it difficult to meet the treatment needs of complex wounds such as diabetic wounds.Thymol is a natural compound extracted from plants with antibacterial,antioxidant,anti-inflammatory and other biological activities.It is a strategy to construct multifunctional hydrogels by adding natural bioactive agents to hydrogels.Therefore,it is of great scientific significance and clinical value to design a multifunctional hydrogel dressing with strong tissue adhesion,controllable swelling,excellent mechanical properties,antibacterial,antioxidant and good biocompatibility for the treatment of diabetic wounds.In this study,firstly,a composite hydrogel with strong tissue adhesion,controllable swelling and excellent mechanical properties was synthesized by using sulfobetaine methacrylate（SBMA）as the base material and quaternized chitosan methacrylate（QCSMA）and gelatin methacrylate（Gel MA）as the biocompatible crosslinker.Then,thymol was nano-encapsulated by amphiphilic block copolymer F127,and loaded into hydrogel matrix to form a multifunctional hydrogel.Finally,the multifunctional hydrogel was used to repair the wounds of diabetic mice,to observe its effect on the healing of diabetic wounds and explore the possible mechanism,in order to provide a new strategy and direction for the treatment of diabetic wounds.Part 1 Synthesis and physical characterization of tissue adhesive hydrogel Objective:To design and construct a composite hydrogel which with strong tissue adhesion,controllable swelling and excellent mechanical properties.Methods:Firstly,QCSMA and Gel MA were prepared by the reaction of quaternary ammonium salt chitosan（QCS）,gelatin（Gel）with methacrylic anhydride,and characterized by fourier transform infrared spectroscopy（FTIR）and ¹H Nuclear Magnetic Resonance（¹H-NMR）.Then,using SBMA as hydrogel substrate,QCSMA and Gel MA as biocompatible crosslinkers to regulate the hydrogel network,a new composite SQG hydrogel was constructed.At the same time,different formulations of SQG hydrogel were synthesized by adjusting the component ratio.The microstructures of the hydrogels were observed by SEM;The swelling properties of hydrogels were measured by weighing method;The rheological properties of the hydrogel were measured by frequency scanning and temperature scanning;The tensile mechanics and adhesion of hydrogel were evaluated by universal material testing machine.Results:QCSMA and Gel MA were synthesized and characterized by FTIR and ¹H-NMR,which indicated that new chemical molecular structures were produced.The degrees of substitution of methacrylate were 6%and 43%,respectively.SQG hydrogel was successfully constructed by"one pot method".SQG hydrogel had a three-dimensional network porous structure,with pores ranging from 5μm to 50μm.The swelling property of SQG hydrogel was controllable,and the equilibrium swelling rate of S₄Q₄G₄ hydrogel was（120.1±4.61）%.Rheological test showed that the modulus of hydrogel（G’）of all the hydrogels was higher than the loss modulus（G"）,and all of them could maintain the colloidal state,showing the characteristics of elastic materials.The mechanical properties of SQG hydrogel were positively correlated with the concentration of SBMA;Compared with other formulations of hydrogels,S₄Q₄G₄ hydrogel showed stronger tensile mechanical properties.With the increase of SBMA,the adhesion of SQG hydrogel gradually improved,and the adhesion of S₄Q₄G₄ hydrogel was the strongest,with the adhesion strength to pig skin,glass,rubber and PTFE reaching 38.2 k Pa,25.4 k Pa,20.1 k Pa and 6.6 k Pa respectively.Conclusion:SQG hydrogels with strong tissue adhesion,controllable swelling and excellent mechanical properties were successfully constructed,among which S₄Q₄G₄ had the best performance.Part 2 Synthesis and characterization of SQG hydrogel containing thymol Objective:To prepare a multifunctional hydrogel containing thymol nano micelles base on SQG hydrogel.Methods:Firstly,thymol（Thy）was coated with F127 to form drug-loaded nano micelles（F127-Thy）,and the drug loading rate,encapsulation rate,particle size,potential were measured.At the same time,the components were analyzed by FTIR and the morphology was observed by transmission electron microscopy（TEM）.Then,taking S₄Q₄G₄ hydrogel as the framework,the multifunctional hydrogel（SQG-F127-Thy）containing thymol was prepared by introducing F127-Thy nano-micelles.The physical properties of hydrogels,such as microstructure,swelling,rheology,mechanics and viscosity were characterized.The drug relieving effect of hydrogel was observed by dialysis;The antioxidant activity of hydrogel was evaluated by free radical scavenging test;The antibacterial activity of hydrogels was evaluated by direct coincubation method;The biocompatibility of hydrogel was evaluated by dialysis bag method,cell inoculation and CCK-8 test,subcutaneous embedding test,skin irritation test.Results:The drug-loaded nano-micelle F127-Thy was obtained by the thin film hydration method.The encapsulation efficiency was 84.79±2.03%,the drug-loaded rate was7.82±0.17%,the particle size was 26.51±0.67 nm,the PDI was 0.14±0.01 and the Zeta potential was-0.76±0.29.The FTIR further confirmed the encapsulation of F127 to thymol;Under the TEM,it could be seen that the nano micelles were spherical,regular in shape and evenly distributed.The blank hydrogel（SQG-F127）and drug loaded hydrogel（SQG-F127-Thy）were prepared,and the scanning electron microscope showed that the two groups of hydrogels were porous three-dimensional structure;The swelling rate of SQG-F127 was 146.6±10.4%,while that of SQG-F127-Thy was 108.5±10.5%,showed statistical significance（P<0.05）between them;Rheological frequency scanning showed that the hydrogel could maintain its gel state;The tensile mechanics of the two groups of hydrogels were similar,and the strain stress of the hydrogels decreased slightly after loading drugs;For the adhesion to pig skin,SQG-F127 reached a maximum of 36.8 k Pa,and SQG-F127-Thy reached a maximum of 34.1 k Pa.The release of thymol from SQG-F127-Thy reached41.5±2.86%in the first 24 hours,reached a balance on the 5th and 6th days,and the cumulative release reached 83.9±2.08%on the 6th day.Compared with F127-Thy,SQG-F127-Thy had stronger free radical scavenging activity,which was enhanced with the time.The killing rates for Escherichia coli（E.coli）and Staphylococcus aureus（S.aureus）were 72.8±6.9%and 79.3±2.6%in SQG-F127 hydrogel group,93.6±2.3%and 95.1±2.8%in SQG-F127-Thy,respectively,and showed statistical significance（P<0.05）between them.The hemolysis rate of F127-Thy was 1.4±0.1%,and of which the SQG-F127-Thy was about 1.6±0.2%,which was far lower than the national standard of 5%,showing good blood compatibility;Under the confocal laser scanning microscope,fibroblasts adhered evenly and compactly to the surface of the hydrogel,CCK-8 assay experiment showed that the cell proliferation rates of SQG-F127 and SQG-F127-Thy were 95.8±1.7 and 94.1±2.0%respectively,showing no obvious toxicity and good cytocompatibility;Both groups of hydrogels did not cause serious inflammation when implanted subcutaneously in mice,and had good histocompatibility;The hydrogel did not cause sensitization when contacting with mouse skin,and had good skin compatibility.Conclusion:A drug sustained-release hydrogel SQG-F127-Thy containing thymol has been successfully prepared,which had the characteristics of strong skin tissue adhesion,excellent antibacterial,antioxidant and good biocompatibility.Part 3 Adhesive hydrogel containing thymol promotes wound repair in diabetic miceObjective:To investigate the effect of SQG-F127-Thy hydrogel on wound healing in diabetic mice and its related mechanism.Methods:Firstly,diabetic mice models were established by intraperitoneal injection of streptozotocin（STZ）and then randomly divided into Control group,SQG-F127group and SQG-F127-Thy group.After the skin defects were made,they were covered with gauze,blank hydrogel and drug-loaded hydrogel respectively.Then,the effect of hydrogel on the healing of diabetic wound was evaluated by observing the wound healing rate and histomorphologic changes of H&E and Masson staining at different time points after trauma;The expressions of Nrf2 and HO-1 in the wound tissue were further observed by immunohistochemistry method,the expressions of Nrf2 and HO-1 m RNA in the wound tissue were detected by q RT-PCR,the protein expressions of Nrf2,HO-1,TNF-a and IL-6 in the wound tissue were detected by Western blot,and the contents of MDA,SOD and GSH in each wound tissue were observed by colorimetry to explore the possible mechanism of action.Results:The skin defect models of diabetic mice were successfully constructed and experimental interventions were carried out.On the 7th,14th and 21th days after trauma,the wound healing rate,granulation tissue thickness and re-epithelialization rate in SQG-F127-Thy group were higher than those in Control group（P<0.01）,and also higher than those in SQG-F127 group（P<0.05）;Masson staining showed that the collagen deposition and collagen fiber formation were more obvious in SQG-F127-Thy group.Immunohistochemical staining showed that the expression of Nrf2 in wound tissue of SQG-F127-Thy group was significantly higher than that in control group and SQG-F127 group at all three time points（P<0.01）;On the 7th day of trauma,the expression of HO-1 in SQG-F127-Thy group was significantly higher than that in Control group and SQG-F127 group（P<0.01）;On the 14th and 21st days of trauma,it decreased slightly but was still higher than that in the other two groups（P<0.05）.QRT-PCR showed that the expression of Nrf2 m RNA in SQG-F127-Thy group was significantly higher than that in Control group and SQG-F127 group on the7th day after trauma（P<0.01）,and slightly decreased on the 14th and 21st days,but still higher than that in Control group（P<0.05）;At all three time points after trauma,the expression of HO-1 m RNA in wound tissue of SQG-F127-Thy group was significantly higher than that of Control group（P<0.01）.Western blot analysis showed that the expressions of Nrf2 and HO-1 in the wound tissue of SQG-F127-Thy group was significantly higher than that of Control group and SQG-F127 group,while the expressions of TNF-αand IL-6 were significantly lower.Compared with Control group and SQG-F127 group,the contents of SOD and GSH in wound tissue of SQG-F127-Thy group were significantly increased at each time point（P<0.01）,while MDA was significantly decreased（P<0.05）.Conclusion:SQG-F127-Thy hydrogel can accelerate diabetic wound healing,promote granulation and epithelial regeneration.This may be achieved by activating the Nrf2/HO-1 signaling pathway,inhibiting wound inflammation,and increasing wound antioxidant levels.