Carbon Monoxide Releasing Hydrogel For Diabetic Wound Healing

The hyperglycemic environment in chronic wounds caused by diabetes and the intermediate or final products of glucose metabolism can trigger severe inflammatory responses,which are one of the main reasons for the refractory wound healing,and have become one of the major challenges in the field of skin repair.Hydrogel is a biomaterial with a three-dimensional network structure formed by cross-linking of water-soluble polymers.It has a microstructure similar to that of the extracellular matrix,has good biocompatibility,and can provide a good microenvironment for wound repair.Carbon monoxide（CO）is a signal-regulating gas molecule in the body,which has many important biol ogical functions such as anti-inflammatory,antibacterial,and anti-apoptosis,but its release is poorly controllable.Based on this,this study uses polymer hydrogel as a CO release platform to achieve rapid therapeutic effect by locally regulating the in flammatory response and angiogenesis of diabetic wounds.The following research work is specifically carried out:（1）Construction and properties of carbon monoxide sustained-release hydrogel（CO@HAG）In this paper,sulfhydryl-functionalized hyaluronic acid molecules（HA-SH）were synthesized by EDC coupling reaction,and then hyaluronic acid/silver ion hydrogels（HAG）were prepared by the metal coordination interaction between sulfhydryl groups and silver ions.The CO-releasing compound,manganese carbonyl（Mn2（CO）10）,was loaded into the above hydrogels to prepare CO@HAG.The gel rheological test results showed that the addition of Mn₂（CO）₁₀ increased the storage modulus of HAG from～2000 Pa to～4500 Pa after gelation for two hours.CO@HAG can be successfully restored to its original state within 60 min afte r cutting,and can also be molded into different shapes,showin g good self-healing properties and plasticity.The results of colony count and bacterial CCK-8 experiments showed that CO@HAG had good antibacterial activity against both Gram-negative bacteria（E.coil）and Gram-positive bacteria（S.aureus）.Gas release experiments show that CO@HAG hydrogels can continuously release CO in response to hydrogen peroxide（H₂O₂）stimulation,and the amount of gas released depends on the H ₂O₂concentration.The H₂O₂ scavenging experiment showed that CO@HAG hydrogel can achieve longer antioxidant effects than HAG hydrogel.（2）In vivo study of diabetic wound healingIn this work,the skin repairing function of CO@HAG hydrogel was investigated through a full-thickness injury model of type I diabetic rats.In vivo experiments showed that at 16 days after gel treatment,the wound retention rates of CO@HAG group,HAG group and untreated group were 6.59±0.39%,12.87±2.76%,26.20±4.80%,respectively.Histomorphological results showed that on the7th day after treatment,the average thickness of granulation tissue in CO@HAG group was 946.12±65.42μm,while that in HAG group and untreated group was696.5±74.2μm and 461.46±18.85μm,respectively.At the same time,the average scar lengths were 8517.45μm（untreated group）,7220.62μm（HAG group）,5992.28μm（CO@HAG group）and the average epidermal thickness was 122.98μm（untreated group）,115.66μm（HAG group）,176.23μm（CO@HAG group）respectively.Immunofluorescence results showed that compared with the HAG group and the untreated group,the expression of M1 macrophage marker（i NOS）in the CO@HAG group was significantly decreased,while the M2 macrophage marker（CD206）and vascular endothelial marker（CD31）were significantly increased.Using ELISA to detect in situ cytokine concentrations in skin tissue,the results showed that,compared with the HAG group,the concentration of pro-inflammatory factor IL-6 was significantly decreased and the concen tration of anti-inflammatory factor IL-10 was significantly increased 10 days after CO@HAG treatment.RT-q PCR experiments showed that compared with the untreated group,the expressions of IL-6 and TNF-αwere significantly down-regulated and the expressions of IL-4 and IL-10 were significantly up-regulated at 7 days after CO@HAG treatment,and the relative m RNA levels ofα-SMA,a functional vascular marker,were significantly up-regulated at 10 days after CO@HAG treatment.The above results indicate that CO@HAG has good functions of promoting diabetic skin wound repair,anti-inflammatory and promoting angiogenesis.In summary,we prepared a CO sustained-release hydrogel CO@HAG with self-healing plasticity by using metal-ligand coordination chemistry.It has good plasticity,antibacterial properties,self-healing properties and antioxidant properties,and can continuously release CO,can effectively inhibit excess ive or persistent inflammation,promote the formation of new blood vessels,promote the repair of diabetic skin wounds,and has good clinical applications potential.