Biodegradable Hydrogels With Photodynamic Antibacterial Activity Promote Wound Healing And Mitigate Scar Formation

Objective: Bacterial proliferation and disordered extracellular matrix(ECM)at the wound site are major reasons for delayed healing and abnormal scarring.The development of new multifunctional dressing materials that can effectively prevent scar formation without delaying wound healing remains a challenge.In this study,we construct a verteporfin-loaded biodegradable hydrogel(VP-gel)using hyaluronic acid and thiol-terminated 4-arm poly(ethylene oxide).The VP-gel sustainably releases small doses of verteporfin in the wound microenvironment that generates reactive oxygen species(ROS)under red light irradiation to kill bacteria efficiently,and promote the scarless repair of infected wounds.Methods: 1)We successfully added methacrylic acid units to hyaluronic acid and characterized the product by 1H NMR spectroscopy;2)The hyaluronic acid hydrogel was characterized by transmission electron microscopy and rotational rheometer.Measure properties such as rheology,swelling and degradability of hyaluronic acid hydrogels;3)We observed the cytotoxicity of hyaluronic acid hydrogels by Calcein(AM)/propidium iodide(PI)fluorescent staining;4)Using the MTT method to test the cytotoxicity of the components of verteporfin hydrogel;5)The characteristic peaks of verteporfin were tested with a UV-visible spectrophotometer,and the release performance of VP-gel was detected;6)Learning the production of reactive oxygen species of verteporfin through singlet oxygen detection;7)Observation of cell morphologies to understand the inhibitory effect of verteporfin on the proliferation of 3T3 cells and the anti-scar mechanism of verteporfin by Western blot;8)Observing the antibacterial effect of VP-gel through the inhibition zone experiment,and conducting scanning electron microscope experiments on bacteria to understand the antibacterial mechanism of VP;9)Constructing a mouse full-thickness wound model infected by methicillin-resistant Staphylococcus aureus to evaluate the antibacterial and anti-scar effect of VP-gel in vivo;10)Through H&E staining,Masson’s trichrome staining,immunohistochemical analysis,and wound bacterial counts on the healed skin,the antibacterial,anti-scarring,and toxicity of VP-gel were comprehensively analyzed.Results: 1)We successfully synthesized hyaluronic acid hydrogel with good biocompatibility,good swelling and degradability matching wound healing;2)The VPgel sustainably releases small doses of verteporfin generates reactive oxygen species(ROS)under red light irradiation;3)VP could regulate TGF-β family-induced cellular responses and the downstream signaling molecule Smad2 in fibroblasts to reduce myofibroblast differentiation,promoting ECM reconstruction and scarless wound healing;4)Scanning electron microscopy and inhibition zone experiments show that verteporfin hydrogel can produce good antibacterial effect by destroying the membrane structure of Gram-positive bacteria;5)The immunohistochemical examination of mouse fullthickness wound healing model and histomorphology showed that the wound area of VPgel(Light)group had reduced inflammatory cells,moderate thickness of epithelium,fewer and thinner collagen fibers,and organized collagen gaps.It has a good accelerating effect on the healing of infected wounds.Conclusion: VP-gel with anti-scarring,antibacterial activity,and enhanced infection wound healing ability shows great potential in the treatment of scar healing for infected wounds.