Experimental Study Of The Mg²⁺/NMN-loaded,Antibacterial,and Antioxidant Hydrogel In Promoting Diabetic Wound Healing

BackgroundDiabetes is a chronic metabolic disorder.In 2021,there were approximately 537 million people worldwide with diabetes,about 20%of whom suffer from hard-to-heal diabetic wounds.Globally,about 2.5%to 15%of the healthcare budget is spent on diabetes treatment annually,with a significant portion allocated to the treatment of diabetic wounds.The high blood sugar environment in diabetic patients affects wound healing,leading to cellular dysfunction,persistent inflammation,and microcirculation disorders,all of which delay wound healing.Additionally,the complex wound microenvironment,such as excessive Reactive Oxygen Species（ROS）,bacterial infection,persistent inflammation,impaired synthesis of Nicotinamide Adenine Dinucleotide（NAD⁺）,and angiogenesis disorders,continues to make the treatment of diabetic wounds a challenging clinical problem.Current traditional treatments include blood sugar control,debridement,skin flap transplantation,and wound dressings,but the outcomes are still not satisfactory.Therefore,developing effective treatment targets and materials for diabetic wounds,based on their pathogenesis,is crucial for improving patients’quality of life and reducing medical costs.Magnesium ions(Mg²⁺)play crucial physiological roles in the human body,including involvement in cellular metabolism and reducing inflammatory responses.Our research group’s preliminary studies have shown that Mg²⁺possesses significant antimicrobial properties,yet an excess of Mg²⁺may lead to cytotoxicity.NAD⁺,also known as Coenzyme I,is an important coenzyme in the tricarboxylic acid cycle,participating in energy metabolism.It has been observed that NAD⁺levels decrease in diabetic patients.Although this decrease can be corrected by supplementing with Nicotinamide Mononucleotide（NMN）,the direct precursor of NAD⁺,the efficacy of NMN in promoting diabetic wound healing remains uncertain.Our earlier research found that NMN supplementation can reduce ROS levels in HUVEC cells induced by high glucose,and enhance HUVEC cell proliferation,migration,and angiogenesis.Further,in vivo experiments indicated that wound healing in diabetic mice is delayed compared to healthy mice,with reduced NAD⁺levels in wound tissues.Peri-wound injection of NMN significantly accelerated wound healing in diabetic mice,but it did not address issues such as the short half-life of NMN,its lack of antimicrobial activity,and complications arising from the injection,including peri-wound swelling,drug leakage,and low drug concentration at the wound center.Hydrogels are polymer materials with a three-dimensional network structure.They are characterized by good biocompatibility,effective adhesiveness,adjustable mechanical strength,oxygen permeability,and high water content（70-95%）,making them extensively studied and applied in wound healing.The three-dimensional network structure facilitates cell adhesion,proliferation,and migration,as well as the loading of active factors and drugs for sustained release.Therefore,to address the challenges associated with Mg²⁺and NMN in diabetic wound treatment,this study proposes a novel in-situ injectable hydrogel based on poly（glycerol sebacate）co-poly（ethylene glycol）grafted with catechol prepolymers（PEGSD）and quaternized chitosan（QCS）.This hydrogel further incorporates NMN and Mg²⁺(QP/NMN/Mg²⁺),ensuring the long-term release of NMN and Mg²⁺through electrostatic and coordination interactions.By combining these two components within the hydrogel,a dual function is anticipated:antibacterial and anti-inflammatory on one hand,and promotion of cellular energy metabolism on the other,thereby accelerating the healing process of Type II diabetic wounds.AimBased on the type II diabetes wound model,this study aimed to develop a multifunctional hydrogel with biocompatibility,adhesion,antibacterial,slow release of NMN and Mg²⁺,clearance of ROS,and promotion of angiogenesis:1.The mechanism of NMN in the treatment of diabetic wounds.2.Physical and chemical properties of hydrogel and its adaptability to diabetic wounds.3.The effect and safety of the hydrogel in promoting diabetic wound healing.4.The mechanisms underlying tissue repair promotion.MethodsStudy on the role of NMN in diabetic wound healing.We constructed a Type II diabetic mouse model by inducing pancreaticβ-cell necrosis in mice using a high-fat and high-sugar diet and streptozotocin（STZ）and created full-thickness skin defects with a diameter of 8mm.The NAD⁺content in the wound tissues of diabetic and healthy mice was determined using an NAD⁺assay kit.The healing speed of wounds in both groups of mice was recorded with a digital camera,and the inflammation,granulation formation,and collagen deposition in the wound tissues were assessed by HE and Masson staining.CD31 immunofluorescence staining was used to evaluate angiogenesis in the wounds.The biological effects of NMN on endothelial cells were assessed using the CCK-8 cell proliferation assay,scratch assay,and angiogenesis assay.The changes in ROS and NAD⁺levels in HUVEC cells under high glucose conditions after NMN treatment were measured,and NMN was injected into the wounds to evaluate its impact on diabetic wound healing.Preparation and evaluation of NMN/Mg²⁺hydrogel.Polyethylene glycol（PEG,Mn=2000）,glycerol,sebacic acid,and 3,4-dihydroxyhydrocinnamic acid were mixed and subjected to a melt polycondensation reaction without a catalyst to form the PEGSD prepolymer precursor.Chitosan（1g）reacted with GTMAC（glycidyltrimethylammonium chloride）at 55℃for 18 hours to yield quaternized chitosan（QCS）.QCS（20 mg/m L）and PEGSD（100 mg/m L）were dissolved and mixed,followed by the addition of Mg Cl₂·6H₂O(58.82μg/m L Mg²⁺)and NMN（5 mg/m L）.Under an oxidative system of 0.6%H₂O₂（300μg/m L）and HRP（25μg/m L）,the QP/NMN/Mg²⁺hydrogel was formed.The optimal working concentrations of Mg²⁺and NMN were determined using a CCK-8 kit.The structure and physicochemical properties of the hydrogel were characterized by FT-IR spectroscopy,UV-vis spectroscopy,X-ray photoelectron spectroscopy,¹H NMR spectroscopy,field emission scanning electron microscopy,and rheometry.The injectability,adhesiveness,in vitro degradation,equilibrium swelling ratio,NMN and Mg²⁺release,hemocompatibility,and cytocompatibility of the hydrogel were also tested.The effects of different hydrogels on cell proliferation,migration,angiogenesis,ROS scavenging,antioxidation,and antibacterial capabilities were evaluated.Biocompatibility,efficacy in diabetic wound healing,and mechanisms of the NMN/Mg²⁺hydrogel.Biocompatibility was assessed through subcutaneous implantation of the hydrogel and subsequent evaluation by hematoxylin and eosin（HE）staining.The biocompatibility was further confirmed by staining of major organs,blood biochemistry,and complete blood count tests.The hemostatic properties of the hydrogel were evaluated using mouse tail amputation and liver hemorrhage models.Different hydrogel dressings were applied to wounds in diabetic mice to compare the rates of wound healing.Histological analysis,in vivo ROS staining and immunofluorescence staining were used to assess inflammation（HE staining,ROS,TNF-α）,collagen deposition（Masson staining）,vascularization（CD31 and VEGF）,epidermal formation（HE staining）,and macrophage polarization（CD206/CD86）in the wounds.RNA-seq analysis was conducted to elucidate the molecular mechanisms by which the QP/NMN/Mg²⁺hydrogel promotes wound repair,with validation of related mechanisms through Western blot analysis.ResultsCompared to healthy mice,diabetic mice exhibit prolonged wound healing times.NAD⁺levels in wound tissues are reduced,and diabetic wounds show severe inflammatory responses,decreased collagen deposition,and reduced angiogenesis.In vitro,NMN enhances HUVEC proliferation and migration,increasing intracellular NAD⁺levels.NMN also promotes angiogenesis in HUVEC and reduces the ROS levels induced by high glucose.Injecting NMN around wounds in mice accelerates the healing process in diabetic mice,reduces inflammatory cell infiltration,and promotes collagen deposition and angiogenesis.The QP/NMN/Mg²⁺hydrogel continuously and stably releases NMN and Mg²⁺for up to 14 days and completely degrades after 16 days.This novel in-situ injectable hydrogel exhibits strong tissue adhesiveness,with a porous structure that allows an equilibrium swelling ratio of about 50%,capable of absorbing some wound exudate.The hydrogel significantly promotes the proliferation and migration of HUVEC and human fibroblasts,enhances HUVEC angiogenic function,and reduces the ROS level.Hemolysis tests and live/dead cell staining demonstrate the hydrogel’s excellent biocompatibility.The incorporation of Mg²⁺enhances the hydrogel’s antibacterial capability against Staphylococcus aureus.The hydrogel exhibits good biocompatibility and biosafety.Different bleeding models demonstrate that the QP/NMN/Mg²⁺adhesive hydrogel significantly reduces bleeding in mouse tails and livers.By day 14,the wound closure rate in the QP/NMN/Mg²⁺group was98.2%,in contrast to the control group,which still had substantial wounds.HE staining results show increased fibroblast migration and reduced inflammatory cell infiltration in wound tissues treated with the hydrogel,with a smaller gap between granulation tissue and epidermis and increased collagen deposition.ROS levels and TNF-αexpression in wounds are significantly reduced,with an increase in CD31⁺neovessels,elevated VEGF expression,decreased M1 macrophages,and increased M2 macrophages.The QP/NMN/Mg²⁺hydrogel upregulates the expression of genes related to angiogenesis and wound healing,significantly inhibits the expression of genes related to inflammation,apoptosis,and proteolysis,and activates the Wnt/β-catenin signaling pathway.ConclusionWe have developed a novel injectable hydrogel based on the synergistic effects of NMN and Mg²⁺,featuring adhesive,antibacterial,antioxidative,hemostatic,pro-angiogenic,and biocompatible properties.By continuously delivering NMN and Mg²⁺to the wound site,this hydrogel showed excellent reparative and regulatory functions during the hemostasis,inflammation,and proliferation phases of wound repair,thereby accelerating the healing of diabetic wounds.Consequently,it holds significant clinical application potential.