Research On Poly(Amino Acid) Polysaccharide Hydrogel Repairing Diabetic Refractory Wounds By Modulation Of Immune Microenvironment

BackgroundThe underlying pathogenesis of type I diabetes(T1D)involves the autoimmune destruction of insulin-producing?cells in the pancreas.There is no way to prevent type I diabetes for the moment,and patients can only rely on insulin injections for treatment.If the disease is prolonged,diabetes can cause many complications.Common acute complications include diabetic ketoacidosis and non-ketotic hyperosmolar coma.Among the long-term complications,4-10%of diabetic patients will have a combination of diabetic ulcers,and their risk of death within 5 years will be 2.5 times higher than that of other patients with diabetes alone.The prolonged inflammatory environment within the ulcers increases the level of reactive oxygen species(ROS),exacerbates the destruction of the extracellular matrix(ECM),and accelerates the degradation of growth factors,thereby recruiting more inflammatory cells to secrete chemokines,which together create a vicious cycle of inflammatory responses within the damaged tissue,leading to a prolonged healing process.A variety of FDA-approved dressings are available,but most of them only provide moisturization,environmental isolation,and antimicrobial effects,but do not target the microenvironment within the wound to actively transform it into an anti-inflammatory and growth-promoting state.Diabetic wounds can be caused by neuropathy,angiopathy,or both,and the most important of these causes is the uncontrollable inflammatory response and the production and residence of various harmful substances.As an essential amino acid,L-methionine is involved in a variety of antioxidant activities in the body.The thioether group of its side chain can be oxidized by ROS to a sulfone or sulfoxide group,which then reduces the local content of ROS.Heparin,as a glycosaminoglycan with sulfonic acid group,can adsorb specific pro-inflammatory chemokines such as monocyte chemotactic protein-1(MCP-1)by positive and negative charge.ObjectiveOur goal is to To investigate the effects of ROS-responsive mPEG_2k-b-P(Met₂₅-b-Lys₅)/OHEP(m PML/OHEP)hydrogels on the regulation of the immune microenvironment of diabetic non-healing wounds,and to investigate the effect of repairing wounds from a multi-dimensional perspective,with the hope that it can provide an experimental basis for the development of bioactive wound dressings in the future.MethodsThe experiment is divided into three parts:synthesis and characterization of the physicochemical properties of the hydrogel,application to in vitro cellular experiments to verify its biocompatibility and promotion of cell proliferation and migration,and application to in vivo animal experiments to verify its effect on the repair of diabetic wounds and its ability to modulate the immune microenvironment.The specific research methods are described below:(1)The m PML/OHEP hydrogel system was designed,and the polypeptide was firstly synthesized,then the suitable ratio of polypeptide to glycosaminoglycan was determined,and finally the m PML/OHEP hydrogel was obtained.Subsequently,the temperature-sensitive properties,shear thinning property,self-healing property,secondary structure,microscopic morphology and degradability of the hydrogels were investigated.(2)The safety and biocompatibility of m PML/OHEP hydrogels were investigated by live/dead staining and CCK-8 assay using L929 cells,and their protective effects on cells in ROS environment were verified;the ability of m PML/OHEP to scavenge ROS from L929 cells cultured in hypoxia was detected using DCFH-DA probe;HUVECs was selected to explore the promotion effect of m PML/OHEP on their migration ability.(3)A type I diabetic mouse skin wound model was established and treated with m PML/OHEP.The effect of wound healing was evaluated by H&E staining,Masson staining,immunohistochemical staining and immunofluorescence staining;the polarization of macrophages in skin tissue was detected by flow cytometry after 7 days of treatment;the content of each cytokine in skin tissue was detected by cytometric bead array system;the mechanism of protein level alteration in the process of promoted healing by m PML/OHEP was investigated by the detection of skin tissue proteomics.ResultsIn this study,we successfully synthesized ROS-responsive m PML/OHEP temperature-sensitive hydrogel with good temperature-sensitive properties,excellent shear-thinning and self-healing ability,and stable structure,which has excellent basic properties as an injectable temperature-sensitive hydrogel dressing.In vitro cell experiments have also demonstrated that m PML/OHEP can promote normal cell proliferation and protect cells by reducing the ROS burden in the microenvironment,and also have certain promotion effects on cell migration ability.In animal experiments,m PML/OHEP greatly promoted wound healing,and regenerative skin tissues showed good performance in collagen deposition,granulation thickness,epithelial thickness,and angiogenesis.m PML/OHEP prompted macrophages to polarize toward the M2 phenotype during the inflammatory phase,advancing proliferative repair behavior.The detection of cytokines in tissues revealed a significant reduction of the inflammatory chemokine MCP-1,as well as a decrease in the levels of various pro-inflammatory factors.The results of proteomics showed that m PML/OHEP significantly affected the body's immune microenvironment by influencing tissue regeneration-related pathways,including cellular processes,cell growth,and cell death,and by altering the expression of immune and signal transduction-related proteins.ConclusionThe mPEG_2k-b-P(Met₂₅-b-Lys₅)/OHEP temperature-sensitive hydrogel could alleviate the inflammatory response of diabetic refractory wounds by modulating the immune microenvironment,reducing ROS load,and binding specific chemokines to facilitate the transition to the reconstruction phase.