Study On The Effect Of Antimicrobial Peptide Hydrogel On Diabetic Chronic Wounds

Background Chronic diabetic trauma is one of the serious complications of diabetes mellitus with high incidence,causing severe pain and even endangering patients’ lives.Hydrogel is a macromolecular hydrophilic network material formed by chemical or physical cross-linking of water-soluble polymers,which is called "the closest material to living tissue" due to its high water content and good biocompatibility.Antimicrobial peptides(AMPs)are a class of host defense peptides with broad-spectrum antimicrobial properties,and their unique antimicrobial mechanism minimizes microbial resistance,making them an effective alternative to antibiotics for the treatment of microbial infections.Therefore,the development and application of antimicrobial peptide hydrogels has become a hot research topic for chronic trauma dressings for diabetes.Objective To design a multifunctional antimicrobial peptide hydrogel to promote the healing of diabetic chronic wounds.Method A multifunctional hydrogel was prepared by reacting oxidized konjac glucan(OKGM)with ε-polylysine(EPL)by dynamic Schiff base reaction.Fourier transform infrared spectroscopy(FTIR)was used to study the infrared spectra of konjac dextran(KGM),OKGM,EPL,and OKGM/EPL-3 hydrogels.Scanning electron microscopy(SEM)was applied to study the microstructure of the hydrogels.The swelling rate,gelation time,injectability,self-healing and adhesion properties of the hydrogels were further determined.The energy storage modulus(G’)and loss modulus(G")of hydrogels were examined under different rheological models using a rheometer.Live/dead cell experiments were applied to evaluate the cytocompatibility of the hydrogels.Three models of liver incision,liver injury and tail truncation were used to evaluate the in vivo hemostatic properties of OKGM/EPL-3 hydrogels in male Sprague-Dawley rats(6 weeks,180-220 g,n=9).The bacteriostatic ability of the hydrogel was evaluated by the inhibition ring method and the coating plate method.Finally,a rat whole-layer diabetic trauma model was established to evaluate the therapeutic effect of the hydrogel.The results obeyed normal distribution then expressed by mean ± standard deviation(Mean ± SD),and the variance was flush then compared by two independent samples t-test,otherwise the non-parametric test of rank transformation was applied for comparison.p < 0.05 means the data differences were statistically significant.Results The dynamic Schiff base reaction between-CHO of OKGM and-NH2 of EPL was demonstrated by FTIR spectroscopy,and the OKGM/EPL hydrogel network was successfully constructed.SEM observed that all three different concentrations of OKGM/EPL hydrogels exhibited a significant porous structure,and the diameter of hydrogel pores decreased from 68±10 μm to 30±2 μm with increasing EPL concentration.2 μm,but the density of pores increased.All OKGM/EPL hydrogels swelled rapidly in PBS and showed good swelling properties.The gel formation time of the hydrogels gradually decreased with increasing EPL concentration.okgm/epl hydrogels exhibited excellent mechanical,self-healing,adhesion and cytocompatibility properties.In the rat liver incision model,the blood loss in the OKGM/EPL-3 hydrogel group was 20±7 mg(P<0.001)and the hemostasis time was reduced to 34±9 s(P<0.001).In the rat liver injury model,the OKGM/EPL-3 hydrogel group significantly reduced the blood loss to15±6 mg(P<0.001)and the hemostasis time to 26±11 s.In the rat tail truncation model,the blood loss in the hydrogel group was 9±6 mg and the hemostasis time was also reduced to 17±5 s.The effects of OKGM/EPL hydrogel on Gram-positive Staphylococcus aureus(S.aureus)and gram-negative Escherichia coli(E.coli)and gram-negative Pseudomonas aeruginosa(P.aeruginosa)with broad-spectrum antibacterial properties,and the OKGM/EPL-3 hydrogel had the best antibacterial effect.Direct observation by establishing a whole-layer diabetic wound model in rats revealed that the OKGM/EPL hydrogel group had the best wound healing effect compared with the other three groups(gauze group,sodium zirconium phosphate silver alginate dressing group and OKGM group).On days 7 and 14,hair follicles,sweat ducts and sebaceous glands could be clearly observed in the OKGM/EPL hydrogel group,and the thickness of new granulation tissue formed and collagen deposition were significantly higher than those in the control group.the expression of anti-inflammatory factors,such as IL-4,IL-10,vascular endothelial growth factor(VEGF),transforming growth factor-β(TGF-β1),in the OKGM/EPL hydrogel group was significantly The expression of pro-inflammatory factors,such as IL-6,IL-1β and tumor necrosis factor(TNF-α),was significantly lower in the OKGM/EPL hydrogel group.the fluorescence intensity of CD31 and α-SMA was significantly enhanced in the OKGM/EPL hydrogel group,and the expression of CD206 was significantly higher than in the other groups.Conclusion We prepared a multifunctional hydrogel with OKGM and ε-EPL by dynamic Schiff base reaction.OKGM/EPL hydrogel showed good mechanical properties,injectability,good self-healing properties and excellent adhesion.OKGM/EPL hydrogel may,due to its adhesion,water absorption and electrostatic attraction,in three models of rat liver incision,liver injury and tail truncation OKGM/EPL hydrogel showed excellent hemostatic properties on L929 fibroblasts with good cytocompatibility and proliferation-promoting effects.In addition,OKGM/EPL hydrogels exhibited excellent broad-spectrum antimicrobial properties against S.aureus,E.coli and P.aeruginosa.Most importantly,the hydrogel was able to promote wound healing,modulate macrophage polarization and promote angiogenesis in chronic wound experiments in rats with diabetes mellitus.We believe that OKGM/EPL hydrogel provides a new strategy for the treatment of diabetic chronic wounds and has great prospects for clinical application.