Fabrication Of Carboxymethylcellulose Hydrogel Containing β-cyclodextrin–eugenol Inclusion Complexes For Promoting Diabetic Wound Healing

Objective The difficult healing of wounds caused by diabetes remains a challenging clinical problem.The activation of nuclear factor kappa-B as a key factor contributes to prolonged inflammation and inhibition of angiogenesis.As a natural antioxidant,eugenol may downregulate the lectin-like oxidized low-density lipoprotein receptor-1 and inhibit the activation of nuclear factor kappa-B in endothelial cells.However,eugenol shows poor solubility,pungent odor,and volatility,thus impeding its clinical application as a potential therapeutic agent.This study developed a novel bioactive carboxy-methylcellulose hydrogel loaded with inclusion complexes of eugenol with β-cyclodextrin(eugenol–β-cyclodextrin/carboxymethylcellulose hydrogel)to regulate lectin-like oxidized low-density lipoprotein receptor-1-induced activation of nuclear factor kappa-B to enhance angiogenesis and inhibit inflammation for accelerating diabetic wound healing in vivo.It was found that eugeno–β-cyclodextrin/carboxymethylcellulose hydrogel exhibited remarkable antibacterial activity in vivo and in vitro.Furthermore,it accelerated diabetic wound healing by reducing the lectin-like oxidized low-density lipoprotein receptor-1/nuclear factor kappa-B-induced dysfunction in endothelial cells and promoting angiogenesis.Methods Using β-cyclodextrin eugenol as raw material,eugenol cyclodextrin inclusion compound was prepared by saturated aqueous solution method,and carboxymethyl cellulose sodium eugenol cyclodextrin inclusion compound was used as raw material to prepare eugenol cyclodextrin inclusion compound.Compound hydrogel(eugenol β-cyclodextrin / carboxymethylcellulose hydroge),and prescription optimization based on the release of eugenol cyclodextrin inclusion complex.Scanning electron microscope infrared spectrum scanning,ultraviolet-visible spectrophotometry,and surface morphology structure Characterize the performance,explore the antibacterial effect and mechanism of the dressing through bacterial experiments.In cytology experiments,the eugenol cyclodextrin inclusion complexes were investigated by the tetramethylazozolium microenzyme reaction colorimetric method(MTT method).Effects of human umbilical vein endothelial cells(HUVECs)proliferation;Transwell experiment explores the effect of eugenol cyclodextrin inclusion compounds on the migration ability of HUVECs;angiogenesis experiments to explore the inclusion of eugenol cyclodextrin Effects of biochemicals on angiogenesis;finally,the effects of inclusion complexes on intracellular protein expression levels,including hemagglutinin-like oxidized low-density lipoprotein receptor-1(Western Blot)(LOX-1),nuclear factor κB(NF-κB),tumor necrosis factor-α(TNF-α),inflammatory factor interleukin-6(IL-6),vascular endothelial growth factor(Vascular endothelial growth factor,VEGF for short),In the pharmacodynamic experiments,metal matrix protease 9(MMP-9)was used to observe the wound healing of different administration groups in mouse skin wound models,and to perform H & E staining on the wound tissue to evaluate the quality of wound healing.Results In this paper,a UV spectrophotometric method was established to analyze the eugenol cyclodextrin inclusion complex content.The linear precision recovery was in line with the methodological requirements.According to the release of eugenol cyclodextrin inclusion compound from the hydrogel,the The formulation is optimized,and the finally obtained eugenol cyclodextrin inclusion complex hydrogel has high surface porosity and forms a film.The inclusion complex is evenly dispersed in the hydrogel.The inclusion compound hydrogel prepared by the optimal prescription12 Accumulated hourly release rate reaches 80%.The results of hydrogel microstructure research by SEM show that CMC hydrogel and EG-β-CD/CMC hydrogel both show similar three-dimensional networks and have a large number of regular Pore structure,indicating that filling EG-β-CD into many smaller holes allows rapid water absorption around the wound.In the bacteriostatic experiment,the higher the concentration of EG-β-CD in the hydrogel,the better the bactericidal effect.The bacterial coating experiment intuitively shows that as the concentration of chitosan nanoparticles increases,bacteria decrease.EG-β-CD showed strong antibacterial activity against Gram-negative bacteria(E.coli)and Gram-positive bacteria(Staphylococcus aureus).In cell experiments,EG-β-CD was not cytotoxic to HUVECs by MTT assay,but the proliferation effect was not obvious.EG-β-CD can promote HUVECs migration and angiogenesis.The number of cell migration in EG-β-CD treatment Significantly higher than the free EG treatment group.Angiogenesis experiments also showed that the number of rings of cells treated with EG-β-CD was significantly higher than that of free EG alone.Next,we examined the effect of EG-β-CD on the expression level of cellular proteins through Western Blot.The results showed that compared with the PBS group as a control group,EG and EG-β-CD significantly reduced LOX-1,NF-κB.The expression of NF-κB p65,TNF-a and IL-6 plays an anti-inflammatory effect.EG and EG-β-CD enhanced the expression of p-AKT,p-ERK and VEGF,suggesting that EG also activated PI3K/AKT and ERK signaling in high glucose(HG)-induced HUVECs.This also indicates that matrix metalloproteinase 9(MMP-9)has the highest protein level in the PBS treatment group,indicating that high glucose(HG)induces the overexpression of MMP-9in HUVECs and may cause excessive degradation of the extracellular matrix,thereby delaying wound Heal.The addition of EG and EG-β-CD can effectively down-regulate the expression of MMP-9 in HG-HUVECs,thereby accelerating wound healing.Conclusions In summary,the present study confirmed the role of EG on promoting healing of diabetic wound by exerting antibacterial,anti-inflammatory,and angiogenesis effects.The anti-inflammatory effect of EG–b-CD/CMC hydrogel resulted from the EG-induced LOX-1 inhibition,which may prevent the activation of NF-j B and secretion of pro-inflammatory cytokines.These results showed that the obtained EG–β-CD/CMC hydrogel contributed to the rapid healing of diabetic skin wounds,possibly rendering it a promising therapeutic strategy for the management of diabetic wound.