Preparation Of Nanofiber Mats Loaded With Anemoside B4 For Diabetic Wound Healing Treatment

Objective: With the worldwide prevalence of diabetes,considering the complexity of diabetic wound microenvironment,it is urgent to design and develop a new multifunctional wound dressing for the wound that is difficult to heal in patients with diabetes.In this study,composite electrospinning technology was used to prepare nano-fiber wound materials loaded with anti-inflammatory ingredients,and its ability to treat diabetic wounds was systematically studied.Diabetic wound is a kind of chronic skin disease which is difficult to heal.Anemoside B4(ANE)is a natural active ingredient from Pulsatilla pulsatilla,which has a variety of pharmacological effects.Its application in diabetic wounds has not been reported yet.The purpose of this study was to investigate the effect and related mechanism of ANE on diabetic wound.Methods: In this study,Chitosan(CS)and Poly Vinyl alcohol(PVA),which have a natural hemostatic effect,were used as the substrate materials of nanofiber pads.CSPVA-ANE nanofiber pad was prepared by electrostatic spinning mechanism.We evaluated the morphology,hydrophilicity,tensile properties,drug release,hemostatic ability and biocompatibility of nanofiber pads by scanning electron microscopy,water contact Angle test,mechanical property test,in vitro drug release test,liver bleeding test and material cell co-culture.Based on the above evaluation,nano-fiber pads suitable for diabetic wounds were screened out.In the in vitro experiment,RAW264.7cells were co-cultured with nanofiber mats for 6 h after Lipopolysaccharide(LPS)stimulation.The phenotype of RAW264.7 cells was analyzed by flow cytometry.The wound model of diabetic mice was established in vivo.Photographs were taken and observed every day.On days 7 and 14,the skin tissue at the wound site was taken for H&E and Masson staining to observe the wound healing and collagen deposition.Cell proliferation and angiogenesis were observed by immunofluorescence.Serum levels of inflammatory cytokines IL-6 and TNF-α were measured with biochemical kits.Western blotting method was used to investigate the mechanism of CS-PVA-ANE nanofiber pad accelerating diabetic wound healing by improving inflammation.Results: The CS-PVA-ANE wound dressing had significant water absorption,biomimetic elastic properties,continuous release of ANE and excellent hemostatic properties.In vitro studies showed that CS-PVA-ANE nanofiber mats could significantly inhibit the differentiation of LPS stimulated pro-inflammatory(M1)macrophage subsets,significantly reduce the production of reactive oxygen species(ROS),and significantly reduce the release of inflammatory cytokines.In vivo animal studies have shown that CS-PVA-ANE nanofiber pad can significantly improve wound healing rate,promote good angiogenesis,and promote the deposition of multiple epithelium and collagen matrix in various stages of wound healing.Conclusion: This study indicates that CS-PVA-ANE nanofiber pad can effectively shorten wound healing time by inhibiting inflammatory activity,and is expected to be a candidate material for treating refractory wounds caused by diabetes.