Preparation Of Curcumin-loaded Chitosan Nanoparticles And The Effect On Skin Wound Repair In A Diabetic Rat Model

ObjectiveThe enhancement of the inflammatory response and the infinite extension of the inflammatory phase are the main reasons for the delayed healing of diabetic wounds.Inflammation is induced by a large number of inflammatory mediators secreted by macrophages.When diabetic wounds are in a disordered state,inflammatory factors such as tumor necrosis factor α(TNF-α),nuclear factor kappa-B(NF-κB),interleukin-6(IL-6)and toll-like receptor-4(TLR-4)secreted by macrophages,resulting in a decline in the ability of peripheral endothelial cells to migrate and vascular disease,which ultimately hindered the process of diabetic wound healing.Curcumin(Cur)is an effective anti-inflammatory drug.However,due to the poor solubility and short release time,the treatment effect is significantly reduced.Therefore,there is an urgent need to develop an effective carrier to improve the application limitations of Cur and improve the curative effect of Cur.The objective of this project is to prepare Curcumin-loaded chitosan(CS)nanoparticles(Cur-CS-NPs).Cytological experiments and pharmacodynamic experiments are used to study the inhibitory effect of Cur-CS-NPs on macrophage-mediated inflammation and its effect on wound healing.MethodsIn this experiment,an in vitro content determination method for Cur was established,and the linearity,precision,and recovery of the determination method were determined to meet requirements.As the free amino of CS can react with the anion of sodium tripolyphosphate(TPP),the Cur-CS-NPs were prepared by using ionic crosslinking method using Cur,CS and TPP as raw materials.Screening and optimization of the preparation method of Cur-CS-NPs was performed by single factor investigation experiments.The morphology,particle size,Zeta potential,encapsulation efficiency and in vitro drug release pattern of the prepared Cur-CS-NPs were investigated by transmission electron microscopy,nanometer particle size potential analyzer,and UV-visible spectrophotometer.In cytology experiments,the effect of Cur-CS-NPs on the growth of mouse leukemia cells of monocyte macrophage(RAW 264.7)was investigated by MTT colorimetry.A laser confocal microscope was used to detect the uptake of Cur-CS-NPs by RAW 264.7.The effects of Cur-CS-NPs on the migration ability of HUVECs were investigated by scratch experiments and Transwell experiments on human umbilical vein endothelial cells(HUVECs).Through angiogenesis experiments,the effects of Cur-CS-NPs on angiogenesis were investigated.Western blot was used to detect the expression levels of proteins including inflammatory factors and vascular endothelial growth factor(VEGF)in RAW 264.7 cell supernatants and HUVECs cells treated with Cur-CS-NPs.In animal experiments,a diabetic rat model and a diabetic rat skin wound model were established to evaluate the quality of wound healing and to investigate the curative effect of Cur-CS-NPs on the wound healing process.H&E staining was performed on skin wound tissues obtained at different time periods to explore the changes of various cells and tissues in the wound.ResultsThe linearity,precision,repeatability,and recovery of the quantitative analysis method established in this paper meet the methodological requirements.It is determined that this method can be used for the in vitro determination of Cur.The optimal prescription was determined through univariate analysis experiments,and finally the structure-complete,monodispersed,uniform spherical Cur-CS-NPs were obtained.The average particle diameter of Cur-CS-NPs was 91.28 ± 4.30 nm,and the Zeta potential was 21.9 ± 2.1 m V.In the release experiment,Cur-CS-NPs were released more slowly than Cur-Free,which prolonged the release time of Cur.In cytology experiments,MTT colorimetry was used to verify that Cur-CS-NPs had no significant toxicity to RAW 264.7.And the Cur concentrations used in cytology experiments were determined to be 5 and 45 μg/m L.The uptake of Cur-CS-NPs by RAW 264.7 was observed with a laser confocal microscope.The results showed that Cur-CS-NPs effectively increased the uptake capacity of RAW 264.7 on Cur.In addition,it was confirmed that Cur-CS-NPs can promote the migration and angiogenesis of HUVECs in angiogenesis experiments and cell migration experiments.Western Blot experiments investigated the effect of Cur-CS-NPs on the expression level of cellular proteins.The results showed that Cur-CS-NPs can effectively reduce the secretion of inflammatory factors by macrophages,and increase the expression of proliferative and angiogenic factors in HUVECs.In animal experiments,Cur-CS-NPs significantly improved the speed and quality of wound healing compared to the PBS group and the curcumin-free group(Cur-Free).ConclusionsCur-CS-NPs reduce the inflammatory response caused by RAW 264.7 in diabetic wounds,promote the migration ability and angiogenesis of HUVECs,make diabetic wounds from the inflammatory phase to the proliferative phase, and achieve the effect of accelerating wound healing.Therefore,Cur-CS-NPs may be an effective treatment strategy for accelerating diabetic wound healing by reducing the inflammatory response in diabetic rat skin wound models.