Hydrogels Coated With Curcumin And Copper Ions Regulate Inflammation And Angiogenesis To Accelerate Wound Healing

Background and purposeWound healing is a complex process that usually involves hemostasis,inflammatory proliferation and remodeling of chronic wounds due to the long treatment process The poor quality of tissue regeneration affected by the environment is still a major challenge for clinical nursing,which affects as many as 6.5 million people every year,and the loss is as high as 25 billion dollars.Timely and effective wound management methods are needed to realize the smooth passage of chronic wounds through the inflammatory period,accelerate angiogenesis and proliferation,and promote wound healing.In this work,multifunctional hydrogel is used as wound dressing,which has anti-inflammatory and vascular regeneration functions and can effectively promote wound healing.The whole hydrogel system mainly consists of three parts:carrier module,inflammation transformation module and angiogenesis module.Among them,Matrigel can provide a good carrier as a platform to deliver drugs and small molecules.Curcumin（Cur）can polarize mouse bone marrow derived macrophages（BMDMs,MΦ）from M1 phenotype promoting inflammation to M2 phenotype inhibiting inflammation,and also directly polarize MΦfrom M0 phenotype to M2 phenotype,thus achieving anti-inflammatory function.Copper ions(Cu²⁺)can promote angiogenesis,increase blood oxygen supply and promote tissue regeneration.Functional hydrogel wound dressing has the function of targeted treatment,providing a simple solution to accelerate chronic wound healing,and contributing to the intelligent wound management in the futureMethods1.Aqueous solutions of different concentrations,and its solubility was investigated by UV-visible spectrophotometer After the mother solution of Cur and Cu²⁺were diluted into different concentrations in complete medium,MTT experiment was conducted to explore the range of concentrations in which Cur and Cu²⁺did not inhibit cell proliferation and had no toxic effect on cells,so as to determine the concentration of drugs and molecules contained in the gel.2.Preparation and gel state of Cur and Cu²⁺loaded gels:The hydrogel with the final concentration of Cur and Cu²⁺of 2μg/m L was prepared by diluting the mother solution of copper sulfate pentahydrate and Cur and adding Matrigel.After determining the optimum concentration,the drug loading system was mixed with Matrigel in different proportions according to the final concentration,and incubated at 37℃for 30 minutes.The gel state was verified by reverse experiment,and the experimental results were recorded by photographing.3.Effects of Cu²⁺on HUVEC migration and angiogenesis:HUVEC was cultured with high sugar complete medium containing Cu²⁺and Cu²⁺,and scratches began when the cells converged to 100%at the bottom of the pore plate.After that,serum-free medium was changed to co-culture with cells,and the healing of cells after scratches was observed at 0 h,24 h,48 h,72 h and 96 h.After Matrigel was spread on 96-well plates,HUVEC was mixed with Cu²⁺and Cu²⁺-free high-sugar complete media,respectively,and then inoculated into the well plates.Cell angiogenesis was observed under microscope after 6 h,8 h and 12 h.4.Effect of Cur on polarization of MΦ:MΦwas extracted from mouse bone marrow and stimulated with IL-4,IL-13 or Cur after differentiation and maturation induced by macrophage colony stimulating factor.Or the MΦwas first stimulated by liposolysaccharide（LPS）and interferonγ（IFN-γ）into MΦlike MΦand then stimulated with Cur.The expression of nitric oxide synthase（NO）and arginine（ARG-1）in MΦsamples and MΦsamples were detected by western blot.The expression of m HPRT,TNF-α,IL-1β,NOS2,CD206,Arg1,Fizz and Ym1 with different polarity MΦwere detected by real-time fluorescence quantitative PCR.The expression of MΦmarkers F4/80,CD86 and CD206 with different polarity were detected by flow cytometry and confocal laser.5.Effect of gel loaded with Cur and Cu²⁺in chronic wounds:Wound models of normal mice and diabetic mice were constructed.Dressing was added on day 0,2 and 4, and the changes of wound were recorded by photographing.The mice were sacrificed after anesthesia on day 7 and 14,and the tissue at the wound site was fixed. CD31 immunohistochemistry was used to observe the angiogenesis of wound sites in mice.6.Safety of gel loaded with Cur and Cu²⁺in the treatment of chronic wounds:As mentioned above,the mice were sacrificed after anesthesia on the 7th and 14th days, and each organ was fixed and waxed after dissection.After section dewaxing,HE staining was performed to observe the changes of each organ after dressing application.Consequence1.Cur solubility characterization and biocompatibility:When the concentration of Cur solution was between 1-10μg/m L,the absorbance increased with the increase of the concentration,and when the concentration was 10μg/m L,the absorbance was the highest;When the concentration of copper sulfate pentahydrate was 0-5μg/m L and the concentration of Cur was 0-2μg/m L,the HUVEC survival rate of human umbilical vein endothelial cells was close to 100%with little fluctuation.2.Preparation and gel state of Cur and Cu²⁺loaded gels:Preparation and formation of extracellular matrix like hydrogel texture dressings;Without the addition of Matrigel group,the drug loading system in the tube directly flowed to the tube cap after reversing the EP tube,and the drug loading system with the same final concentration but different proportion of dressing was added to the Matrigel group（2:1 and 1:1）.After reversing the EP tube,the dressing still adhered to the tube bottom.3.Effects of Cu²⁺on HUVEC migration and angiogenesis:As the incubation time increases,in comparison with the control group,the scratching healing ability of the group containing 2μg/m L Cu²⁺in the previous incubation medium was significantly improved,and the difference could be seen at 24 h（P<0.05）.After Matrigel was spread on the 96-well plate,HUVEC was inoculated into the well plate.In compar ison with the control group,angiogenesis was significantly increased in the high-glucose complete medium containing Cu²⁺.The Cu²⁺group showed stronger angiogenesis promotion at 6 h,and continued to form thicker vessels at 8 h and 10 h（P<0.001）.4.Effect of Cur on polarization of MΦ:Western blot showed that the expression of M1Φmarker nitric oxide synthase was significantly increased in LPS group,and the expression of M2Φmarker arginine was significantly increased in LPS+Cur group;The M1Φmarkers tumor necrosis factorα（TNF-α）,IL-1β,nitric oxide synthase Nos2 and M2Φmarkers Arg-1,CD206,YM-1 and Fizz were detected by real-time fluorescence quantitative PCR.The M1Φmarker expression was high in LPS group, while the M2Φmarker expression was low;The results of LPS+Cur,IL-4/IL-13, and Cur groups were reversed（P<0.001）.Flow cytometry showed that the double positive rate of CD86 and F4/80 decreased from 84.5%in LPS group to 4.8%in LPS+Cur group,and the double positive rate of CD206 and F4/80 increased from 3.97%in LPS group to 75.5%in LPS+Cur group（P<0.001）.In addition,the double positive rate of CD206 and F4/80 in Cur group and IL-4/IL-13 group increased by 36.45%and 40.38%,respectively,there was no significant difference.Confocal laser scanning showed the same result.5.Effect of gel loaded with Cur and Cu²⁺in chronic wounds:When the wound models of normal mice and diabetic mice were constructed,the body weight was between 19-21 g,with an overall slight decrease（P<0.05）;Fasting blood glucose level increased to 10±1.2 mmol/L on day 7 in all groups except normal mice group（P< 0.001）;The ratio of wound area also changed significantly.Among the diabetic mice wound groups,compared with other groups,the ratio of wound area in the Matrigel+Cur+Cu²⁺group decreased the most from 100%to about 7%on the 14th day（P<0.05）,which was same as the ratio of wound area in normal mice.Masson staining showed that subcutaneous collagen accumulation and angiogenesis were increased in Matrigel+Cur+Cu²⁺group compared with other diabetic mouse wound groups on day 7;On day 14,compared with other diabetic mouse wound groups,the subcutaneous collagen tissues of Matrigel+Cur+Cu²⁺group were widely distributed.CD31 immunohistochemical staining showed more positive areas in Matrigel+Cu²⁺ and Matrigel+Cur+Cu²⁺groups than in PBS group.6.Safety of gel loaded with Cur and Cu²⁺in the treatment of chronic wounds:H&E staining showed no organic changes in all organs in PBS group and Matrigel+Cur+Cu²⁺group,and the dressing used in this study was safe.ConclusionCur can induce macrophages derived from BMDMs to become M2-like directly from M0,and then transform from M1-like to M2-like after LPS stimulation.Cu²⁺can promote HUVEC migration and tubule formation.Matrigel loaded with Cur and Cu²⁺has good biocompatibility in full-thickness skin defect model of diabetic mice,and can promote cell proliferation and vascular formation.