Study Of GelMA Hydrogel Microneedles Loaded With Human Umbilical Cord Mesenchymal Stem Cell Exosomes And Two-dimensional Nanomaterial Nb₂C In Promoting Diabetic Wound Healing

Purpose: Chronic diabetic wounds are challenging to treat because of their persistent nature and high recurrence rate.Redox imbalance was one of the main pathogenesis of diabetic wound healing.Hyperglycemia and oxidative stress lead to altered macrophage polarization and imbalance of inflammatory and anti-inflammatory factors,thus increasing the risk of microbial colonization,biofilm formation,and infection.Meanwhile diabetic wounds had insufficient angiogenesis,significantly reduced vascular and capillary density,and difficulty in re-epithelialization.Traditional wound drug delivery is characterized by a single functional component,short duration of efficacy,and shallow drug delivery,so there is an urgent need to develop a new multifunctional wound dressing that can precisely deliver therapeutic components to the wound internal environment.Methods: Characterize the basic structure of human umbilical Mesenchymal stem cell Eexosome（Hu MSC-exo）and niobium carbide nanosheets（Nb₂C Nano Sheet,Nb2 C NS）.Microneedles were prepared by two-step method,and the tip morphology characteristics of microneedles were observed under light microscopy and electron microscopy,and the degradation time,mechanical strength and slow-release ability of microneedles were observed.Puncture capacity,in vivo sustained release,and long-term storage capacity for Hu MSC-exo of microneedle were detected.The antioxidant capacity,antibacterial ability,macrophage polarization regulation,epithelialization ability and pro-vascularization ability of Gel MA hydrogel microneedles loaded with Nb2 C NS and Hu MSC-exo and（G@NE MN）were verified in vitro.The ability of G@NE MN to promote wound healing in diabetic mice was observed in vivo.Results: Hu MSC-exo and Nb2 C NS had typical nanostructure characteristics,and Nb2 C NS had good biocompatibility.The tip of the microneedle was conical with 600 μm height;Microneedle punctured up to a depth of approximately 500 μm.Microneedles enabled sustained release and long-term storage of Hu MSC-exo.G@NE MN had good biocompatibility.Nb2 C NS and G@NE MN effectively remove various ROS components;Nb₂C NS had concentration-dependent antibacterial effect;G@NE MN regulated the polarization of macrophages in the direction of M2 and modulated the expression of inflammatory factors;G@NE MN significantly increased the migration of Ha Ca T cells and the migration and angiogenesis of HUVEC cells.G@NE MN promoted wound healing in diabetic mice and regulated the expression of α-SMA/CD31,CD86/CD206,ROS,IL6 and TNF-α proteins in diabetic mice wound.Conclusion: Gel MA microneedles had good tip morphology,biodegradability,mechanical strength,puncture ability,sustained release ability and storage ability.G@NE MN possessed good antioxidant properties,bacteriostatic properties,macrophage regulatory ability,epithelialization and vascularization ability.G@NE MN as a novel multifunctional dressing could effectively promote wound healing in diabetic mice.Part I: Preparation and characterization of Gel MA hydrogel microneedlesPurpose: The aim of this study was to explore the preparation of Gel MA-based hydrogel microneedles and their ability to sustained release,continuous delivery and stable preservation of active substances.Methods: Firstly,the extracted human umbilical cord MSC exosomes（Hu MSC-exo）were characterized by transmission electron microscopy（TEM）,nanoparticle tracking analysis（NTA）and western blotting（WB）,the synthesized Nb2 C nanosheets（Nb₂C NS）were characterized through bright-field and dark-field TEM images,TEM lattice analysis,X-ray diffraction（XRD）,X-ray photoelectron spectroscopy（XPS）and atomic mechanics microscopy（AFM）.The biocompatibility of Nb2 C NS was assessed by monitoring mouse body weight and serum transaminase levels,and observation of H&E staining of major organs of mice.Next,the hydrogel microneedle patch was synthesized by two-step casting mold method through vacuum debubbling and drying.Firstly,the synthetic hydrogel microneedle patch was explored by using different concentrations of Gel MA solution,and the morphological characteristics of the microneedle tip were observed by optical microscopy and scanning electron microscopy（SEM）.Then the depth of microneedle puncture delivery was observed by skin mimic Parafilm puncture,in vivo mouse skin puncture and mouse skin ex vivo H&E staining.Furthermore,the sustained release and continuous delivery of Hu MSC-exo in Gel MA microneedle was followed by the capture of the fluorescence of Hu MSC-exo accumulated in the microneedle extract,the uptake of the fluoresce labeled Hu MSC-exo released by the microneedles in Ha Cat and HUVEC,and the exosomes retained in the skin area of mice In Vivo Imaging Spectrum（IVIS）.Finally,TEM,NTA and WB of Hu MSC-exo under different storage conditions were performed to evaluate the preservation of hydrogel microneedles for active substances.Results: Hu MSC-exo had a typical cup-like bilayer membrane structure,uniform particle size distribution and expression of CD9,CD63,CD81 and TSG101 protein markers.Nb2 C NS contained Nb,C,O and F elements with a monolayer structure,Nb2 C NS displayed a typical low-angle（002）peak and 0.235 nm lattice spacing,its average horizontal size was about 150 nm,and average thickness was about 5 nm.Tail vein injection of Nb2 C NS did not affect weight gain and transaminase levels in mice and was not toxic to major organs.The optimal Gel MA concentration of synthetic hydrogel microneedle patch was confirmed by general morphology,degradation time and mechanical strength to be 10%.The resulting microneedle array was 20×20,the number of needle tips was 400,the needle tips were conical with uniform length and neat arrangement,the needle tip height was 600μm and tip distance was 550μm.Microneedles could penetrate the parafilm and mouse skin to a depth of about 500μm,effectively adhered to the mouse skin and formed micropores.Gel MA microneedles could achieve sustained release of Hu MSC-exo both in vivo and in vitro,and could realize long-term storage for Hu MSC-exo of morphological structure,particle size and protein markers expression at 4°C.Conclusion: Hu MSC-exo and Nb2 C NS had stable and typical structural characteristics,and Nb2 C NS possessed good biocompatibility in mice.Gel MA hydrogel microneedles had good tip morphology,biodegradability,and mechanical strength.The microneedles could form micropore channels in mouse skin for downward delivery to a depth of 500μm.Gel MA hydrogel microneedle patches enabled the continuous delivery and effective storage of Hu MSC-exo.Ultimately,Gel MA was loaded with Hu MSC-exo and Nb2 C NS to synthesize the microneedle patch G@NE MN.Part Ⅱ: Effectiveness and mechanism of G@NE MN in promoting healing of diabetic woundsPurpose: The combination of multiple pathophysiological alterations led to delayed healing of diabetic wounds.Therefore,there is an urgent need to develop new tools for wound healing with multiple functions.Methods: The biocompatibility of G@NE MN was first assessed by cell LIVE/DEAD staining,cytoskeleton morphology staining and flow cytometry apoptosis assay.Next,the ability of Nb2 C NS to scavenge reactive oxygen species（ROS）components was assessed using total antioxidant capacity assay,superoxide dismutase（SOD）activity assay and hydroxyl free radicals（·OH）scavenging ability assay,ROS probe fluorescence staining and ROS flow cytometry were to evaluate the ability of G@NE MN to clear ROS in Ha Ca T and HUVEC.Then Nb2 C NS was co-incubated with Escherichia coli（E.coli）and Staphylococcus aureus（S.aureus）and subjected to OD600 bacterial density assay,plate smearing assay,bacterial LIVE/DEAD staining to assess the antibacterial efficacy of Nb2 C NS.Further,the ability of G@NE MN to regulate macrophage polarization by immunofluorescence staining for macrophage markers CD86/CD206.Quantitative PCR（q PCR）was used to detect the m RNA levels of inflammatory factors（IL-6 and TNF-α）and anti-inflammatory factors（IL-10 and TGF-β）.Cell scratch assay,transwell migration assay and tube formation assay was performed to assess the promotion effect of G@NE MN on epithelialization and vascularization.Mouse diabetic wound model was constructed and treated with microneedle patch,the wound healing status was observed by wound area calculation,H&E staining and Masson staining.The mechanism of G@NE MN in promoting the healing of diabetic wounds was further explored by immunofluorescence staining with α-SMA/CD31,macrophage markers CD86/CD206,ROS,and inflammatory factors IL6 and TNF-α in mouse skin section.Results: Ha Ca T cells and HUVEC cells treated with G@NE MN for 72 h still maintained good cell viability,good skeletal morphology,and no apoptosis;Nb₂C NS could effectively scavenge ·OH and superoxide anions（O2·-）,and G@NE MN could effectively scavenge ROS produced by Ha Ca T cells and HUVEC cells in the oxidative damage model in vitro.Nb2 C NS could effectively inhibit E.coli and S.aureus growth,and the antimicrobial ability was enhanced with the increase of Nb2 C NS concentration.G@NE MN could regulate the macrophages toward M2 polarization and promoted the decrease of IL-6,TNF-α expression and the increase of IL-10,TGF-β expression level;G@NE MN significantly improves the migration of Ha Ca T cells,which also promotes the migration and tube formation of HUVEC cells.G@NE MN treated mice owned better wound healing,good recovery of multilayer epithelium,higher density and regular arrangement of collagen fibers,higher density of skin appendages.G@NE MN group exhibited increased α-SMA/CD31-positive cells,downregulated ratio of CD86/CD206,and significantly reduced number of ROS,IL6 and TNF-α-positive cells.Conclusion: Nb2 C NS possessed intrinsic reducibility,and G@NE MN dispalyed remarkable antioxidant capacity.G@NE MN could modulate macrophages toward M2 polarization with inflammatory regulation.Nb2 C NS demonstrated good antibacterial properties.G@NE MN could promote re-epithelialization and re-vascularization in wound area,and G@NE MN significantly promoted wound healing in diabetic mice.