MCP-1 response type microneedle, microneedle patch and preparation method and application of MCP-1 response type microneedle and microneedle patch

Abstract

The invention relates to the technical field of pharmaceutical preparations, and particularly provides an MCP-1 response type microneedle, a microneedle patch and a preparation method and application of the MCP-1 response type microneedle and the microneedle patch. The MCP-1 response type microneedle loaded glucan modified silver nanoparticles and the heparin modified taurine PLGA microspheres are used for targeted elimination of bacteria and reprogramming of an immune microenvironment. The invention develops a double-layer microneedle system responding to a bacterial microenvironment, and by integrating antibiosis, antioxidation and M2 type macrophage polarization capability into the double-layer microneedle structure, local infection control, immune regulation and tissue regeneration can be synergistically combined, so that the key obstacle of wound repair is effectively overcome, the complex wound healing obstacle can be solved, and the wound healing effect is improved. And particularly, a comprehensive solution is provided for infectious and chronic wounds such as diabetes mellitus and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to an MCP-1 responsive microneedle, a microneedle patch, and a preparation method and application thereof. Background Art

[0002] Diabetic wounds are characterized by persistent inflammation caused by monocyte chemoattractant protein-1 (MCP-1)-mediated immune dysregulation and bacterial biofilm formation, leading to amputation in approximately 30% of cases. Treatment is challenging. There is an urgent need to develop therapeutic strategies that can simultaneously disrupt the MCP-1-driven inflammatory cycle, penetrate the biofilm to achieve effective bactericidal responses, and restore redox balance. Summary of the Invention

[0003] Based on this, it is necessary to provide an MCP-1-responsive microneedle, a microneedle patch, and a preparation method and application thereof. The MCP-1-responsive microneedle can simultaneously break the inflammatory cycle driven by MCP-1, penetrate biofilms to achieve effective sterilization, and restore redox balance.

[0004] The present invention adopts the following technical solutions:

[0005] The present invention provides an MCP-1 responsive microneedle loaded with dextran-modified silver nanoparticles and heparin-modified taurine PLGA microspheres.

[0006] In some embodiments, the needle tip section of the microneedle is loaded with dextran-modified silver nanoparticles, and the non-needle tip section is loaded with heparin-modified taurine PLGA microspheres.

[0007] In some embodiments, the microneedle tip section adopts a hyaluronic acid matrix, and the non-needle tip section adopts a polyvinyl pyrrolidone matrix.

[0008] In some embodiments, the preparation of the dextran-modified silver nanoparticles includes: preparing an aqueous solution containing trisodium citrate and sodium borohydride, adding silver nitrate to react, and collecting silver nanoparticle precipitates; preparing an aqueous suspension of silver nanoparticles, stirring and mixing with the dextran aqueous solution to react, centrifuging and collecting the precipitate, washing, and drying to obtain dextran-modified silver nanoparticles.

[0009] In some embodiments, the preparation of the heparin-modified taurine PLGA microspheres includes: preparing a PLGA oil phase solution, preparing a taurine aqueous phase solution, adding a gelatin aqueous solution after mixing, ultrasonic emulsification, and obtaining an oil emulsion; mixing the oil emulsion with a polyvinyl alcohol aqueous solution, removing impurities, and obtaining taurine PLGA microspheres; suspending the taurine PLGA microspheres in a polyethyleneimine solution for amination, removing impurities, and drying to obtain aminated microspheres; preparing a 2-(N-morpholino)ethanesulfonate buffer solution containing sodium heparin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide, adding the aminated microspheres to react, and removing impurities to obtain.

[0010] The present invention also provides a method for preparing an MCP-1 responsive microneedle, comprising the following steps: preparing dextran-modified silver nanoparticles; preparing heparin-modified taurine PLGA microspheres; dispersing the dextran-modified silver nanoparticles using a hyaluronic acid matrix and dispersing the heparin-modified taurine PLGA microspheres using a polyvinyl pyrrolidone matrix to form the MCP-1 responsive microneedle.

[0011] The present invention provides an MCP-1 responsive microneedle patch, which is prepared by using MCP-1 responsive microneedles.

[0012] Application of the above-mentioned MCP-1 responsive microneedle or the above-mentioned MCP-1 responsive microneedle patch in the preparation of wound repair medicines.

[0013] In some embodiments, the wound is an infectious or chronic wound such as a diabetic wound.

[0014] Compared with the existing technology, the core technical contributions and advantages of this invention are:

[0015] This invention proposes, for the first time, MCP-1-responsive microneedles loaded with dextran-modified silver nanoparticles and heparin-modified taurine PLGA microspheres to target bacterial elimination and reprogram the immune microenvironment. This invention develops a bacterial microenvironment-responsive double-layer microneedle system. By integrating antibacterial, antioxidant, and M2 macrophage polarization capabilities into the double-layer microneedle structure, it synergistically combines local infection control, immune regulation, and tissue regeneration, effectively overcoming key barriers to wound repair and addressing complex wound healing disorders, particularly providing a comprehensive solution for infectious and chronic wounds such as those caused by diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the synthesis route and application regulation process of the microneedles of the present invention; wherein, (A) is a schematic diagram of the synthesis route and structure of the microneedles; (B) is a schematic diagram of the program regulation process when the microneedles are applied to wounds; (C) is a schematic diagram of the microneedle application treatment mechanism.

[0017] Figure 2 Statistical graphs showing the performance of the nanoparticles prepared in the present invention; wherein, (A) is a transmission electron microscope (TEM) image and energy spectrum (EDS) elemental mapping image of silver nanoparticles (Ag nanoparticles, Ag NPs) and glucose-modified silver nanoparticles (Dex / Ag nanoparticles, Dex / Ag NPs); Figure B is a TEM image of Staphylococcus aureus and Escherichia coli after incubation with phosphate buffer solution (PBS, Control), Ag nanoparticles and Dex / Ag nanoparticles for 1 hour (yellow arrows point to the nanoparticles); Figure C is a dynamic light scattering (DLS) statistical test graph of Ag nanoparticles and Dex / Ag nanoparticles; Figure D is a zeta potential test graph of Ag nanoparticles and Dex / Ag nanoparticles; Figure E is a graph showing the zeta potential of Ag nanoparticles and Dex / Ag nanoparticles. Figure 3 shows the full scan X-ray photoelectron spectroscopy (XPS) spectra of Ag nanoparticles and Dex / Ag nanoparticles; Figure F shows the Fourier transform infrared spectroscopy (FTIR) spectra of Ag nanoparticles, Dex and Dex / Ag nanoparticles; Figure G shows the morphological test of planktonic Staphylococcus aureus and Escherichia coli after co-culture with Ag nanoparticles and Dex / Ag nanoparticles using scanning electron microscopy (SEM); Figure H shows the growth curve statistics of Staphylococcus aureus after co-culture with Ag nanoparticles and Dex / Ag nanoparticles of different concentrations.

[0018] Figure 3 Statistical graphs showing the performance of different microspheres prepared in the present invention; (A) shows a schematic diagram of HP-Taurine@MS binding to chemokines and attracting inflammatory monocytes / macrophages to gather around them; (B) shows transmission electron microscopy (TEM) images of microspheres MS, Taurine@MS, and HP-Taurine@MS; (C) shows a statistical graph showing the binding efficiency of HP-Taurine@MS to MCP-1 at different time points; (D) shows the results of macrophage-derived conditioned medium (CM) co-incubated with MS, Taurine@MS, and HP-Taurine@MS for 3 days. After each group of microspheres was replaced every day, the concentration of monocyte chemoattractant protein-1 (MCP-1) was statistically analyzed over time; (E) and (G) are experimental test images of monocyte recruitment by MCP-1-enriched HP-Taurine@MS (monocytes were labeled green with a live-dead cell staining reagent, and yellow arrows indicate microspheres) and relative fluorescence quantitative analysis of macrophages adhering to the surface of microspheres, respectively; (F) and (H) are bright field images and quantitative statistical results of the Transwell migration experiment of L929 cells (n=3), respectively; (I) is a curve of fluorescence co-localization between different microspheres and macrophages.

[0019] Figure 4Statistical graphs of the performance characterization of the microneedles prepared in the present invention; wherein, (A) is a representative photo of (DAg / HTMS-MNs); (B) is a scanning electron microscope (SEM) image of the top surface of DAg / HTMS-MNs; (C) is a top view of DAg / HTMS-MNs, and an enlarged SEM image of the upper and lower parts of the microneedles; (D) is a side view of DAg / HTMS-MNs and a fluorescence image of its double-layer structure; (E) is a top view of DAg / HTMS-MNs and a fluorescence image of its double-layer structure; (F) is a statistical graph of the mechanical strength test of DAg / HTMS-MNs; (G) is a typical image of DAg / HTMS-MNs after application to mouse skin, and a local HE-stained section after the microneedles penetrated the back skin of the mouse; (H) is a graph showing the silver nanoparticles (Ag) in DAg / HTMS-MNs. Figure 3 shows the cumulative release curves of taurine and MNs; (I) Live / dead staining images of L929 cells treated with blank microneedles (MNs), DAg-MNs, HTMS-MNs, and DAg / HTMS-MNs extracts for 1 day and 3 days, respectively; (J) Statistical graph of the relative cell growth rate of L929 cells treated with MNs, DAg-MNs, HTMS-MNs, and DAg / HTMS-MNs extracts for 1 day, 3 days, and 5 days, respectively.

[0020] Figure 5 Figure 1 is a statistical diagram of the performance characterization of microneedles; (A) is a schematic diagram of the structure of the missile-inspired microneedle (MN); (B) is a microscope image showing the process of DAg-HMTS microneedle generating bubbles in phosphate buffer (PBS); (C) is a time-lapse demonstration of the DAg-HMTS microneedle tip being propelled by gas in PBS; (D) is a confocal microscope image of the penetration depth of sodium fluorescein dye released by different microneedles; (E) is a quantitative analysis of the fluorescence intensity of sodium fluorescein dye released by different microneedles at different depths (double-sided t (F) Figure shows a typical photograph of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) colonies after treatment with different microneedle groups; (G) Figure shows a statistical graph of the quantitative analysis of the bacterial survival rate of Staphylococcus aureus and E. coli determined by the colony counting method; (H) Figure shows a scanning electron microscope (SEM) image of Staphylococcus aureus and E. coli biofilms treated with different microneedle groups; (I) Figure shows a three-dimensional confocal laser scanning microscope (3D CLSM) image of Staphylococcus aureus and E. coli biofilms treated with different microneedles (green: live bacteria, red: dead bacteria); (J) Figure shows crystal violet stained photos of Staphylococcus aureus and E. coli biofilms after treatment with different microneedles; (K) Quantitative analysis statistical graph of crystal violet stained biofilms.

[0021] Figure 6Figure 1 is a statistical graph of the experimental results of microneedles and phagocytes; (A) shows the staining results of mitochondrial reactive oxygen species (mtROS) in macrophages activated by H2O2 after macrophages were treated with MNs (blank microneedles), HTMS-MNs, DAg-MNs and DAg / HTMS-MNs for 24 hours (cells cultured in complete medium were used as controls); (B) shows the JC-1 fluorescence images of macrophages treated with extracts of MNs, HTMS-MNs, DAg-MNs and DAg / HTMS-MNs; (C) shows the corresponding JC- 1 Statistical graph of red / green fluorescence intensity ratio; (D) The figure shows the fluorescence images of iNOS and CD206 to show the effects of MNs, HTMS-MNs, DAg-MNs and DAg / HTMS-MNs on the polarization of Raw264.7 cells pretreated with lipopolysaccharide (LPS); (E) The figure shows a typical image of macrophages phagocytosing bacteria (green fluorescence indicates Staphylococcus aureus, red fluorescence indicates macrophages, and blue fluorescence indicates cell nuclei); (F) The figure shows the statistical graph of fluorescence quantitative analysis of phagocytic bacteria; (G) The figure shows a heat map depicting the relative expression levels of iNOS and CD206.

[0022] Figure 7 Statistical graphs of the co-culture experiment results of microneedles and macrophages; among them, (A) is a typical image of a scratch experiment in which the supernatant of the microneedle extract and macrophage co-culture was collected and then co-cultured with human umbilical vein endothelial cells (HUVECs); (B) is a quantitative analysis statistical graph of the scratch closure rate (residual wound area, %); (C) is a typical image of a Transwell migration assay performed on HUVECs after the supernatant of the microneedle extract and macrophage co-culture was collected; (D) is a quantitative analysis statistical graph of migrating cells in the migration assay; (E) is a typical image of a tube formation assay performed on HUVECs after the supernatant of the microneedle extract and macrophage co-culture was collected; (F) is the number of branch points (cells / mm) in the tube formation assay 2 ) quantitative analysis; (G) A typical image of CD31 immunofluorescence staining of HUVECs after collecting the supernatant of microneedle extracts co-cultured with macrophages; (H) A quantitative analysis of the relative fluorescence intensity of CD31; ***p<0.001, ****p<0.0001.

[0023] Figure 8Figure 1 is a statistical graph showing the results of microneedle application in diabetic mouse wound model experiments; (A) shows the construction of diabetic mouse wound model and the schematic diagram of microneedle treatment process; (B) shows images of mouse wounds at different time points after different treatments; (C) shows simulated images of infected wounds at different time points after different treatments; (D) shows representative photos of bacterial colonies in secretions of infected wounds in each group on the 5th day; (E) shows representative H&E staining images of wounds in each group on the 10th day; (F) shows representative H&E staining images of wounds in each group on the 5th day. Representative images of Giemsa staining; (G) Representative images of Masson's trichrome staining of wounds in each group on the 10th day; (H) Quantitative analysis and statistical graph of wound area at different time points; (I) Quantitative analysis and statistical graph of bacterial counts by Giemsa staining; (J) Quantitative analysis and statistical graph of re-epithelialization rate based on H&E staining; (K) Quantitative analysis of collagen deposition in wounds of each group based on image (G); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 9 Figure 2 shows the gene analysis diagram of the microneedle application experiment; (A) is a two-dimensional principal component analysis (PCA) diagram of the correlation between all samples; (B) is a volcano plot of the DAg / HTMS-MNs group and the control group (red indicates up-regulated genes, blue indicates down-regulated genes); (C) is a heat map of differentially expressed genes between the DAg / HTMS MNs group and the control group (red areas indicate up-regulated genes, blue areas indicate down-regulated genes); (D) GO functional enrichment analysis of up-regulated genes; (E) GO functional enrichment analysis of down-regulated genes; (F) KEGG functional enrichment analysis of up-regulated and down-regulated genes; (G) KEGG enrichment terms and their corresponding enrichment chord diagrams.

[0025] Figure 10 Statistical graphs of the microneedle application experimental results; among them, (A) shows the expression images of iNOS and CD206 in the central area of ​​the wound in the control group, MNs group, DAg-MNs group, HTMS-MNs group and DAg / HTMS-MNs group after 10 days of treatment; (B) shows the statistical graph of the relative value of iNOS fluorescence quantitative expression; (C) shows the statistical graph of the relative value of CD206 fluorescence quantitative expression; (D) shows the statistical graph of the fluorescence intensity ratio of M1 macrophages to M2 macrophages; (E) shows the expression images of CD31 in the central area of ​​the wound in the control group, MNs group, DAg-MNs group, HTMS-MNs group and DAg / HTMS-MNs group after 10 days of treatment; (F) shows the statistical graph of the relative value of CD31 fluorescence quantitative expression.

[0026] Figure 11Figure 1 is a statistical graph of the results of the microneedle application experiment; (A) shows the immunohistochemical staining of IL-17 in the central area of ​​the wound in the control group, MNs group, DAg-MNs group, HTMS-MNs group, and DAg / HTMS-MNs group after 10 days of treatment; (B) shows the statistical graph of the relative gene expression level of IL-17; (C) shows the immunohistochemical staining of IL-6 in the central area of ​​the wound in the control group, MNs group, DAg-MNs group, HTMS-MNs group, and DAg / HTMS-MNs group after 10 days of treatment; (D) shows the relative gene expression level of IL-6 (E) The figure shows the immunohistochemical staining of TNF-α in the central area of ​​the wound in the control group, MNs group, DAg-MNs group, HTMS-MNs group, and DAg / HTMS-MNs group after 10 days of treatment; (F) The figure shows the statistical graph of the relative gene expression level of TNF-α; (G) The figure shows the immunohistochemical staining of IL-10 in the central area of ​​the wound in the control group, MNs group, DAg-MNs group, HTMS-MNs group, and DAg / HTMS-MNs group after 10 days of treatment; (H) The figure shows the statistical graph of the relative gene expression level of IL-10.

[0027] Figure 12 Performance characterization diagrams of different microspheres; among them, (A) shows the dynamic light scattering (DLS) data of MS, Taurine@MS, and HP-Taurine@MS; (B) shows the Zeta potential of MS, Taurine@MS, and HP-Taurine@MS; (C) shows the full spectrum scan of X-ray photoelectron spectroscopy (XPS) of MS, Taurine@MS, and HP-Taurine@MS; (D) shows the Fourier transform infrared spectroscopy (FTIR) of taurine, heparin, MS, Taurine@MS, and HP-Taurine@MS.

[0028] Figure 13 Bright field images and quantitative statistical results of Transwell migration experiments of L929 cells with MS, Taurine@MS, and HP-Taurine@M.

[0029] Figure 14 These are the H&E staining results of the main organs (heart, liver, spleen, lung, and kidney) of the control group and DAg-HTMS MNs-treated mice at the end of the experiment.

[0030] Figure 15 Fluorescence microscope images of TNF-α and IL-10 immunofluorescence staining and the corresponding relative quantitative analysis statistical graphs.

[0031] Figure 16 Statistical graph of bacterial killing efficiency of macrophages in the same treatment group.

[0032] Figure 17 This is a statistical chart of bacterial survival rate in wound secretions on the 5th day after treatment.

[0033] Figure 18 The expression heat map of key inflammatory mediators in the control group and DAg / HTMS-MNs group. DETAILED DESCRIPTION

[0034] like Figure 1 As shown, the present invention proposes for the first time an MCP-1 responsive microneedle, which is layered loaded with dextran (Dextran, Dex)-modified silver nanoparticles (Dex / Ag NPs) and heparin-modified taurine PLGA microspheres (HP-Taurine@MS), which can target the elimination of bacteria and reprogram the immune microenvironment.

[0035] Implementing a "global decompression-local enhancement" strategy: i) Heparin's sulfate groups chelate monocyte chemoattractant protein-1 (MCP-1), reducing the overall chemokine gradient within the wound bed, thereby inhibiting the recruitment of inflammatory cells to the entire wound area; ii) The porous microspheres create a local chemokine gradient, which allows macrophages to accumulate in the wound bed and enhance their function; iii) Continuous taurine release reprograms macrophages to a pro-repair M2 phenotype. The design of HP-Taurine@MS demonstrates a key innovation: the sequential recruitment of pro-inflammatory cells to the target site has significant advantages in reducing drug dispersion loss and improving lesion clearance efficiency, which has greater therapeutic significance than simple passive drug diffusion. It has a triple therapeutic mechanism for the treatment of infected wounds:

[0036] (1) The pneumatically driven microneedle tip can quickly penetrate the biofilm. At the same time, the hyaluronidase secreted by bacteria degrades the hyaluronic acid matrix to promote the immediate release of Dex / Ag NPs in the needle tip area. The sulfate group of heparin chelates monocyte chemoattractant protein-1 (MCP-1), reducing the overall chemokine gradient in the wound bed, thereby inhibiting the recruitment of inflammatory cells to the entire wound area, thereby enabling rapid removal of the biofilm and controlling the infection.

[0037] (2) The loaded HP-Taurine@MS functional layer coordinates the regulation of the wound microenvironment through chemokine redistribution. The initial chemokine redistribution breaks the inflammatory cascade reaction and effectively reduces the overall inflammatory burden of the entire wound bed.

[0038] (3) The immunomodulatory capacity was further demonstrated through targeted macrophage recruitment and taurine-mediated metabolic reprogramming. The porous microspheres created a local chemokine gradient that allowed macrophages to accumulate and enhance in the wound bed, and their function reprogrammed macrophages to a pro-repair M2 phenotype.

[0039] Targeted inflammatory cell recruitment enhances local drug delivery and lesion clearance, outperforming passive drug diffusion. Released taurine enhances mitochondrial homeostasis and antioxidant activity, effectively neutralizing excess free radicals while simultaneously inhibiting key inflammatory pathways (IL-17, NF-κB, and TNF signaling). This synergistic effect significantly suppresses proinflammatory cytokine production and neutrophil extracellular trap (NET) formation, thereby reversing the pathological inflammatory microenvironment. Mechanistic studies suggest that bacterial clearance, combined with interruption of the inflammatory cycle, synergistically enhances macrophage function through taurine release. Transcriptome analysis and in vivo validation confirmed significant downregulation of inflammatory markers (MMPs, TNF-α, and IL-17) while upregulating repair factors (IL-10 and CD31). This sustained action provides sustained immune homeostasis, rather than a transient anti-inflammatory effect. Both in vitro and in vivo studies have demonstrated that this effect significantly reduces inflammation, promotes macrophage repolarization, and accelerates wound healing.

[0040] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly. The following embodiments are only used to illustrate the present invention, but are not limited to limiting the scope of the present invention. Based on the specific embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are conventional means well known to those skilled in the art.

[0041] Simplified terminology:

[0042] Silver nanoparticles: Ag NPs. Dextran-modified silver nanoparticles: Dex / Ag NPs. Poly(lactic-co-glycolic acid) (PLGA). PLGA microspheres (MS). Taurine microspheres (Taurine@MS).

[0043] Heparin, abbreviated as HP. Heparin-modified taurine microspheres, abbreviated as HP-Taurine@MS.

[0044] Heparin-coated taurine-loaded porous poly(lactic-co-glycolic acid), abbreviated as HP-Taurine@MSPLGA.

[0045] Hyaluronic acid, abbreviated as HA. Pure hyaluronic acid microneedles (blank microneedles), abbreviated as MNs.

[0046] Hyaluronic acid microneedles with Dex / Ag NPs incorporated only in the top needle area (Dex / Ag NPs microneedles), abbreviated as: DAg-MNs.

[0047] Polyvinyl pyrrolidone microneedles with HP-Taurine@MS incorporated only in the non-top needle area (HP-Taurine@MS microneedles), abbreviated as HTMS-MNs.

[0048] Hyaluronic acid-polyvinyl pyrrolidone microneedles with Dex / Ag NPs incorporated in the ejection region and HP-Taurine@MS incorporated in the non-ejection region, abbreviated as: DAg / HTMS-MNs.

[0049] Example 1 Preparation of Nanoparticles

[0050] This example provides a method for preparing silver nanoparticles (Ag NPs). Citrate-capped silver nanoparticles are synthesized using sodium borohydride as a reducing agent, comprising the following steps: 1.2 mL of trisodium citrate (100 mM) and 4 mL of freshly prepared sodium borohydride (NaBH4, 100 mM) are mixed with 390 mL of water in an ice bath while stirring to obtain an aqueous solution containing trisodium citrate and sodium borohydride. 4 mL of a 10 mM aqueous solution of AgNO3 is added, and the mixture is reacted for 30 minutes. The precipitate is collected by centrifugation, washed three times with ultrapure water, and dried under vacuum to obtain the product.

[0051] This example provides a method for preparing dextran-modified silver nanoparticles (Dex / Ag NPs), comprising the following steps: dissolving 6 mg of Dex in 5 mL of deionized water, mixing the obtained silver nanoparticles with magnetic stirring at room temperature for 5 hours, then collecting the precipitate by centrifugation, washing it three times with ultrapure water, and vacuum drying it.

[0052] Example 2 Preparation of microspheres

[0053] This example provides a method for preparing PLGA microspheres (MS), comprising the following steps: preparing an oil phase by dissolving 50 mg of poly(lactic-co-glycolic acid) (PLGAMw90000) in 2 mL of dichloromethane. Simultaneously, a 10% (w / v) gelatin aqueous solution was prepared by dissolving gelatin in deionized water at 60°C. Subsequently, 2 mL of the PLGA-dichloromethane solution was first mixed with 0.6 mL of a 10% (w / v) gelatin solution using a cell disruptor (20 W power), and the mixture was repeatedly sonicated in a 40°C water bath to form a dense emulsion (oil phase mixture). The oil phase mixture was then mixed with a 5% aqueous solution of polyvinyl alcohol (PVA) and homogenized using a cell disruptor in an ice bath. The resulting mixture was transferred to a beaker and stirred at 600 rpm for 6 hours. The microspheres were collected by centrifugation (2000 rpm, 2 minutes) at a constant temperature of 40°C and then washed to remove residual PVA and gelatin. Finally, the product was dried under vacuum to remove any residual dichloromethane.

[0054] This example provides a method for preparing taurine microspheres (Taurine@MS). This method is similar to the method for preparing PLGA microspheres (MS), with the following differences: First, 2 mL of PLGA-dichloromethane solution was mixed with 0.5 mL of taurine solution (10 mg / mL) using a cell disruptor (20 W power). Then, 0.6 mL of gelatin solution (10%, w / v) was added, and the mixture was repeatedly sonicated in a 40°C water bath to form a dense emulsion (oil phase mixture).

[0055] This embodiment provides a method for preparing heparin-modified taurine microspheres (HP-Taurine@MS), comprising the following steps: resuspending the prepared taurine microspheres (Taurine@MS) in a 1 wt% polyethyleneimine (PEI) solution for amination and incubating at 37°C with stirring (200 rpm) for 6 hours. After the reaction is complete, the aminated microspheres are collected by centrifugation (2000 rpm, 2 minutes) and vacuum dried. 200 mg of sodium heparin, 40 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and 60 mg of N-hydroxysuccinimide (NHS) are dissolved in 5 mL of 2-(N-morpholino)ethanesulfonate (MES) buffer (0.1 M, pH 6.0). The solution is stirred at 37°C and 300 rpm for 15 minutes to activate the heparin. The aminated microspheres are then added to the reaction mixture for surface heparinization. The binding reaction is carried out for 5 hours under continuous stirring. Finally, the solution was collected by centrifugation (2000 rpm, 2 minutes).

[0056] Example 3 Preparation of microneedles

[0057] This example provides a method for preparing blank microneedles (MNs), comprising the following steps: 100 mg of hyaluronic acid (HA) was dissolved in 2 mL of deionized water under magnetic stirring to obtain a uniform solution. Subsequently, 2 mL of this solution was carefully added dropwise to a PDMS microneedle mold (10×10 array, needle length: 600 μm), followed by vacuum-assisted filling to ensure complete occupancy of the cavity. The filled mold was air-dried at room temperature to solidify the microneedles. The complete MNs patch was then carefully demolded to obtain a single-phase microneedle structure.

[0058] This example provides a method for preparing hyaluronic acid microneedles (DAg-MNs) incorporating Dex / Ag NPs only in the top needle region. The method comprises the following steps: 100 mg of hyaluronic acid (HA) and 80 mg of Dex / Ag NPs are co-dissolved in 2 mL of deionized water under magnetic stirring, mixed thoroughly, and 1 mL is carefully dripped into a PDMS microneedle mold (10×10 array, needle length: 600 μm). Vacuum-assisted filling is then performed to ensure complete cavitation. The filled mold is air-dried at room temperature to solidify the upper microneedle layer (Dex / Ag NPs are incorporated only in the top needle region). A solution containing 4% (w / v) citric acid, 20% (w / v) polyvinylpyrrolidone (PVP), and 5% (w / v) sodium bicarbonate in anhydrous ethanol is then prepared. 1 mL of the solution is then introduced into the mold containing the preformed upper microneedle layer. Vacuum-assisted filling and room-temperature drying are also performed. The complete DAg-MN patch is then carefully demolded to yield a dual-phase microneedle structure.

[0059] This example provides a method for preparing polyvinyl pyrrolidone microneedles (HTMS-MNs) incorporating HP-Taurine@MS only in the non-top-pin regions. The method comprises the following steps: 100 mg of hyaluronic acid (HA) was dissolved in 2 mL of deionized water under magnetic stirring and mixed thoroughly. 1 mL of this solution was carefully dropwise added to a PDMS microneedle mold (10×10 array, needle length: 600 μm). Vacuum-assisted filling was then performed to ensure complete cavitation. The filled mold was air-dried at room temperature to solidify the upper microneedle layer. A solution containing 4% (w / v) citric acid, 20% (w / v) polyvinyl pyrrolidone (PVP), and 5% (w / v) sodium bicarbonate in anhydrous ethanol was then prepared, supplemented with HP-Taurine@MS to a concentration of 30 mg / mL. 1 mL of this solution was then added to a preformed upper mold, which was similarly vacuum-assisted filled and dried at room temperature. The completed HTMS-MN patch was then carefully demolded to yield a dual-phase microneedle structure.

[0060] This example provides a method for preparing hyaluronic acid-polyvinyl pyrrolidone microneedles (DAg / HTMS-MNs) in which Dex / Ag NPs are incorporated into the ejection region and HP-Taurine@MS is incorporated into the non-ejection region. The method comprises the following steps: 100 mg of hyaluronic acid (HA) and 80 mg of Dex / Ag NPs are co-dissolved in 2 mL of deionized water under magnetic stirring and dispersed evenly. 1 mL of the solution is carefully dropwise added to a PDMS microneedle mold (10×10 array, needle length: 600 μm). Vacuum-assisted filling is then performed to ensure complete occupancy of the cavity. The filled mold is air-dried at room temperature to solidify the upper microneedle layer. Anhydrous ethanol containing 4% (w / v) citric acid, 20% (w / v) polyvinylpyrrolidone (PVP), and 5% (w / v) sodium bicarbonate was prepared, and HP-Taurine@MS was supplemented to a concentration of 30 mg / mL. 1 mL of the solution was introduced into the pre-formed upper mold, and vacuum-assisted filling and room temperature drying were also performed. The complete DAg / HTMS-MNs patch was then carefully demolded to obtain a dual-phase microneedle structure.

[0061] The prepared products were subjected to characterization tests and efficacy tests.

[0062] The microstructure and elemental composition of Ag NPs and Dex / Ag NPs were characterized using a transmission electron microscope (HT-7800, Hitachi High-Technologies Corporation, Japan) equipped with energy dispersive X-ray spectroscopy (EDS). The zeta potential and particle size of Ag NPs and Dex / Ag NPs were measured using a nanoparticle size and zeta potential analyzer (DLS, Malvern Ceramics Nano ZS90). X-ray photoelectron spectroscopy (XPS, Thermal Science K-Alpha, USA) was used for X-ray photoelectron spectroscopy analysis. Fourier transform infrared spectroscopy (FTIR, Thermal Science Inc. 6700) was used to determine the functional groups of the nanoparticles in the range of 1000–4000 cm⁻¹.

[0063] Targeted bactericidal performance test of Dex / Ag NPs: Staphylococcus aureus and Escherichia coli suspensions (200 μL, 1×10 CFU / mL) were centrifuged (8000 rpm, 5 minutes), washed with PBS, and treated with Ag NPs or Dex / Ag NPs (200 μL, 50 μg / mL) for 1 hour (TEM). After incubation, the bacterial solution was centrifuged and fixed with 2.5% glutaraldehyde at 4°C overnight for TEM analysis. To evaluate the antibacterial activity, the bacterial suspension was incubated with Ag NPs or Dex / Ag NPs for 5 hours. For SEM imaging, the bacteria were dehydrated with a gradient ethanol series (50%, 70%, 80%, 90%, 95% and 100%) and then freeze-dried. In vitro antibacterial assays against planktonic bacteria were performed. Briefly, 100 μL of bacterial suspension with an OD of 0.04 and 100 μL of different concentrations of Ag NPs and Dex / Ag NPs were added to each well of a 96-well plate. During the 18-hour co-incubation period, turbidity measurements were performed to monitor bacterial growth in real time.

[0064] The morphology of HP-Taurine@MS was imaged using a scanning electron microscope (SEM, Nova Nano 450). The zeta potential and particle size of the microspheres were measured using a nanoparticle size and zeta potential analyzer. The elemental composition of the microspheres was determined by X-ray photoelectron spectroscopy. Fourier transform infrared spectroscopy was used to identify the functional groups of the microspheres in the range of 1000-4000 cm⁻¹.

[0065] To assess chemokine binding, HP-Taurine@MS were incubated in a cell culture incubator with medium containing 2% FBS and 1000 ng / mL MCP-1. At 0, 3, 6, 12, 24, and 48 hours after incubation, the culture medium was collected and stored at -80°C until ELISA analysis. Furthermore, changes in MCP-1 concentrations were measured by incubating bone marrow-derived macrophage-conditioned medium with MS, Taurine@MS, and HP-Taurine@MS. Freshly prepared MS, Taurine@MS, and HP-Taurine@MS were replaced daily and co-incubated with conditioned medium for 24 hours under each condition.

[0066] In order to evaluate the recruitment and capture of microspheres by macrophages, a migration assay was performed using a Transwell system (8 μm pore size, Corning, USA). Prior to the experiment, macrophages were activated by pre-treatment with 100 ng / mL LPS for 24 hours to induce pro-inflammatory macrophages in RAW cells. In brief, RAW cells (1×10) were seeded in the upper chamber, and the lower chamber contained a culture medium with the corresponding microspheres. The co-culture system was incubated for 24 hours. Subsequently, the cells on the upper surface of the membrane were gently removed with a cotton swab, and the adherent cells on the lower surface were fixed and stained with 0.5% crystal violet solution. In order to simulate the recruitment ability of the "wound bed", the conditioned medium of each group of upper chambers was collected and placed in the lower chamber, while the other steps were consistent with the previous procedure to evaluate the recruitment effect of the "wound bed" treated with microspheres on macrophages.

[0067] To determine the cell capture ability of the microspheres, each group was first pretreated by incubation with a culture medium containing MCP-1 (10 ng / mL). RAW cells were then suspended and co-cultured with the microspheres in the culture medium. After 24 hours of culture, the microspheres were fixed with paraformaldehyde and stained with DAPI for observation under a fluorescence microscope. In addition, fluorescence colocalization of the microspheres and macrophages was performed.

[0068] The morphological structure of DAg / HTMS-MNs was photographed by scanning electron microscopy (SEM, Nova Nano 450) and fluorescence microscopy. For fluorescence tracking, Dex / Ag NPs were labeled with sodium fluorescein (green emission), while the microspheres were labeled with rhodamine (red emission). These modified components were then used to prepare fluorescently labeled microneedles. Their structural features were subsequently examined by fluorescence microscopy. In order to evaluate the mechanical properties of the microneedles, square patches with a base of 8.5 mm and a height of 0.6 mm were prepared and tested, and the samples were compressed by an electronic universal testing machine (Mester Industrial, CMT6104, Zhongshan Mester Industrial Co., Ltd.) at a moving speed of 0.2 mm / min. In the skin puncture experiment, the skin of normal rats was punctured with microneedles, local photos of the skin were taken, and the tissue was stained with HE.

[0069] To determine nanoparticle release from microneedles, two microneedles were placed in 5 mL of PBS and incubated at 37°C with constant stirring. At predetermined time points, 1 mL of supernatant was collected and 1 mL of PBS was added to maintain the volume. Taurine release from DAg / HTMS-MNs was measured by high-performance liquid chromatography. Silver ion release from DAg / HTMS-MNs was measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7800, USA).

[0070] Biocompatibility testing of the DAg / HTMS-MNs patch: MN extracts were prepared by immersing one microneedle patch in 2 mL of DMEM medium at 37°C with constant agitation for 24 hours. The extracts were then filtered and sterilized for subsequent cell experiments. The in vitro biocompatibility of the biomaterial was assessed using CCK-8 assays and live / dead cell staining. L929 cells were plated at 2 × 10 cells per well. 3 Cells were seeded at a density of 100 cells / mL in 96-well plates and cultured in standard complete DMEM medium or conditioned medium containing MN extract. Relative cell growth rate was determined by measuring the optical density (OD) of cell supernatants on days 1, 3, and 5. In addition, live / dead cell staining was performed on days 1 and 3 to assess cell viability.

[0071] Immunomodulatory ability test of DAg / HTMS-MNs patch:

[0072] The antioxidant capacity of these cells was tested using a DCFH-DA assay kit. Macrophages were incubated with extracts of MNs, Dex / Ag MNs, HTMSMNs, and DAg / HTMS MNs for 24 hours and then treated with hydrogen peroxide (0.3 mM) for 30 minutes. After washing the cells with PBS, the staining working solution was added, incubated in the dark for 20 minutes, and then observed under a microscope. Fluorescence intensity was quantified using ImageJ software. Simultaneously, 264.7 cells were incubated with DMEM complete medium containing extracts of MNs, Dex / Ag MNs, HTMS MNs, and DAg / HTMSMNs for 24 hours and then treated with 0.3 mM hydrogen peroxide for 30 minutes. After aspirating the supernatant and washing the incubated cells with PBS, they were incubated with JC-1 staining working solution at 37°C for 20 minutes. Subsequently, the cells were washed twice with JC-1 dilution buffer and observed under a fluorescence microscope. The red / green fluorescence intensity ratio was calculated using ImageJ software.

[0073] RAW 264.7 macrophages were cultured in complete DMEM medium containing 100 ng / ml lipopolysaccharide (LPS) for 24 hours to establish an inflammatory microenvironment. After LPS stimulation, the medium was replaced with conditioned medium containing microneedle extract and incubation continued for another 48 hours. After treatment, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100, and blocked with 10% bovine serum albumin (BSA). The cells were then incubated with anti-CD206 polyclonal antibodies overnight, followed by incubation with the corresponding fluorescent secondary antibody for 1 hour in the dark. The cell nuclei were counterstained with DAPI. Cell visualization and imaging were performed using a fluorescence microscope. Similarly, immunofluorescence staining was performed using INOS, TNF-α, and IL-10 polyclonal antibodies, and the fluorescence intensity was quantified using Image J software.

[0074] Assessment of Bacterial Phagocytosis and Killing: Fluorescently labeled biofilms were collected and processed into treated biofilm solutions. Prior to the start of the assay, RAW 264.7 macrophages were pretreated with the microneedle extract solutions from the respective experimental groups for 12 hours. To assess bacterial phagocytosis, the pretreated macrophage suspension was mixed with the treated biofilm solution in a 6-well plate and incubated at 37°C for 3 hours. Subsequently, the culture medium was replaced with fresh DMEM containing 200 μg / mL gentamicin and incubated for an additional 1 hour at 37°C to eliminate extracellular bacteria. Following incubation, cells were washed, fixed, and dual-stained with TRITC-phalloidin (for cytoskeletal visualization) and DAPI (for nuclear staining), and all cell images were systematically captured. For intracellular bacterial viability assessment, after removal of antibiotics, 1 mL of 1% Triton X-100 was added to each well to lyse the macrophage membrane and release internalized bacteria, enabling subsequent viability quantification.

[0075] In vitro angiogenic properties of the DAg / HTMS-MNs patch:

[0076] In the cell scratch assay, human umbilical vein endothelial cells (HUVECs) were seeded into 6-well plates. When the cell density reached approximately 90%, a straight line was drawn in the center of each well using a 1 mL pipette tip to create a scratch. Subsequently, the medium in each well was replaced with fresh DMEM complete medium containing the microneedle extract, and co-culture was continued for 24, 48, and 72 hours. Cell migration and proliferation were recorded and imaged at 0, 24, 48, and 72 hours, and wound closure was quantified using ImageJ software.

[0077] In the Transwell migration assay, 100 μL of a suspension containing HUVECs (1×10 cells) was added to DMEM supplemented with 10% FBS and 1% P / S and then placed in the upper chamber of a Transwell chamber (Corning, USA). The Transwell chamber was then placed into a well of a 24-well plate containing 500 μL of each microneedle extract. Standard complete culture medium served as a control. After incubation for 24 hours at 5% CO and 37°C, the cells were fixed, stained with crystal violet solution, and photographed to quantify the number of migrating cells.

[0078] For the in vitro tube formation assay, 100 μL of Matrigel (BD Biosciences, USA) containing growth factors was dispensed into each well of a 24-well plate and incubated at 37°C for 30 minutes. HUVECs were then seeded onto the Matrigel at a density of 2 × 10 cells per well, and microneedle extracts were added to serum-free DMEM. A serum-free medium control served as a control. After 6 hours of culture, cell morphology was observed using a fluorescence microscope, and the number of branch points in the Matrigel was quantified using ImageJ software.

[0079] Human umbilical vein endothelial cells (HUVECs) were incubated with conditioned medium containing the microneedle extracts from each group for 48 hours. Following treatment, cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100, and blocked with 10% bovine serum albumin. Subsequently, cells were incubated with an anti-CD31 polyclonal antibody overnight and then with the corresponding fluorescent secondary antibody for 1 hour in the dark. Cell nuclei were stained with DAPI and imaged by fluorescence microscopy. Finally, CD31 fluorescence intensity was quantified using Image J software, and relative fluorescence intensity was normalized to that of the control group.

[0080] Aerodynamic effect test of DAg / HTMS-MNs patch:

[0081] To evaluate the penetration kinetics of a microneedle (MN) patch, a 1.5wt% agarose gel was used as an in vitro skin model. Penetration behavior was quantified by analyzing the fluorescence intensity distribution across the cross section at different depths after microneedle insertion. Furthermore, the dissolution kinetics of individual microneedle tips in phosphate-buffered saline (PBS) were monitored by microscopic observation.

[0082] In vitro antibacterial performance test of DAg / HTMS-MNs patch:

[0083] To evaluate the antibacterial effect, the ultraviolet-sterilized microneedle patch was immersed in a centrifuge tube containing 3 ml of bacterial culture medium, inoculated with 100 μL of Staphylococcus aureus or Escherichia coli (10 CFU / ml), and then the culture was incubated for 6 hours. The simple culture medium was used as the control group. After incubation, the bacterial culture medium was diluted to 1×10 -5 The diluted bacterial culture was then inoculated onto agar plates. After incubation for 20 hours, the agar plates were photographed and the colonies were counted.

[0084] Biofilms were constructed by adding 500 μL of bacterial suspension to a 24-well plate. After treatment with the various microneedle formulations, the liquid culture medium was aspirated from the wells. The attached biofilms were then fixed with anhydrous ethanol and stained with crystal violet for 20 minutes. After removing the staining solution, the stained biofilms were photographed using a digital camera. To quantitatively assess biofilm mass, the biofilms were dissolved in 30% glacial acetic acid, and the absorbance (OD) at 600 nm was measured using a microplate reader. To visualize the three-dimensional structure of the biofilms, additional biofilms were cultured in confocal culture dishes. After the respective treatments, the biofilms were washed and stained using a live / dead bacterial viability kit for 30 minutes in the dark. The stained biofilms were immediately observed using confocal laser scanning microscopy (CLSM). For scanning electron microscopy (SEM) observation of biofilm morphology after intervention, bacterial samples from all experimental groups were dehydrated using a graded ethanol series (50%, 70%, 80%, 90%, 95%, and 100%) and then lyophilized.

[0085] In vivo healing study of DAg / HTMS-MNs patch on diabetic infected wounds:

[0086] To establish a diabetic chronic wound model infected with Staphylococcus aureus, 50 8-week-old male mice were purchased and fed a high-fat, high-sugar diet for 6 weeks. They were then injected intraperitoneally with a 10g / L streptozotocin solution at a dose of 10mL / kg. Three days after injection, tail vein blood glucose was measured. Mice with blood glucose levels ≥16.7mmol / L for three consecutive days were considered to have successfully established type 2 diabetes and were included in subsequent studies. Each mouse was then anesthetized, and a full-thickness skin defect (8mm in diameter) was created. Each wound was injected with 10μL of Staphylococcus aureus (1×10 CFU / mL) and allowed to rest for 2 days. The mice were then randomly divided into five groups: control, MNs, HTMS-MNs, DAg-MNs, and DAg / HTMS-MNs. Each wound was treated with a different microneedle treatment. A control group received no treatment. Each mouse was housed individually postoperatively, and wound contraction was recorded by photographing at various time points. Five days after treatment, wound exudate from each group was collected with an inoculating loop and plated onto agar plates. After 20 hours, the number of colonies was recorded by taking photos. Wound tissue samples were collected on days 5, 10, and 15 and fixed with 4% paraformaldehyde.

[0087] Histology and immunofluorescence staining:

[0088] To assess wound healing in diabetic mice, wound tissue was collected on days 5, 10, and 15 of wound healing. Sections were first dehydrated with graded alcohol, then embedded in paraffin, and cut into 5 μm sections for hematoxylin and eosin (H&E) staining and Masson's trichrome staining. For immunofluorescence staining, all sections were deparaffinized from day 10 specimens, rehydrated with graded alcohol, and then subjected to antigen retrieval in heated citrate buffer (citrate buffer, pH 6.0) for 5 minutes. Nonspecific antibody binding sites were blocked using a 5% bovine serum albumin (BSA) solution. Sections were then incubated with the primary antibody overnight at 4°C and the secondary antibody for 1 hour at room temperature. Finally, sections were stained with DAPI for 5 minutes and imaged by fluorescence microscopy. The relative fluorescence intensities of CD206, INOS, and CD31 were analyzed using Image J software and then normalized to the control group. Inflammatory responses were assessed by immunohistochemical staining for IL-6, IL-10, IL-17, and TNF-α. To evaluate the potential systemic toxicity of the microneedle patch, hematoxylin-eosin (H&E) staining was performed on major organs (heart, liver, spleen, lung, and kidney) collected on day 15.

[0089] Transcriptome sequencing and data analysis:

[0090] Total RNA was extracted using TRIzol reagent (Invitrogen, California, USA) according to the manufacturer's protocol. RNA purity and quantification were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Libraries were then constructed using the VAHTS Universal V6 RNA-seq Library Preparation Kit according to the manufacturer's instructions. Transcriptome sequencing and analysis were performed by Shanghai Ouyi Biotechnology Co., Ltd.

[0091] Libraries were sequenced on the Illumina Novaseq 6000 platform, generating 150-bp paired-end reads. Raw reads in Fastq format were first processed using Fast p to remove low-quality reads to obtain clean reads. Clean reads were mapped to the reference genome using HISAT2. FPKM and read counts for each gene were calculated using HTSeq-count. Principal component analysis (PCA) was performed using R (v3.2.0) to assess the biological reproducibility of the samples.

[0092] GO and KEGG pathway enrichment analysis were performed on differentially expressed genes (DEGs) based on hypergeometric distribution to screen significantly enriched entries. Histograms, chord plots, and bubble plots of significantly enriched entries were drawn using R (v 3.2.0).

[0093] Gene set enrichment analysis (GSEA) was performed using GSEA software. This analysis uses predefined gene sets to rank genes based on their differential expression between the two sample types and then tests whether the predefined gene sets are enriched at the top or bottom of the ranked list.

[0094] Statistical analysis: All data were expressed as mean ± mean square error and were analyzed using GraphPad Prism 8.0 software. The differences between two groups were evaluated using the t-test, and multiple groups were compared using one-way analysis of variance (ANOVA) followed by Tukey's multivariate comparison test. A p-value of less than 0.05 was considered statistically significant.

[0095] The test statistics are shown in Figures 2 to 18 .

[0096] See also Figure 2 :

[0097] Transmission electron microscopy (TEM) observations showed that the silver nanoparticles were roughly spherical and uniform in size; energy dispersive X-ray spectroscopy (EDS) analysis showed that the elemental composition of the nanoparticles contained silver and carbon. Dex / Ag NPs can be easily dispersed in aqueous solution and maintain a spherical morphology ( Figure 2 Figure A in the figure). Dynamic light scattering (DLS) analysis was used to evaluate the size and stability of the nanoparticles. The results showed that the zeta potentials of unmodified Ag NPs (silver nanoparticles) and dextran-modified Dex / Ag NPs were -21.16±1.46mV and -11.39±0.96mV, respectively. The average particle size of Ag NPs was 20.92nm. After dextran modification, the average particle size of the nanoparticles increased to 32.06nm ( Figure 2 The XPS results show that the Ag spectrum has a clear double peak at 368.2 eV, which confirms the successful synthesis of Ag NPs and Dex / Ag NPs ( Figure 2 The synthesis of Dex / Ag NPs was further confirmed by Fourier transform infrared spectroscopy (FTIR), which not only retained all the characteristic peaks belonging to AgNPs, but also -1 The characteristic peak ( Figure 2F). This may be due to the stretching vibration of the CH bond in the sugar ring. The targeted recognition effect of Dex / Ag NPs on Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) was evaluated using transmission electron microscopy (TEM). It was observed that the untreated Staphylococcus aureus and E. coli showed smooth surfaces and clear boundaries ( Figure 2 B). After the addition of Ag NPs and Dex / Ag NPs, random dispersion of Ag NPs and Dex / Ag NPs was observed, respectively. In contrast, the introduction of Dex / Ag NPs resulted in the observation of a large number of nanoparticles firmly attached to the surfaces of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in an encapsulated manner ( Figure 2 B). These findings provide strong evidence for the targeted recognition ability of Dex / Ag NPs for bacteria. This targeted recognition effect can be attributed to the high specificity of dextran for bacterial outer membrane lectins and its interaction with bacterial surface polysaccharides. Scanning electron microscopy (SEM) showed that compared with the untreated control group, bacteria in groups A and B underwent significant morphological changes, including bacterial wall depression (indicated by red arrows) and local bacterial wall destruction. Notably, group B exhibited denser nanoparticle adhesion (indicated by yellow arrows) and more extensive bacterial envelope damage, indicating that engineered surface functionality enhanced the antibacterial effect by enhancing nano-biointerface interactions ( Figure 2 G). For planktonic bacteria, bacterial growth curves were recorded after treatment with different concentrations of Ag NPs and Dex / Ag NPs for more than 18 h ( Figure 2 H), and found that treatment with Ag NPs at a concentration of only 80 μg / ml completely inhibited visible bacterial growth, while Dex / Ag NPs at a concentration of only 40 μg / ml completely inhibited visible bacterial growth. Compared to silver nanoparticles, dextran-functionalized derivatives exhibited enhanced antibacterial activity due to targeted bacterial function, suggesting their broad potential for precision therapeutic intervention against drug-resistant infections.

[0098] See also Figure 3 、 Figure 12 and Figure 13 :

[0099] The microsphere system exhibits an ingenious chemokine capture mechanism: heparin molecules covalently bound to the surface selectively capture and immobilize the chemokine MCP-1 through high-affinity electrostatic interactions. This biofunctionalization strategy works synergistically with the three-dimensional interconnected porous structure of the microspheres to achieve an orderly cell recruitment process: (i) the porous network maintains a stable chemokine concentration gradient, and (ii) the microspheres act as a biomimetic migration scaffold to guide the recruitment of monocytes / macrophages. The resulting spatially confined enrichment of MCP-1 forms a potent chemotactic field, specifically recruiting inflammatory monocytes to the microspheres, constructing an active interface with targeted immune regulation function ( Figure 3 A). The surface morphology of HP-Taurine@MS was characterized using scanning electron microscopy (SEM). Figure 3 As shown in Figure B, the microspheres exhibit a clear and well-defined porous structure. Subsequent taurine loading and heparin sodium functionalization resulted in a gradual decrease in pore size and pore density. Notably, the HP-taurine@MS composite material maintained its structural porosity, which is particularly important for promoting taurine encapsulation and achieving the capture of inflammatory chemokines. The microsphere system was systematically characterized by dynamic light scattering (DLS) technology to evaluate its particle size distribution and particle stability. The zeta potential test results showed that there were significant differences in the surface charge characteristics of the original microspheres (-21±0.12mV), taurine@MS (-31.5±0.26mV), and HP-taurine@MS (-21±0.12mV). Hydrodynamic diameter analysis showed that the average particle size of the microspheres showed a gradient increase from MS (79.93 μm) to taurine@MS (101 μm) and then to HP-taurine@MS (119.81 μm). This result confirmed the gradual surface modification achieved by taurine loading and heparin sodium modification through regular changes ( Figure 12 A and B). In order to confirm the successful synthesis of the microspheres, the surface composition of the three microspheres was compared by X-ray photoelectron spectroscopy (XPS) analysis. Compared with MS, a sulfur (S) peak (characteristic peak of Taurine) appeared in Taurine@MS, indicating that Taurine has been successfully loaded. In Taurine@MS, sodium heparin sulfate is grafted to the surface of the microspheres through an amide bond. This process can be confirmed by detecting the characteristic sulfur (S) peak of the heparin sulfonic acid group and the nitrogen (N) peak that is not observed in MS. As shown in the FTIR spectrum, the peak change at 1121cm-1 in Taurine@MS is related to the SO stretching vibration in taurine, while the peak change at 1651cm-1 in HP-Taurine@MS may be attributed to the formation of an amide bond, and the peak change at 1230cm-1 is related to the sulfonic acid group in heparin sodium ( Figure 12C and D). The above results indicate that taurine and heparin have been successfully integrated into HP-Taurine@MS. The HP-Taurine@MS is a dual-functional platform that promotes wound healing. Its mechanism of action is to sequester overexpressed chemokines at the site of injury while recruiting and reprogramming monocytes. Heparin was chosen as the surface modification ligand due to its sulfated glycosaminoglycan structure, in which the negatively charged sulfate groups interact electrostatically with the positively charged amino acid residues on the chemokines, thereby achieving efficient chelation. Figure 3 As shown in the binding kinetics in C, MCP-1 (monocyte chemoattractant protein-1) exhibited rapid binding to the functionalized microspheres, capturing most of the chemokines within the first 24 hours. To evaluate this mechanism, we first stimulated macrophages with lipopolysaccharide (LPS) to induce the production of a pro-inflammatory secretome. Conditioned medium containing a complex mixture of inflammatory mediators produced by these activated macrophages was incubated with HP-taurine@MS for 0, 1, 2, and 3 days (HP-taurine@MS was replaced daily). Notably, we observed a gradual decrease in the level of MCP-1 in the conditioned medium, indicating that the microspheres were able to effectively capture chemokines ( Figure 3 D).

[0100] In this study, the ability of HP-Taurine@MS to recruit macrophages from the microenvironment was systematically evaluated by establishing a microsphere-macrophage direct co-culture system and a Transwell migration system. The experimental results showed that HP-Taurine@MS could induce the formation of a unique radial cell aggregation structure ( Figure 3 E), which is significantly different from the dispersed distribution pattern of the MS group and Taurine@MS group. Live / dead cell staining analysis showed that the HP-Taurine@MS surface has a significant cell adhesion advantage, and its fluorescence signal intensity is significantly higher than that of other groups ( Figure 3 G) and highly colocalized with macrophages ( Figure 3 I). More importantly, Transwell migration assay confirmed that the cell recruitment efficiency of HP-Taurine@MS was significantly higher than that of other groups ( Figure 3 F and 3H).

[0101] Subsequently, a "wound bed" culture medium was prepared by incubating the microspheres in the above-mentioned inflammatory conditioned medium. When applied to a monocyte recruitment assay, the "wound bed" culture medium from the microsphere-treated group showed significantly reduced chemotactic activity for macrophages compared to the untreated inflammatory control group ( Figure 13AB). This reduced recruitment correlated with microsphere-mediated MCP-1 depletion in the inflammatory environment, confirming their ability to reduce inflammatory pressure in the wound bed. These findings strongly suggest that HP-Taurine@MS can serve as a multifunctional "bioactive cellular hub," not only providing three-dimensional structural support for cells but also enabling the specific recruitment of endogenous macrophages through active chemotaxis. This property demonstrates its potential in the repair of chronic diabetic wounds.

[0102] See also Figure 4 and Figure 14 :

[0103] DAg / HTMS-MNs showed a clear 10×10 array structure ( Figure 4 A). Scanning electron microscopy (SEM) characterization showed uniform conical microneedles (MNs) with a height of approximately 600 μm and a base diameter of approximately 200 μm ( Figure 4 B). A high-magnification top-view image shows that the needle surface becomes rough (red arrow) due to the incorporation of Dex / Ag NPs in the upper layer (top needle region), while the hemispherical protrusions in the lower layer (lung top needle region, yellow arrow) confirm the successful loading of microspheres ( Figure 4 C). To further verify the bilayer structure, sodium fluorescein (green) and rhodamine (red) were loaded into Dex / AgNPs and microspheres, respectively. Fluorescence microscopy imaging (side view and top view) confirmed that the Dex / Ag NPs emitting green fluorescence in the upper layer and the microspheres emitting red fluorescence in the lower layer had a distinct spatial distribution, clearly demonstrating the biphasic structure of the microneedles ( Figure 4 D and 4E).

[0104] Mechanical testing showed that each microneedle could withstand a maximum force of 0.6 Newtons, significantly exceeding the 0.1 Newton force per needle required to successfully penetrate the skin ( Figure 4 F). After applying the microneedle patch to the back skin of rats, a 10×10 micropore array was observed, and the microneedles were completely embedded in the skin ( Figure 4 G). Histological cross-sectional analysis further confirmed that the microneedles successfully penetrated the epidermis and dermis, indicating their potential for efficient transdermal drug delivery ( Figure 4 G). Drug release studies showed that the microneedles exhibited different kinetic characteristics in vitro, characterized by rapid release of Ag NPs and sustained release of taurine from PLGA microspheres. This differential release behavior stems from the inherent properties of the matrix materials: rapidly dissolving hyaluronic acid (HA) facilitates immediate delivery of Dex / Ag NPs, while PLGA microspheres enable prolonged release of taurine ( Figure 4H). The synergistic combination of these release kinetics achieves dual therapeutic functions - rapid antibacterial effect and sustained immunomodulation. All extracts from microneedle formulations had no adverse effects on L929 cell proliferation ( Figure 4 I), the relative growth rate (RGR) at all time points was consistently over 80% ( Figure 4 J). According to the ISO 10993.5-2009 cytotoxicity grading standard, these results confirmed that all microneedle (MN) groups had no significant cytotoxicity under in vitro conditions. At the same time, 15 days after blank microneedle or DAg microneedle treatment, major organs (heart, liver, spleen, lung and kidney) were collected for histological analysis. Hematoxylin-eosin (H&E) staining showed that no obvious pathological changes were observed in any organ compared with the untreated control group ( Figure 14 A). These findings demonstrate that our microneedle system has excellent biosafety.

[0105] See also Figure 5 :

[0106] A gas-propelled microneedle (MN) system ( Figure 5 A), In phosphate buffered saline (PBS), no obvious morphological changes were observed in the microneedle (MNs) group, while DAg-HTMS microneedles showed rapid generation of bubbles that were concentrated in the lower half of the needle tip ( Figure 5 B), confirming the gas-driven propulsion. Time-lapse imaging further revealed its dynamic process: initially, no bubbles were observed at the base of DAg / HTMS-MNs, but over time, bubbles gradually accumulated and eventually generated sufficient force to lift the tip of DAg / HTMS-MNs upward ( Figure 5 C) This mechanochemical behavior is similar to the principles of missile design, where controlled gas generation allows for directed propulsion.

[0107] Confocal microscopy analysis showed that the dye penetration depth in the DAg / HTMS-MNs group was significantly increased compared with the control group ( Figure 5 D). Quantitative analysis of subcutaneous fluorescence intensity at different depths showed that the distribution patterns of the drugs in the superficial area of ​​0 to 500 μm were similar among all groups; however, when the depth exceeded 500 μm, the average fluorescence intensity of the DAg / HTMS-MNs group at 750 μm and 1000 μm was significantly higher than that of the MN group, demonstrating that the gas propulsion mechanism can effectively promote deep tissue penetration ( Figure 5 E) These results demonstrate that gas-assisted delivery not only enhances penetration depth, but also significantly improves payload distribution in deeper tissue layers.

[0108] Taking advantage of the bactericidal ability of Dex / Ag NPs and the excellent penetration ability of the microneedle system, the anti-biofilm performance was evaluated using the live / dead staining method. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) biofilms were treated with PBS, MNs, HTMS-MNs, DAg-MNs or DAg / HTMS-MNs, and then SYTO 9 / PI staining was performed to distinguish between live (green) and dead (red) bacteria. Figure 5 As shown in Figure 1, the DAg / HTMS-MNs group exhibited the strongest red fluorescence and the least green signal in the entire biofilm matrix, indicating its excellent biofilm removal efficiency. In contrast, the PBS, MNs, and HTMS-MNs groups showed mainly green fluorescence, indicating that their antibacterial effect was limited. Scanning electron microscopy (SEM) characterization showed that compared with the blank group, the biofilm treated with DAg / HTMS-MNs underwent significant morphological changes, manifested by a significant decrease in bacterial density, complete disintegration of the biofilm structure, and characteristic bacterial film depression / rupture ( Figure 5 H).

[0109] The superior anti-biofilm performance of DAg / HTMS-MNs was quantitatively demonstrated by crystal violet staining and standard plate count assay. Figure 5 F) Compared to the DAg-MNs group and the DAg / HTMS-MNs group, the colony counts of Staphylococcus aureus and Escherichia coli biofilms were significantly reduced. Among them, the DAg / HTMS-MNs system demonstrated the best biofilm removal efficiency, which was attributed to the synergistic effect of the bactericidal effect of Dex / Ag NPs and the enhanced penetration ability of the microneedle platform. Plate cloning assay results showed that for Staphylococcus aureus, the bacterial survival rates in the DAg-MNs group and the DAg / HTMS-MNs group were reduced to 10.87±1.89% and 4.23±0.70%, respectively, which were approximately 1 / 10 of those in the PBS group (100±4.56%), the MNs group (99.16±5.96%), and the HTMS-MNs group (99.52±0.54%). The E. coli experiment showed the same trend: the survival rates of the DAg-MNs group and the DAg / HTMS-MNs group decreased to 5.81±1.92% and 0.57±0.17%, respectively, which were significantly lower than those of the other groups ( Figure 5 F and 5G). Crystal violet staining results showed that although both DAg-MNs and DAg / HTMS-MNs could destroy the biofilm structure, DAg / HTMS-MNs showed a stronger killing ability, leaving only fragmented biofilm ( Figure 5J); while the biofilm of the other group remained intact and its thickness did not decrease. Quantitative analysis further confirmed that DAg / HTMS-MNs had the best ability to disperse mature biofilms, outperforming all test groups ( Figure 5 J and 5K).

[0110] See also Figure 6 、 Figure 15 and Figure 16 :

[0111] Raw264.7 cells pretreated with hydrogen peroxide significantly expressed reactive oxygen species (ROS). Subsequently, the cells were treated with PBS, MNs, HTMS-MNs, DAg-MNs, and DAg / HTMS-MNs extracts (the group without treatment served as the control group). Compared with the hydrogen peroxide group (PBS+H2O2 group), MNs group, and DAg-MNs group, the expression of ROS in Raw264.7 cells treated with HTMS-MNs and DAg / HTMS-MNs was significantly reduced ( Figure 6 A).

[0112] Under hydrogen peroxide-induced oxidative stress, the JC-1 aggregate / monomer ratio decreased, indicating mitochondrial depolarization and dysfunction, while HTMS-MNs and DAg / HTMS-MNs were able to restore this aggregate / monomer ratio ( Figure 6 B) Quantitative results of the JC-1 aggregate / monomer ratio showed that HTMS-MNs and DAg / HTMS-MNs were able to maintain mitochondrial homeostasis, and their aggregate / monomer ratios were significantly higher than those of the other groups ( Figure 6 C). Subsequently, based on the regulatory effects of DAg / HTMS-MNs on macrophage reactive oxygen species (ROS) and mitochondrial homeostasis, we further explored the effects of DAg / HTMS-MNs on macrophage polarization. Fluorescence staining images of CD206 (a marker of M2 macrophages) and INOS (a marker of M1 macrophages) and their corresponding expression heat maps showed that HTMS-MNs and DAg / HTMS-MNs extracts could promote macrophage polarization from a pro-inflammatory state to a pro-regenerative phenotype ( Figure 6 D and 6G). This was also confirmed by pro-inflammatory and anti-inflammatory fluorescence staining images and corresponding fluorescence quantitative analysis. HTMS-MNs and DAg / HTMS-MNs extracts promoted IL-10 expression and inhibited TNF-α expression in macrophages ( Figure 15 A). These results suggest that DAg / HTMS-MNs may accelerate ROS clearance, maintain mitochondrial homeostasis, and promote macrophage reprogramming from M1 (pro-inflammatory phenotype) to M2 (pro-repair phenotype).

[0113] When macrophages were treated with the microneedle extracts and co-cultured with Staphylococcus aureus, both the DAg / HTMS-MNs extract and the DAg / HTMS-MNs extract significantly enhanced bacterial phagocytosis compared with the other groups ( Figure 6 E). Fluorescence imaging showed that the treated macrophages strongly internalized pathogens. Quantitative fluorescence analysis showed that the phagocytosis of bacteria by macrophages was significantly enhanced after treatment with HTMS-MNs and DAg / HTMS-MNs extracts compared with the control group ( Figure 6 F). Importantly, these taurine-containing preparations significantly enhanced the intracellular bacterial killing capacity of macrophages—a key determinant in preventing recurrent infections ( Figure 16 A).

[0114] See also Figure 7 :

[0115] To evaluate the angiogenic efficacy of microneedles, this study used human umbilical vein endothelial cells (HUVECs) treated with macrophage-conditioned medium cultured with microneedle extracts to determine whether the angiogenic factors secreted by macrophages can effectively promote angiogenesis in vitro. In the scratch test, the HTMS-MNs group and the DAg / HTMS-MNs group showed a decrease in scratch area at 24 hours, 48 ​​hours, and 72 hours after incubation compared with the control group, the MNs group, and the DAg-MNs group ( Figure 7 A). Quantitative analysis of scratch closure rates confirmed these observations ( Figure 7 B). In addition, in the HUVECs Transwell migration assay after co-culture, the HTMS-MNs and DAg / HTMS-MNs groups showed significantly more migrating cells and higher cell density compared with the control group, MNs group, and DAg-MNs group ( Figure 7 C). Quantitative assessment of migrating cell counts confirmed these results ( Figure 7 D). In addition, in the HUVECs tube formation experiment, the HTMS-MNs and DAg / HTMS-MNs groups showed more dense branch points than the control group, MNs group, and DAg-MNs group ( Figure 7 E). Quantitative evaluation of connected mesh networks also validates these results ( Figure 7 F). In addition, the study used the angiogenesis marker CD31 to evaluate the effect of the microneedle patch on angiogenesis in human umbilical vein endothelial cells (HUVEC). Fluorescence imaging showed that cells in the HTMS-MNs group and DAg / HTMS-MNs group showed stronger CD31 fluorescence intensity compared with the other groups ( Figure 7 G), and quantitative analysis of CD31 fluorescence intensity further confirmed these results, indicating that it performed better in inducing angiogenesis ( Figure 7 H). Taken together, these findings indicate that the DAg / HTMS-MNs patch has significant pro-angiogenic potential.

[0116] See also Figure 8 and Figure 17 :

[0117] Bacterial infection and sustained reactive oxygen species (ROS) production can exacerbate inflammation and impair wound healing. In this study, DAg / HTMS-MNs demonstrated significant antimicrobial activity, pro-angiogenic effects, and antioxidant properties in vitro. To evaluate their therapeutic efficacy in Staphylococcus aureus (S. aureus)-infected skin wounds, we established a diabetic wound model. Figure 8 A summarizes the experimental timeline, including wound creation, treatment administration, healing observation, and sample collection. The antibacterial properties of the microneedle patch were evaluated by collecting skin tissue and exudate around the wound area on day 5. The bacterial colony counts in the DAg-MNs and DAg / HTMS-MNs groups were significantly reduced compared with the control, MNs, and HTMS-MNs groups ( Figure 8 D), the bacterial survival rates were 21±6.2% and 8.15±1.73%, respectively, confirming the effective antibacterial effect of the microneedle patch in vivo ( Figure 17 A). In addition, Giemsa staining was used to assess the bacterial load within the wound. Consistent with the results of colony counts, both the DAg-MNs group and the DAg / HTMS-MNs group showed a significant decrease in bacteria ( Figure 8 F). Notably, the DAg / HTMS-MNs group had the lowest bacterial load, highlighting its superior antibacterial efficacy ( Figure 8 I).

[0118] Thanks to its potent antimicrobial activity and immunomodulatory capacity, DAg / HTMS-MNs significantly accelerated wound healing. The wound area graph showed that the wounds in the DAg / HTMS-MNs group shrank fastest at each designated time point, achieving almost complete wound closure within 15 days of treatment ( Figure 8 B, 8C, and 8H). Histopathological analysis further confirmed the therapeutic effect by evaluating the re-epithelialization rate and collagen deposition. Comparative studies showed that the wound width in the DAg / HTMS-MNs treatment group was significantly reduced compared with the other groups ( Figure 8 E). Quantitative analysis on day 10 showed that the re-epithelialization rate in the DAg / HTMS-MNs group was the fastest (84±7%), significantly exceeding that in the control group (45.36±16.1%), MNs group (60.3±14.3%), HTMS-MNs group (72.3±6.3%), and DAg-MNs group (73.3±6.3%) ( Figure 8J). Masson trichrome staining on day 15 showed that collagen fiber deposition was significantly enhanced, with neatly arranged structures and improved tissue integrity ( Figure 8 G and 8K). Notably, the newly formed tissue exhibited a new hair follicle structure that was highly similar to that of normal skin, indicating a highly effective healing response.

[0119] See also Figure 9 and Figure 18 :

[0120] To further explore the mechanism by which DAg / HTMS-MNs promote wound healing in diabetic patients, we performed RNA-seq analysis on full-thickness skin from the back wounds of mice. Samples were taken from the control group and the DAg / HTMS-MNs group, with three biological replicates in each group. The two-dimensional principal component analysis (PCA) plot clearly showed that the two groups of samples were clearly separated ( Figure 9 A), indicating that there were significant differences in gene expression profiles between the two groups. By comparing the control group with the DAg / HTMS-MNs group, the researchers screened for differentially expressed genes (DEGs). The results showed that a total of 704 genes were upregulated, of which 201 genes were significantly upregulated; 1053 genes were downregulated, of which 530 genes were significantly downregulated ( Figure 9 B). The top 20 differentially expressed genes are highlighted in the heat map ( Figure 9 C).

[0121] To explore the molecular mechanisms underlying the therapeutic effects of DAg / HTMS-MNs, we performed comprehensive Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of differentially expressed genes. GO analysis revealed that biological processes related to wound healing were significantly upregulated in the DAg / HTMS-MNs group, including oxidoreductase activity, positive regulation of cell migration, and collagen-containing extracellular matrix organization ( Figure 9 D). In contrast, genes related to inflammatory response and oxidative stress, such as those involved in immune response, Toll-like receptor signaling pathway, cellular response to lipopolysaccharide, and genes positively regulating NF-κB transcription factor activity, were significantly downregulated ( Figure 9 E) These findings were further confirmed by enrichment analysis and chordal diagrams detailing specific molecular alterations.

[0122] KEGG pathway analysis showed that wound healing-related pathways were significantly upregulated, including AMPK signaling pathway, extracellular matrix (ECM)-receptor interaction pathway and PPAR signaling pathway ( Figure 9F). In contrast, pathways associated with M1 macrophage activation, including the IL-17 signaling pathway, NOD-like receptor signaling pathway, TNF signaling pathway, and Toll-like receptor signaling pathway, were significantly downregulated. Enrichment analysis chord plots and gene heatmaps provided detailed molecular characteristics of these changes ( Figure 9 F and 9G). Notably, matrix metalloproteinases (MMPs)—enzymes that are often elevated in diabetic wounds and contribute to DNA damage and impaired healing by promoting reactive oxygen species (ROS) production—showed a significant reduction. Key inflammatory mediators (Ccl2, Ccl11, Defb3, Tnfrsf1b, and IL6) and proteolytic factors (Mmp9 and Mmp19) were significantly downregulated ( Figure 18 A).

[0123] Through transcriptome sequencing and analysis, we revealed the molecular mechanism by which DAg / HTMS-MNs improve the wound microenvironment. Overall, the overall improvement in the wound microenvironment is attributed to the synergistic effects of DAg / HTMS-MNs' targeted antimicrobial and immunomodulatory effects, which effectively downregulate inflammatory signaling pathways and accelerate wound healing.

[0124] See also Figure 10 and Figure 11 :

[0125] During the healing process of diabetic infected wounds, DAg / HTMS-MNs exhibited significant immunomodulatory and pro-angiogenic properties. Immunofluorescence analysis showed that DAg / HTMS-MNs treatment effectively regulated macrophage polarization: in treated wounds, the expression of the pro-inflammatory M1 marker iNOS was significantly reduced, while the expression of the anti-inflammatory M2 marker CD206 was increased ( Figure 10 AC). By day 10, the M1 / M2 macrophage ratio in the DAg / HTMS-MNs group had dropped below 1, indicating a successful transition from a pro-inflammatory state to a pro-regenerative state ( Figure 10 D). Notably, CD31 immunofluorescence staining showed that neovascularization was significantly enhanced in DAg / HTMS-MNs-treated wounds ( Figure 10 E), the mean fluorescence intensity of the treated group was 2.17±0.05, which was significantly higher than that of all other experimental groups ( Figure 10 F).

[0126] We conducted further histological analysis to deduce the expression levels of interleukin-17 (IL-17), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10), key factors associated with wound healing. Combined with transcriptome sequencing results, we confirmed that DAg / HTMS-MNs promote the healing of chronic diabetic wounds by downregulating the IL-17 signaling pathway. Representative images are shown in Figure 2. Figure 11 AF. The quantitative analysis results were consistent with the qualitative data. Compared with the control group, the expression levels of IL-17, IL-6, and TNF-α in the DAg-MNs and HTMS-MNs treatment groups were significantly decreased, but the decrease was smaller than that in the DAg / HTMS-MNs group ( Figure 11 AF).

[0127] In contrast, compared with the IL-10 levels in the control group, MNs group, DAg-MNs group, and HTMS-MNs treatment group, the DAg / HTMS-MNs treatment group had the highest immunohistochemical positivity rate, with more brown cells and a darker brown color ( Figure 11 G and 11H). This is mainly attributed to the ability of DAg / HTMS-MNs to quickly clear wound infection, reverse oxidative stress in the wound area, inhibit excessive inflammation, and reduce the expression of pro-inflammatory factors, thereby helping diabetic infected wounds exit the inflammatory phase and enter the repair phase.

[0128] In summary, this study, through the development of a dual-layer microneedle system, DAg / HTMS-MNs, presents a groundbreaking "missile-inspired" therapeutic strategy for diabetic wound management. This system synergistically combines targeted antimicrobial action with intelligent immunomodulation. By utilizing dextran-modified silver nanoparticles for deep-tissue biofilm penetration and heparin-coated taurine-loaded microspheres for spatiotemporal chemokine control, the system simultaneously addresses the triple challenges of bacterial resistance, chronic inflammation, and oxidative stress in diabetic wounds. The system's unique biphasic release profile enables rapid bacterial eradication, followed by sustained macrophage reprogramming, effectively breaking the vicious cycle of MCP-1-driven inflammation while promoting tissue regeneration. This innovative approach establishes a new paradigm in wound treatment, overcoming fundamental limitations of conventional therapies through coordinated material design and biological targeting. The successful biofilm disruption, inflammation resolution, and accelerated healing of DAg / HTMS-MNs demonstrates their potential as a promising candidate for clinical translation. The potential "global decompression-local enhancement" strategy provides a versatile platform for addressing other complex inflammatory diseases requiring precise spatiotemporal therapeutic control. Future development should focus on clinical adaptation through personalized manufacturing and real-time monitoring technologies to fully realize its therapeutic potential.

[0129] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. An MCP-1 responsive microneedle, characterized in that: The MCP-1 responsive microneedles are loaded with dextran-modified silver nanoparticles and heparin-modified taurine PLGA microspheres.

2. The MCP-1 responsive microneedle according to claim 1, characterized in that The needle top section of the microneedle is loaded with dextran-modified silver nanoparticles, and the non-needle top section is loaded with heparin-modified taurine PLGA microspheres.

3. The MCP-1 responsive microneedle according to claim 2, characterized in that The needle top section of the microneedle adopts a hyaluronic acid matrix, and the non-needle top section adopts a polyvinyl pyrrolidone matrix.

4. The MCP-1 responsive microneedle according to any one of claims 1 to 3, characterized in that The preparation method of the dextran-modified silver nanoparticles comprises: preparing an aqueous solution containing trisodium citrate and sodium borohydride, adding silver nitrate to react, and collecting silver nanoparticle precipitates; A silver nanoparticle water suspension is prepared, stirred and mixed with a dextran aqueous solution for reaction, and the precipitate is collected by centrifugation, washed, and dried to obtain dextran-modified silver nanoparticles.

5. The MCP-1 responsive microneedle according to any one of claims 1 to 3, characterized in that: The preparation method of the heparin-modified taurine PLGA microspheres comprises: preparing a PLGA oil phase solution and a taurine aqueous phase solution, mixing them, adding a gelatin aqueous solution, and performing ultrasonic emulsification to obtain an oil emulsion; The oil emulsion is mixed with a polyvinyl alcohol aqueous solution for reaction, and impurities are removed to obtain taurine PLGA microspheres; The taurine PLGA microspheres are suspended in a polyethyleneimine solution for amination, impurities are removed, and the microspheres are dried to obtain aminated microspheres. Prepare 2-(N-morpholino)ethanesulfonate buffer containing heparin sodium, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and N-hydroxysuccinimide, add aminated microspheres for reaction, remove impurities, and obtain the product.

6. A method for preparing MCP-1 responsive microneedles, characterized in that: The steps include: Preparation of dextran-modified silver nanoparticles; Preparation of heparin-modified taurine PLGA microspheres; The MCP-1 responsive microneedles are formed by dispersing dextran-modified silver nanoparticles in a hyaluronic acid matrix and dispersing heparin-modified taurine PLGA microspheres in a polyvinyl pyrrolidone matrix.

7. An MCP-1 responsive microneedle patch, characterized in that: It is prepared using MCP-1 responsive microneedles.

8. Use of the MCP-1 responsive microneedle according to any one of claims 1 to 5 or the MCP-1 responsive microneedle patch according to claim 7 in the preparation of wound repair medicines.

9. The use according to claim 8, characterized in that The wound is an infected, chronic wound.

10. The use according to claim 9, characterized in that The wound is a diabetic wound.

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