Konjac glucomannan microneedle patch for diabetic wound healing and preparation method and application thereof

Abstract

The application discloses a konjac glucomannan microneedle patch for diabetic wound healing and a preparation method and application thereof, and belongs to the technical field of medical device preparation. The microneedle patch is composed of a needle tip array and a backing layer, wherein the needle tip array and the backing layer are both formed by an oxidized konjac glucomannan and a mixed aqueous solution of hyaluronic acid, and a CuGA-MOF material is added to the aqueous solution for making the microneedle patch. The microneedle patch provided by the application can treat diabetic wounds in a minimally invasive manner. In vitro and in vivo verification shows that the konjac glucomannan microneedle patch loaded with the CuGA-MOF can inhibit bacterial growth, remove active oxygen, regulate macrophage M2 polarization and promote angiogenesis, and can effectively realize rapid healing of diabetic wounds. In addition, the raw material of the microneedle patch is abundant, the preparation method is simple, the cost is low, the safety is high, and the microneedle patch has a wide application prospect in the field of treating infected wounds and chronic wounds.

Description

Technical Field

[0001] This invention belongs to the field of medical device manufacturing technology, specifically relating to a konjac glucomannan microneedle patch for diabetic wound healing, its preparation method, and its application. Background Technology

[0002] Diabetic foot ulcer (DFU) is a common and serious complication of diabetes, leading to a global cause of non-traumatic amputation due to its high incidence, recurrence, and mortality rates. Improper wound care and poor healing severely impact the health of diabetic patients and impose a heavy medical burden; DFU has become a common challenge for the global medical community. Currently, clinical treatment for DFU primarily focuses on the following aspects: blood glucose control, debridement of necrotic tissue at the wound site, antibiotic treatment for bacterial infection, surgical treatment, dressing therapy, ultrasound therapy, hydrotherapy, hyperbaric oxygen therapy, and negative pressure therapy. The treatment methods vary depending on the severity of the symptoms, but research has not yet demonstrated a significant advantage of any single therapy over others. Therefore, new and effective strategies to promote wound healing are urgently needed.

[0003] The causes of chronic wounds vary, but they share some basic characteristics. Chronic wounds often fail to heal through the normal process and become inflammatory. This healing stagnation is caused by a variety of factors, including insufficient angiogenesis leading to excessive hypoxia, persistent overexpression of pro-inflammatory cytokines, and infection. Therefore, the main strategies for managing chronic wounds are to improve the oxidative stress environment at the wound site caused by excessive accumulation of reactive oxygen species, regulate the immune microenvironment to promote macrophage polarization to the M2 type, and prevent bacterial wound infections.

[0004] Local treatment is a rational strategy for treating chronic wounds. Currently, the main clinical methods for treating chronic wounds include tissue debridement, infection control, topical medication, and bandaging. Generally, most medications only cover the wound surface, but chronic wounds are covered by a layer of hardened skin and necrotic tissue. The wound exudate containing various enzymes washes away the medication locally, significantly reducing its bioavailability. Microneedle patches, due to their unique structure, can overcome these barriers to deliver medication directly into the wound and exert its effects promptly, thus showing great potential in treating chronic wounds.

[0005] In previous studies, materials used to manufacture microneedle patches (MNs) were only used as drug carriers to assist drug delivery, but they did not possess any function themselves. The mannose units in glucomannan can mimic active ligands in vivo and effectively stimulate mannose receptors to polarize macrophages to the M2 phenotype, leading to the secretion of large amounts of pro-regenerative cytokines. Therefore, konjac glucomannan (KGM), as a natural polymer with good biocompatibility and biodegradability, can serve as a functional drug carrier. However, the high viscosity and low solubility of KGM greatly limit its application, resulting in no reports of its formulation into MNs to date. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a konjac glucomannan microneedle patch for diabetic wound healing, its preparation method, and its application. The aim is to effectively achieve antibacterial, reactive oxygen species scavenging, macrophage polarization and inflammatory microenvironment regulation, and angiogenesis promotion, thereby promoting diabetic wound healing.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a microneedle patch for healing diabetic wounds, the microneedle patch comprising a backing layer and a microneedle array; the microneedle patch is made of a biodegradable material, and both the backing layer and the microneedles of the microneedle array contain gallic acid-copper metal-organic framework material.

[0009] Furthermore, the microneedle array is composed of several arrayed microneedles, the bottom end of the microneedles is connected to the backing layer, and the microneedles and the backing layer are perpendicular to each other; the microneedles in the microneedle array are square pyramids with a height of 400-1000μm and a bottom side length of 150-400μm.

[0010] Furthermore, the gallic acid-copper metal-organic framework material comprises particles with an average diameter of 450 nm, made of Cu 2+ It is composed of gallic acid.

[0011] Furthermore, the gallic acid-copper metal-organic framework material in the microneedle patch releases Cu 2+ It has antibacterial properties and promotes angiogenesis. At the same time, the released gallic acid can scavenge excess intracellular reactive oxygen species, thereby regulating the inflammatory microenvironment.

[0012] Furthermore, the preparation method of the gallic acid-copper metal-organic framework material includes the following steps:

[0013] (1) Dissolve gallic acid in a sodium hydroxide aqueous solution with a mass concentration of 4-5% to obtain a gallic acid solution with a mass concentration of 8-9%.

[0014] (2) Add an 8-9% copper chloride aqueous solution dropwise to a gallic acid solution, stir at 90-95℃ for 2-3 hours, and collect the precipitate;

[0015] (3) Wash with water and dry to obtain gallic acid-copper metal-organic framework material.

[0016] Furthermore, the biodegradable material is modified konjac glucomannan or a mixture of modified konjac glucomannan and hyaluronic acid. The mannose units in konjac glucomannan can mimic active ligands in vivo, stimulating macrophage mannose receptors to polarize to the M2 phenotype and regulating macrophage inflammation.

[0017] Furthermore, the modified konjac glucomannan is oxidized konjac glucomannan, and the preparation method of the oxidized konjac glucomannan includes:

[0018] Prepare an aqueous solution of konjac glucomannan, add sodium periodate of the same mass as konjac glucomannan to the solution, react at 40-45℃ in the dark for 6-7 hours, then add ethylene glycol to terminate the reaction, and continue stirring for 2-3 hours. Dialyze the compound with a molecular weight cutoff of 12000 Da, freeze dry, and obtain oxidized konjac glucomannan.

[0019] Furthermore, the relative molecular mass of the hyaluronic acid is between 10,000 and 400,000. The relative molecular mass affects the viscosity of the hyaluronic acid solution; for the same mass of HA, a higher relative molecular mass results in a higher viscosity and a lower concentration of hyaluronic acid solution can be prepared. A defined relative molecular mass ensures that the prepared microneedles have suitable appearance and mechanical properties.

[0020] Secondly, the present invention provides a method for preparing the aforementioned microneedle patch, comprising the following steps:

[0021] S1. Dissolve 0.1-5g of oxidized taro glucomannan and 0-5g of hyaluronic acid in 10mL of deionized water and stir at room temperature until a clear and transparent solution is formed to obtain the microneedle matrix solution.

[0022] S2. Take 0.5-50 mg of gallic acid-copper metal-organic framework material and add it to the microneedle matrix solution. Mix well to obtain a needle suspension.

[0023] S3. Inject the needle suspension into the microneedle mold, form it under negative pressure, dry it, and demold it to obtain a microneedle patch for diabetic wound healing.

[0024] Thirdly, the present invention provides the application of the microneedle patch or the microneedle patch prepared by the preparation method described above in the preparation of diabetic wound healing dressings.

[0025] Furthermore, the diabetic wound healing refers to diabetic foot ulcer wound healing.

[0026] Verification has shown that the microneedle patch prepared by this invention is based on oxidized konjac glucomannan and Cu. 2+ In synergy with gallic acid, it can promote the rapid healing of diabetic wounds by inhibiting bacterial growth, scavenging reactive oxygen species, regulating macrophage polarization, and promoting angiogenesis.

[0027] The present invention provides a konjac glucomannan microneedle patch for diabetic wound healing, the beneficial effects of which are:

[0028] 1. The needle body and base of the microneedle patch of this invention are made of functional natural polymer materials, so that the efficacy of the microneedle patch is not limited to the drug loaded within it. The microneedle patch of this invention has good ability to penetrate biological membranes, can cross barriers to deliver drugs into the wound and exert their effects in a timely manner, and can effectively enhance the deep delivery of drugs.

[0029] 2. The preparation method of the microneedle patch of the present invention has the advantages of abundant raw material sources, simple preparation method, mild reaction conditions and low production cost, and is suitable for industrial-scale production.

[0030] 3. The CuGA-MOF-loaded konjac glucomannan microneedle patch prepared in this invention has multifunctionality. This invention utilizes Cu... 2+ The CuGA-MOF was formed by combining with gallic acid, thus realizing Cu 2+ The sustained release of gallic acid prolonged the duration of drug action and simultaneously reduced Cu. 2+ Cytotoxicity. Through Cu 2+ It resists drug-resistant bacterial infections and promotes angiogenesis. By scavenging reactive oxygen species with gallic acid, it regulates the microenvironment, aiding cell proliferation and tissue regeneration. Furthermore, using konjac glucomannan as a carrier, it exhibits excellent biocompatibility and biodegradability. The mannose units in konjac glucomannan can mimic active ligands in vivo and actively stimulate mannose receptors, polarizing macrophages to the M2 phenotype, leading to the secretion of a large number of pro-regenerative cytokines and effectively promoting wound healing in diabetic patients. Attached Figure Description

[0031] Figure 1 The image shows the characterization of CuGA-MOF prepared in Example 1 of this invention; wherein, (a) is the X-ray diffraction characterization of CuGA-MOF; (b) is the morphology of CuGA-MOF and the distribution of carbon, oxygen and copper elements in CuGA-MOF; and (c) is the particle size distribution of CuGA-MOF.

[0032] Figure 2Characterization images of OKGM and microneedle patches prepared in Example 1 of this invention are shown below; (a) is the 1H NMR spectrum of oxidized konjac glucomannan; (b) is the infrared spectrum of oxidized konjac glucomannan; (c) is the appearance of the microneedle patch under an optical camera; (d) is the local appearance of the microneedles in the microneedle patch under a scanning electron microscope; (e) is the distribution of CuGA-MOF in the microneedles; and (f) is the appearance of the microneedle patch before and after dissolution under an optical microscope.

[0033] Figure 3 This is a diagram showing the in vitro antibacterial test results of the microneedle patch in Application Example 1 of the present invention.

[0034] Figure 4 Examples 2 of this invention demonstrate the antioxidant properties and in vitro cell compatibility of the microneedle patch. (a) shows the in vitro antioxidant performance of different concentrations of CuGA-MOF; (b) shows the intracellular reactive oxygen species scavenging performance of CuGA-MOF and the microneedle patch; (c) shows the cell viability after incubation with different concentrations of CuGA-MOF and the microneedle patch for 48 hours; and (d) shows the cell viability and cell death staining after incubation with different concentrations of CuGA-MOF and the microneedle patch for 24 hours and 48 hours.

[0035] Figure 5 The following are the results of evaluating wound healing in diabetic mice using microneedle patches in Example 3 of this invention: (a) is the result of evaluating wound healing in diabetic mice; (b) is the quantitative result of (a); (c) is the H&E staining image of wound healing tissue in diabetic mice; and (d) is the Masson staining image of wound healing tissue in diabetic mice.

[0036] Figure 6 Example 3 of this invention describes the evaluation of the M2 phenotype of macrophage polarization in the wound of diabetic mice using microneedle patches; (a) shows the immunofluorescence staining of macrophage phenotype distribution in the wound tissue of diabetic mice on days 7 and 14; (b) shows the quantitative results on day 7 in (a); and (c) shows the quantitative results on day 14 in (a). Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, all reagents or instruments used in the embodiments are conventional products that can be purchased through legitimate channels.

[0038] Example 1

[0039] A konjac glucomannan microneedle patch, the preparation method of which includes the following steps:

[0040] (1) Synthesis and characterization of CuGA-MOF: 0.425 g gallic acid was dissolved in 5 mL of an aqueous solution containing 0.22 g sodium hydroxide and stirred until fully dissolved. 0.426 g CuCl2·2H2O was dissolved separately in 5 mL of deionized water and this solution was added dropwise to the gallic acid solution. The mixture was then heated to 90 °C and stirred for 2 hours. After the reaction was completed, the product was centrifuged to collect the brown precipitate, which was repeatedly washed with deionized water. Finally, the particles were freeze-dried and collected to obtain gallic acid-copper metal-organic framework (CuGA-MOF).

[0041] Figure 1 (a) shows the X-ray diffraction pattern of CuGA-MOF, with peaks at 10.2, 13.4, 20.2, 26.7, 28.0, 31.5, 33.5, 41.9 and 42.8°, indicating the polymorphism of the material and demonstrating the successful synthesis of CuGA-MOF.

[0042] Figure 1 (b) shows the morphology of CuGA-MOF and the distribution of carbon, oxygen and copper elements in CuGA-MOF, demonstrating the uniform distribution of carbon, oxygen and copper in CuGA-MOF particles.

[0043] Figure 1 (c) The particle size distribution of CuGA-MOF was determined using a Malvern laser particle size analyzer. The results showed that the average particle size of CuGA-MOF was 457.3 nm and the PDI was 0.156.

[0044] (2) Synthesis and characterization of oxidized konjac glucomannan: 2.5g of konjac glucomannan was dissolved in 250mL of deionized water, and sodium periodate of the same mass as konjac glucomannan was added. The reaction was carried out at 40℃ in the dark for 6 hours. Then, 25mL of ethylene glycol was added to terminate the reaction. The mixture was stirred for another 2 hours. The mixture was then transferred to a dialysis bag with a molecular weight cutoff of 12000Da and dialyzed with deionized water for 3 days. The mixture was then freeze-dried to obtain oxidized konjac glucomannan.

[0045] Figure 2 (a) and Figure 2 (b) are the 1H NMR spectra of oxidized konjac glucomannan. 1 H-NMR spectrum and Fourier transform infrared (FT-IR) characterization. From 1 The ¹H-NMR spectrum shows that, compared to konjac glucomannan (KGM), oxidized konjac glucomannan (OKGM) has additional shift peaks at 8.5 and 9.3 ppm, identified as aldehyde groups. The Fourier transform infrared spectrum shows that, compared to KGM, OKGM has a higher shift peak at 1720 cm⁻¹. -1 There is an absorption peak at this point, which is the absorption peak of the free aldehyde group, which proves that KGM was successfully oxidized to OKGM.

[0046] (3) Preparation and characterization of microneedle patches: 1.0 g of oxidized konjac glucomannan and 1.0 g of hyaluronic acid were accurately weighed and dissolved in 8 mL of deionized water to obtain a microneedle matrix solution with a mass concentration of 20%. 3.2 mg of CuGA-MOF was added to the microneedle matrix solution and mixed evenly to obtain a needle suspension. An appropriate amount of needle suspension was injected into a microneedle mold, placed in a 50 mL centrifuge tube with PDMS inserts, and centrifuged at 4000 rpm for 10 min to ensure that the drug-loaded solution fully enters the needle tip orifice of the microneedle mold. After drying, the microneedle patch (CuGA-MOF@MN) was peeled off from the mold.

[0047] Figure 2 (cf) shows the morphology of the microneedle patch under an optical camera and a scanning electron microscope, and the distribution of CuGA-MOF within the microneedles. SEM images reveal that the tips of CuGA-MOF@MN are pyramidal in shape and orderly arranged on the backing layer. This sharp pyramidal structure ensures that MN can be quickly, non-invasively, and accurately inserted deep into the skin. Elemental mapping of CuGA-MOF within MN was used to evaluate its distribution, showing that Cu is uniformly distributed throughout the MN structure, indicating that CuGA-MOF is uniformly loaded into MN.

[0048] Comparative Example 1

[0049] A konjac glucomannan microneedle patch, the preparation method of which includes the following steps:

[0050] (1) Synthesis of oxidized konjac glucomannan: The method is the same as step (1) in Example 1;

[0051] (2) Preparation of microneedle patches: Accurately weigh 1.0 g of oxidized konjac glucomannan and 1.0 g of hyaluronic acid and dissolve them in 8 mL of deionized water to obtain a microneedle matrix solution with a mass concentration of 20%. Inject an appropriate amount of microneedle matrix solution into a microneedle mold, place it into a 50 mL centrifuge tube with PDMS inserts, and centrifuge at 4000 rpm for 10 min to ensure that the solution fully enters the needle tip holes of the microneedle mold. After drying, peel it off from the mold to obtain the microneedle patch (MN).

[0052] Example 2

[0053] A konjac glucomannan microneedle patch, the preparation method of which includes the following steps:

[0054] (1) Synthesis of oxidized konjac glucomannan: The method is the same as step (1) in Example 1;

[0055] (2) Synthesis of CuGA-MOF: The method is the same as step (2) in Example 1;

[0056] (3) Preparation of microneedle patches: Accurately weigh 1.0 g of oxykonjac glucomannan and 1.0 g of hyaluronic acid and dissolve them in 8 mL of deionized water to obtain a microneedle matrix solution with a mass concentration of 20%. Add 1.6 mg of CuGA-MOF to the microneedle matrix solution and mix well to obtain a needle suspension. Inject an appropriate amount of needle suspension into a microneedle mold, place it in a 50 mL centrifuge tube with PDMS insert, and centrifuge at 4000 rpm for 10 min to ensure that the drug-loaded solution fully enters the needle tip orifice of the microneedle mold. After drying, peel it off from the mold to obtain the microneedle patch.

[0057] Example 3

[0058] A konjac glucomannan microneedle patch, the preparation method of which includes the following steps:

[0059] (1) Synthesis of oxidized konjac glucomannan: The method is the same as step (1) in Example 1;

[0060] (2) Synthesis of CuGA-MOF: The method is the same as step (2) in Example 1;

[0061] (3) Preparation of microneedle patches: Accurately weigh 1.0 g of oxykonjac glucomannan and 1.0 g of hyaluronic acid and dissolve them in 8 mL of deionized water to obtain a microneedle matrix solution with a mass concentration of 20%. Add 0.8 mg of CuGA-MOF to the microneedle matrix solution and mix well to obtain a needle suspension. Inject an appropriate amount of needle suspension into a microneedle mold, place it in a 50 mL centrifuge tube with PDMS insert, and centrifuge at 4000 rpm for 10 min to ensure that the drug-loaded solution fully enters the needle tip orifice of the microneedle mold. After drying, peel it off from the mold to obtain the microneedle patch.

[0062] Application Example 1

[0063] This application example tests the antibacterial ability of the konjac glucomannan microneedle patch prepared in Example 1 according to the following steps.

[0064] The bacterial suspension was co-cultured with the microneedle patch for 4 hours. 100 μL of the bacterial suspension was then transferred to an LB solid agar plate, spread evenly, and incubated in a 37°C incubator for 24 hours. The number of macroscopic colonies was then counted.

[0065] Transfer 100 μL of bacterial suspension to an LB solid agar plate, spread the suspension evenly, place the microneedle patch on the LB solid agar plate, and incubate at 37°C for 24 hours. Observe the inhibition zone.

[0066] Figure 3The figure shows the results of the in vitro antibacterial experiment of the microneedle patch. As can be seen from the figure, the bacteria in the bacterial culture with CuGA-MOF@MN added were completely killed, and a clear inhibition zone appeared around the MN loaded with CuGA-MOF, indicating that CuGA-MOF@MNs have a good antibacterial effect.

[0067] Application Example 2

[0068] This application example tests the in vitro antioxidant capacity and cell biocompatibility of CuGA-MOF obtained in step (2) of Example 1 by following the steps below.

[0069] A certain amount of 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) was weighed and dissolved in anhydrous ethanol to prepare a 0.4 mmol / L DPPH solution. 100 μL was added to each well of a 96-well plate, followed by 100 μL of CuGA-MOF solution at different concentrations (5, 10, 20, 40, 80, 160 μg / mL). After incubation at 37℃ for 30 min, the absorbance was measured at 517 nm using a microplate reader. 100 μL of deionized water was used as a control. The DPPH radical scavenging rate of the samples was calculated.

[0070] Figure 4 (a) shows the antioxidant properties of CuGA-MOF at different concentrations. As can be seen from the figure, the inhibition rate of DPPH free radicals gradually increases with the increase of CuGA-MOF concentration, and the antioxidant rate reaches 90% when the CuGA-MOF concentration is 80 μg / mL.

[0071] Intracellular reactive oxygen species (ROS) were detected using a reactive oxygen species detection kit (DCFH-DA). The specific procedure involved seeding L929 cells (mouse fibroblasts) into 12-well plates (5 × 10⁻⁶ cells per well). 4 Cells were cultured in wells for 24 hours until cell adhesion occurred. Rosup reagent was added to the wells to stimulate cell adhesion for 30 minutes to increase reactive oxygen species levels; no Rosup was added to the negative control group. Subsequently, CuGA-MOF solutions at concentrations of 40 μg / mL and 80 μg / mL, blank microneedle solution, and drug-loaded microneedle reconstitution solution at a concentration of 80 μg / mL (based on CuGA-MOF) were added to the wells. DMEM medium was used as the positive control, and the cells were cultured for 12 hours. DCFH-DA was diluted 1:1000 (v / v) with serum-free culture medium to a final concentration of 10 μmol / L. The cell culture medium was removed, and 500 μL of diluted DCFH-DA was added. The cells were incubated at 37°C for 20 minutes. The cells were washed three times with serum-free cell culture medium to remove any uninfiltrated DCFH-DA. The stained cells were observed using an inverted fluorescence microscope.

[0072] Figure 4 (b) shows the intracellular reactive oxygen species scavenging experiment of CuGA-MOF and microneedle patch. As can be seen from the figure, compared with the positive control, the fluorescence of CuGA-MOF group and CuGA-MOF@MN group is weaker. This is because the effective release of gallic acid in CuGA-MOF enables CuGA-MOF and CuGA-MOF@MN to show good antioxidant capacity in the reactive oxygen species scavenging experiment.

[0073] The cell compatibility of CuGA-MOF and microneedle patches was investigated in vitro using L929 cells. CCK-8 assays and dead / live cell assays were performed in this study.

[0074] CCK-8 experiment: L929 cells were digested with trypsin, counted, and diluted to 5×10⁶ cells. 4 Cells / mL: 100 μL of diluted L929 cell suspension was seeded into 96-well plates, and cell adhesion occurred after 24 h. Reconstituted solutions of CuGA-MOF at concentrations of 10, 20, 40, 80, and 160 μg / mL (calculated as CuGA-MOF) and the microneedle patch prepared in Example 1 were prepared using DMEM complete medium. DMEM medium was used as a blank control. The liquid in the wells was discarded, and 100 μL of the above solutions were added to each well, with six replicates per group. The plates were incubated at 37°C and 5% CO2 for 24 h. CCK-8 was diluted 10-fold with complete medium, the liquid in the plate was discarded, and the diluted CCK-8 was added to the above 96-well plates (100 μL / well). The plates were incubated for another 1 h. The OD value of each well was measured at 450 nm using a microplate reader, and the relative cell viability was calculated.

[0075] Dead / live cell assay: L929 cells were seeded into 12-well plates (5 × 10⁻⁶ cells / well). 4 Cells / wells were cultured in an incubator for 24 h, after which cell adhesion occurred. Cells were then cultured in CuGA-MOF at a concentration of 80 μg / mL (calculated as CuGA-MOF) and in drug-loaded microneedle reconstituted solution at 37℃ and 5% CO2 for 24 h and 48 h, respectively, with DMEM medium used as a blank control. Cells were stained with calcein AM / PI fluorescent dye (dead cells stained red, live cells stained green). Images of stained cells were observed using an inverted fluorescence microscope.

[0076] Figure 4(c) CCK-8 in vitro cell compatibility assays of CuGA-MOF and microneedle patches. As shown in the figure, when the CuGA-MOF concentration increased from 0 mg / mL to 160 mg / mL, the cell viability after 24 h was consistently higher than that of the control group, indicating a certain promoting effect on cell proliferation. The cell viability of the drug-loaded microneedle patch group at a concentration of 80 μg / mL (calculated as CuGA-MOF) was 76.41 ± 8.02%, exhibiting relatively low cytotoxicity.

[0077] Figure 4 (d) Cell viability staining experiment of CuGA-MOF and microneedle patch. As can be seen from the figure, L929 cells were in good condition and spindle-shaped after being cultured for 24h and 48h in the presence of CuGA-MOF and microneedles at a concentration of 80μg / mL (calculated as CuGA-MOF), indicating that CuGA-MOF material and microneedle patch have good cell compatibility.

[0078] Application Example 3

[0079] This application example tests the effect of the konjac glucomannan microneedle patch prepared in Example 1 and Comparative Example 1 on the healing of diabetic wounds according to the following steps.

[0080] Mice were randomly divided into three groups: a normal control group (Normal control), a diabetic control group (Positive control), a blank microneedle group (Comparative Example 1, KGM-MNs group), and a drug-loaded microneedle group (Example 1, CuGA-MOF@MNs group), with 10 mice in each group. The diabetic mouse model was established by a single high-dose intraperitoneal injection of streptozotocin. Three days later, when blood glucose levels stabilized at a hyperglycemic state, a 1.1cm × 1.1cm incision was made on the back of the mouse, indicating successful model establishment. All mice in the diabetic control group, blank microneedle group, and drug-loaded microneedle group were diabetic.

[0081] Normal mouse control group: No treatment was given.

[0082] Microneedle group: The konjac glucomannan microneedle patches prepared in Example 1 and Comparative Example 1 were applied to the wounds cut on the backs of mice, and the treatment was carried out every other day.

[0083] Wound status was photographed and observed daily until the epidermis of the diabetic control mice healed spontaneously. On days 7, 14, and 21, the repaired wound tissue was isolated and immersed in 4% paraformaldehyde solution. Subsequently, it was dehydrated and embedded in paraffin for further tissue sectioning. Hematoxylin-eosin (H&E) and Masson's trichrome staining were performed according to standard procedures to assess skin regeneration, collagen deposition, and angiogenesis, respectively. Macrophage polarization was assessed using immunofluorescence staining with CD68 and CD206, and cells were treated with Phalloidin / DAPI staining.

[0084] Figure 5 (a) and Figure 5 (b) is a graph showing the results of wound healing assessment in diabetic mice. The graph shows that the healing rate of each group is: drug-loaded microneedle group ≥ normal mouse control group > blank microneedle group > diabetic mouse control group. This indicates that the CuGA-MOF-loaded konjac glucomannan microneedle patch has the ability to promote wound healing in diabetic mice.

[0085] Figure 5 (c) and Figure 5 (d) H&E staining and Masson staining images of wound healing tissue in diabetic mice, respectively. On day 7 of treatment, a large number of inflammatory cells were observed in the diabetic mouse control group, while fewer inflammatory cells and a small number of fibroblasts and neovascularization were observed in the drug-loaded microneedle group and the normal mouse control group. On day 14, intact new epithelium was observed in the other three groups except for the diabetic mouse control group, and the number of fibroblasts and neovascularization increased significantly, while the number of inflammatory cells decreased. However, the newly formed epidermal structure in the blank microneedle group was loose, and a small number of inflammatory cells were still observed, indicating a relatively poor healing effect. When the wound healed to day 21, the wounds in all four groups were basically healed, the inflammatory cells had basically disappeared, the number of fibroblasts had decreased, and the fiber bundles were arranged in an orderly manner. At the same time, the new epithelium in the drug-loaded microneedle group and the normal mouse control group became thinner (about 30 μm), and new hair follicles appeared in the new epithelial area, indicating good overall recovery. The new epithelium in the blank microneedle group was thicker (about 70 μm), and there was still an area of ​​about 3100 μm without new hair, indicating that the overall healing was slightly worse than the previous two groups. In the diabetic mouse control group, the thickness of newly formed epithelium was approximately 65 μm, and the length of the area without new hair growth was approximately 4100 μm, indicating the worst healing condition. Masson's trichrome staining revealed that the drug-loaded microneedle group, compared to the blank microneedle group or the diabetic mouse control group, exhibited greater collagen deposition and directional alignment, approaching that of the normal mouse control group. This suggests that the combined treatment significantly improved extracellular matrix reconstruction and tissue remodeling.

[0086] Figure 6Immunofluorescence staining images of macrophage phenotypic distribution in wound tissue of diabetic mice show that, compared with the control group, the percentage of CD206-positive macrophages was significantly increased in both the drug-loaded microneedle group and the blank microneedle group, indicating a reduced inflammatory state. On day 14 of treatment, the proportion of M2 macrophages exceeded that of M1 macrophages in the skin wounds treated with glucomannan, while untreated wounds, whether diabetic or normal, were mainly composed of M1 macrophages. These data indicate that CuGA-MOF@MNs can effectively polarize macrophages recruited at the wound site to the M2 type, thereby improving the local inflammatory response and transforming the tissue microenvironment from inflammatory to reparative.

[0087] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the basic principles of the present invention should be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A microneedle patch for healing diabetic wounds, characterized in that: The microneedle patch includes a backing layer and a microneedle array; the microneedle patch is made of a biodegradable material, and both the backing layer and the microneedles of the microneedle array contain gallic acid-copper metal-organic framework material. The gallic acid-copper metal-organic framework material described herein consists of particles with an average diameter of 450 nm, composed of Cu 2+ Composed of gallic acid; The biodegradable material is modified konjac glucomannan or a mixture of modified konjac glucomannan and hyaluronic acid; The microneedle array consists of several microneedles arranged in an array. The bottom end of each microneedle is connected to the backing layer, and the microneedles and the backing layer are perpendicular to each other. The microneedles in the microneedle array are square pyramids with a height of 400-1000 μm and a bottom end with a side length of 150-400 μm. The preparation method of the gallic acid-copper metal-organic framework material includes the following steps: (1) Dissolve gallic acid in a sodium hydroxide aqueous solution with a mass concentration of 4-5% to obtain a gallic acid solution with a mass concentration of 8-9%. (2) Add an 8-9% copper chloride aqueous solution dropwise to a gallic acid solution, stir at 90-95℃ for 2-3 hours, and collect the precipitate; (3) Wash with water and dry to obtain gallic acid-copper metal-organic framework material; the modified konjac glucomannan is oxidized konjac glucomannan; The preparation method of the oxidized konjac glucomannan includes: Prepare an aqueous solution of konjac glucomannan, add sodium periodate of the same mass as konjac glucomannan to the solution, react at 40-45℃ in the dark for 6-7 hours, then add ethylene glycol to terminate the reaction, and continue stirring for 2-3 hours. Dialyze the compound with a molecular weight cutoff of 12000 Da, freeze dry, and obtain oxidized konjac glucomannan.

2. The microneedle patch according to claim 1, characterized in that, The relative molecular mass of the hyaluronic acid is 10,000 to 400,000.

3. The method for preparing the microneedle patch according to claim 1 or 2, characterized in that, Includes the following steps: S1. Dissolve 0.1-5g of oxidized konjac glucomannan and 0-5g of hyaluronic acid in 10mL of deionized water and stir at room temperature until a clear and transparent solution is formed to obtain the microneedle matrix solution. S2. Take 0.5-50 mg of gallic acid-copper metal-organic framework material and add it to the microneedle matrix solution. Mix well to obtain a needle suspension. S3. Inject the needle suspension into the microneedle mold, form it under negative pressure, dry it, and demold it to obtain a microneedle patch for diabetic wound healing.

4. The application of a microneedle patch according to claim 1 or 2 or a microneedle patch prepared by the preparation method according to claim 3 in the preparation of a diabetic wound healing dressing.

5. The application according to claim 4, characterized in that, The term "diabetic wound healing" refers to the healing of diabetic foot ulcer wounds.

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