Microneedle systems, preparation methods and applications

Abstract

This invention discloses a microneedle system, its preparation method, and its applications. The microneedle system includes a microneedle substrate and a microneedle tip. The microneedle substrate is a cross-linked polyvinyl alcohol hydrogel, and the microneedle tip includes the cross-linked polyvinyl alcohol hydrogel and FGF21 and GLP-1RA encapsulated within the cross-linked polyvinyl alcohol hydrogel. This invention prepares PVA with localized ROS responsiveness. tsPBA The microneedle system co-encapsulates FGF21 and GLP-1RA, enabling them to work synergistically at the wound site to improve endothelial function, promote angiogenesis, and facilitate keratinocyte migration, thereby achieving highly effective treatment of diabetic foot ulcers.

Description

Technical Field

[0001] This invention relates to the field of diabetes treatment technology, and more specifically, to a microneedle system, its preparation method, and its application. Background Technology

[0002] Approximately 25% of people with diabetes are at risk of developing diabetic foot ulcers (DFU). Diabetic foot ulcers are a serious chronic complication of diabetes, with a global prevalence of 6.3%, representing a growing health problem and a leading cause of amputation in diabetic patients. Poor long-term glycemic control and associated lipid metabolism abnormalities in diabetic patients lead to impaired local glucose and lipid metabolism at the wound site, resulting in endothelial dysfunction, local angiogenesis, and keratinocyte migration dysfunction. This is a significant factor contributing to the difficulty in healing diabetic foot ulcers. Therefore, exploring appropriate and effective treatments for repairing diabetic foot ulcers is of great importance.

[0003] Currently, there are several treatment methods for diabetic foot ulcers: 1. Local therapies, including non-surgical debridement agents, immunomodulators, growth factor therapy, metalloproteinase inhibitor therapy, autologous platelet-derived gel therapy, bioengineered skin substitutes, and topical oxygen therapy (TOT).

[0004] 2. Systemic and oral drug therapies, including hyperbaric oxygen therapy (HBOT), oral drug therapy, etc.

[0005] 3. Physical therapy: such as negative pressure wound therapy (NPWT) and intravenous infusion therapy.

[0006] 4. New therapies, such as stem cell therapy, gene therapy, peptide therapy, and neuropeptide therapy.

[0007] 5. Nanotechnology therapy.

[0008] 6. Experimental therapies, such as traction-activated payloads (TrAPs) that locally deliver short interfering RNA to disrupt matrix metalloproteinase-9 in the wound, and hydrogels containing bioactive substances / cells. Hydrogels are composed of hydrophilic polymer chains that absorb large amounts of water without dissolving due to the presence of cross-links. Furthermore, hydrogels can bind bioactive substances and / or cells, achieving sustained release of the active ingredients.

[0009] Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by intestinal L cells. Its main physiological functions include stimulating insulin secretion and release, inhibiting glucagon secretion, promoting the proliferation of pancreatic β cells, and inhibiting their apoptosis. Therefore, GLP-1RA (glucagon-like peptide-1 receptor agonist), a drug targeting GLP-1, was the first to be successfully marketed internationally in 2005. Currently, GLP-1RA has become a new treatment option for patients with type 2 diabetes. Furthermore, studies have shown that GLP-1 plays an important role in the anti-atherosclerosis of patients with type 2 diabetes; GLP-1RA can reduce diabetic foot complications; and GLP-1RA has pleiotropic effects in the healing of diabetic wounds: in vivo (including local application and intraperitoneal injection) and in vitro experiments on diabetic mouse wounds show that GLP-1RA can promote keratinocyte migration; it enhances the angiogenesis capacity of wounds by upregulating the expression of angiogenic factors such as HIF-1α, VEGFR2, and eNOS; and it promotes the healing of diabetic wounds through its AMPK-dependent endothelial protection and angiogenesis-promoting effects. Therefore, GLP-1RA may be a potential target drug for improving diabetic skin ulcers. However, due to the limitations of protein drugs and small molecule peptide drugs, which are easily hydrolyzed by complex environments, the direct local application of GLP-1RA for the treatment of diabetic foot has not been reported. Therefore, designing novel delivery systems and exploring local drug delivery is of great significance.

[0010] In view of this, the present invention is proposed. Summary of the Invention

[0011] The purpose of this invention is to provide a microneedle system, its preparation method, and its application.

[0012] This invention is implemented as follows: In a first aspect, the present invention provides a microneedle system, comprising a microneedle substrate and a microneedle tip, wherein the microneedle substrate is a cross-linked polyvinyl alcohol hydrogel, and the microneedle tip comprises a cross-linked polyvinyl alcohol hydrogel and FGF21 and GLP-1RA encapsulated within the cross-linked polyvinyl alcohol hydrogel. The cross-linked polyvinyl alcohol hydrogel is prepared from polyvinyl alcohol using the compound of Formula I as a cross-linking agent, wherein the compound of Formula I has the following structure: .

[0013] In an optional embodiment, the mass ratio of FGF21 to GLP-1RA is 2:(2.8-3.2).

[0014] In an optional embodiment, the concentration of FGF21 in the microneedle tip is 0.18 mg / mL - 0.22 mg / mL; the concentration of GLP-1RA in the microneedle tip is 0.28 mg / mL - 0.32 mg / mL.

[0015] Secondly, the present invention provides the application of the microneedle system described in any one of the foregoing embodiments in a pharmaceutical preparation for treating diabetic foot ulcers.

[0016] Thirdly, the present invention provides the application of the microneedle system described in any of the foregoing embodiments in a pharmaceutical formulation for upregulating the SIRT1 / AMPK pathway to improve cell viability after high glucose-induced damage.

[0017] Fourthly, the present invention provides a method for preparing a microneedle system according to any one of the foregoing embodiments, wherein FGF21 and GLP-1RA are added to an aqueous solution containing a crosslinking agent and polyvinyl alcohol to obtain a mixed solution, the mixed solution is added to the needle tip of a microneedle mold, and then the mixed aqueous solution of polyvinyl alcohol and crosslinking agent is added to the mold for filling, and then a crosslinking reaction is carried out before demolding to obtain a microneedle system.

[0018] In an optional embodiment, the concentration of polyvinyl alcohol in the mixed solution is 0.06 g / ml - 0.07 g / ml, and the ratio of the crosslinking agent to the concentration of polyvinyl alcohol in the mixed aqueous solution is 1:3.5-4.5.

[0019] In an optional embodiment, bisphenol A and N,N,N,N-tetramethyl-1,3-propanediamine (TMPA) are dissolved in N,N-dimethylamide to obtain a pre-reaction solution, and then the reaction is carried out. The reaction solution is then transferred to tetrahydrofuran to obtain a precipitate, which is the crosslinking agent.

[0020] In an optional embodiment, the concentration of bisphenol A in the pre-reaction solution is 1.15 mmol / ml - 1.2 mmol / ml, and the concentration of TMPA is 0.035 mmol / ml - 0.040 mmol / ml.

[0021] In an optional embodiment, before adding the mixed aqueous solution of polyvinyl alcohol and crosslinking agent to the mold for filling, the mold is first placed under vacuum for 8-12 minutes; and / or after adding the mixed aqueous solution of polyvinyl alcohol and crosslinking agent to the mold for filling, the mold is centrifuged at 1800rpm-2200rpm for 18-22 minutes.

[0022] The present invention has the following beneficial effects: This invention prepares PVA tsPBAThe microneedle system co-encapsulates FGF21 and GLP-1RA, enabling them to exert a synergistic effect on the wound surface, improving endothelial function, promoting angiogenesis, and promoting keratinocyte migration, thereby achieving highly effective treatment of diabetic foot ulcers. The design of this microneedle system for treating diabetic foot ulcers has the following advantages: (1) Co-delivery of FGF21 and GLP-1RA can fully utilize their synergistic effect, resulting in better efficacy than single-drug administration. (2) Microneedle administration has minimal invasiveness to the skin stratum corneum barrier compared to traditional administration methods, while avoiding contact with important nerves and capillaries in the epidermis, reducing pain and tissue damage, and minimizing wound exposure to the external environment. (3) The microneedle patch can controllably maintain the release of therapeutic drugs, and ROS triggers the release of FGF21 and GLP-1RA. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Dissolution of needle tips in microneedle systems with different ratios of tsPBA-PVA; Figure 2 PVA tsPBA Microneedle system preparation steps; Figure 3 PVA tsPBA Morphology of PVA microneedles under optical microscope (A) and scanning electron microscope (B) for microneedle system; Figure 4 PVA tsPBA The distribution of drug loading was observed using an inverted fluorescence microscope on the microneedle system (FITC green label carrier, RhB red label drug, Merge overlay). Figure 5 PVA tsPBA Mechanical strength test of the microneedle system and representative images of the microneedles after applying compressive force; Figure 6 PVA tsPBA Photograph of a microneedle system penetrating the skin of a mouse; Figure 7 PVA tsPBA Drug release kinetics curves of the microneedle system; Figure 8 PVA tsPBA Gromacs molecular simulation of the microneedle system; Figure 9Example 2: Flowchart and wound diagram (A is the flowchart of diabetic wound modeling; B is a representative image of the wound on day 0, day 3, day 7, day 10, and day 14 after treatment with Control, rMN, GLP-1RA@rMN, FGF21@rMN, and FGF21 / GLP-1RA@rMN; C is a wound size diagram of wound healing within 14 days of modeling; D is the statistical data of wound area percentage versus time after different treatments, with the wound area ratio on day 0 defined as 100%, scale bar = 10 μm, and statistical significance indicated by * P<0.05, ** P<0.01, *** P<0.001, I: Control; II: rMN; III: FGF21@rMN; IV: GLP-1RA@rMN; V: FGF21 / GLP-1RA@rMN). Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0026] This embodiment provides a microneedle system, including a microneedle substrate and a microneedle tip. The microneedle substrate is a cross-linked polyvinyl alcohol hydrogel, and the microneedle tip includes a cross-linked polyvinyl alcohol hydrogel and FGF21 and GLP-1RA encapsulated within the cross-linked polyvinyl alcohol hydrogel. The cross-linked polyvinyl alcohol hydrogel is prepared from polyvinyl alcohol using the compound of Formula I as a cross-linking agent, wherein the compound of Formula I has the following structure: .

[0027] Fibroblast growth factor (FGF) is a family of cell signaling proteins. FGF stimulates angiogenesis and proliferation of fibroblasts and is a potent angiogenic factor. FGF21, a member of the FGF superfamily, is produced by metabolically involved tissues such as the liver, adipose tissue, skeletal muscle, and pancreas. It can lower blood glucose, improve lipid profiles, increase insulin sensitivity, promote fatty acid oxidation, and increase energy expenditure through various signaling pathways and mechanisms. Simultaneously, as an angiogenic molecule, FGF21 promotes angiogenesis in the mid-to-late stages of wound healing and is a key regulator of keratinocyte migration and differentiation during wound healing, promoting reepithelialization of the wound by activating autophagy.

[0028] Long-term hyperglycemia leads to elevated levels of advanced glycation end products (AGEs) in the blood. AGEs directly contribute to the formation of high concentrations of reactive oxygen species (ROS). Because ROS overexpression prevents the transition from the inflammatory phase to the proliferative phase, diabetic wounds often fail to heal. TsPBA is a ROS-responsive crosslinking agent that enables microneedle systems (PVA) to... tsPBA The system was prepared to have ROS-responsive and dual drug-loaded microneedle system. Polyvinyl alcohol (PVA), as an FDA-approved substance, has good biocompatibility and biodegradability, and can achieve local release of GLP-1RA and FGF21 in ROS-responsive diabetic foot ulcer wounds, and give full play to the synergistic effect of the two.

[0029] Subcutaneous injection of GLP-1RA is not suitable for all patients with refractory diabetic foot ulcers, and FGF21 applied to the wound surface is often easily degraded by proteases secreted by the inflammatory microenvironment. This invention utilizes a ROS-responsive PVA that co-delivers FGF21 and GLP-1RA. tsPBA Microneedling enables FGF21 and GLP-1RA to work synergistically on diabetic foot ulcers, providing a new treatment strategy for the healing of diabetic foot ulcer wounds.

[0030] In an optional embodiment, the mass ratio of FGF21 to GLP-1RA is 2:(2.8-3.2). Due to PVA tsPBA The release rates of FGF21 and GLP-1RA differ, requiring consideration of PVA release rates. tsPBA The ratio of FGF21 to GLP-1RA was adjusted to regulate the relative release rates of FGF21 and GLP-1RA, thereby enhancing their synergistic effect.

[0031] In an optional embodiment, the concentration of FGF21 in the microneedle tip is 0.18 mg / mL to 0.22 mg / mL; the concentration of GLP-1RA in the microneedle tip is 0.28 mg / mL to 0.32 mg / mL.

[0032] The absolute release rates of FGF21 and GLP-1RA are related to PVA. tsPBA The content of FGF21 and GLP-1RA is related, so it is necessary to reasonably select the concentration of FGF21 and GLP-1RA to adjust their release rate so that FGF21 and GLP-1RA can exert better effects.

[0033] Secondly, the present invention provides the application of the microneedle system described in any one of the foregoing embodiments in a pharmaceutical preparation for treating diabetic foot ulcers.

[0034] Thirdly, the present invention provides the application of the microneedle system described in any of the foregoing embodiments in a pharmaceutical formulation for upregulating the SIRT1 / AMPK pathway to improve cell viability after high glucose-induced damage.

[0035] Fourthly, the present invention provides a method for preparing a microneedle system according to any one of the foregoing embodiments, wherein FGF21 and GLP-1RA are added to an aqueous solution containing a crosslinking agent and polyvinyl alcohol to obtain a mixed solution, the mixed solution is added to the needle tip of a microneedle mold, and then the mixed aqueous solution of polyvinyl alcohol and crosslinking agent is added to the mold for filling, and then a crosslinking reaction is carried out before demolding to obtain a microneedle system.

[0036] In an optional embodiment, the concentration of polyvinyl alcohol in the mixed solution is 0.06 g / ml - 0.07 g / ml, and the ratio of the crosslinking agent to the concentration of polyvinyl alcohol in the mixed aqueous solution is 1:3.5-4.5.

[0037] The presence of polyvinyl alcohol and crosslinking agents affects PVA tsPBA The mechanical properties of microneedles and the release rate of FGF21 and GLP-1RA encapsulated within them are important factors. Excessive concentration leads to greater binding of FGF21 and GLP-1RA, resulting in a relatively slower release rate. Conversely, insufficient concentration leads to less binding of FGF21 and GLP-1RA and makes them more prone to dissolution and deformation, thus affecting the efficacy. Therefore, a reasonable selection is necessary.

[0038] In an optional embodiment, bisphenol A and TMPA are dissolved in N,N-dimethylamide to obtain a pre-reaction solution, and then the reaction is carried out. The reaction solution is then transferred to tetrahydrofuran to obtain a precipitate, which is the crosslinking agent.

[0039] In an optional embodiment, the concentration of bisphenol A in the pre-reaction solution is 1.15 mmol / ml - 1.2 mmol / ml, and the concentration of TMPA is 0.035 mmol / ml - 0.040 mmol / ml.

[0040] In an optional embodiment, before adding the mixed aqueous solution of polyvinyl alcohol and crosslinking agent to the mold for filling, the mold is first placed under vacuum for 8-12 minutes; and / or after adding the mixed aqueous solution of polyvinyl alcohol and crosslinking agent to the mold for filling, the mold is centrifuged at 1800rpm-2200rpm for 18-22 minutes.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] Example 1 This embodiment prepares a PVA tsPBAMicroneedle systems, such as Figure 1 and 2 As shown, the steps are as follows: (1) Preparation of tsPBA crosslinking agent: Bisphenol A (BPA) (4.7 mmol) and TMPA (1.5 mmol) were dissolved in 40 mL of N,N-dimethylamide (DMF) and stirred at 60 °C for 24 hours. Then, the reaction mixture was poured into 100 mL of tetrahydrofuran to precipitate the product. The precipitate was collected by centrifugation and washed three times with 20 mL of THF each time. Finally, the precipitate was vacuum dried at room temperature overnight to obtain tsPBA.

[0043] (2) Evaluation of the effect of different tsPBA contents on the dissolution of microneedle tips: tsPBA solutions of different concentrations were prepared, namely 1% (w / v), 3% (w / v), and 5% (w / v), and mixed with PVA to obtain PVA-tsPBA solutions. The mixed solutions were filled into microneedle molds, centrifuged at 2000 rpm for 20 min, and dried in a drying oven for 12 h to obtain tsPBA-PVA microneedles with different proportions. The microneedle tips were inserted into 3% agar, and the tip morphology was observed and recorded at 0 s, 15 s, 30 s, and 60 s. The results are as follows: Figure 1 As shown.

[0044] (2) Preparation of PVA loaded with FGF21 and GLP-1RA tsPBA Microneedles: Prepare a 10% (w / v) PVA solution and a 5% (w / v) TsPBA solution by mixing them at a volume ratio of 2:1 to obtain a PVA-TsPBA solution. Mix FGF21 and GLP-1RA with the PVA-TsPBA solution to achieve FGF21 and GLP-1RA concentrations of 0.2 mg / mL and 0.3 mg / mL, respectively, and then add the mixture dropwise into a microneedle mold and incubate under vacuum for 10 min. Then, transfer the PVA-TsPBA mixture without added FGF21 and GLP-1RA into the mold and centrifuge at 2000 rpm for 20 min to form a microneedle base.

[0045] The fluorescent microneedles were obtained by replacing FGF21 and GLP-1RA in Example 1 with FITC-labeled FGF21 and Rhodamine B-labeled GLP-1RA, while following the same steps as in Example 1.

[0046] For the PVA containing FGF21 and GLP-1RA in this embodiment tsPBA Characterization of microneedles: (1) Observation of microneedle morphology and drug loading: The basic morphology of the microneedles was observed using an optical microscope and a scanning electron microscope, and the drug loading of the fluorescent microneedles was observed using an inverted fluorescence microscope. The results are as follows: Figure 3 , Figure 4 As shown.

[0047] (2) Testing the mechanical properties of microneedles using a universal testing machine: Take PVA tsPBA The microneedle was placed on a metal plate of an electronic universal testing machine. The metal probe moved downwards at a speed of 0.5 mm / min until the needle body underwent 100% deformation, and the force-displacement curve was obtained. The results are as follows: Figure 5 As shown.

[0048] (3) Testing the penetration performance of the microbeads on mouse skin: PVA tsPBA Microneedles were applied to the mouse skin and held for 1 minute. Methylene blue was then used to stain the skin for 1 minute. The staining depth of the microneedle puncture sites was observed. The results are as follows: Figure 6 As shown.

[0049] (4) Pharmacokinetic assay: The PVA containing FGF21 and GLP-1RA was measured. tsPBA Cumulative drug release rate of microneedles: Fluorescent microneedles were incubated in 500 μL PBS, with 250 μL of PBS collected at each time point. The total amount of drug released was calculated, and an equal amount of fresh PBS was added. Release kinetic curves were plotted, and the test results are shown below. Figure 7 As shown in Table 1, LG is liraglutide (GLP-1RAglutide, GLP-1RA), which is a type of GLP-1RA.

[0050] Table 1. Relevant constants from pharmacokinetic simulations

[0051] Two diffusion mechanisms enable this microneedle system to achieve the desired effect: the preferential and rapid release of LG small molecules improves the microenvironment of diabetic wounds, while the slow release of FGF21 large molecules enhances cellular function.

[0052] (5) Gromacs molecular simulation: The simulation results are as follows Figure 8 As shown, in the upper left and lower left figures, purple represents PVA, and the various colored substances encapsulated within it represent FGF21 or GLP-1; VDW represents van der Waals forces, Coulomb represents Coulomb forces, Total represents the sum of van der Waals forces and Coulomb forces; SASA represents the total surface area of ​​PVA combined with FGF21 or GLP-1.

[0053] according to Figure 8 Molecular simulation results further revealed the PVA encapsulating FGF21 and GLP-1RA. tsPBAThe unique interaction modes between the microneedles, a unique polymer system, and FGF21 and LG are key factors contributing to the differences in their release rates. Specifically, the interaction strength and type between PVA microneedles and FGF21 and LG differ significantly, and these differences directly affect the drug's diffusion behavior and release kinetics within the carrier.

[0054] Example 2 (1) In this embodiment, a PVA is prepared. tsPBA The microneedle system, the steps are as follows: Synthesis of TSPBA: 1 g of 4-(bromomethyl)phenylboronic acid (4.7 mmol) and N,N,N′,N′-tetramethyl-1,3-propanediamine (1.5 mmol) were weighed and dissolved in 40 mL of N,N-dimethylamide (DMF) and stirred at 60 °C for 24 h. The reaction mixture was then poured into 100 mL of tetrahydrofuran (THF) to precipitate the product. The precipitate was collected by high-speed centrifugation (10,000 rpm, 5 min) and washed with THF (3 × 20 mL), and finally dried in a vacuum drying oven.

[0055] Synthesis of FGF21 / GLP-1RA@rMN microneedles: The mold used to manufacture the microneedles was purchased from Taizhou Microchip Medical Technology Co., Ltd. This mold was a 10×10 array with needles 600 μm long, 300 μm in bottom diameter, and 600 μm in tip-to-tip distance. A PVA-TsPBA solution was prepared by mixing 15% PVA and 3% TSPBA at a volume ratio of 5:1. FGF21 and GLP-1RA were mixed with the PVA-TsPBA solution to achieve concentrations of 0.2 mg / mL and 0.3 mg / mL for FGF21 and GLP-1RA, respectively. This mixture was then added dropwise to the microneedle mold and incubated under vacuum for 10 min.

[0056] Synthesis of rMN microneedles: The difference between rMN and FGF21 / GLP-1RA@rMN is that the concentrations of both FGF21 and GLP-1RA are 0 mg / mL.

[0057] Synthesis of GLP-1RA@rMN microneedles: The difference from FGF21 / GLP-1RA@rMN is that the concentration of FGF21 is 0 mg / mL.

[0058] Synthesis of FGF21@rMN microneedles: The difference from FGF21 / GLP-1RA@rMN is that the concentration of FGF21 is 0 mg / mL.

[0059] (2) Establishment of a full-thickness skin resection wound model for diabetes On the evening of day -7 of the experiment, mice were fasted but allowed water. On the morning of day -6, mice were intraperitoneally injected with STZ at a dose of 150 mg / kg to induce a diabetic model. On day -1, blood glucose concentration was measured using tail vein blood collection. Mice with a blood glucose concentration higher than 16.7 mmol / L were randomly identified as diabetic mice and randomly divided into 5 groups of 10 mice each for the formal experiment. On day 0, mice were anesthetized with 1% sodium pentobarbital, and their backs were prepared: hair was shaved, and depilatory cream was evenly applied to fully expose the back skin. Then, two circular full-thickness skin defects with a diameter of 6 mm were prepared on the exposed skin using a punching machine. To eliminate interference from external factors such as wound contraction caused by mouse activity on the healing process, a black silicone ring with an inner diameter of 8 mm, an outer diameter of 16 mm, and a thickness of 0.5 mm was used and attached to the periphery of the wound with double eyelid tape, ensuring that the wound was exactly centered on the silicone ring. Mice were subsequently treated with Control, rMN, GLP-1RA@rMN, FGF21@rMN, and FGF21 / GLP-1RA@rMN on days 0, 3, 7, and 10. After administration, the wounds were sealed with transparent 3M dressings. To reinforce the dressing and prevent it from being scratched or bitten off, a medical bandage was wrapped around the mouse's torso and adjusted to a suitable tightness. Finally, the bandage was sutured to the mouse's back skin using 4-0 sterile sutures to ensure the stability of the entire protective device during the experiment. The wounds were photographed on days 0, 3, 7, 10, and 14 to record the healing progress at different time points. On days 3, 7, and 14, 3-4 mice were randomly selected for back wound sampling, as well as tissue samples from the heart, liver, spleen, lungs, and kidneys. The collected skin tissue was gently washed with physiological saline and laid flat on filter paper. The skin and organs were divided in half: one half was placed in an embedding cassette and immediately immersed in a 4% paraformaldehyde solution for fixation, in preparation for subsequent dehydration, embedding, and sectioning; the other half was transferred to a centrifuge tube, quickly placed on dry ice to freeze, and then stored at -80 ℃ for later use.

[0060] (3) Based on the good biocompatibility, cell migration-promoting ability, and antioxidant capacity of FGF21 / GLP-1RA@rMN in vitro, a diabetic wound model was constructed using C57BL / 6 mice to evaluate its in vivo therapeutic effect. The specific procedure is as follows: Figure 9As shown in Figure A, on day -6, mice that were fasted but allowed water were given an intraperitoneal injection of STZ and continued to be fed. Blood glucose levels were measured on day -1, and mice with a random blood glucose level greater than 16.7 mmol / L were selected for the experiment. On day 0, a 6 mm diameter ring of skin was excised from the back of each mouse, and an 8 mm diameter adhesive band was applied to prevent skin wrinkling. The mice were then randomly divided into five groups: Control, rMN, GLP-1RA@rMN, FGF21@rMN, and FGF21 / GLP-1RA@rMN, designated as group IV, to study the therapeutic effect of FGF21 / GLP-1RA@rMN on diabetic wounds. Figure 9 As shown in Figure B, by day 3, the wound in the FGF21 / GLP-1RA@rMN group had healed by almost half, much faster than the other groups. By day 14, the wound in the FGF21 / GLP-1RA@rMN group had almost completely healed, while the Control group still had obvious unhealed areas. Figure 9 C and Figure 9 The wound statistics chart quantified this: on day 3, the FGF21 / GLP-1RA@rMN group had healed by approximately 50%, while the Control group had healed by approximately 80%. By day 14, the FGF21 / GLP-1RA@rMN group had almost completely healed, while the Control group still had approximately 40% of the wound remaining. Furthermore, the chart shows that the healing effect of the FGF21 / GLP-1RA@rMN group was significantly faster than that of the FGF21@rMN and GLP-1RA@rMN groups, indicating that combined medication can significantly accelerate wound healing.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A microneedle system, comprising a microneedle substrate and a microneedle tip, characterized in that, The microneedle tip comprises cross-linked polyvinyl alcohol hydrogel and FGF21 and GLP-1RA encapsulated within the cross-linked polyvinyl alcohol hydrogel; The cross-linked polyvinyl alcohol hydrogel is prepared from polyvinyl alcohol using a compound of formula I as a cross-linking agent, wherein the structure of compound of formula I is as follows: 。 2. The microneedle system according to claim 1, characterized in that, The mass ratio of FGF21 to GLP-1RA is 2:(2.8-3.2). And / or, the microneedle substrate is a cross-linked polyvinyl alcohol hydrogel.

3. The microneedle system according to claim 1, characterized in that, The concentration of FGF21 in the microneedle tip is 0.18 mg / mL - 0.22 mg / mL; the concentration of GLP-1RA in the microneedle tip is 0.28 mg / mL - 0.32 mg / mL.

4. The use of the microneedle system according to any one of claims 1-3 in a pharmaceutical preparation for treating diabetic foot ulcers.

5. The use of the microneedle system according to any one of claims 1-3 in a pharmaceutical formulation for upregulating the SIRT1 / AMPK pathway to improve cell viability after high glucose-induced damage.

6. A method for preparing the microneedle system according to any one of claims 1-3, characterized in that, FGF21 and GLP-1RA were added to an aqueous solution containing a crosslinking agent and polyvinyl alcohol to obtain a mixed solution. The mixed solution was added to the needle tip of a microneedle mold. Then, a mixed aqueous solution of polyvinyl alcohol and crosslinking agent was added to the mold to fill it. After the crosslinking reaction was carried out, the mold was demolded to obtain a microneedle system.

7. The method for preparing the microneedle system according to claim 6, characterized in that, The concentration of polyvinyl alcohol in the mixed solution is 0.06 g / ml - 0.07 g / ml, and the ratio of the crosslinking agent to the concentration of polyvinyl alcohol in the mixed aqueous solution is 1:3.5-4.

5.

8. The method for preparing the microneedle system according to claim 6, characterized in that, Bisphenol A and N,N,N,N-tetramethyl-1,3-propanediamine were dissolved in N,N-dimethylamide to obtain a pre-reaction solution, which was then reacted. The reaction solution was then transferred to tetrahydrofuran to obtain a precipitate, which was the crosslinking agent.

9. The method for preparing the microneedle system according to claim 8, characterized in that, The concentration of bisphenol A in the pre-reaction solution is 1.15 mmol / ml - 1.2 mmol / ml, and the concentration of N,N,N,N-tetramethyl-1,3-propanediamine is 0.035 mmol / ml - 0.040 mmol / ml; and / or, the reaction temperature is 55℃-65℃, and the reaction time is 22h-26h.

10. The method for preparing the microneedle system according to claim 6, characterized in that, Before filling the mold with the mixed aqueous solution of polyvinyl alcohol and crosslinking agent, vacuum the mold for 8-12 minutes; and / or after filling the mold with the mixed aqueous solution of polyvinyl alcohol and crosslinking agent, centrifuge the mold at 1800rpm-2200rpm for 18-22 minutes.

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