Separable microneedle patch loaded with double natural medicines as well as preparation method and application of separable microneedle patch

Abstract

The invention discloses a double natural drug loaded separable microneedle patch, which comprises a microneedle tip array layer prepared from a polylactic acid-glycolic acid copolymer loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a separable backing layer connected to the back of the microneedle tip array layer, and the separable backing layer is prepared from polyvinyl alcohol and polyvinylpyrrolidone. In addition, the invention further discloses a preparation method and application of the separable microneedle patch loaded with the double natural drugs. According to the invention, Rk3 and DATS are cooperatively loaded in the PLGA microneedle, so that the effects of immunoregulation and angiogenesis promotion are exerted, the problems of local immune imbalance and tissue perfusion disorder in CLI treatment are remarkably improved, and anti-inflammatory and vascular remodeling dual-targeting combined treatment is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, and in particular relates to a detachable microneedle patch loaded with dual natural drugs, and a preparation method and application thereof. Background Art

[0002] Severe lower limb ischemia (CLI) is the most severe clinical manifestation of peripheral arterial disease. It is mainly caused by severe insufficient blood perfusion due to atherosclerosis, which in turn causes persistent pain, ulcers and tissue necrosis, and has extremely high disability and mortality rates. The current mainstream treatment options for CLI include interventional vascular reconstruction (such as drug-eluting stents, artificial vascular bypass surgery), drug therapy, and cell or gene therapy. However, these methods generally have problems such as high vascular restenosis rate, strong trauma, large systemic side effects, and unstable efficacy. In addition, some patients are classified as "no-option CLI" due to surgical contraindications or poor efficacy. Clinical treatment needs have not yet been effectively met.

[0003] Studies have found that the pathogenesis of CLI is not limited to blood flow obstruction, but is closely related to local chronic inflammation and immune imbalance. Inflammation can induce vascular wall thickening and lumen stenosis, further aggravating tissue ischemia and hindering angiogenesis and tissue repair. Therefore, controlling local inflammation and promoting M2 macrophage polarization and angiogenesis through immune regulation strategies have become new treatment directions. Currently, a variety of natural active molecules, protein factors and metal ions have been shown to have the effect of promoting the restoration of blood perfusion in ischemic tissues. However, due to the short half-life of drugs, poor local retention, systemic toxicity and side effects, and low delivery efficiency, their therapeutic effects are still unsatisfactory. Summary of the Invention

[0004] The present invention addresses the shortcomings of the prior art by providing a detachable microneedle patch loaded with dual natural drugs. By synergistically loading Rk3 and DATS within PLGA microneedles, this microneedle patch exerts immunomodulatory and angiogenic effects, significantly alleviating the local immune imbalance and tissue perfusion impairments associated with CLI treatment, achieving dual-targeted "anti-inflammatory and angiogenesis" therapy.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: a detachable microneedle patch loaded with dual natural drugs, characterized in that it includes a microneedle tip array layer made of polylactic acid-glycolic acid copolymer loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a detachable backing layer connected to the back of the microneedle tip array layer, and the detachable backing layer is made of polyvinyl alcohol and polyvinyl pyrrolidone.

[0006] The above-mentioned dual-natural drug-loaded detachable microneedle patch is characterized in that the structure of the microneedle tip array layer is 8×8 to 12×12, with a total area of 100mm 2 ~150mm 2 The total height of the microneedle is 700μm~1200μm, the height of the cone section is 500μm~800μm, the height of the frustum section is 200μm~400μm, and the needle spacing is 600μm~900μm.

[0007] Furthermore, the present invention provides a method for preparing the above-mentioned dual-natural drug-loaded detachable microneedle patch, characterized in that it comprises the following steps:

[0008] Step 1: dissolving poly(lactic-co-glycolic acid) copolymer in an organic solvent to obtain a PLGA solution with a concentration of 20% to 30% (w / v), and then adding rare ginsenoside Rk3 and diallyl trisulfide to obtain a drug-loaded solution; the concentration of Rk3 in the drug-loaded solution is 100 μg / mL to 500 μg / mL, and the concentration of diallyl trisulfide is 5 μg / mL to 25 μg / mL;

[0009] Step 2: Inject the drug-loaded liquid described in step 1 into the microneedle mold, fill the microneedle cavity with the liquid by centrifugation at a centrifugal speed of 3000g to 4000g for 2min to 5min, repeat the injection molding 2 to 5 times, and vacuum dry for 12h to 48h to obtain a solidified microneedle tip array layer;

[0010] Step 3: Polyvinyl alcohol and polyvinyl pyrrolidone are prepared into a 20% to 30% (w / v) aqueous solution in a mass ratio of (3 to 5): 1, and the solution is added dropwise to the surface of the solidified microneedle tip array layer as a backing material. After drying at room temperature for 12 to 48 hours, a detachable backing layer is formed, and the mold is removed to obtain a detachable microneedle patch loaded with dual natural drugs.

[0011] The above method is characterized in that the organic solvent in step 1 is 1,4-dioxane.

[0012] The above method is characterized in that the molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer in step 1 is 45:55 to 60:40.

[0013] The above method is characterized in that the detachable backing layer can be completely dissolved and separated from the microneedle tip array layer within 1 minute to 5 minutes after skin insertion.

[0014] Furthermore, the present invention provides an application of the above-mentioned dual-natural drug-loaded detachable microneedle patch in preparing a platform for treating severe lower limb ischemia.

[0015] The above application is characterized in that the detachable microneedle patch can achieve polarization of macrophages from M0 and M1 types to M2 types in the CLI model, inhibit the activity of TNF and NF-κB pathways, and promote the expression of IL-10 and VEGF.

[0016] The above application is characterized in that the platform includes a detachable microneedle patch loaded with dual natural drugs and low-intensity pulsed ultrasound. The low-intensity pulsed ultrasound can enhance the permeability of tissue gaps and accelerate the formation of H2S nanobubbles through stable cavitation, thereby synergistically promoting the penetration of drugs into deep ischemic tissues.

[0017] The above application is characterized in that the parameters of the low-intensity pulsed ultrasound are: frequency 0.5MHz to 1.5MHz, sound intensity 0.3W / cm 2 ~2.0W / cm 2 , applied intermittently for 5–20 minutes daily, which not only enhances local drug release but also avoids tissue damage.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. The dual-drug synergistic mechanism is clear, localized, and comprehensive: By synergistically loading Rk3 and DATS in PLGA microneedles, the present invention exerts immune regulation and promotes angiogenesis, significantly improving the local immune imbalance and tissue perfusion disorders in CLI treatment, and achieving a dual-targeted combined treatment of "anti-inflammatory + vascular reconstruction";

[0020] 2. Structural innovation to meet the therapeutic needs of controlled penetration and sustained release: This invention adopts a double-layer microneedle structure. The microneedle tip array layer serves as the drug loading area, and the detachable backing layer serves as the support and separation area. The PVA / PVP dissolves rapidly and can be quickly separated after insertion, ensuring that the drug needle body remains in the dermis layer for sustained drug release and improving treatment compliance.

[0021] 3. Combined low-intensity pulsed ultrasound to enhance therapeutic effects: This invention introduces low-intensity pulsed ultrasound physical stimulation, utilizes the stable cavitation effect to promote tissue permeability, and simultaneously accelerates DATS to generate H2S gas, forming a "gas nanomotor" that synergistically promotes drug delivery to deep tissues, increases local drug concentration, and enhances therapeutic effects;

[0022] 4. Good biocompatibility and controllable degradation: The materials used in this invention are all FDA-approved medical-grade biodegradable materials. The microneedles can gradually degrade and release drugs in the tissue, avoiding the problem of foreign body residue and having good biosafety and tissue compatibility.

[0023] 5. Highly convenient application and adaptable to various lesion treatment scenarios: The microneedle patch of the present invention is suitable for self-administration, is easy to operate, and has the potential for modular design. It can be subsequently expanded to the localized precision treatment of other types of ischemic or chronic inflammatory diseases;

[0024] 6. The efficacy is fully verified and has significant clinical translation prospects: Animal experiments have verified that the platform of the present invention can achieve more than 97% blood flow recovery within 21 days, which is significantly better than the single-drug or non-ultrasound control group, and has clear therapeutic effect advantages and translational value.

[0025] The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the structure of the dual-natural drug-loaded detachable microneedle patch of the present invention.

[0027] Figure 2 The figure is a schematic diagram of the process for preparing the dual-natural drug-loaded detachable microneedle patch of the present invention.

[0028] Figure 3 This is a scanning electron microscope (SEM) image of the dual-natural drug-loaded detachable microneedle patch of the present invention.

[0029] Figure 4 These are actual photos and fluorescence imaging images of the dual-natural drug-loaded detachable microneedle patch of the present invention after being inserted into the back skin of a mouse.

[0030] Figure 5 This is a graph showing the mechanical properties of the dual-natural drug-loaded detachable microneedle patch of the present invention.

[0031] Figure 6 This is the in vitro release curve of diallyl trisulfide drug under different experimental conditions.

[0032] Figure 7 This is an SEM image of the microstructural changes of the detachable microneedle patch loaded with dual natural drugs during the microneedle release process of the present invention, showing the morphological evolution of PLGA microneedles during in vitro degradation.

[0033] Figure 8 This is a quantitative analysis chart of the red blood cell hemolysis rate of the detachable microneedle patch loaded with dual natural drugs of the present invention.

[0034] Figure 9 Graph showing the results of HUVECs cell viability and migration ability testing of the dual-natural drug-loaded detachable microneedle patch of the present invention.

[0035] Figure 10Flow cytometric images of the immunophenotype of RAW264.7 cells after treatment with different microneedles.

[0036] Figure 11 This is a quantitative analysis chart of the ELISA test results of RAW264.7 cells.

[0037] Figure 12 These are experimental images and quantitative analysis diagrams showing the promotion of HUVECs cell migration and tube formation after co-incubation of RAW264.7 cells with the dual-natural drug-loaded detachable microneedle patch of the present invention.

[0038] Figure 13 These are the laser speckle imaging images of the mouse lower limb ischemia model and the quantitative analysis images of blood perfusion on the 7th, 14th, and 21st days.

[0039] Figure 14 Immunofluorescence images and quantitative analysis of immune markers CD86 and CD206 in ischemic tissues.

[0040] Figure 15 Immunofluorescence images and quantitative analysis of neovascularization markers α-SMA and CD31 in ischemic tissues.

[0041] Figure 16 This is the transcriptomics analysis result diagram, including PCA analysis, volcano plot, GO enrichment map, KEGG pathway analysis and key gene expression heat map.

[0042] Figure 17 This is the Western blot band diagram and semi-quantitative analysis results of VEGF protein expression in tissues. DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the following examples. These examples are intended only to further illustrate the present invention and are not to be construed as limiting the scope of the present invention. Experimental procedures where specific conditions are not specified in the examples are generally based on conventional conditions, those described in the manual, or those recommended by the manufacturer. The equipment, materials, and reagents used are all commercially available unless otherwise specified.

[0044] The dual-natural drug-loaded detachable microneedle patch of the present invention includes a microneedle tip array layer made of a polylactic acid-glycolic acid copolymer loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a detachable backing layer connected to the back of the microneedle tip array layer. The detachable backing layer is made of polyvinyl alcohol and polyvinyl pyrrolidone, has good mechanical support properties and rapid water solubility, can quickly absorb moisture and dissolve after piercing the skin, and can be separated from the microneedle array layer; the structure of the microneedle tip array layer is 8×8 to 12×12, with a total area of 100mm 2 ~150mm2 The total height of the microneedle is 700μm~1200μm, the height of the cone section is 500μm~800μm, the height of the frustum section is 200μm~400μm, and the needle spacing is 600μm~900μm.

[0045] Example 1: Dual-Natural Drug-Loaded Detachable Microneedle Patch

[0046] The dual natural drug-loaded detachable microneedle patch of this embodiment is as follows Figure 1 As shown, it includes a microneedle tip array layer made of polylactic acid-co-glycolic acid loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a detachable backing layer connected to the back of the microneedle tip array layer, wherein the detachable backing layer is made of polyvinyl alcohol and polyvinyl pyrrolidone; the microneedle body is as shown Figure 3 The structure is shown as a double-segment structure, with the front segment being a conical drug loading area and the rear segment being a truncated cone transition area. The overall geometry is "rocket-shaped" to enhance insertion efficiency and separation from the needle tip backing. The array is arranged in a 10×10 pattern with a total area of 120 mm. 2 The total height of the microneedle is 1000 μm, the height of the cone section is 700 μm, the height of the frustum section is 300 μm, and the needle spacing is 800 μm.

[0047] The preparation process of the dual natural drug-loaded detachable microneedle patch of this embodiment is as follows: Figure 2 As shown, the specific steps include:

[0048] Step 1: dissolving poly(lactic acid-co-glycolic acid) in an organic solvent, 1,4-dioxane, to obtain a 25% (w / v) PLGA solution; then adding rare ginsenoside Rk3 and diallyl trisulfide to obtain a drug-loaded solution; the concentration of Rk3 in the drug-loaded solution is 300 μg / mL, and the concentration of diallyl trisulfide is 15 μg / mL; the molar ratio of lactic acid to glycolic acid in the poly(lactic acid-co-glycolic acid) is 50:50;

[0049] Step 2: Inject the drug-loaded liquid described in step 1 into the microneedle mold, and fill the microneedle cavity with the liquid by centrifugation at a centrifugal speed of 3500g for 3 minutes. Repeat the injection molding 4 times and vacuum dry for 24 hours to obtain a solidified microneedle tip array layer;

[0050] Step 3: Prepare a 25% (w / v) aqueous solution of polyvinyl alcohol and polyvinyl pyrrolidone in a mass ratio of 4:1, and drop it onto the surface of the cured microneedle tip array layer as a backing material. After drying at room temperature for 24 hours, a detachable backing layer is formed, and the mold is removed to obtain a detachable microneedle patch loaded with dual natural drugs.

[0051] The detachable backing layer of this embodiment is inserted into the skin as Figure 4As shown, it can be completely dissolved and separated from the microneedle tip array layer within 1 to 5 minutes.

[0052] The dual-natural drug-loaded detachable microneedle patch of this embodiment can be used to treat critical limb ischemia (Critical Limb Ischemia, CLI) by transdermal delivery of Rk3 and DATS to achieve a synergistic therapeutic effect of local immune regulation and angiogenesis.

[0053] The detachable microneedle patch of this embodiment can polarize macrophages from M0 and M1 to M2 in the CLI model, inhibit the activity of TNF and NF-κB pathways, and promote the expression of IL-10 and VEGF.

[0054] The detachable microneedle patch of this embodiment can be used in conjunction with low-intensity pulsed ultrasound (LIPUS) to enhance drug tissue penetration and local therapeutic effects, and can achieve a blood flow recovery rate of more than 90% in ischemic limbs within 21 days. The parameters of the low-intensity pulsed ultrasound are: frequency 1.0 MHz, sound intensity 0.5 W / cm 2 .

[0055] Example 2: Dual-Natural Drug-Loaded Detachable Microneedle Patch

[0056] The dual natural drug-loaded detachable microneedle patch of this embodiment is as follows Figure 1 As shown, it includes a microneedle tip array layer made of polylactic acid-co-glycolic acid loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a detachable backing layer connected to the back of the microneedle tip array layer, wherein the detachable backing layer is made of polyvinyl alcohol and polyvinyl pyrrolidone; the microneedle body is as shown Figure 3 The structure is shown as a dual-stage structure, with the front section being a conical drug loading area and the rear section being a truncated cone transition area. The overall geometry is "rocket-shaped" to enhance insertion efficiency and separation from the needle tip backing. The array is arranged in an 8×8 pattern with a total area of 100mm. 2 The total height of the microneedle is 700 μm, the height of the cone section is 500 μm, the height of the frustum section is 200 μm, and the needle spacing is 600 μm.

[0057] The preparation process of the dual natural drug-loaded detachable microneedle patch of this embodiment is as follows: Figure 2 As shown, the specific steps include:

[0058] Step 1: dissolving poly(lactic acid-co-glycolic acid) in an organic solvent, 1,4-dioxane, to obtain a 20% (w / v) PLGA solution; then adding rare ginsenoside Rk3 and diallyl trisulfide to obtain a drug-loaded solution; the concentration of Rk3 in the drug-loaded solution is 100 μg / mL, and the concentration of diallyl trisulfide is 25 μg / mL; the molar ratio of lactic acid to glycolic acid in the poly(lactic acid-co-glycolic acid) is 45:55;

[0059] Step 2: Inject the drug-loaded liquid described in step 1 into the microneedle mold, and fill the microneedle cavity with the liquid by centrifugation at a centrifugal speed of 3000g for 5 minutes. Repeat the injection molding twice and vacuum dry for 12 hours to obtain a solidified microneedle tip array layer;

[0060] Step 3: Polyvinyl alcohol and polyvinyl pyrrolidone are prepared into a 20% (w / v) aqueous solution in a mass ratio of 3:1, and the solution is added dropwise to the surface of the cured microneedle tip array layer as a backing material. After drying at room temperature for 12 hours, a detachable backing layer is formed, and the mold is removed to obtain a detachable microneedle patch loaded with dual natural drugs.

[0061] The detachable backing layer of this embodiment can be completely dissolved and separated from the microneedle tip array layer within 1 to 5 minutes after being inserted into the skin.

[0062] The dual-natural drug-loaded detachable microneedle patch of this embodiment can be used to treat critical limb ischemia (Critical Limb Ischemia, CLI) by transdermal delivery of Rk3 and DATS to achieve a synergistic therapeutic effect of local immunomodulation and angiogenesis.

[0063] The detachable microneedle patch of this embodiment can polarize macrophages from M0 and M1 to M2 in the CLI model, inhibit the activity of TNF and NF-κB pathways, and promote the expression of IL-10 and VEGF.

[0064] The detachable microneedle patch of this embodiment can be used in conjunction with low-intensity pulsed ultrasound (LIPUS) to enhance drug tissue penetration and local therapeutic effects, and can achieve a blood flow recovery rate of more than 90% in ischemic limbs within 21 days. The parameters of the low-intensity pulsed ultrasound are: frequency 0.5 MHz, sound intensity 0.3 W / cm 2 .

[0065] Example 3: Dual-Natural Drug-Loaded Detachable Microneedle Patch

[0066] The dual natural drug-loaded detachable microneedle patch of this embodiment is as follows Figure 1As shown, it includes a microneedle tip array layer made of polylactic acid-co-glycolic acid loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a detachable backing layer connected to the back of the microneedle tip array layer, wherein the detachable backing layer is made of polyvinyl alcohol and polyvinyl pyrrolidone; the microneedle body is as shown Figure 3 The structure is shown as a dual-stage structure, with the front section being a conical drug loading area and the rear section being a truncated cone transition area. The overall geometry is "rocket-shaped" to enhance insertion efficiency and separation from the needle tip backing. The array is arranged in 12×12, with a total area of 150mm. 2 The total height of the microneedle is 1200 μm, the height of the conical section is 800 μm, the height of the frustum section is 400 μm, and the needle spacing is 900 μm.

[0067] The preparation process of the dual natural drug-loaded detachable microneedle patch of this embodiment is as follows: Figure 2 As shown, the specific steps include:

[0068] Step 1: dissolving poly(lactic acid-co-glycolic acid) in an organic solvent, 1,4-dioxane, to obtain a 30% (w / v) PLGA solution; then adding rare ginsenoside Rk3 and diallyl trisulfide to obtain a drug-loaded solution; the concentration of Rk3 in the drug-loaded solution is 500 μg / mL, and the concentration of diallyl trisulfide is 5 μg / mL; the molar ratio of lactic acid to glycolic acid in the poly(lactic acid-co-glycolic acid) is 60:40;

[0069] Step 2: Inject the drug-loaded liquid described in step 1 into the microneedle mold, and fill the microneedle cavity with the liquid by centrifugation at a centrifugal speed of 4000g for 2 minutes. Repeat the injection molding 5 times and vacuum dry for 48 hours to obtain a solidified microneedle tip array layer;

[0070] Step 3: Prepare a 30% (w / v) aqueous solution of polyvinyl alcohol and polyvinyl pyrrolidone in a mass ratio of 5:1, and drop it onto the surface of the cured microneedle tip array layer as a backing material. After drying at room temperature for 48 hours, a detachable backing layer is formed, and the mold is removed to obtain a detachable microneedle patch loaded with dual natural drugs.

[0071] The detachable backing layer of this embodiment can be completely dissolved and separated from the microneedle tip array layer within 1 to 5 minutes after being inserted into the skin.

[0072] The dual-natural drug-loaded detachable microneedle patch of this embodiment can be used to treat critical limb ischemia (Critical Limb Ischemia, CLI) by transdermal delivery of Rk3 and DATS to achieve a synergistic therapeutic effect of local immune regulation and angiogenesis.

[0073] The detachable microneedle patch of this embodiment can polarize macrophages from M0 and M1 to M2 in the CLI model, inhibit the activity of TNF and NF-κB pathways, and promote the expression of IL-10 and VEGF.

[0074] The detachable microneedle patch of this embodiment can be used in conjunction with low-intensity pulsed ultrasound (LIPUS) to enhance drug tissue penetration and local therapeutic effects, and can achieve a blood flow recovery rate of more than 90% in ischemic limbs within 21 days. The parameters of the low-intensity pulsed ultrasound are: frequency 1.5 MHz, sound intensity 2.0 W / cm 2 .

[0075] The microneedle patch prepared in Example 1 was used as the experimental object to test various properties of the microneedle patch:

[0076] Example 4: Microneedle Structure and Performance Characterization Method

[0077] The macroscopic appearance of the microneedle patch was observed using a stereo microscope to confirm that the microneedles were evenly arranged and had neat edges. The microneedle morphology was characterized using a scanning electron microscope to observe whether the connection between the tip and base of the needle was clear and complete.

[0078] like Figure 1 and Figure 3 As shown, the microneedle body has a "rocket-shaped" double-segment structure, including a conical front section and a truncated cone-shaped back section. The array is arranged in 10×10, with a total area of 120mm 2 The total height of the microneedle is 1000 μm, the height of the cone section is 700 μm, the height of the frustum section is 300 μm, and the needle spacing is 800 μm.

[0079] Example 5: Verification of the mechanical properties and separation ability of microneedles

[0080] The microneedle patch was fixed on a rigid substrate and subjected to compression performance testing using a material mechanics tester. The test conditions were set as follows: probe pressing speed 0.1mm / s, pressing depth 0.4mm, and recording the single needle puncture force and structural integrity. The results are as follows Figure 5 As shown, the measured single needle penetration force was 0.19 N, and the microneedle retention rate was 100%. The test results show that the average penetration force of the microneedles produced by the present invention is higher than the minimum force required for skin puncture of 0.1 N, and can smoothly penetrate the surface layer of the skin, meeting the skin penetration threshold.

[0081] Then, the microneedle patch was placed on the surface of the mouse's back skin, and constant finger pressure was applied for 5 to 60 seconds. After the backing absorbed moisture and dissolved automatically, the backing was removed and it was observed that all the microneedle tips had successfully penetrated the subcutaneous tissue. Figure 4 The results showed that the backing layer was completely detached, the microneedle tip could be effectively left under the skin, and the remaining needle body was not loose or broken.

[0082] Example 6: In vitro drug release experiment

[0083] The microneedle patches were placed in the following four release media for in vitro release experiments: ①PBS (control group); ②PBS+2mM GSH; ③PBS+LIPUS; ④PBS+2mM GSH+LIPUS, simulating the reducing environment and ultrasonic stimulation conditions in CLI tissue.

[0084] The LIPUS parameters are set to 1.0 MHz frequency and 1.0 W / cm 2 Ultrasound was applied for 10 min daily to promote microneedle structure degradation and drug diffusion.

[0085] Each group of samples was immersed in a 37°C constant temperature shaker for 21 days, with regular sampling and replacement of buffer. The DATS concentration was measured using a UV-visible spectrophotometer, and the H2S production was determined using a hydrogen sulfide detection kit. Figure 6 The morphological evolution of the PLGA microneedle tip during in vitro degradation is shown in Figure 7 shown.

[0086] The results showed that in the group containing GSH and LIPUS, the cumulative release rate of the drug reached 60-80%, and the release curve was stable; the H2S concentration was maintained between 500 and 900 nM, and there was no obvious toxic effect.

[0087] Example 7: Evaluation of blood compatibility and cytocompatibility of microneedle system

[0088] Fresh mouse blood was diluted to a 2% red blood cell suspension and mixed with the aqueous extract of the microneedle patch (concentration of 0.05-0.2 g / mL) and incubated at 37°C for 30-90 minutes. After centrifugation, the supernatant was collected and the absorbance was measured to calculate the hemolysis rate. Figure 8 As shown, the results showed that the hemolysis rate of each group was less than 5%, which was in line with the industry's common judgment standards for hemolytic materials in the biological evaluation of medical devices, indicating that the microneedle patch has good blood compatibility.

[0089] Human umbilical vein endothelial cells (HUVECs) were seeded in 96-well plates and treated with 50 mg / mL of microneedle extract. After 24 to 72 hours of culture, cell proliferation activity was assessed using CCK-8 reagent. AO / EB double staining was used to further assess cell growth.

[0090] The results are as follows Figure 9 As shown, the results showed that no obvious toxic reactions were observed in all treatment groups, the cell survival rate was higher than 95%, and the cell morphology was intact, indicating that the microneedle system has good blood and cell compatibility.

[0091] Example 8: In vitro experiments related to immune regulation

[0092] In order to evaluate the immunomodulatory effect of the microneedle patch of the present invention, a RAW 264.7 cell-related experiment was designed. First, macrophage cell line RAW264.7 was cultured at 5×10 6 Cells were seeded at a density of 100 μg / mL in 6-well cell culture plates and cultured at 37°C, 5% CO₂ for 24 hours. Following incubation, different inducers were added to each well: 100 ng / mL lipopolysaccharide (LPS) was added to one group to induce M1 macrophage polarization; 20 ng / mL interleukin-4 (IL-4) was added to another group to induce M2 macrophage polarization. A standard culture medium (DMEM) group served as a blank control. After 12 hours of induction, the culture medium was replaced with the pre-treated microneedle solution for the MN@Rk3, MN@Rk3@DATS, and MN@All groups, and incubated for an additional 24 hours. The same induction conditions were maintained for the LPS and IL-4 groups until the end of the experiment. After incubation, cells were gently scraped from each well using a cell scraper, washed twice with PBS, and then stained with antibodies. Fluorescently labeled antibodies against the M1 macrophage marker CD86 and the M2 marker CD206 were used for incubation. After staining, flow cytometry was used, and the data were analyzed using FlowJo software to assess the effects of each microneedle treatment group on the polarization (M1 / M2) of RAW264.7 cells. To further validate the microneedle platform's regulatory effect on macrophage secretory function, ELISA was used to measure the expression levels of inflammatory cytokines in the cell supernatants of the different treatment groups.

[0093] The results are as follows Figure 10 、 11 As shown, the results showed that the MN@All group had the best effect of promoting the polarization of M0 and M1 to M2 anti-inflammatory phenotype, and at the same time had the best effect of promoting anti-inflammatory factors and inhibiting the release of pro-inflammatory factors, indicating that this microneedle system has good blood and cell compatibility.

[0094] Example 9: In vitro experiments related to angiogenesis

[0095] To evaluate the effect of the microneedle patch on angiogenesis, a multi-cell co-culture experiment was designed. First, RAW264.7 macrophages were co-cultured with human umbilical vein endothelial cells (HUVECs). Transfer factors indirectly acted to simulate the vascular reconstruction process regulated by the immune microenvironment.

[0096] RAW264.7 cells were treated with the microneedle extract (50 mg / mL) for 24 hours, and the supernatant was collected and used to culture HUVECs. Cell migration (wound wound healing) and angiogenesis (Matrigel tube formation) assays were performed to assess changes in vascular biological behavior.

[0097] In the scratch test, HUVECs were cultured at a density of 1 × 10 5 Cells were seeded at a density of cells / mL in 24-well plates. After the monolayer became confluent, a scratch was made. Serum-free medium containing the treated supernatant was added and cultured for 12 hours before observing migration. The scratch closure rate was recorded and the migration velocity was calculated.

[0098] In the tube formation experiment, 200 μL of Matrigel was spread on a pre-cooled well plate, HUVECs were inoculated, and the microneedle pretreatment supernatant was added. After culturing for 6 to 12 hours, the formed capillary-like structure was observed, and indicators such as tube length and node number were counted.

[0099] The results are as follows Figure 12 As shown, the MN@All treatment group promoted cell migration rate to 70% to 90%, with a complete tubular structure network and dense branches, showing a significant pro-angiogenic effect.

[0100] In this example, RAW cells were treated with 50 mg / mL microneedle extract for 24 h, and the resulting supernatant induced an 83.29% wound closure rate and optimal tube formation in HUVECs.

[0101] Example 10: Animal model construction and treatment effect verification

[0102] C57BL / 6J male mice weighing 18-24 g were used as experimental subjects to establish a lower limb ischemia model to simulate the pathological state of CLI. Under anesthesia, the femoral artery was exposed, the proximal and distal femurs were ligated, and the midsection was cut to create a stable acute ischemic state.

[0103] Immediately after surgery, the microneedle patch prepared in the present invention was applied to the outer skin of the ischemic limb. The experimental groups were divided into the following groups: (1) blank control group (model group); (2) MN@Rk3 group; (3) MN@Rk3@DATS group; (4) MN@All group (combined with LIPUS).

[0104] Laser speckle contrast imaging (LSCI) was used to evaluate blood flow recovery in the ischemic limb on days 1, 7, 14, and 21 after surgery, and the perfusion ratio (ischemic side / contralateral side) was quantified.

[0105] The results are as follows Figure 13As shown, the blood flow recovery rate in the MN@All group was the fastest, with the perfusion ratio reaching 97.7% on the 21st day, which was significantly better than other control groups. At the same time, the limb retention rate evaluation showed that MN@All treatment could significantly reduce the risk of gangrene and amputation.

[0106] Example 11: Modulating the tissue immune microenvironment to promote angiogenesis and verification of its mechanism

[0107] Gastrocnemius muscle tissue was obtained from mice 21 days after surgery, paraffin sections were prepared, and immunofluorescence staining and molecular mechanism analysis were performed. Detection indicators included total macrophages (F4 / 80), M1 phenotype (CD86), M2 phenotype (CD206), and vascular markers (CD31, α-SMA).

[0108] Immunostaining results Figure 14 、 15 The results showed that the MN@All-treated group showed a significant decrease in CD86 positive signals and an increase in CD206 positive signals in the ischemic area, indicating that the immune microenvironment has shifted from an inflammatory state to a repair state. The MN@All group showed the most positive signals for CD31 and α-SMA, indicating a significant increase in the number of new blood vessels.

[0109] Total RNA was further extracted from gastrocnemius muscle tissue for transcriptome sequencing. Figure 16 As shown in the figure, PCA analysis, volcano plot screening and KEGG pathway enrichment analysis showed that the MN@All group significantly inhibited inflammatory signaling pathways such as TNF, NF-κB, and IL-17, and activated pro-angiogenic signaling pathways such as PI3K-Akt and ERK1 / 2.

[0110] In Western blot detection, the experimental results are as follows Figure 17 As shown in the figure, the expression of VEGF protein in the MN@All group was significantly upregulated compared with other groups, verifying the mechanistic basis of vascular reconstruction through immune reprogramming.

[0111] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment based on the technical essence of the invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A detachable microneedle patch loaded with dual natural drugs, characterized in that: The invention comprises a microneedle tip array layer made of polylactic acid-glycolic acid copolymer loaded with rare ginsenoside Rk3 and diallyl trisulfide, and a detachable backing layer connected to the back of the microneedle tip array layer, wherein the detachable backing layer is made of polyvinyl alcohol and polyvinyl pyrrolidone.

2. The detachable microneedle patch loaded with dual natural drugs according to claim 1, characterized in that: The structure of the microneedle tip array layer is 8×8 to 12×12, with a total area of 100mm 2 ~150mm 2 The total height of the microneedle is 700μm~1200μm, the height of the cone section is 500μm~800μm, the height of the frustum section is 200μm~400μm, and the needle spacing is 600μm~900μm.

3. A method for preparing the dual-natural drug-loaded detachable microneedle patch according to claim 1, characterized in that: The following steps are involved: Step 1: dissolving poly(lactic-co-glycolic acid) copolymer in an organic solvent to obtain a PLGA solution with a concentration of 20% to 30% (w / v), and then adding rare ginsenoside Rk3 and diallyl trisulfide to obtain a drug-loaded solution; the concentration of Rk3 in the drug-loaded solution is 100 μg / mL to 500 μg / mL, and the concentration of diallyl trisulfide is 5 μg / mL to 25 μg / mL; Step 2: Inject the drug-loaded liquid described in step 1 into the microneedle mold, fill the microneedle cavity with the liquid by centrifugation at a centrifugal speed of 3000g to 4000g for 2min to 5min, repeat the injection molding 2 to 5 times, and vacuum dry for 12h to 48h to obtain a solidified microneedle tip array layer; Step 3: Polyvinyl alcohol and polyvinyl pyrrolidone are prepared into a 20% to 30% (w / v) aqueous solution in a mass ratio of (3 to 5): 1, and the solution is added dropwise to the surface of the solidified microneedle tip array layer as a backing material. After drying at room temperature for 12 to 48 hours, a detachable backing layer is formed, and the mold is removed to obtain a detachable microneedle patch loaded with dual natural drugs.

4. The method according to claim 3, characterized in that The organic solvent in step 1 is 1,4-dioxane.

5. The method according to claim 3, characterized in that The molar ratio of lactic acid to glycolic acid in the polylactic acid-glycolic acid copolymer in step 1 is 45:55 to 60:

40.

6. The method according to claim 3, characterized in that The detachable backing layer can be completely dissolved and separated from the microneedle tip array layer within 1 to 5 minutes after being inserted into the skin.

7. Use of the dual-natural drug-loaded detachable microneedle patch as claimed in claim 1 in the preparation of a device for treating severe lower limb ischemia.

8. The use according to claim 7, characterized in that The detachable microneedle patch can polarize macrophages from M0 and M1 to M2 in the CLI model, inhibit the activity of TNF and NF-κB pathways, and promote the expression of IL-10 and VEGF.

9. The use according to claim 7, characterized in that The platform includes a dual-natural drug-loaded detachable microneedle patch and low-intensity pulsed ultrasound.

10. The use according to claim 9, characterized in that The parameters of the low-intensity pulsed ultrasound are: frequency 0.5 MHz to 1.5 MHz, sound intensity 0.3 W / cm 2 ~2.0W / cm 2 .

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