A microneedle patch for promoting diabetic wound healing and its preparation method

Abstract

The present invention relates to the field of pharmaceutical technology, and specifically discloses a microneedle patch for promoting the healing of diabetic wounds and a preparation method thereof. The microneedle patch includes a backing, graphene oxide adsorbed with cobalt ions dispersed inside the backing, microneedles provided on the backing, and hydrogen-releasing nanoparticles dispersed inside the microneedles. The hydrogen-releasing nanoparticles are mesoporous silica nanoparticles loaded with the hydrogen precursor drug ammonia borane. The microneedle patch for promoting the healing of diabetic wounds provided by the present invention combines microneedle transdermal drug delivery and nanomaterials to continuously release hydrogen locally at the wound surface to relieve inflammation. At the same time, hydrogen reduces graphene oxide to release cobalt ions to promote angiogenesis, has excellent photothermal antibacterial effects, effectively inhibits bacterial infection at the wound surface, and promotes the healing of diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a microneedle patch for promoting the healing of diabetic wounds and a preparation method thereof. Background Art

[0002] The non-healing of chronic wounds is one of the major threats to global health. In particular, about 15-25% of diabetic patients will develop diabetic foot ulcers. At present, the understanding of the complex wound healing process is still limited, and there is still a lack of effective treatment for chronically non-healing wounds.

[0003] The wound healing process generally consists of four stages: hemostasis, inflammation, proliferation, and remodeling. Bacterial infection is the most common and inevitable problem in wound healing. When a wound is infected, bacteria will cause a continuous inflammatory response at the infected site, which will further delay the healing process in the inflammatory stage. Therefore, alleviating wound inflammation is the key to promoting wound healing.

[0004] Related studies have found that hydrogen can neutralize hydroxyl radicals under acute oxidative stress conditions, can selectively neutralize strongly toxic free radicals such as hydroxyl radicals and peroxynitrite anions, and produce a very effective antioxidant effect. Hydrogen can not only reduce the level of reactive oxygen species (ROS), but also stimulate macrophages to polarize into an anti-inflammatory M2 phenotype, indicating excellent anti-inflammatory properties. Therefore, it is very meaningful to develop a microneedle patch that can continuously release hydrogen to alleviate wound inflammation.

[0005] Wound dressings can cover the wound and provide a temporary barrier against external infections, and can also act as an inductive template to guide the reorganization of skin cells and subsequent infiltration and integration of host tissues, showing a significant impact on wound healing. An ideal skin wound dressing needs to meet the following requirements: (1) good tissue compatibility, without causing toxicity or inflammation; (2) good moisture retention, capable of maintaining a moist environment for the wound, promoting cell hydration, and having a certain absorption effect on wound exudate; (3) sufficient physical and mechanical strength to ensure its integrity and avoid external bacterial invasion caused by material breakage; (4) appropriate surface microstructure and biochemical properties to promote cell adhesion, proliferation, and differentiation. Hydrogels have become the most competitive candidates for wound dressings due to their good hydrophilicity, biocompatibility, and three-dimensional (3D) network structure similar to the extracellular matrix (ECM). And the functions of hydrogels have also changed from single physical coverage or single function to the current combination of multiple functions, and show a further trend of intelligentization. However, the existing administration methods of wound dressings mainly rely on drug penetration, resulting in slow drug action and low absorption rate. Summary of the Invention

[0006] The objective of the present invention is to construct a microneedle patch for promoting the healing of diabetic wounds. Mesoporous silica (MSN) is used as a drug carrier to load the hydrogen prodrug ammonia borane (AB) to obtain nano hydrogen-releasing particles. The nano hydrogen-releasing particles are uniformly mixed with PVP hydrogel as the microneedle preparation solution. Graphene oxide (GO) has a high photothermal conversion efficiency, and a large number of oxygen-containing groups are modified on the surface of GO, which can be used to adsorb metal ions. In the present invention, GO adsorbs cobalt ions to obtain GO-Co 2+ , which is uniformly mixed with PVA hydrogel as the backing preparation solution. The microneedle and the backing preparation solution are added to the PDMS mold, and the microneedle patch is prepared by the method of casting. The PVP aqueous solution has sufficient mechanical strength to pierce the skin after drying, and the PVA aqueous solution has good toughness to fit the skin after drying. After the microneedle patch of the present invention pierces the skin, the tip of the needle quickly dissolves, releasing nano hydrogen-releasing particles. AB continuously releases hydrogen through hydrolysis to relieve wound surface inflammation. At the same time, the reducibility of hydrogen can reduce the oxygen-containing groups on the surface of GO, release cobalt ions, and promote wound surface angiogenesis. Under the irradiation of near-infrared light, the local temperature of the wound surface rises, which can effectively inhibit bacterial infection, thereby achieving the purpose of promoting the healing of diabetic wounds.

[0007] The technical solution of the present invention is realized through three steps.

[0008] (1) Microneedle preparation solution

[0009] 50 mg of mesoporous silica is added to 10 ml of ammonia borane solution with a concentration of 0.5 - 2 mol / L, magnetically stirred for 12 h, and then centrifuged at a speed of 10000 rpm for 10 min to remove the supernatant. The ammonia borane attached to the surface is ultrasonically cleaned with ethanol and deionized water, and then placed in an oven at 60 °C for drying to prepare hydrogen-releasing nanoparticles.

[0010] (2) Weigh 50 - 200 mg of nano hydrogen-releasing particles and magnetically stir them with 1 ml of PVP aqueous solution for 2 h to uniformly disperse the nano hydrogen-releasing particles in the PVP aqueous solution to obtain the microneedle preparation solution;

[0011] (3) Backing preparation solution

[0012] Prepare 20 ml of an aqueous solution of Co(NO3)2·4H2O with a concentration of 0.25 - 1 mol / L, weigh 5 mg of graphene oxide, magnetically stir for 12 h, then centrifuge at a speed of 10000 rpm for 10 min to remove the supernatant, and ultrasonically clean with ethanol and deionized water to prepare GO-Co 2+ .

[0013] (4) Weigh 5 - 10 mg of GO-Co 2+ and magnetically stir it with 1 ml of PVA aqueous solution for 2 h to make GO-Co 2+It is uniformly dispersed in the PVA aqueous solution to obtain a backing preparation solution;

[0014] (5) Preparation of microneedles by the casting method

[0015] PDMS and a curing agent are mixed evenly at 10:1 (w / w) and poured onto the microneedle male mold. After curing in an oven at 70 °C, it is demolded to obtain a microneedle female mold.

[0016] The microneedle male mold is prepared from a photosensitive resin as a raw material through 3D printing technology.

[0017] The microneedle preparation solution is dropped onto the PDMS female mold, and the microneedle mold is subjected to negative pressure vacuum treatment for 30 min by an air pump to completely fill the needle holes with the microneedle preparation solution. After removing the excess microneedle preparation solution, the backing preparation solution is then dropped into the mold. After drying in an oven at 40 °C for 12 h, it is taken out and demolded to obtain a microneedle patch.

[0018] Advantages of the present invention:

[0019] (1) The present invention realizes the local release of hydrogen by microneedle transdermal drug delivery, solving the problem that hydrogen has low solubility and cannot act locally on the wound.

[0020] (2) The present invention loads graphene oxide adsorbed with cobalt ions on the backing layer. Utilizing the reducibility of hydrogen, the oxygen-containing functional groups on the surface of graphene oxide are reduced, promoting the release of cobalt ions to promote angiogenesis at the wound surface.

[0021] (3) After piercing the skin, the present invention rapidly dissolves and continuously releases hydrogen locally at the wound surface to relieve inflammation in the early stage of wound healing. At the same time, the continuous release of hydrogen reduces the oxygen-containing functional groups on the surface of graphene oxide, releasing cobalt ions, promoting angiogenesis during the tissue remodeling period, and realizing the intelligent regulation of wound healing. Description of the drawings

[0022] Figure 1 It is the SEM image of the nano-hydrogen-releasing particles in Example 1, Figure 1 in which, (A) MSN; (B) AB@MSN;

[0023] Figure 2 It is the ATR-FTIR spectrum of the nano-hydrogen-releasing particles in Example 1;

[0024] Figure 3 It is the hydrogen release amount diagram of Example 1, Example 2, and Example 3;

[0025] Figure 4 It is the evaluation of the biocompatibility of fibroblasts of the nanoparticles in Example 1;

[0026] Figure 5Evaluation of the biocompatibility of the nanoparticles in Example 1 with macrophages;

[0027] Figure 6 Evaluation of the anti-inflammatory effect of the nanoparticles in Example 1;

[0028] Figure 7 For the ATR-FTIR spectrum of GO-Co 2+ in Example 5;

[0029] Figure 8 For the morphology of the PDMS negative mold in Example 8;

[0030] Figure 9 For the SEM image of the microneedle patch in Example 8;

[0031] Figure 10 For the stereomicroscope image of the microneedle patch in Example 8;

[0032] Figure 11 For the hydrogen release amount diagram of the microneedle patch in Examples 8, 9, and 10;

[0033] Figure 12 For the heating curve diagram of the microneedle patch in Example 8;

[0034] Figure 13 For the antibacterial experiment diagram of the microneedle patch in Examples 8 and 11;

[0035] Figure 14 For the heating curve diagram of the microneedle patch in Examples 8 and 11;

[0036] Figure 15 Evaluation of the effect of the microneedle patch on vascular endothelial cell proliferation in Comparative Example 1;

[0037] Figure 16 Evaluation of the pro-angiogenic effect of the microneedle patch in Examples 8 and Comparative Example 2. Detailed implementation manners

[0038] The present invention will be specifically described below in conjunction with the examples.

[0039] Example 1

[0040] Preparation of nano hydrogen-releasing particles

[0041] Add 50 mg of mesoporous silica to 10 ml of an ammonia borane solution with a concentration of 1 mol / L, magnetically stir for 12 h, then centrifuge at a speed of 10,000 rpm for 10 min to remove the supernatant. Use ethanol and deionized water to ultrasonically clean for 10 min to wash away the ammonia borane attached to the surface, and place it in an oven at 60 °C for drying to obtain hydrogen-releasing nanoparticles.

[0042] (1) Morphological Characterization of Hydrogen-Release Nanoparticles

[0043] After sputtering gold on the prepared hydrogen-release nanoparticles, a field emission scanning electron microscope (FE-SEM, ZEISS, Germany, SUPRA55) was used to observe the morphology of the hydrogen-release nanoparticles. As Figure 1 shown, the mesoporous silica before and after loading ammonia borane are both spheres with uniform size.

[0044] (2) Test by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR)

[0045] An ATR-FTIR test was performed on the hydrogen-release nanoparticles using a Fourier transform infrared spectrometer (NICOLET IS 10), and the infrared spectrum obtained is as Figure 2 shown. The vibration peak of MSN appears at 1088 cm -1 for the hydrogen-release nanodrug AB@MSN, and the vibration peak of ammonia borane appears at 2330 cm -1 , indicating that MSN has successfully loaded the hydrogen prodrug ammonia borane as a carrier.

[0046] Example 2

[0047] Preparation of Hydrogen-Release Nanoparticles

[0048] 50 mg of mesoporous silica was added to 10 ml of ammonia borane solution with a concentration of 0.5 mol / L. After magnetic stirring for 12 h, it was centrifuged at 10000 rpm for 10 min to remove the supernatant. It was ultrasonically treated with ethanol and deionized water for 10 min to wash the ammonia borane attached to the surface, and then placed in an oven at 60 °C for drying to prepare hydrogen-release nanoparticles.

[0049] Example 3

[0050] Preparation of Hydrogen-Release Nanoparticles

[0051] 50 mg of mesoporous silica was added to 10 ml of ammonia borane solution with a concentration of 2 mol / L. After magnetic stirring for 12 h, it was centrifuged at 10000 rpm for 10 min to remove the supernatant. It was ultrasonically treated with ethanol and deionized water for 10 min to wash the ammonia borane attached to the surface, and then placed in an oven at 60 °C for drying to prepare hydrogen-release nanoparticles.

[0052] Measurement of Hydrogen Release Amount of Hydrogen-Release Nanoparticles

[0053] The hydrogen-release nanoparticles were dispersed in 25 ml of phosphate buffer solution. After 24 h, it was centrifuged at 10000 rpm for 10 min, and a dissolved hydrogen tester (ENH-2000, RUSTLEX, Japan) was used to test the hydrogen release amount of the sample. After the measurement, a new phosphate buffer solution was added, and the value of the release amount was recorded every 24 h and a new phosphate buffer solution was added.

[0054] The results are as Figure 3 shown. Compared with the 0.5 M ammonia borane solution, the amount of drug loaded using 1 and 2 M ammonia borane solutions is higher, the hydrogen release amount is higher, and the release time is more persistent.

[0055] Example 4

[0056] Weigh 4 mg of silica (MSN) and silica-loaded ammonia borane (AB@MSN) respectively, and ultrasonically disperse them in 4 ml of DMEM medium to obtain a nanoparticle suspension of 1 mg / ml. Dilute it respectively to obtain nanoparticle suspensions of 50, 100, and 200 μg / ml.

[0057] Inoculate fibroblast L929 into a 48-well plate with a cell density of 3000 cells / well. After the cells adhere for 24 h, aspirate the medium, add the nanoparticle suspension, and after treating for 48 h, aspirate the medium and add CCK8. Incubate in an incubator for 2 h and then measure the absorbance at 450 nm using a microplate reader.

[0058] The results are as Figure 4 shown. When MSN and AB@MSN are co-cultured with fibroblasts, the cell proliferation rate is comparable to that of the control group, indicating that MSN and AB@MSN have good biocompatibility.

[0059] Inoculate macrophage RAW264.7 into a 24-well plate with a cell density of 20000 cells / well. After the cells adhere for 24 h, aspirate the medium, add the nanoparticle suspension, and after treating for 48 h, aspirate the medium and add CCK8. Incubate in an incubator for 2 h and then measure the absorbance at 450 nm using a microplate reader.

[0060] The results are as Figure 5 shown. When MSN and AB@MSN are co-cultured with macrophages, the cell proliferation rate is comparable to that of the control group, indicating that MSN and AB@MSN have good biocompatibility.

[0061] Inoculate fibroblast L929 into a 24-well plate with a cell density of 50000 cells / well. After the cells adhere for 24 h, aspirate the medium, add 1.2 mg / ml H2O2 solution and treat for 2 h, then wash with PBS, add the nanoparticle suspension, and after culturing for 24 h, aspirate the medium and add CCK8. Incubate in an incubator for 2 h and then measure the absorbance at 450 nm using a microplate reader.

[0062] The results are as Figure 6 shown. AB@MSN has good antioxidant performance and can effectively scavenge ROS.

[0063] Example 5

[0064] Preparation of GO-Co by Adsorbing Cobalt Ions on Graphene Oxide 2+

[0065] Prepare 20 ml of Co(NO3)2·4H2O solution with a concentration of 250 mM. Weigh 5 mg of graphene oxide and add it to the cobalt nitrate solution. After magnetic stirring for 12 h, centrifuge at a speed of 10000 rpm for 10 min, remove the supernatant, wash ultrasonically with ethanol and deionized water, and dry in an oven at 60 °C to obtain GO-Co 2+ 。

[0066] GO-Co 2+ Testing by Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR) of GO-Co

[0067] Perform ATR-FTIR testing using a Fourier transform infrared spectrometer (NICOLET IS 10) to obtain an infrared spectrum as shown Figure 7 After adsorbing cobalt ions, the oxygen-containing groups on the surface of graphene oxide are significantly reduced

[0068] Example 6

[0069] Preparation of GO-Co by Adsorbing Cobalt Ions on Graphene Oxide 2+

[0070] Prepare 20 ml of Co(NO3)2·4H2O solution with a concentration of 500 mM. Weigh 5 mg of graphene oxide and add it to the cobalt nitrate solution. After magnetic stirring for 12 h, centrifuge at a speed of 10000 rpm for 10 min, remove the supernatant, wash ultrasonically with ethanol and deionized water, and dry in an oven at 60 °C to obtain GO-Co 2+ 。

[0071] Example 7

[0072] Preparation of GO-Co by Adsorbing Cobalt Ions on Graphene Oxide 2+

[0073] Prepare 20 ml of Co(NO3)2·4H2O solution with a concentration of 1000 mM. Weigh 5 mg of graphene oxide and add it to the cobalt nitrate solution. After magnetic stirring for 12 h, centrifuge at a speed of 10000 rpm for 10 min, remove the supernatant, wash ultrasonically with ethanol and deionized water, and dry in an oven at 60 °C to obtain GO-Co 2+ 。

[0074] Example 8

[0075] Preparation of Microneedle Patches

[0076] (1) Weigh 200 mg of the nano hydrogen - releasing particles described in Example 1, and magnetically stir them with 1 ml of PVP aqueous solution for 2 h to uniformly disperse the nano hydrogen - releasing particles in the PVP aqueous solution, obtaining a microneedle preparation solution;

[0077] (2) Weigh 5 mg of GO - Co described in Example 5 2+ , and magnetically stir it with 1 ml of PVA aqueous solution for 2 h to obtain a backing preparation solution;

[0078] (3) Mix PDMS and the curing agent evenly at a ratio of 10:1 (w / w), pour the mixture onto the microneedle male mold, cure it in an oven at 70 °C and then demold to obtain a microneedle female mold. The prepared microneedle female mold is as Figure 8 shown.

[0079] The microneedle male mold is prepared by 3D printing technology using photosensitive resin as the raw material.

[0080] Use a pipette to suck 500 μl of the microneedle preparation solution and drop it into the PDMS mold. Perform negative pressure vacuum pumping on the microneedle mold through an air pump to completely fill the needle holes with the microneedle preparation solution, and remove the excess microneedle preparation solution outside the tip chamber. Use a pipette to suck 300 μl of the backing preparation solution and drop it into the mold. After drying in an oven at 40 °C for 12 h, take it out and demold to obtain the microneedle patch AB@MSN - GO / Co 2+ -MN.

[0081] Morphology characterization of the microneedle patch

[0082] After sputtering gold on the microneedle patch, fix the microneedle patch on an inclined sample stage with conductive glue, and observe the morphology of the microneedle patch using a field - emission scanning electron microscope (FE - SEM, ZEISS, Germany, SUPRA55), as Figure 9 shown.

[0083] Place the microneedle patch obliquely and observe the morphology of the microneedle patch using a stereomicroscope (RH - 2000), as Figure 10 shown.

[0084] After being completely dried, the microneedle patch prepared by this method has a flat substrate, good flexibility, sharp needle tips, and a complete structure.

[0085] Measurement of hydrogen release amount of the microneedle patch

[0086] Place the prepared microneedle patch in 25 ml of phosphate - buffered solution. After 24 h, centrifuge it at a speed of 10000 rpm for 10 min. Use a dissolved hydrogen tester (ENH - 2000, RUSTLEX, Japan) to test the hydrogen release amount of the sample. After the measurement, change the solution, record the value of the release amount every 24 h and change the solution. As Figure 11 shown, the microneedle patch can continuously release hydrogen within 10 days.

[0087] Temperature rise effect test of microneedle patch

[0088] Irradiate the microneedle patch described in Example 8 with an 808 near-infrared laser emitter (LWIRL808-12W-6P) for 6 min, with a power of 1.5 W / cm 2 , and use an infrared thermometer gun to record the temperature every 60 s. The temperature rise curve is as Figure 12 shown.

[0089] The temperature of the microneedle patch rose from room temperature to 55 °C within 6 min, indicating that the microneedle patch loaded with GO-Co 2+ also has good photothermal conversion efficiency.

[0090] Antibacterial effect test of microneedle patch

[0091] Sterilize the microneedle patches described in Example 8 and Example 11 by irradiating them with ultraviolet light for 30 min. Place the microneedle patches in a 12-well plate on a bacteria workbench, and inoculate Escherichia coli (E. coli) on the microneedle patches at a density of 10 5 . After culturing for 1 day in a bacteria incubator, irradiate with near-infrared light of 1.5 W / cm 2 for 5 min, and then put it back into the bacteria incubator for culturing for two hours. Then use a pipette gun to remove the supernatant and add MTT, and incubate in the incubator for 2 h. Add DMSO, shake well on a shaker, transfer to a 96-well plate, and measure the absorbance at 570 nm using a microplate reader. Set 3 parallel samples for each sample. The antibacterial effect is as Figure 13 shown.

[0092] Irradiate the microneedle patches described in Example 8 and Example 11 with an 808 near-infrared laser emitter for 6 min, with a power of 1.5 W / cm 2 , and use an infrared thermometer gun to record the temperature every 60 s. The temperature rise curve is as Figure 14 shown.

[0093] The temperature of the microneedle patch rose from room temperature to 57 °C within 6 min. As the concentration of the loaded GO-Co 2+ increased, the temperature of the microneedle patch also increased.

[0094] Irradiate the microneedle patch with an 808 near-infrared laser emitter for 6 min, with powers of 1.2 and 1.8 W / cm respectively 2 , and use an infrared thermometer gun to record the temperature every 60 s. The temperature rise curve is as Figure 12 shown.

[0095] At a power of 1.8 W / cm 2Under the irradiation of near-infrared light, the temperature of the microneedle patch increased from room temperature to 63 °C within 6 min, and the temperature of the microneedle patch increased with the increase of the near-infrared power.

[0096] Example 9

[0097] (1) Weigh 50 mg of the nano hydrogen-evolving particles described in Example 1, and magnetically stir with 1 ml of PVP aqueous solution for 2 h to uniformly disperse the nano hydrogen-evolving particles in the PVP aqueous solution to obtain a microneedle preparation solution;

[0098] (2) Weigh 5 mg of GO-Co described in Example 5 2+ , and magnetically stir with 1 ml of PVA aqueous solution for 2 h to obtain a backing preparation solution;

[0099] (3) Mix PDMS and curing agent evenly at 10:1 (w / w), pour them onto the microneedle male mold, cure them in an oven at 70 °C and then demold to obtain a microneedle female mold. The prepared microneedle female mold is as Figure 8 shown.

[0100] Use a pipette to suck 500 μl of the microneedle preparation solution and drop it into the PDMS mold. Perform negative pressure vacuuming on the microneedle mold through an air pump to make the microneedle preparation solution completely fill the needle holes, and remove the excess microneedle preparation solution outside the tip chamber. Use a pipette to suck 300 μl of the backing preparation solution and drop it into the mold. Take it out after drying in an oven at 40 °C for 12 h, and demold to obtain the microneedle patch AB@MSN-GO / Co 2+ -MN.

[0101] Example 10

[0102] (1) Weigh 100 mg of the nano hydrogen-evolving particles described in Example 1, and magnetically stir with 1 ml of PVP aqueous solution for 2 h to uniformly disperse the nano hydrogen-evolving particles in the PVP aqueous solution to obtain a microneedle preparation solution;

[0103] (2) Weigh 5 mg of GO-Co described in Example 5 2+ , and magnetically stir with 1 ml of PVA aqueous solution for 2 h to obtain a backing preparation solution;

[0104] (3) Mix PDMS and curing agent evenly at 10:1 (w / w), pour them onto the microneedle male mold, cure them in an oven at 70 °C and then demold to obtain a microneedle female mold. The prepared microneedle female mold is as Figure 8 shown.

[0105] Use a pipette to aspirate 500 μl of the microneedle preparation solution and add it dropwise to the PDMS mold. Perform negative pressure vacuum pumping on the microneedle mold through an air pump to completely fill the needle holes with the microneedle preparation solution, and remove the excess microneedle preparation solution outside the tip chamber. Use a pipette to aspirate 300 μl of the backing preparation solution and add it dropwise to the mold. After drying in an oven at 40 °C for 12 h, take it out and demold to obtain the microneedle patch AB@MSN-GO / Co 2+ -MN.

[0106] Place the microneedle patches described in Example 8, Example 9, and Example 10 into 25 ml of phosphate buffer solution. After 24 h, centrifuge at a speed of 10000 rpm for 10 min. Use a dissolved hydrogen tester (ENH-2000, RUSTLEX, Japan) to test the hydrogen release amount of the sample. After the measurement, change the solution, record the value of the release amount every 24 hours and change the solution, as Figure 11 shown, as the concentration of the nano-hydrogen-releasing particles increases, the hydrogen release amount also increases.

[0107] Example 11

[0108] (1) Weigh 200 mg of the nano-hydrogen-releasing particles described in Example 1, and magnetically stir with 1 ml of PVP aqueous solution for 2 h to uniformly disperse the nano-hydrogen-releasing particles in the PVP aqueous solution to obtain the microneedle preparation solution;

[0109] (2) Weigh 10 mg of GO-Co described in Example 5 2+ , and magnetically stir with 1 ml of PVA aqueous solution for 2 h to obtain the backing preparation solution;

[0110] (3) Mix PDMS and the curing agent evenly at a ratio of 10:1 (w / w) and pour it onto the microneedle male mold. After curing in an oven at 70 °C, demold to obtain the microneedle female mold. The prepared microneedle female mold is as Figure 8 shown.

[0111] Use a pipette to aspirate 500 μl of the microneedle preparation solution and add it dropwise to the PDMS mold. Perform negative pressure vacuum pumping on the microneedle mold through an air pump to completely fill the needle holes with the microneedle preparation solution, and remove the excess microneedle preparation solution outside the tip chamber. Use a pipette to aspirate 300 μl of the backing preparation solution and add it dropwise to the mold. After drying in an oven at 40 °C for 12 h, take it out and demold to obtain the microneedle patch AB@MSN-GO / Co 2+ -MN.

[0112] Comparative Example 1

[0113] (1) Weigh 5 mg of GO-Co described in Example 5, Example 6, and Example 7 2+ , and magnetically stir with 1 ml of PVA aqueous solution for 2 h to uniformly disperse GO-Co 2+ in the PVP aqueous solution to obtain the backing preparation solution;

[0114] (2) The preparation of the microneedle patch was the same as that in Example 8.

[0115] The microneedle patch was immersed in 2 ml of F-12 medium for 24 h to obtain the extract of the microneedle patch.

[0116] Human umbilical vein endothelial cells (HUVECs) were seeded into a 24-well plate at a cell density of 20,000 cells / well. After the cells adhered for 24 h, the medium was aspirated, and the diluted 8-fold extract of the microneedle patch was added. After culturing for 3 days and 7 days, the medium was aspirated, CCK8 was added, and after incubation in an incubator for 2 h, the absorbance at 450 nm was measured using a microplate reader.

[0117] The results were as Figure 15 shown. Compared with the control group, cobalt ions at 250 mM promoted the proliferation of vascular endothelial cells, while cobalt ions at 500 mM and 1000 mM inhibited the proliferation of vascular endothelial cells.

[0118] Comparative Example 2

[0119] (1) Weigh 200 mg of the nano-hydrogen releasing particles described in Example 1, and magnetically stir with 1 ml of PVP aqueous solution for 2 h to uniformly disperse the nano-hydrogen releasing particles in the PVP aqueous solution, obtaining the microneedle preparation solution;

[0120] (2) Weigh 5 mg of GO, and magnetically stir with 1 ml of PVA aqueous solution for 2 h to obtain the backing preparation solution;

[0121] (3) PDMS and the curing agent were mixed evenly at a ratio of 10:1 (w / w) and poured onto the microneedle male mold. After curing in an oven at 70 °C and demolding, the microneedle female mold was obtained.

[0122] Use a pipette to aspirate 500 μl of the microneedle preparation solution and drop it into the PDMS mold. The microneedle mold was subjected to negative pressure vacuum treatment for 30 min by an air pump to completely fill the needle holes with the microneedle preparation solution, and the excess microneedle preparation solution outside the tip chamber was removed. Use a pipette to aspirate 300 μl of the backing preparation solution and drop it into the mold. After drying in an oven at 40 °C for 12 h, take it out and demold to obtain the microneedle patch AB@MSN-GO-MN.

[0123] The microneedle patches described in Example 8 and Comparative Example 2 were immersed in 2 ml of F-12 medium for 24 h to obtain the extracts of the microneedle patches.

[0124] Human umbilical vein endothelial cells (HUVECs) were seeded into 24-well plates at a cell density of 20,000 cells / well. After the cells adhered for 24 h, the culture medium was aspirated, and the extract of the microneedle patch diluted 8-fold was added. After culturing for 3 days and 7 days, the culture medium was aspirated, CCK8 was added, and after incubation in an incubator for 2 h, the absorbance at 450 nm was measured using a microplate reader.

[0125] The results are as Figure 16 shown. The microneedle patch has good biocompatibility. At the same time, the introduction of cobalt ions promotes the proliferation of vascular endothelial cells, indicating that the microneedle patch has an angiogenesis-promoting effect.

Claims

1. A microneedle patch for promoting the healing of diabetic wounds, characterized in that, The microneedle patch includes a backing, graphene oxide adsorbed with cobalt ions dispersed inside the backing, microneedles disposed on the backing, and hydrogen-releasing nanoparticles dispersed inside the microneedles, wherein the hydrogen-releasing nanoparticles are mesoporous silica nanoparticles loaded with the hydrogen-precursor ammonia borane.

2. The microneedle patch according to claim 1, wherein The height of the microneedles is 800 μm, the diameter is 400 μm, and within the range where the side length of the backing is 15 mm, the number of microneedles is 100.

3. A preparation method of a microneedle patch for promoting the healing of diabetic wounds, characterized in that, The preparation method comprises the following steps: (1) Loading ammonia borane into mesoporous silica to prepare the hydrogen-releasing nanoparticles; (2) Uniformly dispersing the hydrogen-releasing nanoparticles in an aqueous solution of polyvinylpyrrolidone to obtain a microneedle preparation solution; (3) Graphene oxide adsorbs cobalt ions to obtain GO-Co 2+ ; (4) Disperse GO-Co 2+ uniformly in an aqueous solution of polyvinyl alcohol to obtain a backing preparation solution; (5) Pouring polydimethylsiloxane onto the microneedle male mold to prepare a microneedle female mold, injecting the microneedle preparation solution and the backing preparation solution into the polydimethylsiloxane female mold, drying and demolding to obtain the microneedle patch.

4. The preparation method of the microneedle patch according to claim 3, wherein Step (1) is to stir 50 mg of mesoporous silica with 10 ml of an ammonia borane solution with a concentration of 0.5 - 2 mol / L for 12 h, then centrifuge to remove the supernatant, ultrasonically clean the surface-attached ammonia borane with ethanol and deionized water, and dry in an oven at 60 °C to obtain the hydrogen-releasing nanoparticles.

5. The preparation method of the microneedle patch according to claim 3, characterized in that, Step (2) is to add 50 - 200 mg of the hydrogen-releasing nanoparticles to 1 ml of an aqueous PVP solution and stir for 2 h to uniformly disperse the hydrogen-releasing nanoparticles in the PVP aqueous solution to obtain the microneedle preparation solution.

6. The method for preparing the microneedle patch according to claim 3, wherein Step (3): Add 5 mg of graphene oxide into 20 ml of cobalt ion solution with a concentration of 0.25 - 1 mol / L, stir for 12 h to allow GO to adsorb cobalt ions in the solution, centrifuge at a speed of 10000 rpm for 10 min, remove the supernatant, and ultrasonically clean with ethanol and deionized water to obtain GO-Co 2+ .

7. The preparation method of the microneedle patch according to claim 3, wherein Step (4) is to add 5 - 10 mg of GO-Co 2+ to 1 ml of aqueous PVA solution and stir for 2 h to uniformly disperse GO-Co 2+ in the aqueous PVA solution to obtain a backing preparation solution.

8. The preparation method of the microneedle patch according to claim 3, wherein The microneedle male mold in step (5) is prepared by 3D printing technology using photosensitive resin as the raw material.

9. The preparation method of the microneedle patch according to claim 3, characterized in that, The microneedle female mold is prepared by mixing PDMS and a curing agent at a ratio of 10:1 (w / w), pouring the mixture evenly onto the microneedle male mold, curing in an oven at 70 °C and then demolding to obtain the microneedle female mold.

10. The preparation method of the microneedle patch according to claim 3, characterized in that, In step (5), the microneedle preparation solution is dropped onto the PDMS female mold, and the microneedle mold is subjected to negative pressure vacuum treatment for 30 min by an air pump to completely fill the needle holes with the microneedle preparation solution, remove the excess microneedle preparation solution, then drop the backing preparation solution into the mold, dry in an oven at 40 °C for 12 h, take out, and demold to obtain the microneedle patch.

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