Double-layer microneedle patch for diabetic wounds and method of making the same

Abstract

The application discloses a double-layer microneedle patch for diabetic wounds and a preparation method thereof. The double-layer microneedle patch for diabetic wounds comprises a needle tip part and a backing part. The needle tip part is prepared by a needle tip preparation system through vacuum drying and ultraviolet crosslinking. The backing part is prepared by a backing solution through vacuum drying and ultraviolet crosslinking. The needle tip preparation system takes nanoparticles loaded with glucose oxidase and a polyvinyl alcohol solution as main raw materials. The backing solution takes a photoinitiator and gelatin grafted with an angiogenic peptide and a double bond as main raw materials. The double-layer microneedle patch can eliminate the dense barrier of a biofilm through a double-enzyme cascade reaction, kill bacteria wrapped in the biofilm, and gradually release the polypeptide to promote angiogenesis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a double-layer microneedle patch for diabetic wounds and a preparation method thereof. BACKGROUND

[0002] Chronic non-healing wounds have become a major burden on global health care. Studies have shown that more than 60% of chronic wounds are related to biofilms, which produce a persistent inflammatory response, damage the regeneration of epithelium and granulation tissue, and also seriously affect the healing of diabetic wounds due to problems such as reduced local extracellular matrix (ECM), blocked blood vessels and granulation tissue neogenesis. Biofilm is a complex bacterial community formed by planktonic bacteria adhering to the wound surface and then forming extracellular polymeric substances (EPS) including proteins, extracellular DNA (eDNA) and exopolysaccharides. EPS provides a natural protective barrier for bacteria, making it difficult for general antimicrobial agents to effectively penetrate into the biofilm, which poses a great challenge to the treatment of chronic wounds.

[0003] A chemical dynamic therapy based on Fenton reaction has attracted extensive research in anticancer, antibiofilm and sterilization because it does not require auxiliary equipment and can convert endogenous H2O2 into toxic ROS. However, the formation of ROS is not ideal due to the low level of endogenous H2O2 in vivo. Among the numerous nanomaterials with Fenton effect, metal-organic frameworks (MOFs) based on iron, copper, cobalt and other metals are considered as a promising candidate due to their pH sensitivity and low cytotoxicity. In addition, the excellent storage capacity of MOF enables it to encapsulate other materials to enhance the limited Fenton reaction by self-produced H2O2. However, it still cannot fully meet the rapid healing needs of diabetic wounds.

[0004] Gelatin methacryloyl (GelMA) is widely used in wound repair due to its excellent cell compatibility, biodegradability and non-immunogenicity. At the same time, GelMA can be easily functionalized with various active substances such as growth factors, polypeptides, etc., thereby solving the problems of short half-life and rapid release. For example, QHREDGS as a new type of pro-angiogenic peptide, when covalently linked to GelMA, its release time is significantly prolonged, and the ability to promote wound healing is greatly improved. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a double-layer microneedle patch for diabetic wounds and a preparation method thereof to overcome the deficiencies of the prior art. The double-layer microneedle patch of the present application can eliminate the dense barrier of biofilm through a double-enzyme cascade reaction, kill bacteria wrapped in the biofilm, and gradually release polypeptides to promote angiogenesis.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is: a double-layer microneedle patch for diabetic wounds, characterized in that it comprises a needle tip part and a backing part, the needle tip part is prepared by a needle tip preparation system through vacuum drying and ultraviolet crosslinking, the backing part is prepared by a backing solution through vacuum drying and ultraviolet crosslinking, the needle tip preparation system takes nanoparticles loaded with glucose oxidase and a polyvinyl alcohol solution as main raw materials, and the backing solution takes a photoinitiator and gelatin grafted with an angiogenic peptide and a double bond as main raw materials.

[0007] In addition, the present application also provides a method for preparing the above-mentioned double-layer microneedle patch for diabetic wounds, characterized in that it comprises:

[0008] Step one, providing a negative mold microneedle array mold;

[0009] Step two, providing a needle tip preparation system, specifically including: adding alpha-amylase to a cooled polyvinyl alcohol solution to obtain a mixed system A, and adding a nanoparticle-containing system to the mixed system A to obtain the needle tip preparation system; the nanoparticle-containing system is a nanoparticle-containing system obtained by dispersing nanoparticles loaded with glucose oxidase in deionized water;

[0010] Step three, providing a backing solution, specifically including: dissolving a photoinitiator in water, and adding gelatin grafted with an angiogenic peptide and a double bond to obtain the backing solution;

[0011] Step four, filling the negative mold microneedle array mold, specifically including: placing the needle tip preparation system in the negative mold microneedle array mold of step one, so that the needle tip preparation system enters the needle tip part of the negative mold microneedle array mold, vacuumizing, and adding the backing solution;

[0012] Step five, vacuum drying the filled negative mold microneedle array mold of step four for 12-24 hours, demolding, and irradiating under a UV lamp for 1-3 minutes to obtain the double-layer microneedle patch for diabetic wounds.

[0013] The above-mentioned method, characterized in that in step two, the mass of polyvinyl alcohol is 20-300 times the mass of alpha-amylase, the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 0.2-0.3 g / mL, and the molecular weight of polyvinyl alcohol is 9000-10000; the volume of the nanoparticle-containing system is 100-330 times the mass of polyvinyl alcohol, the volume unit of the nanoparticle-containing system is μL, and the mass unit of polyvinyl alcohol is g.

[0014] The method, characterized in that, in step three, the mass percentage of the photoinitiator in the backing solution is 0.05-0.1%, and the mass percentage of the gelatin grafted with the pro-angiogenic peptide and the double bond is 20%; the photoinitiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0015] The method, characterized in that, in step four, the volume of the backing solution is 5 times the volume of the needle tip preparation system.

[0016] The method, characterized in that, in step two, the preparation method of the glucose oxidase-loaded nanoparticles comprises:

[0017] Step 101, completely dispersing the iron-containing nanoparticles in water under ultrasonic conditions to obtain a dispersion system;

[0018] Step 102, adding glucose oxidase to the dispersion system in step 101, stirring at 4°C in the dark for 6-12 hours, centrifuging, washing, and vacuum drying to obtain glucose oxidase-loaded nanoparticles.

[0019] The method, characterized in that, in step 101, the concentration of the iron-containing nanoparticles in the dispersion system is 50-200 μg / mL; in step 102, the mass of the glucose oxidase is 0.05-0.2 times the mass of the iron-containing nanoparticles; and the molecular weight of the glucose oxidase is 150 kDa, and the enzyme activity is 100-250 u / mg.

[0020] The method, characterized in that, the preparation method of the iron-containing nanoparticles comprises:

[0021] Step 201, dissolving FeCl3·6H2O and terephthalic acid in N,N-dimethylformamide to obtain a reaction system; the amount of substance of the terephthalic acid is 0.5-1 times the amount of substance of FeCl3·6H2O;

[0022] Step 202, reacting the reaction system in step 201 at a temperature of 120°C for 6-12 hours, cooling, centrifuging and washing, and vacuum drying to obtain iron-containing nanoparticles.

[0023] The method, characterized in that, in step three, the preparation method of the gelatin grafted with the pro-angiogenic peptide and the double bond comprises:

[0024] Step 301, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide and N-hydroxysuccinimide to a gelatin solution to obtain system A;

[0025] Step 302, adding polypeptide to the system A in step 301 to obtain system B, stirring and reacting the system B, dialysis, freeze-drying to obtain gel material;

[0026] Step 303, dissolving the gel material in PBS to obtain system C, adding methacrylic anhydride to the system C, stirring and reacting, terminating, dialysis after terminating the reaction, freeze-drying to obtain gelatin grafted with pro-angiogenic peptide and double bond.

[0027] The method has the following advantages: the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the system A in step 301 is 0.5-0.7 mg / mL, the concentration of N-hydroxysuccinimide is 0.5-0.7 mg / mL; the concentration of polypeptide in the system B in step 302 is 0.3 mg / mL, and the molecular weight of the polypeptide is 827.79; and the mass of methacrylic anhydride in step 303 is 0.1-0.3 times the mass of the gel material.

[0028] Compared with the prior art, the present application has the following advantages:

[0029] 1. The double-layer microneedle patch can eliminate the dense barrier of the biofilm through a double-enzyme cascade reaction, kill the bacteria wrapped in the biofilm, and gradually release the polypeptide to promote angiogenesis.

[0030] 2. In the preparation method, gelatin methacryl Gel-Q-M is used as a raw material, and the cell compatibility, biodegradability and non-immunogenicity are fully utilized to functionalize the active substance, so that the problems of short half-life and rapid release of the active substance are solved, the release time is prolonged, and the wound healing ability is promoted.

[0031] 3. The double-layer microneedle patch contains alpha-amylase in the needle tip, which can degrade polysaccharides in the extracellular polymer of the biofilm, destroy the structure of the biofilm, and achieve the purpose of bacterial exposure.

[0032] 4. The double-layer microneedle patch has nanoparticles loaded with glucose oxidase wrapped on the needle tip, which can generate ROS by utilizing the glucose obtained by degrading the biofilm and the glucose at the wound site itself, without the addition of exogenous H2O2, to kill bacteria and eliminate the biofilm.

[0033] 5. The backing part of the microneedle patch is made of modified gelatin grafted with polypeptide and crosslinked by ultraviolet light, which can effectively achieve slow release of polypeptide to promote angiogenesis and achieve the purpose of wound repair.

[0034] The technical solutions of the present application will be further described in detail below in combination with the drawings and examples.

[0035] Drawings of the specification

[0036] Figure 1 This is a scanning electron microscope (SEM) schematic diagram of the glucose oxidase-loaded nanoparticles (M@G) of Example 2.

[0037] Figure 2 This is a schematic diagram of the UV-Vis spectral analysis results of nanoparticles MIL-101 in Example 1 and nanoparticles (M@G) loaded with glucose oxidase in Example 2.

[0038] Figure 3 This is a schematic diagram of the glucose oxidation-promoting performance of the glucose oxidase-loaded nanoparticles (M@G) in Example 2.

[0039] Figure 4 This is a schematic diagram of parameter changes during the cascade reaction of the glucose oxidase-loaded nanoparticles (M@G) in Example 2.

[0040] Figure 5 This is a schematic diagram of the 1H NMR characterization results of Gel-QM in Example 5.

[0041] Figure 6 This is a schematic diagram of the antibacterial and antibiofilm properties of the microneedles in Example 5.

[0042] Figure 7 This is a bacterial morphology diagram after testing the antibacterial and antibiofilm properties of the microneedles in Example 5.

[0043] Figure 8 The residual biomass after in vitro anti-biofilm testing of the microneedles in Example 5.

[0044] Figure 9 The remaining colony count after the in vitro anti-biofilm test of the microneedles in Example 5.

[0045] Figure 10 This is a schematic diagram of the intradermal dissolution performance of the double-layer microneedles in Example 5.

[0046] Figure 11 This is a schematic diagram of the mechanical properties of the double-layer microneedles in Example 5.

[0047] Figure 12 This is a schematic diagram of mouse skin puncture using a double-layered microneedle in Example 5.

[0048] Figure 13 This is a schematic diagram illustrating the in vitro angiogenesis-promoting properties of the double-layer microneedle patch in Example 5. Detailed Implementation

[0049] Example 1-1

[0050] This embodiment provides a method for preparing iron-containing nanoparticles (MIL-101), including:

[0051] Step one, FeCl3.6H2O and terephthalic acid (H2BDC) are dissolved in 50ml N,N-dimethylformamide (DMF) to obtain a reaction system; in the reaction system, the concentration of FeCl3.6H2O is 0.1mmol / mL, and the concentration of terephthalic acid is 0.05mmol / mL;

[0052] Step two, the reaction system is transferred to a reaction kettle containing a polytetrafluoroethylene lining, the reaction kettle containing the reaction system is placed in an oven at 120℃ for reaction for 12h, cooled to room temperature, the post-reaction system is washed with N,N-dimethylformamide and anhydrous ethanol respectively for 3 times by centrifugation, and dried in a vacuum drying box to obtain the iron-containing nanoparticles (MIL-101).

[0053] Example 1-2

[0054] The embodiment provides a preparation method of iron-containing nanoparticles (MIL-101), which comprises the following steps:

[0055] Step one, FeCl3.6H2O and terephthalic acid (H2BDC) are dissolved in 50ml N,N-dimethylformamide (DMF) to obtain a reaction system; in the reaction system, the concentration of FeCl3.6H2O is 0.1mmol / mL, and the concentration of terephthalic acid is 0.1mmol / mL;

[0056] Step two, the reaction system is transferred to a reaction kettle containing a polytetrafluoroethylene lining, the reaction kettle containing the reaction system is placed in an oven at 120℃ for reaction for 6h, cooled to room temperature, the post-reaction system is washed with N,N-dimethylformamide and anhydrous ethanol respectively for 3 times by centrifugation, and dried in a vacuum drying box to obtain the iron-containing nanoparticles (MIL-101).

[0057] Example 1-3

[0058] The embodiment provides a preparation method of iron-containing nanoparticles (MIL-101), which comprises the following steps:

[0059] Step one, FeCl3.6H2O and terephthalic acid (H2BDC) are dissolved in 50ml N,N-dimethylformamide (DMF) to obtain a reaction system; in the reaction system, the concentration of FeCl3.6H2O is 0.1mmol / mL, and the concentration of terephthalic acid is 0.06mmol / mL;

[0060] Step two, transfer the reaction system to a reaction kettle containing a polytetrafluoroethylene lining, place the reaction kettle containing the reaction system in an oven at 120°C and react for 10h, cool to room temperature, centrifugally wash the post-reaction system with N,N-dimethylformamide and anhydrous ethanol for 3 times respectively, and dry in a vacuum drying box to obtain the iron-containing nanoparticles (MIL-101).

[0061] Example 2-1

[0062] The present example provides a method for preparing glucose oxidase-loaded nanoparticles (M@G), comprising:

[0063] Step one, completely disperse the iron-containing nanoparticles described in Example 1-1 in water under ultrasonic conditions to obtain a dispersion system; the concentration of the iron-containing nanoparticles in the dispersion system is 100 μg / mL;

[0064] Step two, add glucose oxidase to the dispersion system, stir at 4°C in the dark for 10h, centrifuge, wash with PBS for 3 times to remove the unloaded enzyme, and vacuum dry to obtain glucose oxidase-loaded nanoparticles (M@G); the mass of the glucose oxidase is 0.1 times the mass of the iron-containing nanoparticles; the molecular weight of the glucose oxidase is about 150 kDa, and the enzyme activity is 100-250 u / mg, which is purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0065] Figure 1 It is a scanning electron microscope diagram of the glucose oxidase-loaded nanoparticles (M@G) of Example 2-1. According to Figure 1 It can be seen that the glucose oxidase-loaded nanoparticles (M@G) have a particle size of about 700 nm and a spindle structure.

[0066] Figure 2 It is a UV-visible spectrum analysis result diagram of the nanoparticles MIL-101 of Example 1 and the glucose oxidase-loaded nanoparticles (M@G) of Example 2-1. According to Figure 2 It can be seen that the glucose oxidase-loaded nanoparticles (M@G) show new absorption bands at about 1640 cm -1 and 3280 cm + , which are respectively attributed to the NH4 -1 bending vibration and N-H band in the enzyme structure, indicating the successful loading of glucose oxidase.

[0067] Figure 3The schematic diagram of the glucose oxidase-promoting performance of the glucose oxidase-loaded nanoparticles (M@G) of Example 2-1. The test method comprises: mixing the glucose oxidase-loaded nanoparticles with water, ultrasonic treatment, preparing a suspension of different concentrations, adding a glucose solution for reaction respectively, using TMB colorimetry to test the spectrum at 652 nm, detecting the generation of hydroxyl radicals in the reaction system, wherein Figure 3 a is the fixed M@G concentration and different glucose content, Figure 3 b is the fixed glucose content and different M@G concentration. According to Figure 3 It can be seen that when the M@G concentration is 100 μg / mL, the absorption peak intensity at 652 nm is obviously enhanced with the increase of glucose content, and the content of ·OH radicals in the reaction system is also enhanced. Similarly, under the same glucose concentration, the ·OH radicals also show obvious M@G concentration dependence.

[0068] Figure 4 The schematic diagram of the parameter change in the cascade reaction of the glucose oxidase-loaded nanoparticles (M@G) of Example 2-1. The test method comprises: dispersing M@G in a glucose solution to obtain a system with different concentrations of dispersed M@G, placing the system with dispersed M@G in a 37℃ water bath for 24h, measuring the pH value of the system during the reaction by a pH meter, and the results are shown in Figure 4 a; placing the above-mentioned system with dispersed M@G in a 37℃ water bath for 24h, taking out the system at a preset time interval during the process, placing it in a PBS buffer (pH=5.4) containing horseradish peroxidase, reacting for 10 min, adding TMB, recording the characteristic absorbance of the colored TMB, and calculating the amount of H2O2 generated according to the standard curve, and the results are shown in Figure 4 b, wherein the volume of the PBS buffer containing horseradish peroxidase is 1.5 times the volume of the system taken out, the activity of the horseradish peroxidase is 1 U / mL, the volume of the PBS buffer is 5 times the volume of the horseradish peroxidase, and the volume of the TMB is equal to the volume of the horseradish peroxidase; placing the above-mentioned system with dispersed M@G in a 37℃ water bath for 24h, taking out 0.5 mL of the system at a preset time interval during the process, adding 1.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent respectively, heating at 100℃ for 5 min, then quickly transferring to cold water for 20 min, and measuring the UV-vis absorbance at 540 nm to obtain the consumption of glucose. Figure 4 a is the pH change of the system during the cascade reaction. According to Figure 4 a, it can be seen that within 12 hours of reaction, the pH value continuously decreases from 5.8 to 4.2, indicating that glucose acid continuously generates during the reaction, and the pH value of the system decreases, providing suitable acidity for the subsequent reaction.Figure 4 b is a schematic diagram of the change of the amount of intermediate H2O2 in the system during the cascade reaction. According to Figure 4 b It can be seen that the amount of intermediate H2O2 gradually increases as the reaction proceeds. Figure 4 c is the change of glucose content during the cascade reaction. According to Figure 4 b It can be seen that the content of glucose gradually decreases as the reaction proceeds, and the faster the glucose is consumed, the higher the M@G concentration is. This indicates that glucose can be continuously oxidized to generate gluconic acid and hydrogen peroxide, which can provide suitable acidity and substrate for Fenton reaction.

[0069] Example 2-2

[0070] This embodiment provides a preparation method of glucose oxidase-loaded nanoparticles (M@G), comprising:

[0071] Step one, under ultrasonic conditions, the iron-containing nanoparticles described in Example 1-2 are completely dispersed in water to obtain a dispersion system; the concentration of iron-containing nanoparticles in the dispersion system is 50 μg / mL;

[0072] Step two, glucose oxidase is added to the dispersion system, stirred at 4°C in the dark for 6h, centrifuged, washed with PBS for 3 times to remove the unloaded enzyme, vacuum dried to obtain glucose oxidase-loaded nanoparticles (M@G); the mass of glucose oxidase is 0.2 times the mass of iron-containing nanoparticles; the molecular weight of the glucose oxidase is about 150 kDa, and the enzyme activity is 100-250 u / mg, which is purchased from Shanghai Yuan Ye Biological Technology Co., Ltd.

[0073] The performance of the glucose oxidase-loaded nanoparticles (M@G) of this embodiment is basically the same as that of Example 2-1.

[0074] Example 2-3

[0075] This embodiment provides a preparation method of glucose oxidase-loaded nanoparticles (M@G), comprising:

[0076] Step one, under ultrasonic conditions, the iron-containing nanoparticles described in Example 1-3 are completely dispersed in water to obtain a dispersion system; the concentration of iron-containing nanoparticles in the dispersion system is 200 μg / mL;

[0077] Step two, glucose oxidase was added to the dispersion system, stirred at 4℃ for 12h in the dark, centrifuged, washed with PBS for 3 times to remove the unloaded enzyme, vacuum dried to obtain glucose oxidase loaded nanoparticles (M@G); the mass of the glucose oxidase was 0.05 times the mass of the iron-containing nanoparticles; the molecular weight of the glucose oxidase was about 150kDa, and the enzyme activity was 100-250u / mg purchased from Shanghai Yuan Ye Biotechnology Co., Ltd.

[0078] The performance of the glucose oxidase loaded nanoparticles (M@G) of the present embodiment is basically consistent with that of Example 2-1.

[0079] Example 3-1

[0080] The present embodiment provides a preparation method of gelatin grafted with pro-angiogenic peptide and double bond, comprising:

[0081] Step one, gelatin was dissolved in water to obtain a gelatin solution with a mass percentage of 10%;

[0082] Step two, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were added to the gelatin solution, stirred for 20min to obtain system A; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the system A was 0.6mg / mL, and the concentration of N-hydroxysuccinimide was 0.6mg / mL; the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were purchased from Aladdin Reagent Co., Ltd.;

[0083] Step three, polypeptide was added to the system A to obtain system B, and the system B was stirred and reacted at 37℃ for 3h, and then the reacted system was placed in a dialysis bag with a molecular weight of 7000Da for dialysis for one week, and then freeze-dried to obtain a gelatin product, denoted as Gel-Q; the concentration of the polypeptide in the system B was 0.3mg / mL, and the sequence of the polypeptide was QHREDGS (glutamine-histidine-arginine-glutamic acid-aspartic acid-glycine-serine), and the molecular weight was 827.79; the stirring rate in the stirring reaction was 500rpm; and the gelatin product was white foam;

[0084] Step four, dissolve the gelatin in PBS with pH 7.4 at 50℃ to obtain system C, slowly add methacrylic anhydride to the system C, stir the reaction at 50℃ for 2h, terminate the reaction, dialyze the system after termination of the reaction in a 7000Da dialysis bag for one week, freeze-dry to obtain gelatin grafted with pro-angiogenic peptide and double bond, recorded as Gel-Q-M; the mass percentage of gelatin in the system C is 10%; the mass of methacrylic anhydride is 0.2 times of the mass of gelatin; the termination of the reaction can be adding PBS at 40℃ to the system after the reaction, the mass of PBS used for termination of the reaction is 2 times of the mass of PBS used for dissolving gelatin; the stirring rate of the reaction is 500rpm.

[0085] Figure 5 The schematic diagram of 1H NMR characterization results of gelatin grafted with pro-angiogenic peptide and double bond in this example is shown. Figure 5 It can be seen that the peak of 5.2-5.6ppm is attributed to H on the double bond in methacrylamide, indicating that the double bond grafting is successful.

[0086] Example 3-2

[0087] This example provides a preparation method of gelatin grafted with pro-angiogenic peptide and double bond, comprising:

[0088] Step one, dissolve gelatin in water to obtain a gelatin solution with a mass percentage of 10% of gelatin;

[0089] Step two, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution, stir for 20min to obtain system A; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the system A is 0.5mg / mL, and the concentration of N-hydroxysuccinimide is 0.5mg / mL;

[0090] Step three, add polypeptide to the system A to obtain system B, stir the system B at 37℃ for 3h, dialyze the system after the reaction in a 7000Da dialysis bag for one week, freeze-dry to obtain gelatin; the concentration of polypeptide in the system B is 0.3mg / mL, and the sequence of the polypeptide is QHREDGS (glutamine-histidine-arginine-glutamic acid-aspartic acid-glycine-serine), with a molecular weight of 827.79; the stirring rate in the stirring reaction is 500rpm;

[0091] Step four, dissolve the gelatin in PBS with pH 7.4 at 50℃ to obtain system C, slowly add methacrylic anhydride into the system C, stir the reaction at 50℃ for 2h, terminate the reaction, dialyze the system after termination of the reaction in 7000Da dialysis bag for one week, freeze-dry to obtain gelatin grafted with pro-angiogenic peptide and double bond, recorded as Gel-Q-M; the mass percentage of gelatin in the system C is 10%; the mass of methacrylic anhydride is 0.1 times of the mass of gelatin; the termination of the reaction can be adding PBS at 40℃ into the system after the reaction, the mass of PBS used for termination of the reaction is 2 times of the mass of PBS used for dissolving gelatin; the stirring rate of the reaction is 500rpm.

[0092] Example 3-3

[0093] The embodiment provides a preparation method of gelatin grafted with pro-angiogenic peptide and double bond, comprising the following steps:

[0094] Step one, dissolve gelatin in water to obtain a gelatin solution with a mass percentage of 10% of gelatin;

[0095] Step two, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide into the gelatin solution, stir for 20min to obtain system A; the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in the system A is 0.7mg / mL, and the concentration of N-hydroxysuccinimide is 0.7mg / mL;

[0096] Step three, add polypeptide into the system A to obtain system B, stir the system B at 37℃ for 3h, dialyze the system after the reaction in 7000Da dialysis bag for one week, and freeze-dry to obtain gelatin; the concentration of polypeptide in the system B is 0.3mg / mL, the sequence of the polypeptide is QHREDGS (glutamine-histidine-arginine-glutamic acid-aspartic acid-glycine-serine), and the molecular weight is 827.79; the stirring rate in the stirring reaction is 500rpm;

[0097] Step four, dissolve the gel in PBS with pH 7.4 at 50℃ to obtain system C, slowly add methacrylic anhydride to the system C, stir at 50℃ for 2h, terminate the reaction, dialyze the system after termination of the reaction in a 7000Da dialysis bag for one week, freeze-dry to obtain gel grafted with pro-angiogenic peptide and double bond, recorded as Gel-Q-M; the mass percentage of gel in the system C is 10%; the mass of methacrylic anhydride is 0.3 times the mass of gel; the termination of the reaction can be adding PBS at 40℃ to the system after the reaction, the mass of PBS used for termination of the reaction is 2 times the mass of PBS used for dissolving the gel; the stirring rate is 500rpm.

[0098] Example 4

[0099] The embodiment provides a preparation method of a double-layer microneedle patch for diabetic wounds, comprising the following steps:

[0100] Step one, pour polydimethylsiloxane into a master mold of a microneedle array, demold after curing and forming to obtain a negative mold microneedle array mold; the structure of the negative mold microneedle array mold is opposite to that of the master mold of the microneedle array;

[0101] Step two, dissolve 1g of polyvinyl alcohol in 5mL of deionized water under the condition of 80℃ water bath to obtain a polyvinyl alcohol solution, cool, add 5mg of α-amylase to the cooled polyvinyl alcohol solution to obtain a mixed system A; the molecular weight of the polyvinyl alcohol is 9000-10000;

[0102] Step three, add the glucose oxidase-loaded nanoparticles in example 2-2 to deionized water to obtain a nanoparticle-containing system with a glucose oxidase-loaded nanoparticle concentration of 5mg / mL;

[0103] Step four, take 100μL of the nanoparticle-containing system and add it to the mixed system A in step two to obtain a needle tip preparation system;

[0104] Step five, dissolve the photoinitiator 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959) in water, add Gel-Q-M in example 3-2 until completely dissolved to obtain a backing solution; the mass percentage of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone in the backing solution is 0.05%, and the mass percentage of Gel-Q-M is 20%;

[0105] Step six, 20 μL of the tip preparation system described in step four is placed in the negative mold microneedle array mold, and the tip preparation system enters the tip part of the negative mold microneedle array mold, vacuum suction is performed to remove the excess tip preparation system, and 200 μL of the backing solution described in step five is added;

[0106] Step seven, the filled negative mold microneedle array mold in step six is placed in a 37℃ vacuum drying box for drying for 12h, demolding is performed, and light irradiation is performed under a UV lamp for 1min to obtain a double-layer microneedle patch for diabetic wounds; the UV lamp has a UV light wavelength of 365nm, and the light intensity can be 1.5-10mw / cm 2 .

[0107] Example 5

[0108] The embodiment provides a preparation method of a double-layer microneedle patch for diabetic wounds, comprising:

[0109] Step one, polydimethylsiloxane is cast in a master mold of a microneedle array, and after curing and forming, demolding is performed to obtain a negative mold microneedle array mold; the negative mold microneedle array mold is opposite in structure to the master mold of the microneedle array;

[0110] Step two, 1.5g of polyvinyl alcohol is dissolved in 5mL of deionized water under the condition of a 80℃ water bath to obtain a polyvinyl alcohol solution, which is cooled, 50mg of α-amylase is added to the cooled polyvinyl alcohol solution, and mixing is uniformly performed to obtain a mixed system A; the molecular weight of the polyvinyl alcohol is 9000-10000;

[0111] Step three, the glucose oxidase-loaded nanoparticles described in example 2-1 are added to deionized water to obtain a nanoparticle-containing system with a glucose oxidase-loaded nanoparticle concentration of 5mg / mL;

[0112] Step four, 500 μL of the nanoparticle-containing system is taken and added to the mixed system A described in step two to obtain a tip preparation system;

[0113] Step five, a photoinitiator 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone (I2959) is dissolved in water, and Gel-Q-M of example 3-1 is added until complete dissolution to obtain a backing solution; the mass percentage content of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone in the backing solution is 0.1%, and the mass percentage content of Gel-Q-M is 20%;

[0114] Step six, 20 μL of the tip preparation system described in step four was placed in the negative mold microneedle array mold, and the tip preparation system entered the tip part of the negative mold microneedle array mold. Vacuum was applied to remove the excess tip preparation system. 200 μL of the backing solution described in step five was added.

[0115] Step seven, the filled negative mold microneedle array mold of step six was placed in a 37°C vacuum drying oven for 24 hours. After demolding, the microneedle patch was irradiated under a UV lamp for 3 minutes. The UV lamp had a wavelength of 365 nm and an intensity of 1.5-10 mw / cm 2 , denoted as WMN.

[0116] Comparative Example 1

[0117] This comparative example provides a method for preparing an α-amylase-loaded microneedle patch α-Amylase MN, comprising:

[0118] Step one, 1.25 g of polyvinyl alcohol was dissolved in 5 mL of deionized water under the condition of a 80°C water bath. After cooling, 250 μL of the nanoparticle-containing system described in step three of Example 5 was added until it was uniformly dispersed. Centrifugation was performed to remove bubbles, and a microneedle preparation solution was obtained. The molecular weight of the polyvinyl alcohol was 10000.

[0119] Step two, the microneedle preparation solution was added to the PDMS negative mold mold using a syringe. Vacuum was applied to fill the PDMS negative mold mold with the microneedle preparation solution. After 24 hours of ventilation and drying, the microneedle patch was demolded. The microneedle patch loaded with glucose oxidase-loaded nanoparticles was denoted as M@G MN.

[0120] Comparative Example 2

[0121] This comparative example provides a method for preparing a microneedle patch loaded with glucose oxidase-loaded nanoparticles, comprising:

[0122] Step one, 1.25 g of polyvinyl alcohol was dissolved in 5 mL of deionized water under the condition of a 80°C water bath. After cooling, 250 μL of the nanoparticle-containing system described in step three of Example 5 was added until it was uniformly dispersed. Centrifugation was performed to remove bubbles, and a microneedle preparation solution was obtained. The molecular weight of the polyvinyl alcohol was 10000.

[0123] Step two, the microneedle preparation solution was added to the PDMS negative mold mold using a syringe. Vacuum was applied to fill the PDMS negative mold mold with the microneedle preparation solution. After 24 hours of ventilation and drying, the microneedle patch was demolded. The microneedle patch loaded with glucose oxidase-loaded nanoparticles was denoted as M@G MN.

[0124] Comparative Example 3

[0125] The comparative example provides a preparation method of a microneedle patch loaded with a- amylase and glucose oxidase loaded nanoparticles, comprising:

[0126] Step one, under the condition of 80°C water bath, 1.25g polyvinyl alcohol was dissolved in 5mL deionized water, cooled, 25mg a-amylase was added, mixed, 250μL nanoparticle containing system described in step three of example 5 was added until uniformly dispersed, centrifuged to remove bubbles, and a microneedle preparation solution was obtained; the molecular weight of the polyvinyl alcohol was 10000;

[0127] Step two, the microneedle preparation solution was added to the PDMS negative mold mold using a syringe, vacuum was applied to fill the microneedle preparation solution into the PDMS negative mold mold, and the mold was dried in a ventilated environment for 24h, demolded, and a microneedle patch loaded with glucose oxidase loaded nanoparticles was obtained, denoted as MN Tip.

[0128] Comparative example 4

[0129] The comparative example provides a preparation method of a Gel-Q-M microneedle patch, comprising:

[0130] Step one, 5mg photoinitiator 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1- propanone (I2959) was placed in 5mL deionized water and ultrasonicated until dissolved, 1g Gel-Q-M described in example 3-1 was added and placed in a 37°C water bath environment until dissolved, centrifuged to remove bubbles, and a microneedle preparation solution was obtained;

[0131] Step two, the microneedle preparation solution was added to the PDMS negative mold mold using a syringe, vacuum was applied to fill the microneedle preparation solution into the PDMS negative mold mold, and the mold was dried in a ventilated environment for 24h, demolded, and irradiated under ultraviolet conditions for 3min, and a Gel-Q-M microneedle patch was obtained, denoted as MN Base.

[0132] Comparative example 5

[0133] The comparative example provides a preparation method of a polyvinyl alcohol microneedle patch PVA MN without loading any substance, comprising:

[0134] Step one, under the condition of 80°C water bath, 1.25g polyvinyl alcohol was dissolved in 5mL deionized water, centrifuged to remove bubbles, and a microneedle preparation solution was obtained; the molecular weight of the polyvinyl alcohol was 9000-10000;

[0135] Step two, the microneedle preparation solution was added to the PDMS negative mold mold using a syringe, vacuum was applied to fill the microneedle preparation solution into the PDMS negative mold mold, and the mold was dried in a ventilated environment for 24h, demolded, and a microneedle patch loaded with glucose oxidase loaded nanoparticles was obtained, denoted as PVA MN.

[0136] Performance evaluation:

[0137] Figure 6 This is a schematic diagram illustrating the antibacterial and antibiofilm properties of the microneedles in Example 5. The testing method included using methicillin-resistant Staphylococcus aureus (MRSA) as a representative strain at a concentration of 10... 5 Microneedles from Examples 5, 3, and 4 were added to MRSA bacterial suspensions at CFU / mL, respectively. Glucose solution with a final concentration of 1 mg / mL was added to each suspension, and the suspensions were incubated together at 37°C in a shaker for 24 hours. The absorbance at 600 nm was measured. After serial dilution, 100 μL of the diluted bacterial suspension was evenly spread onto agar medium and incubated at 37°C for 24 hours. Bacterial growth was observed and photographed. The results are as follows: Figure 6 As shown. According to Figure 6 It can be seen that, compared with the blank group, the MN Base group (corresponding to the microneedles of Example 4) did not show antibacterial properties, while the MN Tip group (corresponding to the microneedles of Example 3) and the WMN group (corresponding to the microneedles of Example 5) showed a significant reduction in colony count, indicating that when M@G or a backing with angiogenesis effect is loaded into the microneedle patch, there is a significant enhancement in antibacterial ability.

[0138] Figure 7 This image shows the bacterial morphology after testing the antibacterial and anti-biofilm properties of the microneedles in Example 5. The test method included: taking 10 μL of the bacterial culture co-cultured in the above incubator and dropping it onto a coverslip; drying for 5–10 min; adding paraformaldehyde to fix the bacteria; and then, after 2 h, dehydrating the bacteria with gradients of 50%, 75%, 90%, and 95% ethanol solutions, respectively. The bacteria were then sputter-coated with gold for SEM observation of the morphology of the bacteria after different treatments. The results are shown below. Figure 7 As shown. According to Figure 7 As can be seen, similar to the control group, the bacteria in the MN Base group had smooth, intact spherical cell structures, and most were alive. In the MN Tip and WMN groups, it was clearly observed that the cell membrane structure of many bacteria was completely destroyed, and intracellular substances were significantly leaked out. This is likely due to the effective killing of bacteria by the large amount of ROS generated by M@G. This indicates that the microneedles of the present invention can effectively kill bacteria characterized by rapid spread, wide prevalence, and easy development of multidrug resistance.

[0139] Figure 8 The residual biomass after the in vitro anti-biofilm test of the microneedles in Example 5 is shown. The test method includes: first, transferring single colonies of MRSA from solid lysogenic broth (LB) agar plates to TSB medium rich in 3% w / v NaCl and 0.5% w / v glucose, respectively, and incubating with shaking at 37°C until their OD... 600 The value reaches ~0.5 (approximately equal to 10). 8CFU / mL was used to obtain the MRSA system. To obtain a biofilm, 500 μL of MRSA system (1 × 10⁻⁶ CFU / mL) was added. 8 CFU / mL was added to 48-well plates and incubated at 37°C for 24 hours. After removing the culture medium, a biofilm was observed at the bottom of the wells. Unattached bacteria were gently washed away three times with physiological saline. 300 μL of microneedle-containing extract was added to each of the 48-well plates with biofilm formation. After incubation at 37°C for 24 hours, the liquid in the wells was discarded, and the treated biofilm was rinsed three times with physiological saline to remove airborne bacteria. The preparation method of the microneedle-containing extract included: placing dried microneedles in PBS to obtain a microneedle system with a concentration of 1 g / mL; incubating the microneedle system at 37°C for 24 hours; removing undissolved portions to obtain the microneedle extract. The 48-well plate was placed under a fume hood for 20 min to dry the residual biofilm. Then, 500 μL of 0.1% crystal violet was added to each well to stain the biofilm for 15 min. The crystal violet was discarded, and the plate was washed three times with physiological saline to remove unbound crystal violet. Subsequently, 500 μL of 95% ethanol was added to each well to dissolve the dye. The absorbance was measured at 595 nm using a microplate reader to obtain the biomass of the residual biofilm. The results are as follows: Figure 8 As shown. According to Figure 8 As can be seen, compared with the blank group, the residual biomass of α-amylase MN (Comparative Example 1) decreased to 58%, showing a significant effect on biofilm degradation; the residual amount of M@G MN (Comparative Example 2) remained at 89%, indicating that although it exhibited excellent antibacterial ability, its dispersing effect on biofilm was weak; the microneedles (MNTip) of Comparative Example 3 and the microneedles (WMN) of the present invention both had significantly lower biofilm residual amounts, around 40%. This may be because after α-amylase and M@G bind to the biofilm, α-amylase degrades the EPS structure, exposing the originally encapsulated bacteria and making them sensitive. The ROS generated by the cascade reaction of glucose and M@G can more easily contact the bacterial cell membrane to exert an antibacterial effect, reducing bacterial reproduction and adhesion, thus achieving the effect of eliminating biofilm. The combination of peptide-functionalized gelatin as a microneedle backing does not affect the anti-biofilm effect of MN.

[0140] Figure 9 The remaining colony count after the in vitro biofilm test of the microneedles in Example 5 is shown. The test method is the plate test, which specifically includes: washing the treated biofilm three times with PBS, followed by two cycles of vortexing (5 minutes) and sonication (10 minutes) to isolate the attached bacteria into PBS. After serial dilution, 100 μl of the suspension is plated on LB agar medium, incubated at 37°C for 24 hours, photographed, and the colony count is shown. Figure 9 As shown. According to Figure 9It can be seen that a large number of bacteria still exist in the blank group biofilm, and the relative number of bacteria in the α-amylase MN (comparative example 1) is significantly reduced. This is because the destruction of the EPS structure reduces the enrichment and adhesion of bacteria, and a large number of bacteria are easily taken away by PBS, so the number of remaining bacteria is greatly reduced compared with the blank group. The M@G group (comparative example 2) still has 85% residual bacteria, which may be due to the fact that the biofilm provides a strong protective barrier for bacteria, and the destruction of the biofilm by ROS is limited, making it difficult for nanoparticles to directly act on the surface of bacteria. The number of residual bacteria of microneedles (MNTip) of comparative example 3 and microneedles (WMN) of the application is significantly reduced, which may be due to the fact that when α-amylase and M@G act together, the catalytic reaction of the double-enzyme cascade first destroys the EPS structure, and the bacteria lose the “protective layer” and become sensitive, and active oxygen is fully antibacterial, achieving sterilization and biofilm elimination.

[0141] Figure 10 The schematic diagram of the intradermal dissolving performance of the double-layer microneedle of example 5 is shown. The test method comprises the following steps: taking fresh mouse skin, cutting the skin block, absorbing the surface moisture with filter paper, fixing it on a foam board, placing the microneedle to be tested on the mouse skin, pressing it to pierce for 20 s, taking the microneedle out with tweezers at 1 min and 3 min, respectively, and observing the dissolution of the microneedle tip under a vertical microscope. The results are shown in Figure 10 . According to Figure 10 It can be seen that the dissolution rate of the microneedle tip increases with time, and most of the tips have dissolved after 3 min, indicating that the microneedle tip of the application has a rapid dissolving performance, which is beneficial to the rapid release of antibacterial and antibiofilm materials.

[0142] Figure 11 The schematic diagram of the mechanical performance of the double-layer microneedle of example 5 is shown. The test method comprises the following steps: placing the microneedle to be tested upside down on the stainless steel circular small platform of the tensile testing machine, setting the running speed of the tensile testing machine to 1 mm / min, and moving towards the large circular platform at a constant speed, continuously measuring the change of displacement and stress of the microneedle in the process, obtaining the displacement and stress curve to calculate the mechanical performance, and the results are shown in Figure 11 . According to Figure 11 It can be seen that when the displacement is 400 μm, the mechanical strength of the drug-loaded microneedle (double-layer microneedle of example 5) is 0.39 N / needle, which is slightly higher than that of the non-drug-loaded microneedle (comparative example 5), and higher than the minimum penetration force required for normal skin penetration, which is 0.058 N per needle. It indicates that the double-layer microneedle of the application is sufficient to penetrate the skin.

[0143] Figure 12The mouse skin puncture schematic diagram of the double-layer microneedle of Example 5. The puncture experiment was carried out on the mouse skin in vitro, which specifically included: taking fresh mouse skin in physiological saline, absorbing the surface moisture with filter paper, and fixing it on the foam board. After 3 min, the microneedle patch was removed, and the schematic diagram was as follows Figure 12 . According to Figure 12 It can be seen that the mouse skin has a complete array of microneedle holes, which proves that the microneedle patch prepared by the method has good skin puncture effect.

[0144] Figure 13 The in vitro pro-angiogenic performance schematic diagram of the double-layer microneedle patch of the double-layer microneedle of Example 5. The test method included: adding 300 μL Matrigel to each well of a 24-well plate, placing it in a 37℃ incubator for 30 min, until it solidified, and then inoculating human umbilical vein endothelial cells HUVEC (2×10 4 cells / well) on the solidified Matrigel matrix, adding microneedle extract to the experimental group, and not adding any substance to the blank group. After 6 hours of culture (5% CO2, 37℃), the cells were imaged using a microscope, and the number of branches per square millimeter was measured using ImageJ, and the results were as shown in Figure 13 . The preparation method of the microneedle extract included: placing dried microneedles in culture medium to obtain a microneedle system with a microneedle concentration of 1 g / mL, placing the microneedle system in culture medium at 37℃ for 24 hours, removing the undissolved part, and obtaining the microneedle extract.

[0145] According to Figure 13 It can be seen that compared with the blank group, the MN Base group of Comparative Example 4 and the WMN group of the present example both significantly increased the branch points and the average tube length, the branch points were 1.7 times and 1.8 times that of the blank group, respectively, and the average tube length was 1.75 times and 1.86 times that of the blank group, respectively, which showed the ability of the double-layer microneedle of the present example to promote vascular formation of endothelial cells.

[0146] Example 6

[0147] The present example provides a preparation method of a double-layer microneedle patch for diabetic wounds, which includes:

[0148] Step one, pouring polydimethylsiloxane into the master mold of the microneedle array, demolding after curing and forming, to obtain a negative microneedle array mold; the structure of the negative microneedle array mold is opposite to that of the master mold of the microneedle array;

[0149] Step two, under the condition of 80℃ water bath, 1.25g polyvinyl alcohol was dissolved in 5mL deionized water to obtain a polyvinyl alcohol solution, which was cooled, 25mg of α-amylase was added to the above cooled polyvinyl alcohol solution, and mixed uniformly to obtain a mixed system A; the molecular weight of the polyvinyl alcohol is 9000-10000;

[0150] Step three, the glucose oxidase loaded nanoparticles described in example 2-3 were added to deionized water to obtain a nanoparticle containing system with a concentration of glucose oxidase loaded nanoparticles of 5 mg / mL;

[0151] Step four, 250 μL of the nanoparticle containing system described in step two was added to the mixed system A described in step two to obtain a tip preparation system;

[0152] Step five, the photoinitiator 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1- propanone (I2959) was dissolved in water, and the Gel-Q-M of example 3-3 was added until completely dissolved to obtain a backing solution; the mass percentage of 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone in the backing solution was 0.08%, and the mass percentage of Gel-Q-M was 20%;

[0153] Step six, 20 μL of the tip preparation system described in step four was placed in the negative mold microneedle array mold, and the tip preparation system was allowed to enter the tip part of the negative mold microneedle array mold, vacuum was applied, and the excess tip preparation system was removed by suction, and 200 μL of the backing solution described in step five was added;

[0154] Step seven, the filled negative mold microneedle array mold of step six was placed in a 37°C vacuum drying oven for 18 h, demolded, and irradiated under a UV lamp for 2 min to obtain a double-layer microneedle patch for diabetic wounds; the UV light wavelength of the UV lamp was 365 nm, and the light intensity could be 1.5-10 mw / cm 2 .

[0155] The performance of the double-layer microneedle patch in this example was basically the same as that of example 5.

[0156] The above description is only a preferred embodiment of the present application, and does not limit the present application in any way. Any simple modification, change, and equivalent structural change made according to the technical essence of the present application to the above embodiments are still within the protection scope of the technical solution of the present application.

Claims

1. A method for preparing a double-layer microneedle patch for diabetic wounds, characterized in that, The bilayer microneedle patch includes a needle tip and a backing. The needle tip is formed by vacuum drying and ultraviolet crosslinking of a needle tip preparation system. The backing is formed by vacuum drying and ultraviolet crosslinking of a backing solution. The needle tip preparation system uses nanoparticles loaded with glucose oxidase and polyvinyl alcohol solution as the main raw materials. The backing solution uses photoinitiator and gelatin grafted with angiogenic peptides and double bonds as the main raw materials. The preparation method of the bilayer microneedle patch includes: Step 1: Provide a female mold microneedle array mold; Step 2: Providing a needle tip preparation system, specifically including: adding α-amylase to a cooled polyvinyl alcohol solution to obtain a mixed system A; adding a nanoparticle-containing system to the mixed system A to obtain the needle tip preparation system; the nanoparticle-containing system is a nanoparticle-containing system obtained by dispersing glucose oxidase-loaded nanoparticles in deionized water; the preparation method of the glucose oxidase-loaded nanoparticles includes: Step 101: Under ultrasonic conditions, the iron-containing nanoparticles are completely dispersed in water to obtain a dispersion system; Step 102: Add glucose oxidase to the dispersion system described in step 101, stir at 4°C in the dark for 6-12 hours, centrifuge, wash, and vacuum dry to obtain glucose oxidase-loaded nanoparticles. Step 3: Providing a backing solution, specifically including: dissolving a photoinitiator in water, adding gelatin grafted with angiogenic peptides and double bonds to obtain a backing solution; the preparation method of the gelatin grafted with angiogenic peptides and double bonds includes: Step 301: Add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to the gelatin solution to obtain system A; Step 302: Add the pro-angiogenic peptide to system A described in step 301 to obtain system B. Stir system B and then dialyze and freeze-dry it to obtain a gel. Step 303: Dissolve the gel in PBS to obtain system C. Add methacrylic anhydride to system C and stir the reaction to terminate it. Dialyze the system after the reaction is terminated and freeze-dry it to obtain gelatin grafted with angiogenic peptides and double bonds. Step 4: Filling the female mold microneedle array mold, specifically including: placing the needle tip preparation system into the female mold microneedle array mold described in Step 1, so that the needle tip preparation system enters the needle tip part of the female mold microneedle array mold, drawing a vacuum, and adding the backing solution; Step 5: After filling the microneedle array mold in Step 4, vacuum dry it for 12-24 hours, demold it, and irradiate it under ultraviolet light for 1-3 minutes to obtain a double-layer microneedle patch for diabetic wounds.

2. The method for preparing a double-layer microneedle patch for diabetic wounds according to claim 1, characterized in that, In step two, the mass of polyvinyl alcohol is 20 to 300 times the mass of α-amylase, the concentration of polyvinyl alcohol in the polyvinyl alcohol solution is 0.2 to 0.3 g / mL, and the molecular weight of polyvinyl alcohol is 9000 to 10000; the volume of the nanoparticle-containing system is 100 to 330 times the mass of polyvinyl alcohol, the volume unit of the nanoparticle-containing system is μL, and the mass unit of polyvinyl alcohol is g.

3. The method for preparing a double-layer microneedle patch for diabetic wounds according to claim 1, characterized in that, In step three, the photoinitiator in the backing solution has a mass percentage of 0.05-0.1%, and the gelatin grafted with angiogenic peptides and double bonds has a mass percentage of 20%; the photoinitiator is 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

4. The method for preparing a double-layer microneedle patch for diabetic wounds according to claim 1, characterized in that, In step four, the volume of the backing solution is five times the volume of the needle tip preparation system.

5. The method for preparing a double-layer microneedle patch for diabetic wounds according to claim 1, characterized in that, The concentration of iron-containing nanoparticles in the dispersion system described in step 101 is 50–200 μg / mL; the mass of glucose oxidase described in step 102 is 0.05–0.2 times the mass of iron-containing nanoparticles; the molecular weight of glucose oxidase is 150 kDa, and the enzyme activity is 100–250 U / mg.

6. The method for preparing a double-layer microneedle patch for diabetic wounds according to claim 1, characterized in that, The method for preparing the iron-containing nanoparticles includes: Step 201: Dissolve FeCl3·6H2O and terephthalic acid in N,N-dimethylformamide to obtain a reaction system; the amount of terephthalic acid is 0.5 to 1 times the amount of FeCl3·6H2O. Step 202: React the reaction system described in step 201 at 120°C for 6-12 hours, cool, centrifuge and wash, and vacuum dry to obtain iron-containing nanoparticles.

7. The method for preparing a double-layer microneedle patch for diabetic wounds according to claim 1, characterized in that, In step 301, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in system A is 0.5–0.7 mg / mL, and the concentration of N-hydroxysuccinimide is 0.5–0.7 mg / mL; in step 302, the concentration of the angiogenic peptide in system B is 0.3 mg / mL, and the molecular weight of the angiogenic peptide is 827.79; in step 303, the mass of methacrylic anhydride is 0.1–0.3 times the mass of the gel.

Images