External degradable microneedle patch as well as preparation method and application thereof

Abstract

The invention relates to an external degradable microneedle patch as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method comprises the following steps: S1, preparing powdery CS-TA; s2, preparing a powdery Cu-TA nano enzyme; s3, adding the Cu-TA nano-enzyme and glucose oxidase GOD into water, stirring at 1-4 DEG C in an inert gas atmosphere, centrifuging, and re-suspending in water to obtain a GOD-coated Cu-TA compound enzyme solution; s4, adding CS-TA, hyaluronic acid, polyvinylpyrrolidone, L-arginine and a basic fibroblast growth factor into the GOD (at) Cu-TA compound enzyme solution, and preparing a microneedle mother solution; s5, preparing an aqueous solution containing CS-TA, hyaluronic acid and polyvinylpyrrolidone to obtain a substrate mother solution; and S6, filling a microneedle mold cavity of a mold with the microneedle mother solution to form enzyme series microneedles arranged in an array, then pouring a substrate mother solution to form a substrate, drying, and demolding to obtain the external degradable microneedle patch.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a topical biodegradable microneedle patch, its preparation method, and its application. Background Technology

[0002] In recent years, diabetes, as a chronic metabolic disease, has seen a year-on-year increase in the number of patients worldwide, becoming a serious public health problem. Among the many complications of diabetes, diabetic foot is a relatively common and serious one. Diabetic foot refers to the lesions of blood vessels and nerves in the foot distal to the ankle joint in diabetic patients, leading to insufficient blood supply to the foot, abnormal sensation, and symptoms such as ulceration and infection. In severe cases, it can affect muscles and bones, leading to tissue necrosis and even amputation. Long-term hyperglycemia is the basis for the development of diabetic foot. It can damage vascular endothelial cells, causing microvascular disease, thickening of the blood vessel walls, and narrowing of the lumen, thus leading to ischemia and hypoxia in the foot tissues. At the same time, hyperglycemia can also damage nerves, causing neuropathy, resulting in decreased or absent sensation in the foot. Patients are prone to foot skin damage due to factors such as trauma, friction, and pressure without realizing it, thus forming ulcers and other lesions. In addition, diabetic patients have abnormal immune function and reduced wound healing ability. Once foot infection occurs, the condition can easily deteriorate rapidly and is difficult to control.

[0003] Clinically, treatment for diabetic foot mainly includes blood glucose control, improving blood circulation, anti-infection, and wound management. Pharmacologically, antibiotics such as cephalosporins and carbapenems are used to control infection, vasodilators such as beraprost improve blood circulation, and neurotrophic drugs such as mecobalamin and alpha-lipoic acid protect nerves. However, these treatments and drugs have many drawbacks and side effects. Long-term use of antibiotics can easily lead to dysbiosis and increased drug resistance; vasodilators may cause adverse reactions such as headaches and palpitations; antiplatelet drugs increase the risk of bleeding; and while neurotrophic drugs have fewer adverse reactions, their long-term therapeutic effects lack sufficient evidence. Furthermore, traditional wound management methods such as debridement and dressing changes have limited efficacy for refractory wounds, and equipment such as negative pressure wound therapy is expensive. Therefore, developing a new therapy that can effectively promote the healing of diabetic foot wounds, has high biocompatibility, and is easy to translate into clinical practice is of great significance for improving the prognosis and quality of life of diabetic foot patients.

[0004] Microneedles, as a novel transdermal drug delivery technology, offer advantages such as being painless, minimally invasive, and easy to operate. They effectively promote transdermal drug penetration and increase drug concentration in the skin and subcutaneous tissues, providing a new drug delivery route for the treatment of diabetic foot. Nanozymes, on the other hand, are a class of nanomaterials with enzyme-mimicking properties. They can mimic the activity of various biological enzymes, such as superoxide dismutase (SOD) and catalase (CAT), enabling multi-enzyme cascades and biological reaction cascades, demonstrating unique potential in the complex pathological environment of diabetic foot. Microneedle patch delivery of nanozymes allows for precise delivery of nanozymes and synergistic factors to the diabetic foot wound. The catalytic system built around nanozymes effectively removes reactive oxygen species (ROS) from the wound, reduces inflammation, and promotes tissue repair and regeneration. Simultaneously, combined with microneedle technology, nanozymes can form cascade reaction systems with natural enzymes such as glucose oxidase (GOD), achieving cascade catalytic reactions and further enhancing therapeutic effects. This combined application is expected to overcome the shortcomings of traditional treatment methods, providing a new, efficient, safe, and easily clinically translatable strategy for the treatment of diabetic foot.

[0005] Currently, the core technological bottleneck in this field lies in the difficulty of simultaneously achieving long-term adhesion and deep penetration. Furthermore, existing drugs have limited functions, neglecting the multidimensional regulation of wound infection inhibition, inflammatory factor modulation, and angiogenesis promotion, particularly lacking effective solutions for slow-healing wounds. Therefore, there is an urgent need to develop novel treatment strategies that combine long-term adhesion, deep penetration, and multifunctional regulation to achieve efficient repair and regeneration of diabetic foot wounds. Summary of the Invention

[0006] The purpose of this invention is to overcome the existing technical problems in this field and to achieve a biodegradable microneedle patch with long-lasting adhesion, deep penetration and multifunctional control, as well as its preparation method and application.

[0007] In a first aspect, the present invention provides a method for preparing a topical biodegradable microneedle patch, the method comprising the following steps:

[0008] S1. Dissolve chitosan (CS) in dilute hydrochloric acid, adjust the pH to 3.0-4.0, add tannic acid and hydrogen peroxide, stir in the dark until the reaction is complete, dialyze the reaction product and freeze dry to obtain powdered CS-TA.

[0009] S2. Dissolve copper chloride and tannic acid in water, add sodium carbonate solution dropwise, adjust the pH to 8.0-9.0, and then stir at room temperature under an inert gas atmosphere until the reaction is complete. After centrifugation, washing with water and drying, the reaction product is obtained as powdered Cu-TA nanozyme.

[0010] S3. Add Cu-TA nanozyme and glucose oxidase GOD to water, stir at 1-4℃ under an inert gas atmosphere, centrifuge and resuspend in water to obtain GOD@Cu-TA composite enzyme solution;

[0011] S4. Add CS-TA, hyaluronic acid (HA), polyvinylpyrrolidone (PVP-K30), L-arginine (L-Arg) and basic fibroblast growth factor (bFGF) to the GOD@Cu-TA complex enzyme solution to prepare microneedle stock solution;

[0012] S5. Prepare an aqueous solution containing CS-TA, hyaluronic acid and polyvinylpyrrolidone to obtain the base stock solution;

[0013] S6. The microneedle mother liquor is filled into the microneedle cavity of the mold to form an array of enzyme tandem microneedles, and then the base mother liquor is poured in to form a base. After drying, the mold is removed to obtain the external biodegradable microneedle patch.

[0014] In some embodiments of the present invention, in step S1, the mass ratio of chitosan to tannic acid is 1:(0.1-0.5).

[0015] In some embodiments of the present invention, the final concentration of hydrogen peroxide in the reaction system of step S1 is 0.1-1 mol / L.

[0016] In some embodiments of the present invention, in step S2, the mass ratio of copper chloride to tannic acid is (0.5-1):(0.9-1.8).

[0017] In some embodiments of the present invention, in step S3, the mass ratio of Cu-TA nanozyme to glucose oxidase is (0.1-100):1.

[0018] In some embodiments of the present invention, the concentration of CS-TA in the microneedle stock solution is 2-18 mg / mL, the concentration of hyaluronic acid is 2-18 mg / mL, the concentration of polyvinylpyrrolidone is 2-8 mg / mL, the concentration of GOD@Cu-TA complex enzyme is 0.2-3 mg / mL, the concentration of L-arginine is 0.5-5 mg / mL, and the concentration of basic fibroblast growth factor is 0.02-0.2 mg / mL.

[0019] In some embodiments of the present invention, the concentration of CS-TA in the base mother liquor is 2-18 mg / mL, the concentration of hyaluronic acid is 2-18 mg / mL, and the concentration of polyvinylpyrrolidone is 2-8 mg / mL.

[0020] In a second aspect, the present invention provides a topical biodegradable microneedle patch prepared according to the above-described preparation method.

[0021] In some embodiments of the present invention, the enzyme tandem microneedles are in the shape of a square pyramid with a height of 400-500 μm and a base thickness of 110-200 μm.

[0022] A third aspect of the present invention provides the application of the above-described topical biodegradable microneedle patch in the preparation of adjunctive treatment materials for diabetic foot.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) This invention addresses the problems of high glucose levels, easy infection, and difficulty in healing in existing diabetic foot wounds. It designs a microneedle patch with a microneedle array, integrating the compound enzyme GOD@Cu-TA, L-arginine, and basic fibroblast growth factor into the microneedle site. This achieves a reaction cascade based on enzyme tandem and the synergistic effect of multiple components. It innovatively combines functions such as ROS clearance, NO-promoting healing, and accelerated angiogenesis to achieve precise drug delivery to the lesion site. The synergistic effect of multiple components achieves multidimensional regulation of wound infection inhibition, inflammatory factor regulation, and angiogenesis promotion. At the same time, the combination of the characteristics of compound enzymes and microneedles achieves long-term release and improves the therapeutic effect.

[0025] (2) In this invention, chitosan and hyaluronic acid are used to form the microneedle body, which has antibacterial properties to reduce wound infection. The biodegradability of the microneedles is adjusted by doping with PVP to meet the needs of use under different conditions. The biodegradable microneedles can achieve long-term adhesion while avoiding secondary damage caused by microneedle peeling after wound healing. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the process of preparing microneedles using a mold, as provided in an embodiment of the present invention.

[0027] Figure 2 This is a transmission electron microscope image of the Cu-TA nanozyme provided in Example 2 of the present invention;

[0028] Figure 3 The hydration particle size diagram of the Cu-TA nanozyme provided in Example 2 of this invention;

[0029] Figure 4 The results of SOD enzyme activity (left) and CAT enzyme activity (right) of Cu-TA nanozyme in Test Example 2 are shown.

[0030] Figure 5 The figure shows the results of high performance liquid chromatography (HPLC) detection of the residual amount of glucose in the GOD@Cu-TA reaction system as a function of reaction time.

[0031] Figure 6This shows a view of the amount of NO generated by the oxidation of L-arginine under different glucose concentrations using GOD@Cu-TA;

[0032] Figure 7 Optical images and scanning electron microscope images of the microneedle patch prepared in Example 6;

[0033] Figure 8 The image shows the antibacterial effect of the microneedle patches prepared in Examples 4-6 against Staphylococcus aureus and Escherichia coli as determined by the plating method.

[0034] Figure 9 These are photographs of wounds in diabetic mice at different times after appropriate treatment. Detailed Implementation Plan

[0035] The following specific embodiments further illustrate the above-mentioned content of the present invention in detail, but do not imply that the embodiments limit the present invention.

[0036] The present invention will be further illustrated below with specific embodiments.

[0037] The reagents, materials, and instruments used in the following examples are as follows:

[0038] Reagents: copper chloride, tannic acid, chitosan, hyaluronic acid, PVP-K30, hydrogen peroxide, glucose oxidase (GOD), L-arginine (L-Arg), basic fibroblast growth factor (bFGF), agarose, peptone, and cell culture medium;

[0039] Materials: dialysis bags, PDMS microneedle templates, SOD activity assay kits, NO assay kits, cell proliferation and toxicity assay kits;

[0040] Instruments: forced-air drying oven, magnetic stirrer, vacuum drying oven, high-speed centrifuge, stereo microscope, inverted microscope, scanning electron microscope, transmission electron microscope, ultrasonic cleaner, Malvern dynamic light scattering instrument (DLS).

[0041] An embodiment of the first aspect of the present invention provides a method for preparing a topical biodegradable microneedle patch, the method comprising the following steps:

[0042] S1. Dissolve chitosan in dilute hydrochloric acid, adjust the pH to 3.0-4.0, add tannic acid and hydrogen peroxide to obtain a mixed solution, stir in the dark until the reaction is complete, dialyze the reaction product and freeze dry to obtain powdered CS-TA; wherein, the preferred mass ratio of chitosan to tannic acid is 1:(0.1-0.5), and the final concentration of hydrogen peroxide in the mixed solution is (0.1-1) mol / L.

[0043] S2. Dissolve copper chloride and tannic acid in water, add sodium carbonate solution dropwise, adjust the pH value to 8.0-9.0, and then stir at room temperature under an inert gas atmosphere until the reaction is complete. After centrifugation, washing with water and drying, the reaction product is obtained as powdered Cu-TA nanozyme; wherein, the preferred mass ratio of copper chloride to tannic acid is (0.5-1):(0.9-1.8).

[0044] S3. Add Cu-TA nanozyme and glucose oxidase GOD to water, stir at 1-4℃ under an inert gas atmosphere, centrifuge and resuspend in water to obtain GOD@Cu-TA composite enzyme solution; wherein, the preferred mass ratio of Cu-TA nanozyme to glucose oxidase is (0.1-100):1.

[0045] S4. Add CS-TA, hyaluronic acid, polyvinylpyrrolidone, L-arginine, and basic fibroblast growth factor to the GOD@Cu-TA complex enzyme solution to prepare a microneedle stock solution; the preferred concentrations of each component in the microneedle stock solution are: CS-TA concentration of 2-18 mg / mL, hyaluronic acid concentration of 2-18 mg / mL, polyvinylpyrrolidone concentration of 2-8 mg / mL, GOD@Cu-TA complex enzyme concentration of 0.2-3 mg / mL, L-arginine concentration of 0.5-5 mg / mL, and basic fibroblast growth factor concentration of 0.02-0.2 mg / mL.

[0046] S5. Prepare an aqueous solution containing CS-TA, hyaluronic acid and polyvinylpyrrolidone to obtain a base mother liquor; the preferred concentrations of each component in the base mother liquor are: CS-TA concentration of 2-18 mg / mL, hyaluronic acid concentration of 2-18 mg / mL, and polyvinylpyrrolidone concentration of 2-8 mg / mL.

[0047] S6. Fill the microneedle stock solution into the microneedle cavity of the mold to form an array of enzyme tandem microneedles, then pour in the base stock solution to form the base, and after drying, demold to obtain the topical biodegradable microneedle patch. The specific operation process is as follows: Figure 1 As shown.

[0048] Example 1: Synthesis of CS-TA

[0049] Dissolve 1 g of CS in 100 mL of pure water. Then, add a small amount of HCl until the CS is completely dissolved. Adjust the pH of the CS solution to 4.0. Next, add 0.1 g of tannic acid (TA). After the TA dissolves, add hydrogen peroxide to a final concentration of 1 mol / L. Transfer the mixture to a round-bottom flask and stir in the dark for 24 hours, followed by dialyzing in the dark. The dialysis method involves boiling a dialysis bag with an MWCO content of 8000-14000 Da for 10-25 minutes, then adding the supernatant from centrifugation to the dialysis bag. Dialyze in the dark for 36-48 hours using a 0.001 mol / L HCl solution, changing the solution three times at the 2nd, 6th, and 24th hours. Lyophilize to obtain CS-TA powder.

[0050] Example 2: Synthesis of Cu-TA nanozymes

[0051] Dissolve 1 g CuCl2 and 1.8 g TA in 100 ml of ultrapure water. Then add sodium carbonate solution (10 mM / L) and adjust the pH of the solution to 8.0 with vigorous stirring. Purge with nitrogen for 15 min to remove some air from the solution. Stir at room temperature for 24 h, then centrifuge at 10,000 rpm for 15 min. Collect the nanosheet precipitate. Wash with deionized water three times, dry, and obtain Cu-TA nanozyme powder. Prepare Cu-TA nanozyme aqueous solutions of specific concentrations.

[0052] Example 3: Synthesis of GOD@Cu-TA complex enzyme

[0053] 100 mg Cu-TA nanozyme and 10 mg glucose oxidase (GOD) were dissolved in 50 mL of pure water, and then 50 mL of PBS buffer was added to adjust the pH to 7.4. Nitrogen gas was purged for 10 min to remove some air from the solution, and the mixture was stirred at 4°C for 12 h. The solution was then centrifuged at 12000 rpm for 15 min. The precipitate was collected, lyophilized to obtain GOD@Cu-TA, and solutions of specific concentrations were prepared.

[0054] Example 4

[0055] (1) Prepare microneedle stock solution

[0056] The concentrations of CS-TA in the microneedle stock solution were 4 mg / mL, HA was 16 mg / mL, PVP-K30 was 2 mg / mL, GOD@Cu-TA was 1 mg / mL, L-arginine was 2.5 mg / mL, and basic fibroblast growth factor was 0.05 mg / mL.

[0057] (2) Preparation of base stock solution

[0058] In the base stock solution, the concentration of CS-TA was 4 mg / mL, the concentration of HA was 16 mg / mL, and the concentration of PVP-K30 was 2 mg / mL.

[0059] (3) Preparation of biodegradable microneedle patches for external use

[0060] Take 200 μL of microneedle stock solution and spread it evenly on the surface of a 15×15 microneedle array polydimethylsiloxane (PDMS) mold. Place the PDMS mold in a 5 mL vacuum device, evacuate to -0.1 kPa, maintain for 5 min, release the gas, take out the mold, and place it in a 45℃ drying oven for 10 min to allow the air bubbles to float out. Repeat this process 3 times to ensure that the mixed solution completely fills the mold cavity. Use a tool to scrape off the excess solution on top of the mold.

[0061] Then, 100 μL of the base stock solution was poured onto a PDMS mold filled with the microneedle stock solution to form a base layer. After performing the vacuum operation described above, the mold was placed in a drying oven at 37°C for 12 hours until the microneedle array was completely dried. The microneedle array was then demolded to obtain an enzyme-tandem biodegradable microneedle patch for external use. The obtained microneedle array has a close-packed structure, with the needle tip shape being a four-sided pyramid, the needle tip height being 400-500 μm, and the base thickness being 110-200 μm.

[0062] Example 5

[0063] Compared with Example 4, the concentrations of each substance in the microneedle mother liquor and the base mother liquor are different, but the other steps are the same.

[0064] The concentrations of each substance in the microneedle stock solution are as follows:

[0065] The concentrations of CS-TA in the microneedle stock solution were 4 mg / mL, HA was 16 mg / mL, PVP-K30 was 2 mg / mL, GOD@Cu-TA was 1.5 mg / mL, L-arginine was 3 mg / mL, and basic fibroblast growth factor was 0.1 mg / mL.

[0066] The concentrations of each substance in the base mother liquor are as follows:

[0067] In the microneedle tip layer stock solution, the concentration of CS-TA was 4 mg / mL, the concentration of HA was 16 mg / mL, and the concentration of PVP-K30 was 2 mg / mL.

[0068] Example 6

[0069] Compared with Example 4, the concentrations of each substance in the microneedle mother liquor and the base mother liquor are different, but the other steps are the same.

[0070] The concentrations of each substance in the microneedle stock solution are as follows:

[0071] The concentrations of CS-TA in the microneedle stock solution were 4 mg / mL, HA was 16 mg / mL, PVP-K30 was 2 mg / mL, GOD@Cu-TA was 1.5 mg / mL, L-arginine was 3 mg / mL, and basic fibroblast growth factor was 0.2 mg / mL.

[0072] The concentrations of each substance in the base mother liquor are as follows:

[0073] In the base stock solution, the concentration of CS-TA was 4 mg / mL, the concentration of HA was 16 mg / mL, and the concentration of PVP-K30 was 2 mg / mL.

[0074] Comparative Example 1 (without GOD@Cu-TA)

[0075] Compared to Example 6, the microneedle mother solution did not contain GOD@Cu-TA nanozyme, but the remaining steps were the same.

[0076] Comparative Example 2 (without L-Arg)

[0077] Compared to Example 6, the microneedle stock solution did not contain L-Arg, but the other steps were the same.

[0078] Comparative Example 3 (without bFGF)

[0079] Compared to Example 6, the microneedle stock solution did not contain bFGF, but the remaining steps were the same.

[0080] The synthesized Cu-TA, GOD@Cu-TA, and microneedle patches obtained in Examples 1-3 and Comparative Examples 1-3 were characterized using the following methods:

[0081] Test Example 1: Transmission electron microscopy image and hydration particle size diagram of Cu-TA nanozyme

[0082] Morphological characterization of Cu-TA nanozymes: Cu-TA nanozymes were dispersed in water and sonicated for 30 minutes to ensure uniform dispersion. A suitable amount of the dispersion was dropped onto a copper grid and allowed to air dry at room temperature. Characterization was performed using a TALOS transmission electron microscope (TEM). The accelerating voltage was set to 200 kV. The results are shown below. Figure 2 As shown in the figure. Experimental results show that Cu-TA exhibits a plate-like structure without obvious aggregation, indicating that Cu-TA has good dispersibility. Furthermore, Cu-TA nanozyme was dispersed in deionized water to prepare a 0.1 mg / mL solution. The hydrated particle size was determined using a Malvern dynamic light scattering (DLS) instrument, with the temperature set at 25℃ and the measurement angle at 90 degrees. Each sample was measured three times, and the average value was taken. Figure 3As shown, the hydrated particle size of Cu-TA exhibits good stability at different concentrations, with an average hydrated particle size of 154 nm and a narrow particle size distribution. This indicates that Cu-TA has good solubility and stability in aqueous solution and can maintain its nanoscale particle size under physiological conditions, which is beneficial for its application in biological systems.

[0083] Test Example 2: Detection of Superoxide Dismutase (SOD) and Catalase (CAT) Activities in Cu-TA Nanozyme Solution

[0084] SOD enzyme activity assay: Methionine, riboflavin, nitroblue tetrazolium, and Cu-TA nanozyme solutions of different concentrations were mixed and irradiated with light for 30 minutes. The absorbance values ​​at 400nm-800nm ​​were then measured. The results are shown in the figure. Figure 4 The left figure shows that the Cu-TA nanozyme solution exhibits good superoxide anion scavenging ability. In the reaction, the concentrations were methionine (10 mM), riboflavin (20 μM), and nitroblue tetrazolium (0.1 μM).

[0085] CAT enzyme activity assay: The CAT enzyme activity of Cu-TA was detected by measuring the UV absorption change of hydrogen peroxide at 240 nm. 0-12 mM hydrogen peroxide solutions were prepared, and a standard curve was determined. Subsequently, 80 μg / mL Cu-TA was incubated with 20 mM H₂O₂ at 37℃ for different times, and the absorbance at 240 nm was measured to calculate the CAT activity. The results are shown below. Figure 4 The right figure shows that Cu-TA has a good ability to decompose H2O2 and can effectively remove ROS substances accumulated in diabetic foot ulcers.

[0086] Test Example 3: Monitoring the change in glucose residue in the GOD@Cu-TA reaction system over reaction time using high performance liquid chromatography.

[0087] Glucose degradation activity assay: GOD@Cu-TA (1 mg / mL) was mixed with glucose solution (20 mM) and reacted at 37℃ for different times. After centrifugation, the supernatant was collected, filtered, and the remaining glucose concentration was determined by high performance liquid chromatography. The glucose concentration degraded by GOD@Cu-TA at different reaction times was calculated. The results are as follows: Figure 5 As shown, the results indicate that GOD@Cu-TA can effectively break down glucose and reduce glucose concentration at the wound site.

[0088] Test Example 4: Amount of NO generated by the oxidation of L-arginine by GOD@Cu-TA at different glucose concentrations

[0089] NO generation detection: The activity of GOD@Cu-TA in oxidizing L-arginine to NO under different glucose conditions was detected using a NO detection kit. 1 mg / mL GOD@Cu-TA was mixed with 0-20 mM glucose solution and 0.3 mg / mL L-arginine solution, incubated for 48 h, centrifuged, and the results were calibrated using a NO detection kit. The results are shown below. Figure 6 As shown in the figure. Experimental results indicate that GOD@Cu-TA can oxidize NO generated from L-arginine in the presence of glucose, and the amount of NO generated increases with increasing glucose concentration.

[0090] Test Example 5: Optical images and scanning electron microscope images of the microneedle patch prepared in Example 6.

[0091] Microneedle morphology: The morphological characteristics of the microneedle patches prepared in Example 6 were characterized using a camera and a scanning electron microscope (SEM), resulting in morphological photographs and SEM images of the microneedle patches, as shown below. Figure 7 As shown in the electron microscope image, the fabricated microneedle array has a close-packed structure with the needle tips shaped like square pyramids.

[0092] Test Example 6: Antibacterial Experiment of Microneedle Patches Prepared in Examples 4-6

[0093] Antibacterial Experiment: The antibacterial activity of the microneedle patch was evaluated using *Escherichia coli* and *Staphylococcus aureus*. Fresh single colonies of *Escherichia coli* ATCC 25922 and *Staphylococcus aureus* ATCC 6538 were inoculated into liquid culture medium and cultured at 37°C with shaking until the logarithmic development phase (approximately 18–24 h). The bacterial suspension was then adjusted to a suitable concentration (10^6 CFU / mL) with sterile physiological saline. The bacterial suspension obtained in the above steps was mixed with blank PBS solution (control group) or microneedles from Examples 4-6 and incubated at 37°C for 24 h. The mixed and incubated solution was further diluted and inoculated onto the surface of solid agarose culture dishes and incubated at 37°C for 24 h. The culture dishes were removed, photographed, and the bacterial counts were performed to calculate the antibacterial rate. The results are shown below. Figure 8 As shown. Experimental results show that the microneedle patches prepared in Examples 4-6 of this invention have good inhibitory ability against both Gram-negative and Gram-positive bacteria, and can effectively remove bacteria from wounds.

[0094] Test Example 7: Effects of microneedle patches from Examples 4-6 and Comparative Examples 1-3 on wound healing in diabetic mice

[0095] Diabetic mouse model experiment: All animal studies were approved by the Animal Protection and Use Committee of Nantong University. These animals were raised and cared for according to the animal care guidelines established by the animal facilities of Nantong University. ICR mice aged 6-8 weeks and weighing 18-22g were selected and acclimatized for 1-7 days before the experiment. To induce diabetes, streptozotocin (STZ) was injected intraperitoneally into the mice at a dose of 50mg / kg. After injection, the mice were placed in cages and allowed to eat and drink normally, with 10% glucose solution used instead of drinking water until the end of the injection cycle. After 4 consecutive days of injection and 7 days after STZ injection, blood glucose levels were measured twice consecutively using a blood glucose meter. If blood glucose was ≥16.8mmol / L and typical clinical symptoms of diabetes appeared—polydipsia, polyphagia, polyuria, and weight loss—the model was considered successfully established. After blood glucose remained elevated for 7 days, a wound model was constructed.

[0096] Experimental study on the treatment of infected wounds in diabetic mice: A 10 mm diameter wound was created on the back of each successfully modeled mouse using a perforation bio-instrument. Each wound was then infected with 50 μL of Staphylococcus aureus at a concentration of 1 × 10^7 cfu / mL. The diabetic mice with infected wounds were randomly divided into 5 groups of 8 mice each: (1) control group, (2) experimental group 1 (mice treated with microneedles as described in Example 6), (3) experimental group 2 (mice treated with microneedles as described in Comparative Example 1), (4) experimental group 3 (mice treated with microneedles as described in Comparative Example 2), and (5) experimental group 4 (mice treated with microneedles as described in Comparative Example 3). Wounds were photographed with a digital camera on days 0, 7, and 11. The photographs are shown below. Figure 9 As shown, this was used to evaluate wound healing efficacy. Experimental results indicate that enzyme-tandem biodegradable microneedle patches have a good therapeutic effect on an infected diabetic wound model, significantly reducing the risk of reinfection, promoting collagen deposition, and accelerating skin regeneration, demonstrating promising application prospects.

[0097] This invention provides a method for preparing and applying a biodegradable microneedle patch based on enzyme tandem. It combines chitosan (CS), hyaluronic acid (HA), copper-tannic acid (Cu-TA) nanozyme, glucose oxidase (GOD), L-arginine (L-Arg), and basic fibroblast growth factor (bFGF) to achieve antioxidant, antibacterial, and tissue repair-promoting functions. Through the biodegradable microneedle substrate and microneedle body, a synergistic effect is achieved, enabling rapid degradation and long-term therapeutic effects. It exhibits good biocompatibility and clinical application prospects, and is expected to provide a new and effective method for the treatment of diabetic foot.

[0098] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing an external use degradable microneedle patch, characterized by, The preparation method comprises the following steps: S1. Dissolve chitosan in dilute hydrochloric acid, adjust the pH to 3.0-4.0, add tannic acid and hydrogen peroxide, stir in the dark until the reaction is complete, freeze-dry the reaction product after dialysis to obtain powdered CS-TA; S2. Dissolve copper chloride and tannic acid in water, drop in sodium carbonate solution, adjust the pH to 8.0-9.0, then stir at room temperature under inert gas atmosphere until the reaction is complete, centrifuge, wash with water, and dry to obtain powdered Cu-TA nanoscale enzyme; S3. Add Cu-TA nanoscale enzyme and glucose oxidase GOD to water, stir under inert gas atmosphere at 1-4℃, resuspend in water after centrifugation to obtain GOD@Cu-TA composite enzyme solution; S4. Add CS-TA, hyaluronic acid, polyvinylpyrrolidone, L-arginine and basic fibroblast growth factor to the GOD@Cu-TA composite enzyme solution to prepare a microneedle mother liquor; S5. Prepare an aqueous solution containing CS-TA, hyaluronic acid and polyvinylpyrrolidone to obtain a base mother liquor; S6. Fill the microneedle mother liquor into the microneedle mold cavities of the mold to form enzyme cascade microneedles arranged in an array, then pour the base mother liquor to form a base, dry and demold to obtain the degradable microneedle patch for external use.

2. The production method according to claim 1, characterized by, In step S1, the mass ratio of chitosan to tannic acid is 1:(0.1-0.5).

3. The preparation method according to claim 1, characterized in that, In the reaction system of step S1, the final concentration of hydrogen peroxide is 0.1-1 mol / L.

4. The production method according to claim 1, characterized by, In step S2, the mass ratio of copper chloride to tannic acid is (0.5-1):(0.9-1.8).

5. The method of claim 1, wherein, In step S3, the mass ratio of Cu-TA nanoscale enzyme to glucose oxidase is (0.1-100):

1.

6. The method of claim 1, wherein, In the microneedle mother liquor, the concentration of CS-TA is 2-18 mg / mL, the concentration of hyaluronic acid is 2-18 mg / mL, the concentration of polyvinylpyrrolidone is 2-8 mg / mL, the concentration of GOD@Cu-TA composite enzyme is 0.2-3 mg / mL, the concentration of L-arginine is 0.5-5 mg / mL, and the concentration of basic fibroblast growth factor is 0.02-0.2 mg / mL.

7. The preparation method according to claim 1, characterized in that, In the base mother liquor, the concentration of CS-TA is 2-18 mg / mL, the concentration of hyaluronic acid is 2-18 mg / mL, and the concentration of polyvinylpyrrolidone is 2-8 mg / mL.

8. A degradable microneedle patch for external use prepared by the preparation method of any one of claims 1-7.

9. The degradable microneedle patch for external use according to claim 8, wherein The shape of the enzyme cascade microneedle is a quadrangular pyramid, the height is 400-500 μm, and the base thickness is 110-200 μm.

10. Use of the degradable microneedle patch for external use of claim 8 in the preparation of a material for the auxiliary treatment of diabetic foot.

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