A multifunctional microneedle patch for the treatment of oral ulcers, its preparation method and application

Abstract

This invention provides a multifunctional microneedle patch for the treatment of oral ulcers, its preparation method, and its application, belonging to the field of biomedical materials technology. The multifunctional microneedle patch for the treatment of oral ulcers of this invention consists of a substrate and microneedles arrayed on one side of the substrate; the microneedles are loaded with drugs A and B, and the substrate is loaded with drug C. Based on the principle of sequential drug release, the multifunctional microneedle patch of this invention first dissolves and releases the drug to achieve analgesia, while the microneedles continue to remain in the oral mucosa, slowly swelling and releasing the drug to achieve a repair effect, thus solving the problem of existing oral ulcer treatments involving multiple causes and the inability of drugs to remain in the oral cavity for extended periods.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a multifunctional microneedle patch for the treatment of oral ulcers, its preparation method, and its application. Background Technology

[0002] Recurrent aphthous stomatitis (RAS) is the most common oral mucosal lesion, characterized by pain and recurrent ulceration. Generally, younger patients, women, and individuals of higher socioeconomic status are more prone to RAS, with an average prevalence of 20%. These lesions are benign and self-limiting, but are often chronic and frequently recurrent, leading to impaired oral function and quality of life. The etiology of RAS is unclear. It is considered a multifactorial disease. Several etiological mechanisms, including hematopoietic deficiency, particularly folic acid and iron, microbial infections, mechanical trauma, chemicals such as anionic detergents, genetic factors, and stress or anxiety, have been shown to be associated with RAS. In recent years, immune dysfunction has also been considered the most probable cause of RAS.

[0003] Treatment for RAS primarily involves the use of topical or systemic corticosteroids, anti-inflammatory drugs, and immunomodulators. Unlike the skin, the oral cavity lining is a moist, highly dynamic environment, constantly eroded by endogenous saliva and exogenous food. Furthermore, chewing, speaking, swallowing, and even changes in facial expressions all trigger movement of the tongue and oral mucosa. These complex challenges often render topical treatments with protective materials and medications ineffective because they have a very short residence time on the mucosal surface. Clinically used topical preparations such as solutions, powders, ointments, polymer films, and hydrogels are often diluted or washed away by saliva within one hour. This is much shorter than the optimal timeframe; repair of RAS requires at least 12–24 hours of treatment.

[0004] When applied, microneedle patches create numerous micropores within the skin and mucous membranes. Due to their tiny size, the microneedles only pierce the surface layer of the skin and mucous membranes, without stimulating nerve endings beneath the skin. Therefore, they cause no additional pain to the patient, making them a minimally invasive, painless, and highly effective transdermal therapy, widely used for transdermal drug delivery. In skin and mucous membrane wound repair, microneedling therapy, through transdermal drug delivery, can improve drug utilization and the contact area between the dressing and the wound, thereby enhancing the repair effect.

[0005] Therefore, developing a microneedle patch loaded with drugs targeting the causes of ulcers and capable of prolonging the duration of drug action is of great significance for expanding the treatment of RAS, improving patient acceptance, and enhancing the efficacy of RAS. Summary of the Invention

[0006] The purpose of this invention is to provide a multifunctional microneedle patch for the treatment of oral ulcers, its preparation method, and its application. Based on the principle of sequential drug release, the multifunctional microneedle patch of this invention first dissolves and releases the drug to achieve analgesia, while the microneedles remain in the oral mucosa, slowly swelling and releasing the drug to achieve a repairing effect. This addresses the problem of existing oral ulcer treatments involving multiple causes and the inability of drugs to remain in the oral cavity for extended periods.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] The present invention provides a multifunctional microneedle patch for the treatment of oral ulcers, the multifunctional microneedle patch comprising a base and microneedles arrayed on one side of the base; the microneedles are loaded with drugs A and B, and the base is loaded with drug C.

[0009] Preferably, drug A is a drug that promotes mucosal repair; drug B is a drug that promotes angiogenesis; and drug C is a pain reliever.

[0010] Preferably, drug A includes any one of bone marrow mesenchymal stem cell-derived exosomes, topical recombinant epidermal growth factor, and vitamin A; drug B includes any one of folic acid, vascular endothelial growth factor, and tanshinone polyphenolic acid; and drug C includes any one of lidocaine, bupivacaine, and tetracaine.

[0011] Preferably, the microneedle is pyramidal in shape; the height of the microneedle is 500-800 μm, and the ratio of the side length of the base of the microneedle to the height of the microneedle is 1:(1.5-3).

[0012] The present invention also provides a method for preparing the above-mentioned multifunctional microneedle patch, comprising the following steps:

[0013] (1) Dissolve the microneedle matrix material, drug A, drug B, and photoinitiator in phosphate buffer to obtain a microneedle matrix material solution;

[0014] (2) Dissolve the substrate material and drug C in phosphate buffer to obtain a substrate material solution;

[0015] (3) Pour the microneedle matrix material solution into the microneedle cavity of the mold, and then dry and cure it with ultraviolet light to form microneedles; continue to add the matrix material solution into the mold where the microneedles have been formed, and then dry and cure it to obtain the multifunctional microneedle patch.

[0016] Preferably, the microneedle matrix material includes methacrylamide chitosan or methacrylamide carboxymethyl chitosan; the substrate matrix material includes any one of gelatin, polyvinylpyrrolidone, and polyvinyl alcohol; and the photoinitiator is lithium phenyl 2,4,6-trimethylbenzoylphosphonate or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

[0017] Preferably, in the microneedle matrix material solution, the concentration of the microneedle matrix material is 10-30 mg / mL, and the concentration of the photoinitiator is 0.002-0.004 mg / mL; in the substrate matrix material solution, the concentration of the substrate matrix material is 50-300 mg / mL.

[0018] Preferably, the wavelength of the ultraviolet light in step (3) is 400-315 nm; and the curing time of the ultraviolet light is 3-6 min.

[0019] Preferably, the drying and curing temperature in step (3) is 37-40°C, and the drying and curing time is 6-12 hours.

[0020] The present invention also provides the application of the above-mentioned multifunctional microneedle patch in the preparation of a pharmaceutical formulation for treating oral ulcers.

[0021] This invention provides a multifunctional microneedle patch for the treatment of oral ulcers, its preparation method, and its application. Compared with the prior art, this invention has the following specific advantages:

[0022] (1) The multifunctional microneedle patch of the present invention has sequential drug release characteristics. When applied to the mucosal wound, the base first rapidly dissolves to release the loaded drug, relieving wound pain; this is the immediate drug release portion. The microneedles detach from the base and remain in the oral mucosa, gradually releasing the loaded drug as the microneedle matrix material swells and degrades. The main components of the microneedles can induce angiogenesis, promote epithelial repair, and regulate mucosal microenvironment inflammation, thereby accelerating the wound healing process; this is the sustained drug release portion. This can solve the problem of multiple causes of oral ulcers and the inability of drugs to remain in the oral cavity for a long time, achieving combined treatment of oral mucosal diseases with multiple drugs, improving treatment efficacy and reducing adverse reactions.

[0023] (2) The multifunctional microneedle patch of the present invention not only has the characteristic of stepwise drug release, but also has good mechanical properties. Its mechanical strength is mainly provided by the matrix material, and the loading of drugs has little effect on its mechanical strength. In vitro puncture experiments show that the multifunctional microneedle patch provided by the present invention can penetrate the oral mucosal epithelium of rats and has the potential to deliver drugs to the basal layer cells of the mucosa and the nearby connective tissue.

[0024] (3) The matrix material of the multifunctional microneedle patch of the present invention also has antibacterial properties, which can reduce the adverse effects of complex oral flora on ulcer wounds and promote wound healing more quickly.

[0025] (4) The multifunctional microneedle patch for oral ulcers described in this invention can be loaded with two or more different drugs. This invention tested the drug loading and release curve of the multifunctional microneedle patch through experiments. The results showed that the in vitro drug release curve showed the stepwise release characteristics of the multifunctional microneedle patch.

[0026] (5) The multifunctional microneedle patch of the present invention is easy to use. When the multifunctional microneedle patch is inserted into the oral mucosa, the base first dissolves and releases the drug quickly. At the same time, the dissolution of the base causes the microneedle patch to separate and remain in the oral mucosa. As the microneedle matrix material swells and degrades, the drug continues to be slowly released in the oral mucosa. It has the potential to be a safe carrier for drug delivery and has good clinical application prospects.

[0027] (6) The present invention also provides a method for preparing the above-mentioned multifunctional microneedle patch for the treatment of oral ulcers. The method is simple to operate, mild, and does not require high temperature, which is beneficial to protect the activity of the loaded drug. At the same time, the drug loading and the type of loaded drug can be flexibly adjusted according to the actual application requirements. Attached Figure Description

[0028] Figure 1 The image shows the overall morphology of the multifunctional microneedle patch prepared in Example 1.

[0029] Figure 2 The image shows a scanning electron microscope (SEM) image of the multifunctional microneedle patch prepared in Example 1.

[0030] Figure 3 The mechanical property test results are for the multifunctional microneedle patch and the blank microneedle patch prepared in Example 1.

[0031] Figure 4 The in vitro transdermal experiment of the multifunctional microneedle patch prepared in Example 1 is shown in (a) as an oral observation of the rat oral mucosa after microneedle insertion, (b) as the needle hole mark of the rat oral mucosa after the base dissolves, and (c) as a tissue section of the rat oral mucosa 6 hours after insertion.

[0032] Figure 5 The images show the colony growth of Escherichia coli and Candida albicans after treatment with the multifunctional microneedle patch prepared in Example 1 and the blank microneedle patch.

[0033] Figure 6 The results of antibacterial analysis of the multifunctional microneedle patch and blank microneedle patch prepared in Example 1 against Escherichia coli and Candida albicans are shown.

[0034] Figure 7 The results show the experimental effects of the multifunctional microneedle patch and blank microneedle patch prepared in Example 1 on promoting the migration of human oral keratinocytes (HOKs).

[0035] Figure 8 The results show the experimental findings of promoting angiogenesis in human umbilical vein endothelial cells (HUVECs) using the multifunctional microneedle patch and blank microneedle patch prepared in Example 1.

[0036] Figure 9 Morphological observation of the multifunctional microneedle patch prepared for Example 1 and the blank microneedle patch on days 0, 2, 5 and 8 after treatment of oral ulcers.

[0037] Figure 10 Immunofluorescence staining images of cytokeratin 5 (CK5), cytokeratin 13 (CK13), vascular endothelial growth factor (VEGF), and myeloperoxidase (MPO) after oral ulcer treatment using the multifunctional microneedle patch and blank microneedle patch prepared in Example 1. Detailed Implementation

[0038] The present invention provides a multifunctional microneedle patch for the treatment of oral ulcers, the multifunctional microneedle patch comprising a base and microneedles arrayed on one side of the base; the microneedles are loaded with drugs A and B, and the base is loaded with drug C.

[0039] In this invention, the multifunctional microneedle patch preferably consists of a substrate and microneedles arranged in an array on one side of the substrate.

[0040] In this invention, the microneedles are preferably loaded with drug A and drug B.

[0041] In this invention, the substrate is preferably loaded with drug C.

[0042] In this invention, drug A is preferably a drug that promotes mucosal repair, and more preferably includes any one of bone marrow mesenchymal stem cell-derived exosomes, topical recombinant epidermal growth factor, and vitamin A, and even more preferably bone marrow mesenchymal stem cell-derived exosomes.

[0043] In this invention, drug B is preferably a drug that promotes angiogenesis, more preferably including any one of folic acid, vascular endothelial growth factor, and tanshinone polyphenolic acid, and even more preferably folic acid-iron tetroxide nanoparticles.

[0044] In this invention, the drug C is preferably an analgesic, more preferably including any one of lidocaine, bupivacaine, and tetracaine, and even more preferably lidocaine.

[0045] In this invention, the microneedles are preferably pyramidal, and more preferably square pyramidal, which can increase the mechanical strength of the microneedles.

[0046] In this invention, the height of the microneedle is preferably 500-800 μm, and more preferably 800 μm.

[0047] In this invention, the ratio of the bottom side length of the microneedle to the height of the microneedle is preferably 1:(1.5~3), and more preferably 1:1.5, which can ensure that the microneedle has good mechanical strength.

[0048] The present invention also provides a method for preparing the above-mentioned multifunctional microneedle patch, comprising the following steps:

[0049] (1) Dissolve the microneedle matrix material, drug A, drug B, and photoinitiator in phosphate buffer to obtain a microneedle matrix material solution;

[0050] (2) Dissolve the substrate material and drug C in phosphate buffer to obtain a substrate material solution;

[0051] (3) Pour the microneedle matrix material solution into the microneedle cavity of the mold, and then dry and cure it with ultraviolet light to form microneedles; continue to add the matrix material solution into the mold where the microneedles have been formed, and then dry and cure it to obtain the multifunctional microneedle patch.

[0052] In this invention, a microneedle matrix material, drug A, drug B, and photoinitiator are dissolved in a phosphate buffer solution to obtain a microneedle matrix material solution.

[0053] In this invention, to avoid adverse effects on the drug activity during microneedle preparation, the microneedle matrix material is preferably a photo-initiated polymer with a three-dimensional network structure that is biodegradable and biocompatible. The degradation performance of the matrix material can be adjusted by modifying its molecular weight and degree of cross-linking; generally, the lower the molecular weight and degree of cross-linking of the microneedle matrix material, the faster the drug delivery rate. To ensure that the active microbial community in the ulcer wound does not accelerate wound deterioration, the microneedle matrix material is preferably an antibacterial hydrogel. The microneedle matrix material preferably includes methacrylamide chitosan or methacrylamide carboxymethyl chitosan, more preferably methacrylamide carboxymethyl chitosan. Methacrylamide chitosan or methacrylamide carboxymethyl chitosan has biodegradability, biocompatibility, antibacterial properties, and immune-enhancing functions, and can cross-link to form a hydrogel network structure under ultraviolet light irradiation. The drug loaded in the methacrylamide chitosan or methacrylamide carboxymethyl chitosan network structure will be slowly released as the hydrogel swells.

[0054] In this invention, the molecular weight of the methacryloyl carboxymethyl chitosan is preferably 150-250 kDa.

[0055] In this invention, the substrate material includes any one of gelatin, polyvinylpyrrolidone, and polyvinyl alcohol, with gelatin being more preferred. Gelatin is a protein obtained by partial hydrolysis of collagen, possesses homology with collagen, exhibits good biocompatibility, and is water-soluble. Using gelatin as the substrate material allows for rapid dissolution in oral saliva and immediate release of analgesics, while also detaching from the microneedle body.

[0056] In this invention, the molecular weight of the gelatin is preferably 100-200 kDa.

[0057] In this invention, the photoinitiator is preferably lithium phenyl 2,4,6-trimethylbenzoylphosphonate or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone, and more preferably lithium phenyl 2,4,6-trimethylbenzoylphosphonate.

[0058] In this invention, the concentration of the microneedle matrix material in the microneedle matrix material solution is preferably 10-30 mg / mL, and more preferably 20 mg / mL.

[0059] In this invention, the concentration of the photoinitiator in the microneedle matrix material solution is preferably 0.002 to 0.004 mg / mL, and more preferably 0.0025 mg / mL.

[0060] In this invention, the loading of drug A and drug B in the microneedle matrix material solution is preferably determined with reference to the dosage of oral ulcer treatment drugs in actual applications.

[0061] In this invention, the substrate material and drug C are dissolved in phosphate buffer to obtain a substrate material solution.

[0062] In this invention, the concentration of the substrate material in the substrate solution is preferably 50-300 mg / mL, and more preferably 100 mg / mL.

[0063] In this invention, the loading of drug C in the substrate material solution is preferably determined with reference to the dosage of oral ulcer treatment drugs in actual applications.

[0064] In this invention, the microneedle matrix material solution is poured into the microneedle cavity of a mold, and then dried and cured under ultraviolet light to initiate the polymerization of the microneedle matrix material to form microneedles.

[0065] In this invention, the mold is preferably a polydimethylsiloxane mold.

[0066] In this invention, the polydimethylsiloxane mold preferably includes an array of pyramidal microneedle cavities and a square base cavity communicating with the open end of the microneedle cavity.

[0067] In this invention, after the microneedle matrix material solution enters the microneedle cavity of the mold, it is preferably centrifuged to ensure that the solution uniformly fills the microneedle cavity and to recover the solution that did not enter the microneedle cavity.

[0068] In this invention, the centrifugation speed is preferably 2500-4000 rpm, and more preferably 3000 rpm.

[0069] In this invention, the centrifugation time is preferably 3 to 8 minutes, and more preferably 5 minutes.

[0070] In this invention, the drying temperature is preferably no more than 40°C, and more preferably 37°C.

[0071] In this invention, the drying time is preferably 0.5 to 2 hours, and more preferably 1 hour.

[0072] In this invention, the wavelength of the ultraviolet light is preferably 400-315 nm, and more preferably 365 nm.

[0073] In this invention, the UV curing time is preferably 3 to 6 minutes, and more preferably 5 minutes.

[0074] The present invention continues to add the substrate material solution to the mold in which the microneedles are formed, and after drying and curing, demolding is performed to obtain the multifunctional microneedle patch.

[0075] In this invention, after the substrate material solution enters the mold, it is preferably centrifuged to fill the mold with the solution.

[0076] In this invention, the centrifugation speed is preferably 2500-4000 rpm, and more preferably 3000 rpm.

[0077] In this invention, the centrifugation time is preferably 3 to 8 minutes, and more preferably 5 minutes.

[0078] In this invention, the drying and curing temperature is preferably 37-40°C, and more preferably 37°C.

[0079] In this invention, the drying and curing time is preferably 6 to 12 hours, and more preferably 6 hours.

[0080] The present invention also provides the application of the above-mentioned multifunctional microneedle patch in the preparation of a pharmaceutical formulation for treating oral ulcers.

[0081] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0082] Example 1

[0083] This embodiment provides a multifunctional microneedle patch for the treatment of oral ulcers. Using bone marrow mesenchymal stem cell-derived exosomes, folic acid-ferric oxide nanoparticles, and lidocaine as hydrophilic drug models, a sequential drug-release multifunctional microneedle patch for the treatment of oral ulcers is prepared. The preparation process steps are as follows:

[0084] (1) Methacrylamide carboxymethyl chitosan (CMCSMA, molecular weight 150-250 kDa), lithium phenyl 2,4,6-trimethylbenzoylphosphonate (LAP) as photoinitiator, bone marrow mesenchymal stem cell-derived exosomes (MSCs-exo) and folic acid-Fe3O4 nanoparticles (FA-Fe3O4NPs) were dissolved in phosphate buffer, thoroughly mixed, and allowed to stand to remove bubbles to obtain a microneedle matrix material solution; in the microneedle matrix material solution, the concentration of CMCSMA was 20 mg / mL, the concentration of LAP was 0.0025 mg / mL, the concentration of MSCs-exo was 200 μg / mL, and the concentration of FA-Fe3O4 NPs was 10 μg / mL;

[0085] (2) Dissolve gelatin (molecular weight of 100-200kDa) and lidocaine in phosphate buffer, mix thoroughly and dissolve, let stand to remove air bubbles, and obtain a substrate material solution; in the substrate material solution, the concentration of gelatin is 100mg / mL and the concentration of lidocaine is 20mg / mL.

[0086] (3) A polydimethylsiloxane (PDMS) mold is used. The mold includes arrayed quadrangular pyramidal microneedle cavities and a square base cavity connected to the opening end of the microneedle cavity. The bottom surface of the base cavity is 12.4mm×12.4mm and the thickness is 1.5mm. The array in the mold is an 11×11 matrix with a total of 121 microneedle cavities. The height of the microneedle is 800μm, the side length of the pyramidal base is 400μm, and the distance between the tips is 800μm.

[0087] Add the microneedle matrix material solution to the mold, centrifuge at 3000 rpm for 5 min to ensure the solution uniformly fills the microneedle cavity, recover the solution that did not enter the microneedle cavity, place the mold in a 37℃ forced-air drying oven to dry for 1 h to remove some of the moisture in the solution, and then place the mold under a UV (365nm) light source for 5 min to initiate CMCSMA polymerization to form microneedles.

[0088] Then, add the substrate material solution to the mold, centrifuge at 3000 rpm for 5 minutes to fill the mold with the solution, place the mold in a 37°C forced-air drying oven for 6 hours to remove some of the moisture in the solution and then solidify it. Carefully peel it off from the mold to obtain a multifunctional microneedle patch.

[0089] In this embodiment, a microneedle matrix material solution without MSCs-exo and FA-Fe3O4 NPs and a substrate matrix material solution without lidocaine were also prepared, and a blank microneedle patch was prepared according to the above steps.

[0090] Test Example 1

[0091] This experiment examined the morphology and structure of the multifunctional microneedle patch prepared in Example 1. The specific process is as follows:

[0092] The morphology and structure of the multifunctional microneedle patch prepared in this embodiment were observed and its dimensions were measured using a stereomicroscope and a scanning electron microscope. The multifunctional microneedle patch was placed on a sample stage, and its overall morphology was observed using a stereomicroscope. The results are as follows: Figure 1 As shown in the figure. The multifunctional microneedle patch was attached to the sample stage, sputter-coated with gold for 60 seconds, and then purged with nitrogen. The microstructure was observed using a scanning electron microscope, and the microneedle size was measured. The results are as follows. Figure 2 As shown.

[0093] from Figure 1 and Figure 2 As can be seen, the multifunctional microneedle patch prepared in this embodiment is a solid quadrangular pyramidal microneedle. The microneedle has a uniform and complete structure, a sharp needle shape, a height of about 800 μm, a bottom side length of 400 μm, and a distance of about 800 μm between adjacent needle tips.

[0094] Test Example 2

[0095] This experiment measured the mechanical properties of the multifunctional microneedle patch and the blank microneedle patch prepared in Example 1. The specific process is as follows:

[0096] The mechanical strength of the multifunctional microneedle patch and the blank microneedle patch prepared in Example 1 was determined using a universal tensile testing machine. The multifunctional microneedle patch and the blank microneedle patch prepared in Example 1 were placed horizontally on a stainless steel platform, with the microneedle tips pointing vertically upwards. A 100N pressure sensor was moved vertically downwards at a constant speed (10 μm / s) to continuously apply pressure to the microneedles until a preset load (20N) was reached, at which point the pressure application was stopped. The compression displacement and pressure experienced by the microneedles during the compression process were continuously recorded, and a change curve was plotted, as shown in the figure. Figure 3As shown, the curves of both the multifunctional microneedle patch (Multi CMCSMN) and the blank microneedle patch (CMCSMN) are rising and continuous, indicating that they both underwent continuous deformation without breakage. When the compression displacement reaches 0.6 mm, the multifunctional microneedle patch and the blank microneedle patch prepared in Example 1 both withstood a pressure of 17 N, exhibiting similar mechanical strength, which is greater than the mechanical strength required to pierce the skin (≈0.1 N). This indicates that drug loading has virtually no impact on the mechanical strength of the multifunctional microneedle patch.

[0097] Experimental Example 3

[0098] This experiment uses an in vitro transdermal assay to study the ability of the multifunctional microneedle patch prepared in Example 1 to penetrate the oral mucosa. The specific process is as follows:

[0099] The multifunctional microneedle patch prepared in Example 1 was vertically inserted into the oral mucosa of rats (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) using a force of 50 N. Figure 4 As shown in (a), keep it for 10 minutes to allow the substrate to melt. Figure 4 As shown in (b). Six hours after insertion, the rats were euthanized, and the mucosal tissue was harvested, embedded in an OCT scanner, and rapidly frozen. Sections were prepared using a rotary sectioning machine to a thickness of 10 μm, stained with hematoxylin and eosin, and then the tissue cross-sections were observed under an optical microscope, as shown in (b). Figure 4 As shown in (c). From Figure 4 As shown in (c), after 6 hours, the penetration depth of the multifunctional microneedle patch in the oral mucosa was approximately 100–150 μm, but it still maintained a certain pyramidal shape, and degraded material was visible at the pinhole location. This indicates that the multifunctional microneedle patch prepared in Example 1 has the ability to penetrate the oral mucosa, can break through the physiological barrier of the mucosal epithelium, and directly deliver drugs to the mucosal tissue.

[0100] Test Example 4

[0101] This experimental example studies the antibacterial properties of the multifunctional microneedle patch prepared in Example 1 and a blank microneedle patch. The specific process is as follows:

[0102] Using *Escherichia coli* (ATCC35218) and *Candida albicans* (SC5314) as experimental subjects, the bacteria were centrifuged at 5000 rpm for 10 min, and the pellets were collected. *E. coli* was resuspended in LB broth, and *Candida albicans* was resuspended in Sabouraud broth. The multifunctional microneedle patch from Example 1 and the blank microneedle patch were added to the transwell chambers of a 24-well plate, and co-cultured with 1 mL of *E. coli* or *Candida albicans* culture (the bacterial culture was diluted to 10⁻⁶ oz). 6CFU / mL). Positive controls were 1 mL of *E. coli* or *Candida albicans* culture without any culture material. After co-incubation at 37°C for 24 h, a portion of the co-culture medium was extracted from each group's wells and diluted 10⁻⁶ CFU / mL. -5 10 -6 10 -7 10 -8 10 -9 10 -10 0.1 ml of each dilution was inoculated onto an agar plate and incubated at 37°C for 1 day. Colony formation was then observed. The dilution concentration of *E. coli* in each group was 10-1. -10 Candida albicans at a dilution concentration of 10 -5 The colony formation at that time is as follows Figure 5 As shown, compared with the positive control, CMCSMA, as the matrix material for both the multifunctional microneedle patch and the blank microneedle patch, exhibits significant antibacterial properties.

[0103] The bacterial concentrations in the co-culture media of each group were statistically analyzed, and a one-way ANOVA was performed to assess the antibacterial effect. The results are as follows: Figure 6 As shown, both the multifunctional microneedle patch and the blank microneedle patch exhibit significant antibacterial activity against Escherichia coli (p<0.001), while there is no significant difference between the two groups of microneedles. Against Candida albicans, the multifunctional microneedle patch (p<0.001) showed better antibacterial effect than the blank microneedle patch (p<0.01), possibly because the loaded FA-Fe3O4 NPs have certain antibacterial properties.

[0104] Experimental Example 5

[0105] This experiment investigated the ability of the multifunctional microneedle patch prepared in Example 1 and the blank microneedle patch to promote the migration of human oral keratinocytes (HOKs). The specific process is as follows:

[0106] Cell migration assays were performed using a Transwell co-culture system. This experiment used 5 × 10⁶ cells / year. 4 Personal oral keratinocytes (purchased from ATCC) were seeded onto 24-well plate slides and allowed to congregate. Before scratching, the cells were incubated overnight in medium containing 2% FBS. Then, a damaged cell monolayer was rapidly created using a plastic pipette tip. Transwell dishes containing the multifunctional microneedle patch from Example 1 and a blank microneedle patch were quickly placed in the wells of the plate, suspending the microneedle patch but wetting it in the cell culture medium. The blank control consisted of cells that had only been scratched without any added material.

[0107] At 6 hours, cells were imaged using an optical microscope to observe the effect of drug release from microneedles on cell migration ability. Cell imaging is as follows: Figure 7As shown, compared with the blank control and blank microneedle patch, the multifunctional microneedle patch closed the damaged intercellular spaces at 6 hours and had a faster ability to promote cell migration, which is of great significance for accelerating wound healing.

[0108] Experimental Example 6

[0109] This experiment investigated the ability of the multifunctional microneedle patch prepared in Example 1 and the blank microneedle patch to promote angiogenesis in human umbilical vein endothelial cells (HUVECs). The specific process is as follows:

[0110] Cell angiogenesis assays were performed using a Transwell co-culture system. 5 × 10⁶ cells were cultured... 4 Personal umbilical vein endothelial cells (purchased from ATCC) were seeded on 24-well plate slides and allowed to adhere for 30 minutes. Transwell dishes containing the multifunctional microneedle patch from Example 1 and a blank microneedle patch were then placed in the wells, suspending the microneedle patch in the cell culture medium. The blank control consisted of human umbilical vein endothelial cells without any added materials.

[0111] After culturing for 0, 6, and 12 hours, cells were transferred to new plates and fixed with 4% paraformaldehyde for 10 minutes. Then, the cells were infiltrated with 0.3% Triton X-100 for 5 minutes, washed three times with PBS, and incubated with rhodamine (Yeason, China) at room temperature for 30 minutes. Finally, the cells were incubated with DAPI (Invitrogen, USA) for 5 minutes. Cell images were captured using a confocal microscope to observe the effect of drug release from microneedles on cell tube-forming ability. Figure 8 As shown, compared with the blank control and blank microneedle patch, the multifunctional microneedle patch formed a denser vascular network, which helps to promote wound vascularization and facilitate the transport of nutrients.

[0112] Experimental Example 7

[0113] This experimental example studies the therapeutic effects of the multifunctional microneedle patch prepared in Example 1 and the blank microneedle patch on oral ulcers. The specific process is as follows:

[0114] The protocol implemented in animals in this embodiment was approved by the Biomedical Ethics Committee of Peking University (Ethics Approval No.: LA2022384). Fifteen healthy male Sprague Dawley rats (240–260 g, 8 weeks old, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were selected for the experiment. The oral mucosa of the selected rats showed no inflammation or other pathological signs. After anesthesia with 1% sodium pentobarbital (40 mg / kg), round filter paper (5 × 5 mm) soaked in 70% acetic acid was placed on the oral mucosa for 3 minutes to induce oral ulcers. Two days later, the rats were randomly divided into three groups of five. Multifunctional microneedle patches and blank microneedles were applied to the oral mucosal ulcers in each group, with untreated rats serving as a blank control. Gross observations were performed on days 0, 2, 5, and 8. The morphology of the oral mucosal ulcers in each group is as follows: Figure 9 As shown in the figure, compared with the blank control and the blank microneedle patch, the multifunctional microneedle patch rapidly reduced the wound area, achieving faster ulcer repair.

[0115] Rats were sacrificed on day 8, and oral mucosal tissue surrounding the ulcers was collected for histological and immunofluorescence analysis. Cytokeratin 5 (CK5) and cytokeratin 13 (CK13) were used to assess epithelial regeneration, and vascular endothelial growth factor (VEGF) was used to assess mucosal vascularization. Myeloperoxidase (MPO) immunofluorescence staining was used to assess the inflammatory response. The assessment results are as follows: Figure 10 As shown, the multifunctional microneedle patch-treated rat oral mucosa exhibited higher immunofluorescence areas of cytokeratin 5 (CK5), cytokeratin 13 (CK13), and vascular endothelial growth factor (VEGF), indicating that the multifunctional microneedle patch promoted better epithelial regeneration and mucosal vascularization. Simultaneously, the lower immunofluorescence staining area of ​​myeloperoxidase (MPO) suggests that the multifunctional microneedle patch effectively inhibited immune-related inflammation in oral ulcers.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multifunctional microneedle patch for the treatment of oral ulcers, characterized in that, The multifunctional microneedle patch consists of a substrate and microneedles arranged in an array on one side of the substrate; the microneedles are loaded with drugs A and B, and the substrate is loaded with drug C; Drug A is a drug that promotes mucosal repair; drug B is a drug that promotes angiogenesis; drug C is a pain reliever. Drug A includes any one of bone marrow mesenchymal stem cell-derived exosomes, topical recombinant epidermal growth factor, and vitamin A; Drug B includes any one of folic acid, vascular endothelial growth factor, and tanshinone polyphenolic acid; Drug C includes any one of lidocaine, bupivacaine, and tetracaine.

2. The multifunctional microneedle patch according to claim 1, characterized in that, The microneedle is pyramidal in shape; the height of the microneedle is 500–800 μm, and the ratio of the side length of the base of the microneedle to its height is 1: (1.5～3)。 3. A method for preparing the multifunctional microneedle patch according to claim 1 or 2, characterized in that, Includes the following steps: (1) Dissolve the microneedle matrix material, drug A, drug B, and photoinitiator in phosphate buffer to obtain a microneedle matrix material solution; (2) Dissolve the substrate material and drug C in phosphate buffer to obtain a substrate material solution; (3) Pour the microneedle matrix material solution into the microneedle cavity of the mold, and then dry and cure it with ultraviolet light to form microneedles; continue to add the matrix material solution into the mold where the microneedles have been formed, and then dry and cure it to obtain the multifunctional microneedle patch.

4. The preparation method according to claim 3, characterized in that, The microneedle matrix material includes methacrylamide chitosan or methacrylamide carboxymethyl chitosan; the substrate matrix material includes any one of gelatin, polyvinylpyrrolidone, and polyvinyl alcohol; the photoinitiator is lithium phenyl 2,4,6-trimethylbenzoylphosphonate or 2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone.

5. The preparation method according to claim 4, characterized in that, In the microneedle matrix material solution, the concentration of the microneedle matrix material is 10–30 mg / mL, and the concentration of the photoinitiator is 0.002–0.004 mg / mL; in the substrate matrix material solution, the concentration of the substrate matrix material is 50–300 mg / mL.

6. The preparation method according to claim 5, characterized in that, The wavelength of the ultraviolet light in step (3) is 400-315 nm; the curing time of the ultraviolet light is 3-6 min.

7. The preparation method according to claim 6, characterized in that, The drying and curing temperature in step (3) is 37-40°C, and the drying and curing time is 6-12 hours.

8. The use of a multifunctional microneedle patch according to claim 1 or 2, or a multifunctional microneedle patch prepared by the preparation method according to any one of claims 3 to 7, in the preparation of a pharmaceutical formulation for treating oral ulcers.

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