Oral soluble microneedle patch based on nano-enzyme synergy as well as preparation method and application of oral soluble microneedle patch

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an oral soluble microneedle patch based on nano-enzyme synergy as well as a preparation method and application of the oral soluble microneedle patch. A mixed solution containing CMCS, PVA, Se-SCDs, APS and VEGF is used as a microneedle tip layer matrix, a mixed solution containing CMCS, PVA and Se-SCDs is used as a microneedle substrate layer matrix, and the oral soluble microneedle patch with gradient degradation capacity is prepared. The tip of the microneedle patch removes active oxygen through Se-SCDs, APS promotes immunoregulation, VEGF accelerates angiogenesis, and healing of ulcer wounds is promoted; and the microneedle patch substrate simultaneously plays the functions of resisting bacteria and regulating oral microbiome. Due to the gradient degradation design, the micro-needle patch can protect the wound surface and reduce external stimulation, and meanwhile, the gradual degradation of the micro-needle substrate reduces foreign body sensation in the oral cavity; the tip of the microneedle is slowly degraded, so that long-range release of Se-SCDs, APS and VEGF is realized, and synergistic treatment is realized. Experiments prove that the oral ulcer healing rate is remarkably improved, and high biological safety and clinical transformation value are achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a soluble microneedle patch for oral use based on nanozyme synergy, its preparation method, and its application. Background Technology

[0002] Recurrent oral ulcers (RPU) are a common oral mucosal disease characterized by recurrent round or oval ulcers on the oral mucosa, accompanied by significant pain, severely impacting patients' eating, speech, and quality of life. The etiology of RPU is unclear, but studies suggest it is closely related to multiple factors, including immune abnormalities, genetic factors, endocrine disorders, psychological factors, nutritional deficiencies, and local oral trauma. Current mainstream research suggests that the core pathogenesis of RPU is a vicious cycle of immunity-inflammation-oxidative stress: T-lymphocyte-mediated mucosal damage triggers a burst of pro-inflammatory factors (TNF-α, IL-1β), while local reactive oxygen species (ROS) rapidly accumulate, significantly inhibiting superoxide dismutase / catalase (SOD / CAT) enzyme activity. This is accompanied by the formation of ulcerative pseudomembranes, causing persistent tissue damage. Furthermore, the further colonization of pathogenic bacteria in the oral ulcers leads to oral microbiome dysbiosis and biofilm formation, secreting extracellular polysaccharide barriers that hinder drug penetration, further contributing to difficulty in healing.

[0003] Currently, clinical treatment for RPU primarily aims to relieve pain, promote healing, and reduce recurrence frequency, but there is no cure. Traditional treatments include topical application of anti-inflammatory and antibacterial drugs (such as corticosteroid ointments and antibiotic mouthwashes), as well as systemic supplementation with nutrients and immunomodulators. Currently, there are many types of oral ulcer treatments on the market. Common ones include powders such as Bingpengsan and Xileisan, which are applied directly to the ulcer surface. Although they can clear heat and detoxify, reduce swelling and relieve pain, they are easily diluted and washed away by saliva, and the effect is short-lived, requiring frequent use. Mouthwashes such as Kangfuxin liquid and compound chlorhexidine mouthwash can clean the mouth and kill bacteria and reduce inflammation, but the effect is limited and the effect is not good for severe ulcers. Patches such as Ruyiketie contain ingredients such as dexamethasone acetate, which can act directly on the affected area for a continuous effect, but the oral mucosal stratum corneum barrier hinders drug absorption, and long-term use can cause mucosal atrophy as a side effect. Sprays such as watermelon frost spray contain a variety of Chinese medicine ingredients that can clear heat and detoxify, but the drug distribution is uneven and it is highly irritating to the mucosa. Films such as propolis oral films can cover the ulcer and protect the wound, but they are easily displaced or fall off due to saliva washing, affecting the efficacy. Gels also have poor adhesion and low transdermal efficiency, making it impossible to achieve the purpose of continuous drug delivery and long-term treatment.

[0004] Microneedling is an emerging transdermal drug delivery technology that uses tiny needles to briefly penetrate the stratum corneum of the skin, creating microchannels that significantly enhance drug permeability and allow for direct delivery to the lesion site. Microneedle patches offer advantages such as improved drug permeability, reduced dosage and side effects, personalized treatment, and improved patient compliance. They overcome the limitations of traditional transdermal drug delivery methods, which often struggle to penetrate the skin barrier. Furthermore, nanozyme technology, a natural enzyme mimicry technology based on nanomaterials, simulates the catalytic properties of natural enzymes through the unique physicochemical properties of nanomaterials, exhibiting unique catalytic activity and stability. It has been widely applied in biomedicine, environmental science, food processing, and other fields. The ROS scavenging, antioxidant, and antibacterial functions of nanozymes can reduce the side effects of mucosal atrophy and bacterial resistance caused by the overuse of hormones and antibiotics. In the treatment of oral ulcers, the combination of nanozymes and microneedling technology has broad potential applications. By loading nanozymes into microneedle patches, precise drug delivery and long-term release can be achieved, while simultaneously promoting tissue repair. This combined treatment strategy not only improves treatment efficacy but also reduces medication dosage and side effects, providing patients with a painless and highly effective new treatment option. Furthermore, this innovative treatment method is expected to overcome the limitations of traditional treatments, offering a completely new solution for the clinical treatment of oral ulcers, and possesses significant clinical application value and market potential.

[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 and neglect the multidimensional regulation of oxidative stress, immunity, and the microbiome, particularly lacking effective solutions for recurrent, difficult-to-heal ulcers. Therefore, there is an urgent need to develop innovative formulations that combine mechanical puncture, ROS clearance, immune modulation, and timely release to meet clinical treatment needs. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical bottlenecks in this field and provide a soluble microneedle patch for oral use based on nanozyme synergy, its preparation method and application, and to provide an oral ulcer microneedle patch and its preparation method that integrates long-lasting adhesion and deep penetration, ROS clearance, immune regulation and time-sequential release functions.

[0007] The first aspect of the present invention provides a method for preparing a soluble microneedle patch for oral use based on nanozyme synergy, comprising the following steps:

[0008] S1. Citric acid, mercaptoethylamine and sodium selenite are dissolved in water and stirred at room temperature under an inert gas atmosphere. Then, a hydrothermal reaction is carried out. After centrifugation and dialysis, the reaction product is used to obtain Se-SCDs nanozyme and Se-SCDs nanozyme solution is prepared.

[0009] S2. Prepare a microneedle tip layer stock solution containing polyvinyl alcohol (PVA), carboxymethyl chitosan (CMCS), Se-SCDs nanozyme, astragalus polysaccharide and vascular endothelial growth factor;

[0010] S3. Prepare a microneedle base layer stock solution containing polyvinyl alcohol, carboxymethyl chitosan and Se-SCDs nanozymes;

[0011] S4. The microneedle tip layer mother liquid is filled into the tip cavity of the microneedle mold, and then the microneedle base layer mother liquid is poured onto the microneedle mold that has been filled with the tip layer. After drying, the mold is demolded to obtain the oral soluble microneedle patch with microneedle array based on nanoenzyme synergy.

[0012] In some embodiments of the present invention, in step S1, the mass ratio of citric acid, mercaptoethylamine and sodium selenite is (4-8):(0.1-1):(0.02-0.08).

[0013] In some embodiments of the present invention, in step S1, the temperature of the hydrothermal reaction is 120-150 °C and the reaction time is 150-300 min.

[0014] In some embodiments of the present invention, the concentration of carboxymethyl chitosan in the microneedle tip layer mother liquor is greater than that in the microneedle base layer mother liquor, and the concentration of polyvinyl alcohol in the microneedle tip layer mother liquor is less than that in the microneedle base layer mother liquor.

[0015] In some embodiments of the present invention, the concentration of carboxymethyl chitosan in the microneedle tip layer mother solution is 6-10 mg / mL, the concentration of polyvinyl alcohol is 5-9 mg / mL, the concentration of Se-SCDs nanozyme is 0.2-1 mg / mL, the concentration of astragalus polysaccharide is 0.5-3 mg / mL, and the concentration of vascular endothelial growth factor is 0.05-0.2 mg / mL.

[0016] In some embodiments of the present invention, the concentration of carboxymethyl chitosan in the microneedle base layer mother solution is 2-6 mg / mL, the concentration of polyvinyl alcohol is 9-13 mg / mL, and the concentration of Se-SCDs nanozyme is 0.2-1 mg / mL.

[0017] In some embodiments of the present invention, step S2 specifically involves: preparing a polyvinyl alcohol-carboxymethyl chitosan aqueous solution, a Se-SCDs nanoenzyme solution, and an astragalus polysaccharide solution, respectively, and then sequentially adding the Se-SCDs nanoenzyme solution, the astragalus polysaccharide solution, and vascular endothelial growth factor to the polyvinyl alcohol-carboxymethyl chitosan aqueous solution to obtain the microneedle tip layer mother solution.

[0018] A second aspect of the present invention provides a soluble microneedle patch for oral use based on nanozyme synergy, wherein the soluble microneedle patch for oral use based on nanozyme synergy is prepared by the above-described preparation method.

[0019] In some embodiments of the present invention, the microneedle array has a tip shape of a square pyramid, with a tip height of 400-600 μm, a tip width of 8-12 μm, a substrate thickness of 120-220 μm, and a tip spacing of 400-600 μm.

[0020] A third aspect of the present invention provides the application of the above-described nanozyme-based soluble microneedle patch for oral use in the preparation of a medicament or medical device for relieving and / or improving oral ulcers.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This invention addresses the problem that existing oral ulcer drugs are difficult to achieve both long-term adhesion and deep penetration, and have a single treatment path. It designs a microneedle patch with a microneedle array, integrating Se-SCDs, Astragalus polysaccharide APS and VEGF at the microneedle site to achieve synergistic effects of multiple components. It innovatively combines functions such as mechanical puncture, ROS removal, immune regulation and accelerated angiogenesis to achieve precise drug delivery to the lesion site and comprehensively regulate the oxidative stress-immune-microbiome in the oral ulcer treatment process. At the same time, it combines the characteristics of nanozymes to achieve long-term release and improve the treatment effect.

[0023] (2) In this invention, by adjusting the concentration of the microneedle tip mother liquid and the microneedle base layer mother liquid PVA and CMCS, the gradient degradation of the microneedle tip and the microneedle base can be achieved, which can protect the wound and reduce external stimulation, and gradually degrade and reduce the feeling of foreign body in the mouth, while achieving long-term drug release and reducing the frequency of use. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the microneedle patch fabrication process;

[0025] Figure 2 Transmission electron microscopy images of Se-SCDs and statistical plots of hydrated particle size obtained by dynamic light scattering (DLS);

[0026] Figure 3 Morphological features of PVA-CMCS microneedle patches and scanning electron microscope (SEM) images;

[0027] Figure 4 The graph shows the results of SOD enzyme activity detection and hydroxyl radical scavenging ability assessment.

[0028] Figure 5Figure showing the results of CAT enzyme activity detection and hydrogen peroxide removal capacity assessment.

[0029] Figure 6 This is a graph showing the results of a cytotoxicity experiment.

[0030] Figure 7 Figure showing the evaluation results of the dissolution and swelling experiment of PVA-CMCS microneedle patch;

[0031] Figure 8 Figure 1 shows the evaluation results of PVA-CMCS microneedle patch degradation experiments with different CMCS concentration ratios.

[0032] Figure 9 The results of evaluating the antibacterial activity of PVA-CMCS microneedle patches using Escherichia coli and Staphylococcus aureus are shown in the figure.

[0033] Figure 10 Figure showing experimental results of PVA-CMCS microneedle patches promoting ulcer wound healing;

[0034] Figure 11 A bar chart showing the recovery rate of PVA-CMCS microneedle patches in promoting ulcer wound healing. 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] In a specific embodiment of the first aspect of the present invention, a method for preparing a soluble microneedle patch for oral use based on nanozyme synergy is provided, comprising the following steps:

[0037] S1. Citric acid, mercaptoethylamine and sodium selenite are dissolved in water and stirred at room temperature under an inert gas atmosphere. Then, a hydrothermal reaction is carried out. After centrifugation and dialysis, the reaction product is used to obtain Se-SCDs nanozyme and Se-SCDs nanozyme solution is prepared.

[0038] S2. Prepare a microneedle tip layer stock solution containing polyvinyl alcohol (PVA), carboxymethyl chitosan (CMCS), Se-SCDs nanozyme, astragalus polysaccharide and vascular endothelial growth factor;

[0039] S3. Prepare a microneedle base layer stock solution containing polyvinyl alcohol, carboxymethyl chitosan and Se-SCDs nanozymes;

[0040] S4. The microneedle tip layer mother liquid is filled into the tip cavity of the microneedle mold, and then the microneedle base layer mother liquid is poured onto the microneedle mold that has completed the tip layer filling. After drying, the mold is demolded to obtain the oral soluble microneedle patch with microneedle array based on nanoenzyme synergy.

[0041] In some embodiments of the present invention, step S1 uses citric acid, mercaptoethylamine, and sodium selenite to prepare Se-SCDs nanozymes. The preferred mass ratio of citric acid, mercaptoethylamine, and sodium selenite is (4-8):(0.1-1):(0.02-0.08).

[0042] In some embodiments of the present invention, step S1 specifically includes the following steps:

[0043] S11. Dissolve citric acid, mercaptoethylamine, and sodium selenite in pure water, purge with nitrogen to expel some of the air from the solution, and then stir at room temperature until the mixture is homogeneous to obtain a mixed solution; the nitrogen purging time is preferably 5-15 min.

[0044] For example, the nitrogen gas can be introduced for 15 minutes and the stirring time can be 15 minutes.

[0045] S12. Transfer the mixed solution prepared in step S11 to a polytetrafluoroethylene (PTFE) liner, then insert the PTFE liner into a stainless steel reactor and seal it. Place the sealed stainless steel reactor in an oven for reaction. After the reaction is complete, cool to room temperature, centrifuge to collect the supernatant, and dialyze the supernatant through a 500 Da dialysis bag to obtain solid Se-SCDs nanozymes. The oven temperature (i.e., the hydrothermal reaction temperature) is preferably 120-150 °C, and the reaction time is preferably 150-300 min.

[0046] In some embodiments of the present invention, step S2 prepares a microneedle tip layer stock solution containing polyvinyl alcohol (PVA), carboxymethyl chitosan (CMCS), Se-SCDs nanozymes, astragalus polysaccharide and vascular endothelial growth factor. e-SCDs mimic the activity of SOD / CAT dual enzymes to clear ROS, astragalus polysaccharide APS promotes immune regulation, VEGF accelerates angiogenesis, and multiple components work synergistically to comprehensively regulate oxidative stress-immunity-microbiome.

[0047] In some specific embodiments of the present invention, step S2 specifically involves: preparing polyvinyl alcohol-carboxymethyl chitosan aqueous solution, Se-SCDs nanoenzyme solution and astragalus polysaccharide solution respectively, and then sequentially adding Se-SCDs nanoenzyme solution, astragalus polysaccharide solution and vascular endothelial growth factor to the polyvinyl alcohol-carboxymethyl chitosan aqueous solution to obtain the microneedle tip layer mother solution.

[0048] In some specific embodiments of the present invention, the polyvinyl alcohol-carboxymethyl chitosan aqueous solution is obtained by mixing a polyvinyl alcohol aqueous solution and a carboxymethyl chitosan aqueous solution.

[0049] In some specific embodiments of the present invention, the method for preparing polyvinyl alcohol-carboxymethyl chitosan aqueous solution is as follows: at room temperature, carboxymethyl chitosan (CMCS) is added to distilled water and stirred until a homogeneous solution is formed to obtain a CMCS aqueous solution; polyvinyl alcohol (PVA) is added to distilled water and stirred until a homogeneous solution is formed to obtain a PVA aqueous solution; the prepared CMCS aqueous solution and PVA aqueous solution are uniformly mixed to obtain a PVA-CMCS aqueous solution.

[0050] In some embodiments of the present invention, the concentration of carboxymethyl chitosan in the microneedle tip layer mother solution is greater than that in the microneedle base layer mother solution, and the concentration of polyvinyl alcohol in the microneedle tip layer mother solution is less than that in the microneedle base layer mother solution. Under this concentration setting, the degradation rate of the microneedle base in the resulting microneedle patch is faster, while the degradation rate of the microneedle tip is slightly slower, achieving gradient degradation. This not only protects the wound and reduces external irritation but also gradually degrades to reduce the feeling of a foreign body in the mouth, while simultaneously achieving long-term drug release.

[0051] In some specific embodiments of the present invention, the concentration of carboxymethyl chitosan in the microneedle tip layer mother solution is 6-10 mg / mL, the concentration of polyvinyl alcohol is 5-9 mg / mL, the concentration of Se-SCDs nanozyme is 0.2-1 mg / mL, the concentration of astragalus polysaccharide is 0.5-3 mg / mL, and the concentration of vascular endothelial growth factor is 0.05-0.2 mg / mL.

[0052] In some specific embodiments of the present invention, the concentration of carboxymethyl chitosan in the microneedle base layer mother solution is 2-6 mg / mL, the concentration of polyvinyl alcohol is 9-13 mg / mL, and the concentration of Se-SCDs nanozyme is 0.2-1 mg / mL.

[0053] In some specific embodiments of the present invention, the specific process of step S4 is as follows: Figure 1 As shown, it includes the following steps:

[0054] S41. Fill the PDMS microneedle tip layer mother liquor onto the surface of the PDMS microneedle mold. Then, place the PDMS mold in a centrifuge and centrifuge three times at 5000 r / min, each time for 10 min. After each centrifugation, remove the mold and place it in a 45 ℃ drying oven for 10 min to allow air bubbles to float out. After the above three-step centrifugation method, the microneedle tip layer mother liquor completely fills the tip mold cavity. Use a tool to scrape off the excess solution on top of the mold.

[0055] S42. Then, the microneedle base layer mother liquor is poured onto the microneedle mold that has been filled with the needle tip layer. The same centrifugation operation is performed to spread the microneedle base layer mother liquor evenly. Then, it is dried and demolded to obtain a soluble microneedle patch with nano-enzyme synergy and microneedle array.

[0056] In a second aspect of the present invention, a soluble microneedle patch for oral use based on nanozyme synergy is provided, wherein the soluble microneedle patch for oral use based on nanozyme synergy is prepared by the above-described preparation method.

[0057] In some embodiments of the present invention, the microneedle array has a tip shape of a square pyramid, a tip height of 400-600 μm, a tip width of 8-12 μm, a substrate thickness of 120-220 μm, and a tip spacing of 400-600 μm.

[0058] In a third aspect of the present invention, the above-described nanozyme-based soluble microneedle patch for oral use is provided for the preparation of a medicament or medical device for relieving and / or improving oral ulcers.

[0059] The present invention will be further illustrated below with specific embodiments.

[0060] The reagents, materials, and instruments used in the following examples are as follows:

[0061] Reagents: Citric acid, mercaptoethylamine, sodium selenite, carboxymethyl chitosan, polyvinyl alcohol, astragalus polysaccharide, vascular endothelial growth factor, hydrogen peroxide, agarose, peptone, cell culture medium;

[0062] Materials: dialysis bags, PDMS microneedle templates, SOD activity assay kits, cell proliferation and toxicity assay kits;

[0063] Instruments: forced-air drying oven, PTFE-lined stainless steel reaction vessel, stereo microscope, inverted microscope, scanning electron microscope, transmission electron microscope, ultrasonic cleaner, Malvern dynamic light scattering instrument (DLS).

[0064] Synthesis of Se-SCDs nanozymes

[0065] 5 g citric acid, 0.8 g mercaptoethylamine, and 0.06 g sodium selenite were dissolved in 40 mL of pure water. Nitrogen gas was introduced for 15 min to purge some air from the solution, and then the mixture was stirred at room temperature for 15 min. The solution was then transferred to a 50 mL polytetrafluoroethylene (PTFE) liner, which was then inserted into a stainless steel reactor and sealed. The reactor was placed in an oven at 150 °C for 150 min. After cooling, the solution was centrifuged at 8000 rpm for 20 min. The supernatant was collected and dialyzed in a 500 Da dialysis bag. The dialysis method involved boiling the 500 Da dialysis bag in water for 10 min, then adding the collected supernatant to the dialysis bag and dialyzing in water for 48 h. The water was changed three times at the 2nd, 6th, and 24th hours to obtain solid Se-SCDs nanozymes. Transmission electron microscopy images and hydration particle size distributions of Se-SCDs nanozymes are shown below. Figure 2 As shown.

[0066] Se-SCDs were dispersed in ethanol 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) with an accelerating voltage set to 200 kV. The TEM image of Se-SCDs is shown below. Figure 2 The left figure shows the experimental results. The results show that Se-SCDs are regular spherical with uniform particle size and good dispersibility. The particle size is about 2 nm, and no obvious agglomeration phenomenon was observed, indicating that Se-SCDs have good monodispersity.

[0067] Se-SCDs were dispersed in deionized water to prepare solutions of different concentrations. The hydrated particle size was determined using a Malvern dynamic light scattering (DLS) instrument. The temperature was set to 25℃, the measurement angle to 90 degrees, and each sample was measured three times, with the average value taken. A statistical chart of the hydrated particle size of Se-SCDs was obtained, as shown below. Figure 2 The results are shown in the right figure. The results show that the hydrated particle size of Se-SCDs exhibits good stability at different concentrations, with an average hydrated particle size of 6 nm and a narrow particle size distribution. This indicates that Se-SCDs have good solubility and stability in aqueous solution and can maintain their nanoscale particle size under physiological conditions, which is beneficial for their application in biological systems.

[0068] Example 1

[0069] (1) Preparation of microneedle tip layer stock solution

[0070] In the microneedle tip layer mother solution, the concentrations of carboxymethyl chitosan were 8 mg / mL, polyvinyl alcohol was 7 mg / mL, Se-SCDs nanozyme was 0.4 mg / mL, astragalus polysaccharide was 1 mg / mL, and vascular endothelial growth factor was 0.1 mg / mL.

[0071] (2) Preparation of microneedle basement mother solution

[0072] In the mother liquor of the microneedle base layer, the concentration of carboxymethyl chitosan was 4 mg / mL, the concentration of polyvinyl alcohol was 11 mg / mL, and the concentration of Se-SCDs nanozyme was 0.4 mg / mL.

[0073] (3) Preparation of soluble microneedle patches for oral use with nanozyme synergy

[0074] Take 200 μL of the microneedle tip layer 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 centrifuge tube, and then centrifuge the tube three times at 5000 rpm for 10 min each time. After each centrifugation, remove the mold and place it in a 45 ℃ drying oven for 10 min to allow air bubbles to float to the surface. After the above three-step centrifugation method, the microneedle tip layer stock solution completely fills the mold cavity. Use a tool to scrape off any excess solution from the top of the mold.

[0075] Then, 100 μL of the microneedle base layer stock solution was poured onto a PDMS mold filled with the microneedle tip layer stock solution to form a base layer. The mold was then placed in a drying oven at 37 ℃ for 12 h until the microneedle array was completely dried. The microneedle array was then demolded to obtain a nanozyme-synergistic soluble microneedle patch (Se-SCDsMN) for oral use. The obtained microneedle array has a close-packed structure, with a four-sided pyramidal tip shape, a tip height of 550 μm, a tip width of 10 μm, a base thickness of 200 μm, a tip spacing of 500 μm, and a microneedle patch size of 1 cm * 1 cm.

[0076] The morphological features of PVA-CMCS microneedle patches were characterized using a stereomicroscope (JSZ6S, Jiangnan New Optics, Jiangsu, China) and a scanning electron microscope (SEM). Morphological photographs and SEM images of the PVA-CMCS microneedle patches were obtained, such as... Figure 3 As shown.

[0077] Example 2

[0078] Compared with Example 1, the concentrations of each substance in the microneedle tip layer mother solution and the microneedle base layer mother solution are different, while the other steps are the same.

[0079] The concentrations of each substance in the mother liquor of the microneedle tip layer are as follows:

[0080] The concentrations of carboxymethyl chitosan, polyvinyl alcohol, Se-SCDs nanozyme, astragalus polysaccharide, and vascular endothelial growth factor were 10 mg / mL, 5 mg / mL, 0.8 mg / mL, 2 mg / mL, and 0.2 mg / mL, respectively.

[0081] The concentrations of each substance in the mother liquor of the microneedle basal layer are as follows:

[0082] The concentration of carboxymethyl chitosan was 5 mg / mL, the concentration of polyvinyl alcohol was 10 mg / mL, and the concentration of Se-SCDs nanozyme was 0.8 mg / mL.

[0083] Example 3

[0084] Compared with Example 1, the concentrations of each substance in the microneedle tip layer mother solution and the microneedle base layer mother solution are different, while the other steps are the same.

[0085] The concentrations of each substance in the mother liquor of the microneedle tip layer are as follows:

[0086] The concentrations of carboxymethyl chitosan, polyvinyl alcohol, Se-SCDs nanozyme, astragalus polysaccharide, and vascular endothelial growth factor were 0.05 mg / mL.

[0087] The concentrations of each substance in the mother liquor of the microneedle basal layer are as follows:

[0088] The concentration of carboxymethyl chitosan was 6 mg / mL, the concentration of polyvinyl alcohol was 9 mg / mL, and the concentration of Se-SCDs nanozyme was 0.2 mg / mL.

[0089] Comparative Example 1 (without Se-SCDs)

[0090] Compared to Example 1, the mother liquor of the microneedle tip layer did not contain Se-SCDs nanozymes, but the other steps were the same.

[0091] Comparative Example 2 (without APS)

[0092] Compared to Example 1, the mother liquor of the microneedle tip layer does not contain APS, while the other steps are the same.

[0093] Comparative Example 3 (without VEGF)

[0094] Compared to Example 1, the mother liquor of the microneedle tip layer did not contain VEGF, but the other steps were the same.

[0095] Comparative Example 4

[0096] (1) Prepare carboxymethyl chitosan-polyvinyl alcohol aqueous solution as microneedle stock solution.

[0097] The concentration of carboxymethyl chitosan (CMCS) in the microneedle mother liquor was 2 mg / mL, and the concentration of polyvinyl alcohol (PVA) was 13 mg / mL.

[0098] (2) Preparation of pure PVA-CMCS soluble microneedle patches

[0099] Take 200 μL of the microneedle stock solution prepared in step (1) and spread it evenly on the surface of a 15×15 microneedle array polydimethylsiloxane (PDMS) mold. Place the PDMS mold in a 5 mL centrifuge tube, and then put the centrifuge tube into a centrifuge. Centrifuge three times at 5000 rpm for 10 min each time. After each centrifugation, remove the mold and place it in a 45 ℃ drying oven for 10 min to allow air bubbles to float out. After the above three-step centrifugation method, the microneedle stock solution completely fills the mold cavity. Use a tool to scrape off the excess solution on top of the mold.

[0100] Then, 100 μL of the microneedle stock solution prepared in step (1) was poured onto the PDMS mold filled with the microneedle stock solution to form a base layer. The mold was then placed in a drying oven and dried at 37 ℃ for 12 h until the microneedle array was completely dried. After the microneedle array was demolded, a pure methyl chitosan-polyvinyl alcohol (PVA-CMCS) soluble microneedle patch was obtained. The obtained microneedle array has a close-packed structure, with the needle tip shape being a four-sided pyramid, the needle tip height being 550 μm, the needle tip width being 10 μm, the base thickness being 200 μm, the needle tip spacing being 500 μm, and the microneedle patch size being 1 cm * 1 cm.

[0101] Comparative Examples 5-8

[0102] Compared with Comparative Example 4, the concentrations of CMCS and PVA in the microneedle stock solution were different, while the other steps were the same.

[0103] Comparative Example 5 Comparative Example 6 Comparative Example 7 Comparative Example 8 CMCS concentration (mg / mL) 4 6 8 10 PVA concentration (mg / mL) 11 9 7 5

[0104] The synthesized Se-SCDs and the microneedle patch prepared in Example 1 were characterized using the following methods:

[0105] Test Example 1: Enzyme Activity Detection of Se-SCDs Solution

[0106] SOD activity was detected using a Novel Bioassay Test Kit (NBT). Se-SCDs solution was mixed with a xanthine and xanthine oxidase reaction system, NBT was added, and the mixture was incubated at 37°C for a period of time. The absorbance at 560 nm was then measured. SOD activity units were calculated using standards, and the results of SOD enzyme activity detection and hydroxyl radical scavenging ability assessment were obtained, as shown in the figure. Figure 4As shown in the figure. The results indicate that Se-SCDs exhibit good superoxide anion radical scavenging ability, and also have good reactive oxygen species scavenging ability.

[0107] CAT enzyme activity assay: The activity of Se-SCDs was detected using the ultraviolet absorption of hydrogen peroxide. A 0-12 mM hydrogen peroxide solution was prepared, and a standard curve was determined. Se-SCDs were then added, incubated for 1 h, and the absorbance at 240 nm was measured. CAT activity was calculated, and the results of CAT enzyme activity assay and hydrogen peroxide scavenging ability assessment were obtained, as shown in the figure. Figure 5 As shown in the figure. The results indicate that Se-SCDs have a good ability to decompose H2O2 and can effectively remove ROS substances accumulated in oral ulcer sites.

[0108] Test Example 2: Cytotoxicity Experiment

[0109] Cell proliferation and cytotoxicity were determined using the Cell Counting Kit-8 assay. HorF cells were grown in 96-well plates at a seeding density of 10^4 cells / well. Cells in the exponential growth phase were incubated for 24 h with different groups (Se-SCDs, microneedle patches obtained in Examples 1-3), followed by incubation with the CCK8 assay for 2 h. Cell viability was measured at 450 nm. Cell viability (%) = [OD (treatment group) - OD (blank group)] / [OD (control group) - OD (blank group)] × 100%. The results of the cytotoxicity assay are as follows: Figure 6 As shown, the results indicate that the Se-SCDs prepared in this invention and the microneedle patches obtained in Examples 1-3 have good biocompatibility.

[0110] Test Example 3: Microneedle Swelling Experiment

[0111] The microneedle patch prepared in Example 1 was inserted into an agarose hydrogel. The microneedle patch absorbed water and swelled within the agarose hydrogel, reaching swelling equilibrium in approximately 14 minutes, with a final swelling rate of about 200%. Its swelling curve is shown below. Figure 7 As shown.

[0112] Test Example 4: Microneedle Gradient Degradation Experiment

[0113] The PVA-CMCS microneedle patches prepared in Comparative Examples 4-8 were inserted into agarose hydrogels. The microneedle patches absorbed water and swelled within the agarose hydrogels. The degradation process of the microneedles was observed, and the bar graph is shown below. Figure 8As shown. Subsequent studies found that the PVA-CMCS microneedle patch dissolved slowly over different time periods, eventually dissolving completely into the agarose hydrogel. Furthermore, the dissolution time was positively correlated with the CMCS content. The microneedle patch utilizes the slow dissolution of chitosan to deliver the desired drug for a long time, while its swelling properties allow it to adhere to the skin and prevent detachment.

[0114] Test Example 5: Antibacterial Experiment

[0115] Using double-distilled water as a control, the antibacterial ability of samples from Comparative Examples 1-3 and Sample 3 was tested by plate coating and counting experiments.

[0116] Preparation of bacterial suspension: 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 phase (approximately 18–24 h); the bacterial suspension concentration was further adjusted to a suitable concentration (10^6 CFU / mL) with sterile physiological saline.

[0117] Plate coating and counting: The bacterial suspension obtained in the above steps was mixed with the sterile filtrate of the test sample and incubated at 37°C for 24 h. The mixed and incubated solution was further diluted 10^5 times, and the diluted solution was inoculated onto the surface of solid agarose petri dishes and incubated at 37°C for 24 h. The petri dishes were removed, photographed, and the bacterial count was calculated. The results are shown below. Figure 9 As shown in the figure. Experimental results show that the microneedle patch prepared in the embodiments of the present invention has good inhibitory ability against both Gram-negative and Gram-positive bacteria, and can effectively regulate the oral microbiome and restore a healthy oral microenvironment.

[0118] Test Example 6: Wound Healing Promotion Experiment

[0119] Using double-distilled water as a control sample, wound healing promotion experiments were conducted on the samples prepared in Comparative Examples 1-3 and Examples 1-3.

[0120] Microscopic imaging: Samples were dissolved in 1 ml of FBS-free cell culture medium, filtered, and sterile filtrate was obtained. HorF cells were then seeded into 6-well plates and cultured in cell culture medium containing 10% FBS. When the cells reached 80% confluence, they were scratched using a 200 μL pipette tip. Intervention was performed using FBS-free cell culture medium and sterile filtrate from each microneedle patch group. Images were observed under a light microscope at 0 and 24 h post-intervention. Edge mapping was further performed using ImageJ software. The experimental results of microneedle patches promoting ulcer wound healing are shown below. Figure 10 As shown.

[0121] Quantitative migration rate statistics: The wound size was quantified using ImageJ software. Microscopic images were loaded into ImageJ software, and the migration rate (%) was quantitatively calculated according to the formula: migration rate (%) = (scraping distance - post-growth distance) / scraping distance × 100%. Figure 11 As shown in the figure. Experimental results indicate that microneedle technology enables precise drug delivery to the lesion site, while the combination of nanozyme properties achieves long-term release, effectively improving treatment efficacy. Se-SCDs mimic SOD / CAT dual-enzyme activity to scavenge ROS, Astragalus polysaccharide APS promotes immune regulation, and VEGF accelerates angiogenesis. The synergistic effect of multiple components comprehensively regulates oxidative stress, immunity, and the microbiome, achieving multifunctional synergistic therapy and promoting ulcer wound healing.

[0122] This invention provides a method for preparing and applying a nanozyme-synergistic microneedle patch for oral cavity. It combines the antibacterial, antioxidant, and tissue repair-promoting functions of nanozymes, astragalus polysaccharides, and VEGF. Through a gradient-degrading 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 treating oral ulcers. Many methods and approaches can be used to implement this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing a soluble microneedle patch for oral use based on nanozyme synergy, characterized in that, The preparation method includes the following steps: S1. Citric acid, mercaptoethylamine and sodium selenite are dissolved in water and stirred at room temperature under an inert gas atmosphere. Then, a hydrothermal reaction is carried out. After centrifugation and dialysis, the reaction product is used to obtain Se-SCDs nanozyme and Se-SCDs nanozyme solution is prepared. S2. Prepare a microneedle tip layer stock solution containing polyvinyl alcohol, carboxymethyl chitosan, Se-SCDs nanozyme, astragalus polysaccharide and vascular endothelial growth factor; S3. Prepare a microneedle base layer stock solution containing polyvinyl alcohol, carboxymethyl chitosan and Se-SCDs nanozymes; S4. The microneedle tip layer mother liquid is filled into the tip cavity of the microneedle mold, and then the microneedle base layer mother liquid is poured onto the microneedle mold that has completed the tip layer filling. After drying, the mold is demolded to obtain the oral soluble microneedle patch with microneedle array based on nanoenzyme synergy.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of citric acid, mercaptoethylamine, and sodium selenite is (4-8):(0.1-1):(0.02-0.08).

3. The preparation method according to claim 1, characterized in that, In step S1, the temperature of the hydrothermal reaction is 120-150 ℃, and the reaction time is 150-300 min.

4. The preparation method according to claim 1, characterized in that, The concentration of carboxymethyl chitosan in the microneedle tip layer mother liquor is greater than that in the microneedle base layer mother liquor, and the concentration of polyvinyl alcohol in the microneedle tip layer mother liquor is less than that in the microneedle base layer mother liquor.

5. The preparation method according to claim 1, characterized in that, The mother liquor of the microneedle tip layer contains 6-10 mg / mL of carboxymethyl chitosan, 5-9 mg / mL of polyvinyl alcohol, 0.2-1 mg / mL of Se-SCDs nanozyme, 0.5-3 mg / mL of astragalus polysaccharide, and 0.05-0.2 mg / mL of vascular endothelial growth factor.

6. The preparation method according to claim 1, characterized in that, In the mother liquor of the microneedle base layer, the concentration of carboxymethyl chitosan is 2-6 mg / mL, the concentration of polyvinyl alcohol is 9-13 mg / mL, and the concentration of Se-SCDs nanozyme is 0.2-1 mg / mL.

7. The preparation method according to claim 1, characterized in that, Step S2 specifically involves preparing a polyvinyl alcohol-carboxymethyl chitosan aqueous solution, a Se-SCDs nanoenzyme solution, and an astragalus polysaccharide solution, respectively. Then, the Se-SCDs nanoenzyme solution, the astragalus polysaccharide solution, and vascular endothelial growth factor are added sequentially to the polyvinyl alcohol-carboxymethyl chitosan aqueous solution to obtain the microneedle tip layer mother solution.

8. A soluble microneedle patch for oral use based on nanozyme synergy, characterized in that, The oral soluble microneedle patch based on nanozyme synergy is prepared by the preparation method according to any one of claims 1-7.

9. The oral soluble microneedle patch based on nanozyme synergy according to claim 8, characterized in that, The microneedle array has a pyramidal tip shape, a tip height of 400-600 μm, a tip width of 8-12 μm, a substrate thickness of 120-220 μm, and a tip spacing of 400-600 μm.

10. The use of the nanozyme-based soluble microneedle patch for oral use according to claim 9 in the preparation of a drug or medical device for relieving and / or improving oral ulcers.

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