A response microneedle patch based on clostridium butyricum and gastrodin and a preparation method thereof

Abstract

This invention proposes a responsive microneedle patch based on Clostridium butyricum and gastrodin, and its preparation method, relating to the field of biomedical materials. It includes a microneedle substrate and a drug loading system. The microneedle substrate is a sodium alginate-3APBA covalently modified network, and the drug loading system includes dexamethasone, gastrodin, and Clostridium butyricum dormant spores, with gastrodin covalently grafted onto the microneedle substrate; furthermore, each 1 cm... 2 The loading amounts of each component in the single microneedle patch are: dexamethasone 0.3-2 mg, gastrodin 4-20 mg, and Clostridium butyricum dormant spores 1×10⁻⁶. 6 -3.5×10 8 CFU. This invention constructs a highly stable ROS-responsive drug delivery system through stepwise functionalization and graft modification of sodium alginate, enabling on-demand, controllable, and efficient deep drug delivery and synergistic treatment of RA, thereby improving the effectiveness, safety, and long-term efficacy of RA treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, and more specifically, to a responsive microneedle patch based on Clostridium butyricum and gastrodin and its preparation method. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic synovitis, progressive joint damage, and systemic complications. The lesion site is characterized by a microenvironment with a large number of inflammatory cells infiltrating and elevated reactive oxygen species (ROS).

[0003] Currently, systemic drug delivery in clinical practice (oral and injection) suffers from problems such as low local bioavailability in joints and difficulty in maintaining effective concentrations. Existing soluble microneedles mainly rely on passive diffusion for drug release, which has high tissue penetration resistance and limited drug penetration depth and distribution range, making it difficult to meet the treatment needs of deep joint lesions.

[0004] Furthermore, single-drug therapy struggles to simultaneously achieve rapid anti-inflammatory effects and long-term immune regulation: glucocorticoids (such as dexamethasone) have a rapid onset of action but significant long-term side effects; natural active ingredients (such as gastrodin) are suitable for long-term anti-inflammatory and antioxidant effects but have a slow onset of action. Directly encapsulating live bacteria in microneedles also presents challenges such as difficulty in preserving activity, poor standardization, and insufficient safety. Therefore, a composite product is needed that can balance rapid anti-inflammatory effects, long-term conditioning, live bacteria stability, and precise ROS-responsive drug delivery to improve the effectiveness, safety, and long-term efficacy of RA treatment. Summary of the Invention

[0005] The purpose of this invention is to provide a responsive microneedle patch based on Clostridium butyricum and gastrodin and its preparation method. It constructs a highly stable ROS-responsive drug release system through stepwise functionalization and graft modification of sodium alginate, so as to achieve on-demand, controllable, efficient deep drug delivery and synergistic treatment for RA, thereby improving the effectiveness, safety and long-term efficacy of RA treatment.

[0006] The technical problem solved by this invention is achieved by the following technical solution.

[0007] In a first aspect, embodiments of this application provide a responsive microneedle patch based on Clostridium butyricum and gastrodin, comprising a microneedle substrate and a drug loading system. The microneedle substrate is a sodium alginate-3APBA covalently modified network, and the drug loading system comprises dexamethasone, gastrodin, and Clostridium butyricum dormant spores, with gastrodin covalently grafted onto the microneedle substrate.

[0008] per 1cm 2 The loading amounts of each component in the single microneedle patch include: dexamethasone 0.3-2 mg, gastrodin 4-20 mg, and Clostridium butyricum dormant spores 1×10⁻⁶. 6 -3.5×10 8 CFU.

[0009] Secondly, embodiments of this application provide a method for preparing the above-mentioned responsive microneedle patch based on Clostridium butyricum and gastrodin, comprising the following steps: S1. Dissolve medical-grade sodium alginate in PBS buffer and stir at room temperature for 12 h until completely dissolved. Then, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence and activate in the dark for 30 min. Then, slowly add 3-aminophenylboronic acid and stir at a uniform speed at room temperature in the dark for 12 h to obtain a modified sodium alginate solution. S2. Gastrodin was added to the modified sodium alginate solution and stirred at room temperature in the dark for 12 h. After stirring, the mixture was dialyzed for 24 h and then freeze-dried under vacuum to obtain a white powder SA-APBA-Ga ROS-responsive drug-loaded complex. S3. Take standardized Clostridium butyricum live bacterial tablets, grind and disperse them in physiological saline, centrifuge, wash and adjust the spore concentration to the preset value with physiological saline; S4. Dissolve the SA-APBA-Ga ROS-responsive drug-loaded complex in deionized water, stir in a 60°C water bath until completely dissolved, and then cool to room temperature; add dexamethasone powder and stir for 30 min, and finally add the purified Clostridium butyricum spore suspension and stir slowly for 10 min to obtain the composite drug-loaded matrix solution. S5. Slowly inject the composite drug-loaded matrix solution into the microneedle array mold, let it stand to remove bubbles, then centrifuge at 4000 r / min for 15 min, let it stand again for 30 min, then add calcium chloride solution for cross-linking and curing for 20 min, and dry at room temperature in the dark for 12 h. After demolding, the responsive microneedle patch based on Clostridium butyricum and gastrodin can be obtained.

[0010] In step S1, the mass ratio of medical-grade sodium alginate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and 3-aminophenylboronic acid is 20:2:1.5:3.

[0011] In step S2, the dialysis process also includes changing the dialysate every 6 hours.

[0012] In step S3, the centrifugation process includes first centrifuging at 3000 r / min for 5 min to remove tablet excipient impurities, then centrifuging at 8000 r / min for 10 min to enrich the dormant spore precipitate, and finally resuspending the spores in sterile physiological saline to a concentration of 7 × 10⁻⁶. 9 CFU / mL.

[0013] In step S4, the mass ratio of the SA-APBA-Ga ROS-responsive drug-loaded complex to dexamethasone powder is 10000:1.

[0014] In step S5, the microneedle array mold is a 1 cm × 1 cm PDMS microneedle array mold.

[0015] Thirdly, embodiments of this application provide the application of the above-mentioned responsive microneedle patch based on Clostridium butyricum and gastrodin in the preparation of products for treating rheumatoid arthritis.

[0016] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention employs a stepwise covalent grafting modification process with sodium alginate to anchor gastrodin in a sodium alginate-3APBA covalently modified network, forming an integrated and stable drug release system of "carrier-response group-drug". This completely solves the problems of drug leakage and disordered drug release in traditional mixed drug-loaded systems. It utilizes the high ROS microenvironment of lesions to specifically trigger the cleavage of borate ester bonds for drug release, thereby improving the effective utilization rate of the drug and significantly reducing systemic toxic side effects. 2. This invention, through the synergistic effect of dexamethasone and gastrodin, can achieve rapid relief of acute inflammation within 30 minutes and long-term regulation of immune imbalance within 14 days, solving the problems of slow onset of action or long-term side effects of single drugs; and through the in-situ continuous hydrogen production of Clostridium butyricum to construct microscopic driving force, the penetration depth of traditional microneedle drugs is increased from 800 μm to 1000 μm, completely solving the defects of traditional microneedle in shallow penetration, uneven distribution of drugs in deep lesions, and ineffective treatment; 3. This invention uses standardized Clostridium butyricum dormant spores to replace live bacteria, which can store live bacteria at room temperature, solving the problems of difficulty in preserving the activity of traditional live bacteria microneedles, poor standardization, and low safety. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 These are morphological images of different microneedles in embodiments of the present invention; Figure 2 The graphs show the release of dexamethasone and gastrodin from the microneedle patch prepared in Example 1 of this invention under different ROS environments. Figure 3 This is a graph showing the hydrogen production capacity of the microneedle patch prepared in Example 1 of the present invention; Figure 4 Analysis diagram of the effective delivery depth of different microneedles in embodiments of the present invention; Figure 5 A schematic diagram illustrating the mechanism of action of the microneedle patch prepared in Example 1 of this invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0021] In all embodiments of the invention, each English character represents: RA: Rheumatoid Arthritis; ROS: Reactive Oxygen Species; GA: Gastrodin; 3-APBA: 3-Aminophenylboronic acid; EDC: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; NHS: N-hydroxysuccinimide.

[0022] Example 1 This embodiment provides a detailed method for preparing a responsive microneedle patch based on Clostridium butyricum and gastrodin, with the following specific steps: S1. Weigh 2 g of medical-grade sodium alginate (molecular weight 300 kDa, purity ≥98%) and dissolve it in 100 mL of PBS buffer (pH=5.5). Stir magnetically at room temperature for 12 h until completely dissolved to obtain a homogeneous sodium alginate solution. Then, add 0.2 g of EDC and 0.15 g of NHS as amide crosslinking activators sequentially and activate in the dark for 30 min to precisely activate the carboxyl groups of sodium alginate molecules. Subsequently, slowly add 300 mg of 3-APBA and stir uniformly at room temperature in the dark for 12 h. Through the amide reaction, the amino group of 3-APBA is covalently coupled to the carboxyl group of sodium alginate to prepare a boric acid functionalized modified sodium alginate solution (SA-APBA solution).

[0023] It is important to note that the entire reaction process must be kept away from light and air to prevent the functional groups from oxidizing and becoming ineffective.

[0024] S2. Add 900 mg of gastrodin powder to the SA-APBA solution after the above reaction is completed. Stir continuously at room temperature in the dark for 12 h. The boronic acid groups on the surface of SA-APBA specifically bind to the vicinal diol structure of gastrodin, forming ROS-sensitive boronic acid ester bonds, thus completing the stable anchoring grafting of gastrodin. After the reaction, dialyze the solution in deionized water at room temperature for 24 h using a dialysis bag with a molecular weight cutoff of 1000 Da. Change the dialysate every 6 h to thoroughly remove unreacted 3-APBA, gastrodin, and cross-linking agent impurities. After dialysis, freeze-dry under vacuum to obtain a white powdery SA-APBA-Ga ROS-responsive drug-loaded complex, which is then stored at -20℃ in a sealed container in the dark for later use.

[0025] S3. Take commercially standardized Clostridium butyricum live bacterial tablets, grind and pulverize them in a sterile laminar flow hood, disperse them in sterile physiological saline, centrifuge at low speed (3000 r / min, 5 min) to remove tablet excipients and impurities, centrifuge at high speed (8000 r / min, 10 min) to enrich the dormant spore precipitate, and wash three times with sterile physiological saline to remove residual impurities. Finally, resuspend the spores in sterile physiological saline and adjust the spore concentration to 7 × 10⁻⁶. 9 CFU / mL, refrigerate at 4℃ for later use.

[0026] S4. Weigh 2 g of the SA-APBA-Ga complex powder prepared above, dissolve it in 100 mL of sterile deionized water, stir at low speed in a 60℃ water bath until completely dissolved, and cool to room temperature to obtain a 2% (w / v) functionalized sodium alginate drug-loaded solution. Accurately weigh 75 mg of dexamethasone powder, add it to the solution, and stir at low speed for 30 min to ensure uniform physical dispersion in the matrix system; finally, slowly add 10 mL of purified Clostridium butyricum spore suspension (containing 7 × 10⁶ live spores). 9 (CFU), gently stir at low speed for 10 min to avoid high-speed shearing that could cause spore destruction and inactivation, and finally obtain a uniform and stable three-component composite drug-loaded matrix solution.

[0027] S5. Slowly and uniformly inject 0.5 mL of the composite drug-loaded matrix solution into a 1 cm × 1 cm PDMS microneedle array mold (single needle height 600 μm, base diameter 200 μm), let it stand for 5 min to remove air bubbles, ensuring complete filling of the needle cavities without gaps. Place the mold in a high-speed centrifuge and centrifuge at 4000 r / min for 15 min to allow the matrix solution to settle fully and uniformly fill the needle cavities. After centrifugation, let it stand at room temperature for 30 min, then slowly add 0.5% sterile calcium chloride solution for in-situ cross-linking and curing for 20 min to fully cross-link and shape the sodium alginate gel network, improving the strength and stability of the microneedle structure. Then place the cured microneedle mold in a sterile constant temperature drying oven and dry at 25℃ in the dark for 12 h to completely remove moisture and ensure the stability of the microneedle structure. After drying, gently demold to obtain a responsive microneedle patch based on Clostridium butyricum and gastrodin. Based on the previously selected solution ratio, the prepared 1 cm 2 The loading amounts of each component in the single microneedle patch are: dexamethasone 0.3-2 mg, gastrodin 4-20 mg, and Clostridium butyricum dormant spore viable count 1×10⁻⁶. 6 -3.5×10 8 CFU. In the preparation methods mentioned above, those skilled in the art can obtain single-piece microneedle patches with different drug loadings by adjusting the ratio.

[0028] Example 2 The steps in this embodiment are largely the same as those in Embodiment 1, with the following differences: In step S1, equal amounts of 3-APBA, gastrodin, and spore suspension are directly mixed into the sodium alginate solution, without using a covalent stepwise grafting method.

[0029] Performance verification ① First, the morphology of the microneedles (denoted as SBGDC) prepared in Example 1 was verified and compared with that of pure sodium alginate microneedles (SA-BA) and microneedles without butyric acid bacteria (SBGD). Figure 1 It can be seen that the SA-BA microneedles are sharp and conical with a smooth surface; the SBGD microneedles have a gentle taper and complete layered texture on the surface; the SBGDC microneedles have completely blunt and rounded tips, and a large number of disordered wrinkles are generated on the surface, significantly increasing the surface roughness. The texture of the microneedle tips gradually deepens with the addition of functional components, indicating that the dormant spores of Clostridium butyricum are uniformly dispersed in the microneedles, and the wrinkled structure provides them with attachment sites.

[0030] ② Using the microneedle patch prepared in Example 1 as a sample, the release amounts of dexamethasone and gastrodin (Ga) under different ROS environments were measured, and a control group (PBS) was set up simultaneously. The experimental steps included immersing the prepared SBGDC microneedles in H2O2 solutions of different concentrations (0.1, 0.5, 1, 2 mM) to simulate the ROS environment, and then taking the supernatant at different time periods to test the changes in the UV absorption peak. The in vitro release kinetics results are as follows: Figure 2 As shown. By Figure 2 (a) As can be seen, Dex exhibited rapid release behavior under different incubation conditions: the curves of each group highly overlapped, and the cumulative release level approached a plateau within 10 min. The Dex release curves under different H2O2 concentrations (0.1–2.0 mM) showed no significant differentiation, indicating that the release behavior of Dex was independent of the H2O2-induced oxidation response. This phenomenon stems from the physical blending of Dex within the microneedle matrix; the microneedles rapidly swelled and dissolved upon contact with the liquid phase, driving the rapid diffusion and release of Dex, a process unaffected by the ROS concentration in the system.

[0031] Figure 2 (b) shows the cumulative release curves of Ga under different oxidative microenvironments. In the PBS blank system (SBGDC-PBS), the borate ester bond structure is stable, and Ga release is significantly limited, with a cumulative release rate of only about 45% after 70 h of incubation. The introduction of H2O2 can effectively trigger Ga release, and the release behavior shows a clear concentration dependence: as the H2O2 concentration increases, the cumulative release rate of Ga increases synchronously with the final release level. All H2O2 treatment groups showed a rapid release phase in the first 10 h, followed by a slowdown in the release rate, entering a sustained and stable release phase. This result confirms that ROS can specifically induce the cleavage of borate ester bonds, thereby achieving the controlled dissociation of Ga.

[0032] The combined drug release results of the two groups indicate that SBGDC microneedles can achieve differentiated temporal release of two active substances: Dex achieves rapid release through microneedle matrix dissolution, quickly exerting an immediate anti-inflammatory effect; Ga achieves ROS-responsive long-term release through the oxidative cleavage of borate ester bonds, adapting to the continuous treatment needs of chronic inflammatory lesions. This synergistic release mode is expected to simultaneously meet the therapeutic goals of rapid intervention in acute inflammation and long-term regulation of lesion sites.

[0033] ③ Using the microneedle patch prepared in Example 1 as a sample, its hydrogen production capacity was measured at different times. To test whether the live butyric acid bacteria patch had hydrogen production capacity, the prepared SBGDC microneedles were immersed in a sealed container in PBS solution and incubated at 37 degrees Celsius for 24 h to test the gas produced in the sealed container. The experimental results are as follows: Figure 3As shown, the substrate conversion was low in the initial stage of the reaction (4 h), with an average cumulative hydrogen production of 3.6 mL. After the reaction time was extended to 12 h, the substrate continued to convert, and the hydrogen production increased to 5.9 mL, with the hydrogen production rate remaining at a high level. By 16 h, the cumulative hydrogen production of the system reached 8.6 mL. When the reaction time was further extended to 24 h, the total hydrogen production was not significantly different from that at 16 h, proving that hydrogen production could continue in the system at this time, and that the hydrogen production tended to stabilize after 16 h.

[0034] To investigate the in-situ hydrogen production-mediated active deep drug delivery capability of microneedle systems, this study set up the microneedles (SBGDC) prepared in Example 1 as the experimental group, and simultaneously set up two control groups: SABA blank microneedles (without hydrogen-producing live bacteria and therapeutic drugs) and SBAC microneedles (loaded with hydrogen-producing live bacteria, without Dex and Ga). Rhodamine B was used as the fluorescent tracer molecule, and agarose gel was used to simulate the subcutaneous tissue microenvironment. Laser confocal scanning was used to observe the distribution characteristics of fluorescence signals along the depth direction, quantitatively evaluating the longitudinal drug delivery depth of different microneedle platforms. The relevant results are as follows: Figure 4 As shown, the SABA blank microneedle group, lacking hydrogen-producing live bacteria and active drugs, lacked hydrogen-driven propulsion. Rhodamine B penetrated into the tissue solely through passive molecular diffusion, with the fluorescence signal essentially disappearing at a depth of approximately 800 μm, resulting in a maximum effective delivery depth of about 800 μm. The SBAC microneedles, loaded only with hydrogen-producing live bacteria and containing no Dex or Ga, activated the live bacteria in the local microenvironment after insertion into the simulated tissue, continuously generating hydrogen in situ. The driving force of hydrogen formation effectively promoted the migration of tracer molecules deeper into the tissue, increasing the maximum drug delivery depth to 1000 μm. For the SBGDC microneedles, co-loaded with hydrogen-producing live bacteria, Dex, and Ga, the system retained the complete in-situ hydrogen production mechanism of the live bacteria, ultimately achieving a delivery effect similar to that of the SBAC microneedles, with a maximum delivery depth also reaching 1000 μm. These results confirm that the co-loaded Dex and Ga do not significantly affect the physiological activity of the hydrogen-producing live bacteria, and the microneedle system can stably maintain the hydrogen-mediated active permeation-enhancing effect.

[0035] In summary, the results show that blank microneedles without live bacteria loading rely solely on passive diffusion, limiting the depth of drug penetration into tissues. Introducing hydrogen-producing live bacteria allows for the in-situ generation of hydrogen gas, significantly enhancing deep drug delivery. More importantly, co-loading the anti-inflammatory active components Dex and Ga into the system does not weaken the active delivery function mediated by live bacteria, laying the foundation for synergistic deep lesion drug delivery.

[0036] Based on this, those skilled in the art can use this application to prepare products for treating rheumatoid arthritis, which can achieve on-demand, controllable, and highly efficient deep drug delivery and synergistic treatment for rheumatoid arthritis, improving the effectiveness, safety, and long-term efficacy of RA treatment.

[0037] In summary, this invention provides a responsive microneedle patch based on Clostridium butyricum and gastrodin, and its preparation method. It employs a stepwise covalent grafting modification process with sodium alginate to anchor gastrodin within a sodium alginate-3APBA covalently modified network, forming an integrated and stable drug release system of "carrier-responsive group-drug". This completely solves the problems of drug leakage and disordered drug release in traditional mixed-load drug delivery systems. It utilizes the high ROS microenvironment of lesions to specifically trigger the cleavage of borate ester bonds for drug release, thereby improving drug utilization efficiency and significantly reducing systemic toxicity. This invention achieves rapid relief of acute inflammation within 30 minutes and long-term regulation of immune imbalance within 3 days through the synergistic effect of dexamethasone and gastrodin, solving the problems of slow onset of action or long-term side effects of single drugs; and by constructing a microscopic driving force through in-situ continuous hydrogen production by Clostridium butyricum, the penetration depth of traditional microneedle drugs is increased from 800 μm to 1000 μm, completely solving the defects of traditional microneedle in terms of shallow penetration, uneven distribution of drugs in deep lesions, and ineffective treatment. This invention uses standardized Clostridium butyricum dormant spores to replace live bacteria, which can store live bacteria at room temperature, solving the problems of difficulty in preserving the activity of traditional live bacteria microneedles, poor standardization, and low safety.

[0038] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A responsive microneedle patch based on Clostridium butyricum and gastrodin, characterized in that, The invention includes a microneedle substrate and a drug loading system. The microneedle substrate is a sodium alginate-3APBA covalently modified network, and the drug loading system includes dexamethasone, gastrodin, and Clostridium butyricum dormant spores, with the gastrodin covalently grafted onto the microneedle substrate.

2. The responsive microneedle patch based on Clostridium butyricum and gastrodin according to claim 1, characterized in that, per 1cm 2 The loading amounts of each component in the single microneedle patch include: dexamethasone 0.3-2 mg, gastrodin 4-20 mg, and Clostridium butyricum dormant spores 1×10⁻⁶. 6 -3.5×10 8 CFU.

3. A method for preparing a responsive microneedle patch based on Clostridium butyricum and gastrodin as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Dissolve medical-grade sodium alginate in PBS buffer and stir at room temperature for 12 h until completely dissolved. Then, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in sequence and activate in the dark for 30 min. Then, slowly add 3-aminophenylboronic acid and stir at a uniform speed at room temperature in the dark for 12 h to obtain a modified sodium alginate solution. S2. Gastrodin was added to the modified sodium alginate solution, and the mixture was stirred at room temperature in the dark for 12 h. After stirring, the mixture was dialyzed for 24 h and then freeze-dried under vacuum to obtain a white powder SA-APBA-Ga ROS-responsive drug-loaded complex. S3. Take standardized Clostridium butyricum live bacterial tablets, grind and disperse them in physiological saline, centrifuge, wash and adjust the spore concentration to the preset value with physiological saline; S4. Dissolve the SA-APBA-Ga ROS-responsive drug-loaded complex in deionized water, stir in a 60°C water bath until completely dissolved, and then cool to room temperature; add dexamethasone powder and stir for 30 min, and finally add the purified Clostridium butyricum spore suspension and stir slowly for 10 min to obtain the composite drug-loaded matrix solution. S5. Slowly inject the composite drug-loaded matrix liquid into the microneedle array mold, let it stand to remove bubbles, then centrifuge at 4000 r / min for 15 min, let it stand again for 30 min, then add calcium chloride solution for cross-linking and curing for 20 min, and dry at room temperature in the dark for 12 h. After demolding, the responsive microneedle patch based on Clostridium butyricum and gastrodin can be obtained.

4. The preparation method according to claim 3, characterized in that, In step S1, the mass ratio of the medical-grade sodium alginate, the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, the N-hydroxysuccinimide, and the 3-aminophenylboronic acid is 20:2:1.5:

3.

5. The preparation method according to claim 4, characterized in that, In step S2, the dialysis process also includes changing the dialysis fluid every 6 hours.

6. The preparation method according to claim 5, characterized in that, In step S3, the centrifugation step includes first centrifuging at a rate of 3000 r / min for 5 min, and then centrifuging at a rate of 8000 r / min for 10 min, wherein the spore concentration is 7 × 10⁻⁶. 9 CFU / mL.

7. The preparation method according to claim 6, characterized in that, In step S4, the mass ratio of the SA-APBA-Ga ROS-responsive drug-loaded complex to the dexamethasone powder is 10000:

1.

8. The preparation method according to claim 7, characterized in that, In step S5, the microneedle array mold is a 1 cm × 1 cm PDMS microneedle array mold.

9. The use of the responsive microneedle patch based on Clostridium butyricum and gastrodin as described in claim 1 or 2 in the preparation of products for treating rheumatoid arthritis.

Images