Preparation method of a compound microneedle patch of jingang extracellular vesicles

Abstract

This invention discloses a method for preparing a Platycodon grandiflorum extracellular vesicle composite microneedle patch, mainly relating to the field of traditional Chinese medicine. The method includes the following steps: S1. Extracting Platycodon grandiflorum juice and filtering to obtain a filtrate; S2. Centrifuging the filtrate and collecting the supernatant; S3. Separating the vesicle mixture to obtain a Platycodon grandiflorum extracellular vesicle solution; S4. Preparing an activated thioacetate solution; S5. Preparing a thioacetate-luteolin product; S6. Preparing purified Fuc-TK-Lut, i.e., an amphiphilic carrier material; S7. Preparing a blank carrier solution, and mixing the blank carrier solution with a curcumin solution to prepare a ROS-sensitive nanoparticle formulation FTL@Cur; S8. Co-incubating the Platycodon grandiflorum extracellular vesicle FTL@Cur at room temperature; S9. Preparing a matrix solution; S10. Preparing soluble microneedles. The beneficial effects of this invention are as follows: Platycodon grandiflorum extracellular vesicles are co-incubated with a ROS-responsive nanocarrier constructed from fucoidan, thioesterone, and luteolin to obtain a novel intelligent engineered Platycodon grandiflorum extracellular vesicle, which is then loaded into soluble microneedles for the treatment of rheumatoid arthritis.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically a method for preparing a platycodon extracellular vesicle composite microneedle patch. Background Technology

[0002] Traditional Chinese medicine (TCM) is one of the world's oldest herbal medicines, widely used since ancient times. It plays an indispensable role in the prevention and treatment of human diseases, especially chronic diseases such as rheumatoid arthritis. In recent years, with the rapid development of extraction and separation technologies, more and more natural plant active ingredients have come into view. Compared with chemically synthesized drugs, they have advantages such as low toxicity, multiple targets, and stronger synergistic effects, and are therefore widely accepted. However, the poor water solubility, low bioavailability, and short half-life of these active ingredients limit their clinical application.

[0003] With the rapid development of nanomedicine, plant extracellular vesicles have gradually come into view, and combining the active ingredients of natural plants with nanotechnology has become a new development trend. Increasing research shows that combining nanotechnology such as polymer micelles, liposomes, nanocrystals, and microcapsules with the active ingredients of traditional Chinese medicine can improve their inherent drawbacks, and has advantages such as better targeting and synergistic effects, while also reducing toxic side effects and improving bioavailability.

[0004] Extracellular vesicles (EVs) are micro- or nanoscale bioparticles encapsulated in a phospholipid bilayer that regulate cell communication by delivering various biomolecules. Almost all living cells, including plant cells, can secrete EVs. Plant-derived exosome-like nanovesicles (PDENs) have been found to have significant advantages in disease treatment and drug delivery, and are considered to have broad pharmaceutical application prospects. Due to their natural origin and long-standing dietary habits, plant-derived PDENs offer several distinct advantages as therapeutic agents. First, plant extracellular vesicles are derived from most edible plants, and PDENs have been shown to have no detectable toxicity or immunogenicity. Second, PDENs are environmentally friendly due to their green and sustainable plant source, and plants are relatively easy to obtain, allowing for industrial-scale production. Finally, plants do not contain human pathogens, potentially offering greater safety compared to animal-derived exosomes.

[0005] Platycodon grandiflorus, the dried root of the plant Platycodon grandiflorus (family Campanulaceae), has been shown in literature to have the effects of draining pus and relieving carbuncles, clearing the lungs and soothing the throat, relieving cough and resolving phlegm. It possesses pharmacological effects of clearing the lungs, resolving phlegm, soothing the throat, and draining pus. Modern pharmacological studies have also shown that Platycodon grandiflorus has various pharmacological effects such as antipyretic analgesia, anti-inflammatory, anti-ulcer, immunomodulatory, hepatoprotective, antitumor, anti-hyperglycemic, and lipid-regulating effects. Currently, various chemical substances have been obtained from the traditional Chinese medicine Platycodon grandiflorus, with the main active components being triterpenoid saponins and polysaccharides. Based on these advantages, this study has for the first time isolated extracellular vesicles from Platycodon grandiflorus. Drug delivery nanoplatforms based on exosome vesicles have unique advantages in terms of targeting, biocompatibility, and stability. In summary, given the analgesic, anti-inflammatory, and immunomodulatory pharmacological activities of Platycodon grandiflorus, this study will combine the extracellular vesicles isolated from Platycodon grandiflorus with ROS-responsive polymers and soluble microneedles for the treatment of rheumatoid arthritis.

[0006] Luteolin is a natural flavonoid compound found in various traditional Chinese medicines such as chrysanthemum and honeysuckle, and possesses a variety of pharmacological activities. Luteolin inhibits T cell proliferation and activation by suppressing the expression of interleukin-2 and interferon-gamma, inhibits the nuclear translocation of mouse NF-κB and p65 proteins and the phosphorylation of NF-κB inhibitory protein α, and suppresses T cell function through the NF-κB signaling pathway, thereby producing an immunosuppressive effect. Luteolin can also reduce the secretion of MMP-1 and MMP-3 and the expression of IL-6, IL-8, IL-15, and TGF-β in CIA mice, thereby inhibiting RASF proliferation and partially hindering its pathogenicity.

[0007] Numerous researchers both domestically and internationally are investigating the use of microneedles to deliver drugs for the treatment of various diseases, demonstrating promising therapeutic effects. The molecules in soluble microneedles, possessing diverse physicochemical properties, can migrate through the stratum corneum, thereby improving patient compliance. Microneedles penetrate the epidermis, inducing microchannels in the dermis, allowing drugs to further integrate into systemic circulation. Furthermore, the desired drug distribution can be achieved by designing the polymer content of the microneedles or by modifying the manufacturing process. Therefore, combining nano-formulations with microneedles enables targeted accumulation and responsive release of drugs, and enhances direct drug delivery into the skin by bypassing the skin barrier, solving problems associated with traditional transdermal delivery systems.

[0008] Based on this, we co-incubated Platycodon grandiflorum extracellular vesicles with ROS-responsive nanocarriers constructed from fucoidan, thioester, and luteolin to obtain a novel intelligent engineered Platycodon grandiflorum extracellular vesicle, which was then loaded into soluble microneedles for the treatment of rheumatoid arthritis. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a method for preparing a Platycodon grandiflorum extracellular vesicle composite microneedle patch.

[0010] To achieve the above objectives, the present invention employs the following technical solution: A method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch includes the following steps: S1. Add PBS to the platycodon root at a ratio of 1:3, extract the juice from the platycodon root, and filter the filtrate through a gauze. S2. Transfer the filtrate obtained in step S1 to a centrifuge tube, centrifuge at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant. S3. Centrifuge the supernatant obtained in step S2 for another 2 hours, take the precipitate, and resuspend it in PBS; use sucrose density gradient centrifugation to separate the vesicle mixture of different particle sizes, and then resuspend it in PBS to obtain the Platycodon grandiflorum extracellular vesicle solution. S4. Dissolve an appropriate amount of thioacetal in an appropriate amount of organic solvent to obtain a thioacetal solution; weigh an appropriate amount of catalyst EDC and DMAP and dissolve them in an appropriate amount of organic solvent. After they are completely dissolved, mix them with the thioacetal solution and react at a temperature of 38-41℃ for 2.5-3.5h to obtain an activated thioacetal solution. S5. Weigh an appropriate amount of luteolin, dissolve it in an appropriate amount of organic solvent, and after complete dissolution, add the mixed solution to the activated thioacetate solution obtained in step S4. React at room temperature of 25°C for 24 hours to obtain the thioacetate-luteolin (TK-Lut) product, which can be used for subsequent experiments. S6. Dissolve a certain amount of EDC and DMAP in an appropriate amount of organic solvent, add them to the thioacetate-luteolin (TK-Lut) product obtained in step S5, activate the carboxyl group at the other end of the thioacetate at a certain temperature to obtain an activated TK-Lut solution; weigh a certain amount of fucoidan, dissolve it in an appropriate amount of solvent, add it to the activated TK-Lut solution, react at room temperature of 25°C for 24 hours, dialyze and freeze dry to obtain purified Fuc-TK-Lut, i.e., amphiphilic carrier material; S7. Dissolve the amphiphilic carrier material obtained in step S6 to obtain a blank carrier solution; prepare a certain amount of curcumin (Cur) solution, mix the blank carrier solution with the curcumin solution, and after sonication and dialysis, obtain the ROS-sensitive nano-formulation FTL@Cur. Centrifuge and take the supernatant for freeze-drying and storage for subsequent experiments. The molar ratio of the amphiphilic carrier material to curcumin is 10:1. S8. The extracellular vesicles of Platycodon grandiflorus obtained in step S3 are co-incubated with FTL@Cur prepared in step S7 at room temperature for 6 hours to obtain a nano-modified extracellular vesicle of Platycodon grandiflorus with reactive oxygen species (ROS) responsiveness. S9. Weigh an appropriate amount of hyaluronic acid and fucoidan and mix them in a certain proportion. Add 8-11 ml of the co-incubated extracellular vesicles of Platycodon grandiflorus obtained in step S8 and FTL@Cur solution, stir to dissolve, and prepare a matrix solution. S10. Place 0.8-1.2 ml of the matrix obtained in step S9 into a microneedle mold, vacuum dry in a vacuum drying oven, and then air dry to obtain soluble microneedles.

[0011] Furthermore, in step S4, the molar ratio of thioacetate to the EDC and DMAP mixture is 1:1.5.

[0012] Furthermore, in step S5, the molar ratio of thioacetate to luteolin is 1:1.

[0013] Furthermore, in step S6, the molar ratio of fucoidan to TK-Lut is 1.5:1.

[0014] Furthermore, in step S9, the mass ratio of hyaluronic acid to fucoidan is 3:2.

[0015] Furthermore, in step S3, the sucrose gradients are 8%, 30%, 45%, and 60%, respectively.

[0016] Furthermore, the organic solvents used to dissolve the thioacetate solution, EDC solution, and DMAP solution, as well as the organic solvents used to dissolve the luteolin solution and the curcumin solution, are all anhydrous DMSO, and the solvent used to dissolve the fucoidan solution is anhydrous formamide.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention prepares a nano-modified Platycodon grandiflorus extracellular vesicle with reactive oxygen species (ROS) responsiveness by co-incubating it with ROS-responsive nano-formulation. This nano-modified Platycodon grandiflorus extracellular vesicle can load drug components, avoid degradation by gastric acid, and ensure efficacy. 2. By preparing a nano-formulation from fucoidan and luteolin, and loading curcumin into it, the drug can specifically accumulate at the site of inflammation, improving drug water solubility, increasing bioavailability, and treating rheumatoid arthritis more effectively. 3. Utilizing the characteristics of reactive oxygen species and combining them with the microenvironmental features of the inflammatory site, disulfide bonds are specifically broken at the high ROS inflammatory site to achieve the effect of specific drug release at the inflammatory site. 4. Prepare an FTL blank carrier and load curcumin to solve key problems such as poor solubility and low bioavailability of natural Chinese medicinal materials; 5. Loading co-incubated Platycodon grandiflorus extracellular vesicles with FTL@Cur solution into soluble microneedles and using microneedles to deliver drugs can improve the efficiency of transdermal drug delivery, thereby making the treatment effect of rheumatoid arthritis better. Attached Figure Description

[0018] Appendix Figure 1 This is a synthesis route diagram of the amphiphilic carrier material of the present invention.

[0019] Appendix Figure 2 The amphiphilic carrier material of the present invention 1 H-NMR.

[0020] Appendix Figure 3 This is a diagram of the nano-formulation of the present invention.

[0021] Appendix Figure 4 This is a particle size diagram of the nano-formulation of the present invention.

[0022] Appendix Figure 5 This is the potential diagram of the nano-formulation of the present invention.

[0023] Appendix Figure 6 This is a transmission electron microscope image of the nano-formulation of the present invention.

[0024] Appendix Figure 7 This is a transmission electron microscope image of extracellular vesicles of Platycodon grandiflorus cells loaded with ROS-responsive nano-formulations according to the present invention.

[0025] Appendix Figure 8 This is a comparison of 24h and 48h cytotoxicity experiments using the blank vector (FTL) of the present invention.

[0026] Appendix Figure 9 This is a comparison of the cytotoxicity test of the drug-loaded carrier and the free drug in this invention after 24 hours.

[0027] Appendix Figure 10 This is a comparison of the cytotoxicity test of the drug-loaded carrier and the free drug of the present invention after 48 hours.

[0028] Appendix Figure 11 This is the RAW264.7 concentration-dependent uptake experiment of the present invention.

[0029] Appendix Figure 12 This is the morphology of the microneedles of the present invention under a scanning electron microscope.

[0030] Appendix Figure 13 This is the morphology of the microneedles of the present invention under an optical microscope.

[0031] Appendix Figure 14 This refers to the solubility of the microneedles of the present invention in the skin at different times.

[0032] Appendix Figure 15This invention relates to a microneedle puncture skin recovery experiment.

[0033] Appendix Figure 16 The different formulation groups of this invention demonstrate the therapeutic effects on rheumatoid arthritis. Detailed Implementation

[0034] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined in this application.

[0035] Example 1: A method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch, comprising the following steps: 1. Preparation of extracellular vesicles of Platycodon grandiflorus cells: (1) Add PBS to the platycodon root at a ratio of 1:3 (g / mL), extract the juice from the platycodon root, and filter the filtrate through a gauze. (2) Transfer the filtrate to a 50 mL centrifuge tube and centrifuge at 4°C for 20 min at 400 g, 20 min at 800 g, and 20 min at 10000 g. Discard the bottom precipitate and take the supernatant. (3) The supernatant obtained in step (2) was centrifuged at 150,000 g for 2 h, and the precipitate was collected and resuspended in PBS. The mixture of vesicles with different particle sizes was separated by sucrose density gradient centrifugation. 8%, 30%, 45%, and 60% sucrose PBS solutions were prepared respectively. First, 2 mL of resuspension was added to an ultracentrifuge tube, and 2.5 mL of 8% sucrose solution was added to the bottom using a fine needle. Then, the same amount of 30%, 45%, and 60% sucrose PBS solutions were added sequentially using the same method. Finally, resuspension was added to the top layer up to 2 mm from the top of the centrifuge tube. After centrifugation at 150,000 g for 2 h, the mixture band between 30% and 45% sucrose densities was collected, and an equal amount of PBS was added for resuspension. The mixture was centrifuged at 100,000 g for 1 h to wash away the sucrose and collect the precipitate. The precipitate was resuspended in PBS to obtain the Platycodon grandiflorus extracellular vesicle solution, and the membrane protein content was determined using a BCA protein assay kit.

[0036] 2. Preparation of ROS-responsive nanocarriers: Thiocetyl was selected as the highly ROS-sensitive bond and dissolved in anhydrous dimethyl sulfoxide to obtain a thioacetyl solution. EDC and DMAP were dissolved separately in anhydrous dimethyl sulfoxide, and their solutions were mixed with the thioacetyl solution. The mixture was stirred at 40°C for 3 hours to obtain an activated thioacetyl solution for subsequent reactions. The molar ratio of thioacetyl to EDC and DMAP was 1:1.5. Luteolin was dissolved in an appropriate amount of anhydrous dimethyl sulfoxide to obtain a luteolin solution, which was added to the above reaction mixture. The mixture was stirred at room temperature for 24 hours to obtain the preliminary product thioacetyl-luteolin (T). The K-Lut solution was used for subsequent experiments, in which the molar ratio of thioacetate to luteolin was 1:1; appropriate amounts of EDC and DMAP were weighed and dissolved in anhydrous dimethyl sulfoxide, added to the TK-Lut solution, and stirred at 40°C for 3 hours to activate the carboxyl group at the other end of the thioacetate; a certain amount of fucoidan was weighed and dissolved in formamide to obtain a fucoidan solution, which was added to the above reaction and stirred at room temperature for 24 hours, followed by dialyzing for 36 hours using a dialysis bag with a molecular weight cutoff of 2000 Da; after dialysis, the supernatant was centrifuged and lyophilized to obtain FTR, i.e., an amphiphilic carrier material, whose structural formula is shown in Formula 1. 1 H-NMR characterizes the structure, such as Figure 2 As shown.

[0037]

[0038] Formula 1 3. Preparation of ROS-sensitive nanoformulations (FTL @Cur): The amphiphilic carrier material obtained above was dissolved in anhydrous dimethyl sulfoxide at a concentration of 5 mg / mL to obtain a blank carrier solution. A curcumin solution was prepared using anhydrous dimethyl sulfoxide at a concentration of 1 mg / mL. The blank carrier solution and curcumin solution were mixed and sonicated for ten minutes (the molar ratio of carrier material to curcumin was 10:1). The mixture was then dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 2000 Da. After dialysis, the supernatant was collected by centrifugation, filtered through a 0.8 μm microporous membrane, and lyophilized to obtain the ROS-sensitive nano-formulation.

[0039] 4. The above-obtained Platycodon grandiflorum extracellular vesicles are incubated with the above-prepared FTL@Cur at room temperature for 6 hours to obtain a Platycodon grandiflorum extracellular vesicle with reactive oxygen species (ROS) responsiveness.

[0040] 5. Weigh 300 mg of hyaluronic acid and 200 mg of fucoidan and mix them in a 3:2 ratio. Add 10 ml of the co-incubated extracellular vesicles of Platycodon grandiflorus obtained in step S8 and FTL@Cur solution, stir to dissolve, and prepare a matrix solution. Put 1 ml of the matrix into a microneedle mold, vacuum dry in a vacuum drying oven, and air dry to obtain soluble microneedles.

[0041] Example 2: Characterization and performance experiments of ROS-sensitive nanoformulations: 1. Determination of drug loading and encapsulation efficiency in nano-formulations: The nano-formulation prepared in Example 1 is as follows Figure 3 As shown, the encapsulation efficiency and drug loading of FTL@Cur were determined by high performance liquid chromatography. 1 mL of the prepared nano-formulation was diluted to the desired concentration with methanol to break up the emulsion, and then filtered through a 0.22 μm microporous membrane. The absorbance at 425 nm was measured by ultraviolet spectrophotometry, and the drug loading and encapsulation efficiency of the nano-formulation were calculated according to the formula.

[0042] Drug loading (%) = (Drug content in the formulation / Total mass of the formulation) × 100%; Encapsulation efficiency (%) = (Drug content in the formulation / Initial amount of drug added) × 100%.

[0043] Calculations showed that the drug loading of curcumin micelles in Example 1 of the present invention was 6.41%, and the encapsulation rate was 76.73%.

[0044] In this invention, the weight ratio of polymer micelle carrier material to curcumin is 10:1, which is important for this invention.

[0045] 3. FTL@Cur particle size and potential: The prepared FTL@Cur was filtered through a 0.80 μm microporous membrane, and then the particle size and potential of FTL@Cur were measured using a particle size analyzer. The experimental results are as follows. Figure 4 , 5 As shown.

[0046] After passing through a 0.8 μm microporous filter membrane, the particle size is 146.9 nm ± 1.23, and the particle size uniform potential is -22.2 ± 0.49.

[0047] 4. Electron microscopy examination: The prepared FTL@Cur was fixed and stained with copper mesh, and its morphology was photographed using a transmission electron microscope as shown in the figure. Figure 6 As shown in the figure. Transmission electron microscopy of extracellular vesicles of Platycodon grandiflorus cells loaded with ROS-responsive nanoparticles. Figure 7 As shown.

[0048] 5. Cytotoxicity evaluation of blank vector and free drug: The MTT assay was used to determine the cytotoxicity of the culture medium solution containing the blank carrier, evaluating the cytotoxicity of the nano-formulation. The results showed that the FTL blank micelles caused minimal damage to RAW264.7 cells. After 24 hours of culture, the cell viability was still greater than 70% at a FTL blank micelle concentration of 200 μg / mL, and after 48 hours of culture, the cell viability was still greater than 60%. Therefore, it is demonstrated that the FTL blank nanomicelles have very low cytotoxicity and almost no toxicity to nerve cells. The experimental results are as follows: Figure 8 As shown.

[0049] This invention also verified the toxicity of free curcumin to RAW264.7 cells. MTT assays showed that when the concentration of free curcumin reached 20 μg / mL, treating RAW264.7 cells for 24 hours resulted in a cell survival rate of over 75%. The experimental results are as follows: Figure 9 As shown, after treating RAW264.7 cells for 48 hours, the cell viability was over 65%. Experimental results are as follows... Figure 10 As shown in the figure. The two experiments above fully demonstrate the safety of the nanocarrier prepared in this invention.

[0050] 6. Study on cellular uptake and distribution of nano-formulations: The distribution of nano-prepared formulations within RAW264.7 cells after endocytosis was observed using laser confocal microscopy. This invention uses curcumin (Cur) as a fluorescent dye, encapsulating curcumin in our prepared nanomicelles. The phagocytosis by RAW264.7 cells was observed with increasing concentration and compared with free curcumin. The results showed that the cell fluorescence intensity increased with increasing concentration of different formulations, indicating that the uptake behavior of Free Cur and FTL@Cur by RAW264.7 cells is concentration-dependent, and that cells exhibited higher uptake of FTL@Cur. This demonstrates that the nanocarrier micelles have better bioavailability and can achieve better therapeutic effects. Experimental results are as follows: Figure 11 As shown.

[0051] 7. Hyaluronic acid and fucoidan were dissolved in a solution of Platycodon grandiflorus extracellular vesicles, which are responsive to microenvironment, to prepare a drug-loaded microneedle matrix. This matrix was then placed in a mold to prepare soluble microneedles. After air-drying, scanning electron microscopy was performed. Figure 12 As shown; the morphology of microneedles under an optical microscope is as follows Figure 13 As shown; the prepared microneedles were inserted into the skin and removed at 0, 30, 60, 120, and 180 seconds, respectively, and observed under an optical microscope. The dissolution of the microneedles was as follows. Figure 14 As shown; after the microneedles are inserted into the skin, the needle holes heal within ten minutes, as the results are as follows. Figure 15 As shown.

[0052] In summary, this invention addresses the shortcomings of natural product active ingredients, such as ineffective aggregation at inflammatory sites and inability to specifically release drugs. It prepares these active ingredients into nanomaterials, loads them with natural drug active ingredients, and encapsulates them within Platycodon grandiflorum extracellular vesicles through co-incubation. This improves the poor solubility and low bioavailability of the natural product and enhances drug stability. The microenvironment-responsive Platycodon grandiflorum extracellular vesicles constructed in this invention achieve specific drug release at inflammatory sites, improving drug bioavailability while avoiding systemic adverse reactions, thus providing more efficient treatment for rheumatoid arthritis.

Claims

1. A method for preparing a platycodon extracellular vesicle composite microneedle patch, characterized in that: Includes the following steps: S1. Add PBS to the platycodon root at a ratio of 1:3, extract the juice from the platycodon root, and filter the filtrate through a gauze. S2. Transfer the filtrate obtained in step S1 to a centrifuge tube, centrifuge at 4°C using differential centrifugation, discard the bottom precipitate, and take the supernatant. S3. Centrifuge the supernatant obtained in step S2 for another 2 hours, take the precipitate, and resuspend it in PBS; use sucrose density gradient centrifugation to separate the vesicle mixture of different particle sizes, and then resuspend it in PBS to obtain the Platycodon grandiflorum extracellular vesicle solution. S4. Dissolve an appropriate amount of thioacetal in an appropriate amount of organic solvent to obtain a thioacetal solution; weigh an appropriate amount of catalyst EDC and DMAP and dissolve them in an appropriate amount of organic solvent. After they are completely dissolved, mix them with the thioacetal solution and react at a temperature of 38-41℃ for 2.5-3.5h to obtain an activated thioacetal solution. S5. Weigh an appropriate amount of luteolin, dissolve it in an appropriate amount of organic solvent, and after complete dissolution, add the mixed solution to the activated thioacetate solution obtained in step S4. React at room temperature of 25°C for 24 hours to obtain the thioacetate-luteolin (TK-Lut) product, which can be used for subsequent experiments. S6. Dissolve a certain amount of EDC and DMAP in an appropriate amount of organic solvent, add them to the thioacetate-luteolin (TK-Lut) product obtained in step S5, activate the carboxyl group at the other end of the thioacetate at a certain temperature to obtain an activated TK-Lut solution; weigh a certain amount of fucoidan, dissolve it in an appropriate amount of solvent, add it to the activated TK-Lut solution, react at room temperature of 25°C for 24 hours, dialyze and freeze dry to obtain purified Fuc-TK-Lut, i.e., amphiphilic carrier material; S7. Dissolve the amphiphilic carrier material obtained in step S6 to obtain a blank carrier solution; prepare a certain amount of curcumin (Cur) solution, mix the blank carrier solution with the curcumin solution, and after sonication and dialysis, obtain the ROS-sensitive nano-formulation FTL@Cur. Centrifuge and take the supernatant for freeze-drying and storage for subsequent experiments. The molar ratio of the amphiphilic carrier material to curcumin is 10:

1. S8. The extracellular vesicles of Platycodon grandiflorus obtained in step S3 are co-incubated with FTL@Cur prepared in step S7 at room temperature for 6 hours to obtain a nano-modified extracellular vesicle of Platycodon grandiflorus with reactive oxygen species (ROS) responsiveness. S9. Weigh an appropriate amount of hyaluronic acid and fucoidan and mix them in a certain proportion. Add 8-11 ml of the co-incubated extracellular vesicles of Platycodon grandiflorus obtained in step S8 and FTL@Cur solution, stir to dissolve, and prepare a matrix solution. S10. Place 0.8-1.2 ml of the matrix obtained in step S9 into a microneedle mold, vacuum dry in a vacuum drying oven, and then air dry to obtain soluble microneedles.

2. The method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch according to claim 1, characterized in that: In step S4, the molar ratio of thioacetate to the mixture of EDC and DMAP is 1:1.

5.

3. The method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch according to claim 1, characterized in that: In step S5, the molar ratio of thioacetate to luteolin is 1:

1.

4. The method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch according to claim 1, characterized in that: In step S6, the molar ratio of fucoidan to TK-Lut is 1.5:

1.

5. The method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch according to claim 1, characterized in that: In step S9, the mass ratio of hyaluronic acid to fucoidan is 3:

2.

6. The method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch according to claim 1, characterized in that: In step S3, the sucrose gradients are 8%, 30%, 45%, and 60%, respectively.

7. The method for preparing a Platycodon grandiflorus extracellular vesicle composite microneedle patch according to claim 1, characterized in that: The organic solvents used to dissolve the thioacetate solution, EDC solution, and DMAP solution, as well as the organic solvents used to dissolve the luteolin solution and the curcumin solution, are all anhydrous DMSO, and the solvent used to dissolve the fucoidan solution is anhydrous formamide.

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