Dandelion exosome microneedle patch as well as preparation method and application thereof

Abstract

The invention discloses a dandelion exosome microneedle patch as well as a preparation method and application thereof, and the dandelion exosome microneedle patch comprises a microneedle body and a protective layer, the microneedle body is prepared from the following raw materials: dandelion exosome with the concentration of 0.1 to 0.4 mg / mL, sodium alginate with the concentration of 40 to 72 mg / mL, polyethylene glycol with the concentration of 7 to 20 mg / mL, calcium chloride with the concentration of 10 to 25 mg / mL and glutamine transaminase with the concentration of 0.5 to 1.5 U / mL. Through the mutual synergistic effect of the sodium alginate, the polyethylene glycol, the calcium chloride, the glutamine transaminase and the dandelion exosome, the microneedle has excellent compression modulus, can penetrate through necrotic tissues due to relatively strong puncture force, ensures that the dandelion exosome and a medicine can be effectively delivered to a deep focus, and has a good application prospect. The technical problems that necrotic tissue of a diabetic foot ulcer part is tough and a traditional common microneedle is difficult to penetrate through are solved.

Description

Technical Field

[0001] The present invention relates to the field of chronic wound repair, and in particular to a dandelion exosome microneedle patch and a preparation method and application thereof. Background Art

[0002] In the field of chronic wound repair, particularly in the treatment of diabetic foot ulcers, current traditional treatments include functional dressings and drug injections. For dressing therapy, ordinary dressings can only provide limited physical protection for the lesion and fail to effectively treat it. For example, while vaseline gauze dressings can prevent wound drying, they have little effect on promoting tissue repair and controlling infection. Even with the use of dressings with sustained-release drug release, the lack of precise drug release control mechanisms can reduce the compatibility of the drug release rate with the needs of the wound healing stage, leading to poor therapeutic efficacy. In contrast, drug injection therapy allows the drug to act directly on the wound, but multiple injections not only cause great pain to the patient but also increase the risk of infection. Furthermore, the distribution of the injected drug into the body is difficult to precisely control, potentially causing adverse effects on surrounding normal tissues.

[0003] Microneedle patch technology is an emerging drug delivery method that is gradually being used in wound treatment. However, when used to treat diabetic foot ulcers, it is unable to penetrate the thick and tough necrotic tissue, making it difficult for the drugs carried on the microneedles to reach deep lesions and unable to fully exert their therapeutic effects. There is an urgent need to provide a microneedle patch with high mechanical strength. Summary of the Invention

[0004] To overcome the above shortcomings, the purpose of the present invention is to provide a dandelion exosome microneedle patch with high mechanical strength and the ability to effectively penetrate necrotic tissue.

[0005] In order to achieve the above objectives, the technical solution adopted by the present invention is: a dandelion exosome microneedle patch, comprising a microneedle body and a protective layer. The raw materials for preparing the microneedle body include: dandelion exosomes with a concentration of 0.1-0.4 mg / mL, sodium alginate with a concentration of 40-72 mg / mL, polyethylene glycol with a concentration of 7-20 mg / mL, calcium chloride with a concentration of 10-25 mg / mL; and transglutaminase with a concentration of 0.5-1.5 U / mL.

[0006] Furthermore, the above-mentioned method for extracting dandelion exosomes comprises the following steps:

[0007] Step 1: Dandelion Material Selection

[0008] The leaves, roots or stems of healthy dandelions in their vigorous growth stage (4-6 weeks old) were selected as the source of dandelion exosomes. At this stage, the leaf cells are metabolically active, and the dandelion exosomes are produced in high quantities and have stable activity.

[0009] Step 2: Extraction

[0010] ① Pretreatment: Rinse the dandelion material with deionized water 3-5 times to remove surface impurities, then soak and disinfect in 75% ethanol solution for 3-5 minutes. Then rinse with sterile deionized water 2-3 times to completely remove residual ethanol. Cut the disinfected dandelion material into pieces and place them in a homogenizer precooled to 0-5°C containing 0.1M phosphate buffer (PBS, pH=7.4) and 0.5-2% protease inhibitors. The mass volume ratio of dandelion material to buffer is 1:3-7.

[0011] ② Homogenization and centrifugation: At a low temperature (4-10°C), use a homogenizer to homogenize at 8000-15000 rpm for 1-7 minutes to fully break up the dandelion material. Transfer the homogenate to a centrifuge tube and centrifuge at 2000-4000g for 5-20 minutes at 0-5°C to remove large cell fragments and tissue residues, retaining the supernatant. Then transfer the supernatant to an ultracentrifuge tube and centrifuge at 50000-150000g for 30-100 minutes at 0-5°C to allow the dandelion exosomes to precipitate at the bottom of the tube. Carefully discard the supernatant and resuspend the pellet with an appropriate amount of pre-chilled water.

[0012] ③ Purification: The resuspension was filtered through a 0.15-0.35 μm filter membrane to further remove impurities, and then ultracentrifuged (50,000-150,000 g, 0-5°C, 30-100 minutes). Finally, the dandelion exosomes were obtained after resuspending and purification in water.

[0013] The CD44 receptor on the surface of dandelion exosomes mediates targeting of inflammatory sites with greater efficiency than dandelion extract. This allows dandelion exosomes to be precisely concentrated at the site of inflammation in diabetic foot ulcers, avoiding unnecessary effects on normal tissues and resolving the issue of traditional treatments where drugs have difficulty reaching the site of inflammation. Furthermore, dandelion exosomes can inhibit TNF-α secretion, effectively alleviating the inflammatory response, effectively resolving the issue of traditional treatments where drugs have difficulty precisely reaching the site of inflammation and have limited inhibitory effects on inflammatory factors.

[0014] Sodium alginate, as the main matrix material of microneedles, has good biocompatibility and gel-forming properties. The addition of polyethylene glycol (PEG) can improve the flexibility and stability of the matrix. 2+ It will form an "egg-box" structure with sodium alginate, which can enhance the stiffness of the microneedle; and by adding transglutaminase, transglutaminase can catalyze the cross-linking of lysine and glutamine residues brought by gelatin, achieving secondary reinforcement of the mechanical strength of the microneedle; and by adding dandelion exosomes, the mechanical strength of the microneedle patch is further improved.

[0015] The present invention uses the synergistic effect of sodium alginate, polyethylene glycol, calcium chloride, transglutaminase and dandelion exosomes to obtain microneedles that can penetrate necrotic tissue, ensuring that dandelion exosomes and drugs can be effectively delivered to deep lesions, solving the technical problem that necrotic tissue in diabetic foot ulcers is tough and difficult to penetrate by traditional ordinary microneedles.

[0016] Furthermore, the raw materials for preparing the composite solution for the protective layer include gelatin and MMP-9 sensitive peptide segments.

[0017] Furthermore, in the composite solution, the concentration of gelatin is 50-150 mg / mL.

[0018] Furthermore, in the composite solution, the concentration of the MMP-9 sensitive peptide is 2-20 U / mL.

[0019] Among them, MMP-9 sensitive peptide refers to a short peptide sequence that can be specifically cleaved by matrix metalloproteinase-9 (MMP-9).

[0020] The acidic environment (pH 5.5) at the site of inflammation activates the PL1 enzyme, accelerating macropore formation and, consequently, the release of exosomes. This, in turn, acts at the early stages of inflammation. The matrix metalloproteinase (MMP-9) cuts through the gelatin protective layer to precisely control the opening sequence of pores of varying inner diameters, forming a pH / MMP dual-trigger mechanism. This pH / MMP dual-trigger mechanism enables the microneedle patch to precisely control the release of exosomes and drugs based on the specific environment of the inflammation site, improving the targeted and effective treatment while avoiding drug waste and adverse effects on normal tissues.

[0021] Furthermore, the preparation method of the composite solution is as follows: gelatin is added to an appropriate amount of water, stirred and dissolved at 50-70°C, with a stirring speed of 200-300 rpm to prepare a gelatin solution, and the gelatin solution is cooled to 35-45°C, and the MMP-9 sensitive peptide segment is added and stirred evenly to prepare a composite solution.

[0022] Furthermore, the method for preparing the protective layer includes: coating the composite solution on the microneedle body, and obtaining the protective layer after secondary curing.

[0023] Furthermore, the composite solution is added dropwise to a microneedle mold, and the microneedle body is placed in the mold, coating the microneedle body with a uniform layer of the composite solution. The mold is then stored at 33-42°C for 2-3 hours to achieve secondary curing.

[0024] Furthermore, the length of the microneedle body is 1100-1300 μm, preferably 1200 μm, to ensure that it can penetrate the thick necrotic tissue of diabetic foot ulcers.

[0025] Furthermore, the needle tip angle of the microneedle body is 20-30°, preferably 25°, which is conducive to microneedle puncture.

[0026] Furthermore, the distance between adjacent microneedle bodies is 1400-1600 μm, preferably 1500 μm, to avoid excessive skin damage caused by excessive density of microneedles.

[0027] Furthermore, the thickness of the microneedle substrate is 700-900 μm, preferably 800 μm, to ensure the overall stability of the microneedle patch.

[0028] Furthermore, the microneedle body includes a pore, and the inner diameter of the pore ranges from 50 nm to 20 μm.

[0029] Unique pores with varying inner diameters were constructed using pectate lyase PL1 (optimal pH 6.5) carried by dandelion exosomes. This enzyme specifically cleaves the β-1,4 glycosidic bonds of sodium alginate, forming a wide range of pores. These pores include:

[0030] Pores with a larger inner diameter, such as an inner diameter of 20 μm, can provide a rapid release channel for dandelion exosomes, and the exosome release rate can reach 60% within 2 hours. In the early stages of treatment of diabetic foot ulcers, the rapidly released dandelion exosomes can quickly exert their anti-inflammatory and antioxidant effects, timely control wound inflammation, and create good conditions for subsequent repair. Compared with the disadvantage of traditional microneedle patches that cannot quickly provide effective ingredients in the critical early stages of treatment, the dandelion exosome microneedle patch of this application greatly improves the timeliness of treatment.

[0031] Pores with a medium inner diameter, such as 500nm, are sized to accommodate dandelion exosome vesicles (average diameter 150nm), effectively protecting their structural integrity. Maintaining the integrity of dandelion exosomes during their release from the microneedles and their application to the wound surface is crucial for maintaining their biological activity. This protects dandelion exosomes from external interference during storage and release, ensuring their normal biological function.

[0032] Pores with smaller inner diameters, such as 50 nm, are responsible for the sustained release of small-molecule active substances carried by dandelion exosomes, such as flavonoids and polysaccharides. These small-molecule active substances have various benefits during wound repair, such as promoting cell proliferation and regulating immunity. This enables the slow release of these small molecules, maintaining their effective concentration locally within the wound over a longer period of time, and continuously promoting wound repair.

[0033] This pore structure provides dandelion exosomes with an intelligent release mechanism. Furthermore, the enzymatic hydrolysis process avoids the problem of high-temperature freeze-drying in traditional preparation methods, which can easily denature dandelion exosome envelope proteins and impair their function. The enzymatic hydrolysis method used in this invention to form pores can fully unleash the therapeutic potential of dandelion exosomes.

[0034] Furthermore, dandelion exosomes contain taraxasterol and chlorogenic acid, which work synergistically, making dandelion exosomes significantly more effective at scavenging DPPH free radicals than dandelion extract. In diabetic foot ulcer wounds, oxidative stress damages cells and tissues, hindering wound healing. The potent antioxidant capacity of the dandelion exosomes in this invention can effectively scavenge excess free radicals, mitigate oxidative damage, and create a favorable intracellular environment for wound repair.

[0035] Furthermore, the average particle size of dandelion exosomes is 100-150nm, and the Zeta potential is -30-25mV. The surface of dandelion exosome particles has a strong negative charge, which can provide a certain electrostatic repulsion, making the particles relatively stable in the solution.

[0036] A method for preparing a dandelion exosome microneedle patch comprises the following steps:

[0037] S1. Add sodium alginate and polyethylene glycol into water and mix well to prepare a sodium alginate-PEG solution;

[0038] S2. Add the dandelion exosome suspension to the sodium alginate-PEG solution, stir for 5-15 minutes, add calcium chloride solution and transglutaminase to prepare the microneedle matrix;

[0039] S3. Pour the microneedle matrix into the microneedle mold, perform preliminary solidification, prepare a protective layer, dry and demold, and obtain a dandelion exosome microneedle patch.

[0040] Furthermore, in step S1, the specific mixing conditions are: stirring at a speed of 200-400 rpm for 0.5-1.0 h in a constant temperature water bath at 45-60° C., until completely dissolved to form a uniform transparent solution.

[0041] Furthermore, in step S2, the concentration of calcium chloride in the calcium chloride solution is 30-75 mg / mL, and the solvent is water.

[0042] Furthermore, in step S2, calcium chloride solution is slowly added dropwise during stirring to promote the formation of the "egg-box" structure. Meanwhile, transglutaminase is added to promote cross-linking of the matrix in the reaction system.

[0043] Furthermore, in step S3, the microneedle matrix needs to be added dropwise from one end of the mold to ensure that the solution completely fills the microneedle holes and avoids the formation of bubbles. The perfusion process is carried out in a clean bench to ensure a sterile environment.

[0044] Furthermore, in step S3, the specific conditions for preliminary curing are: storing the mold filled with the microneedle matrix at 2-5°C for 0.5-1.0h.

[0045] Further, the mold body is dried and demoulded by transferring the mold to a vacuum drying oven and setting the vacuum degree to 10- 4 -10- 2 mbar, and a temperature of 20-40°C for 15-18 hours to remove moisture from the microneedles and further solidify them. After drying, carefully peel the formed microneedle patch from the mold to obtain the dandelion exosome microneedle patch.

[0046] Application of the dandelion exosome microneedle patch as described above or the dandelion exosome microneedle patch prepared by the preparation method as described above in chronic wound repair. Existing chronic wound repair technologies, especially treatments for diabetic foot ulcers, have significant deficiencies in terms of the accuracy of drug delivery, the protection and release regulation of bioactive components such as exosomes, the mechanical properties of microneedles, and the intelligent response to the special environment of the wound. The present invention effectively solves the problems existing in existing chronic wound repair technologies through the above-mentioned improvements in many aspects, and provides a new, efficient, accurate, and intelligent solution for the treatment of chronic wounds such as diabetic foot ulcers.

[0047] The beneficial effects of the present invention are:

[0048] 1) Through the synergistic effect of sodium alginate, polyethylene glycol, calcium chloride, transglutaminase, and dandelion exosomes, the microneedles possess an excellent compression modulus. The resulting microneedle puncture force can penetrate necrotic tissue, ensuring that dandelion exosomes and drugs can be effectively delivered to deep lesions, solving the technical problem that necrotic tissue in diabetic foot ulcers is tough and difficult for traditional ordinary microneedles to penetrate.

[0049] 2) By adding MMP-9 sensitive peptides to the composite solution, a pH / MMP dual trigger mechanism is formed, which enables the microneedle patch to precisely control the release of exosomes and drugs according to the specific environment of the inflammatory site, thereby improving the targetedness and effectiveness of treatment and avoiding drug waste and adverse effects on normal tissues.

[0050] 3) By adding dandelion exosomes, the CD44 receptor on the surface of dandelion exosomes mediates targeting of inflammatory sites with significantly higher efficiency than dandelion extract. This allows dandelion exosomes to be precisely enriched at the inflammatory site of diabetic foot ulcers, avoiding unnecessary effects on normal tissues and resolving the problem of traditional treatment methods where drugs have difficulty reaching the inflammatory site. At the same time, dandelion exosomes can inhibit TNF-α secretion, effectively reducing the inflammatory response. Therefore, the targeted anti-inflammatory function of the dandelion exosomes of the present invention significantly improves the precision and effectiveness of treatment.

[0051] 4) Dandelion exosomes contain taraxasterol and chlorogenic acid, which have a synergistic effect, making the ability of dandelion exosomes to scavenge DPPH free radicals much higher than that of dandelion extracts. Their powerful antioxidant capacity can effectively scavenge excessive free radicals, reduce oxidative damage, and create a favorable intracellular environment for the repair of diabetic foot ulcer wounds.

[0052] 5) The enzymatic hydrolysis process used in this application effectively reduces the degree of damage to the dandelion exosome envelope protein caused by high-temperature freeze-drying, and protects the exosome activity to the greatest extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0054] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0055] Figure 1 TEM image of dandelion exosomes. DETAILED DESCRIPTION

[0056] To make the above-mentioned objects, features, and advantages of the present invention more readily apparent, specific embodiments of the present invention are described in detail below. The following description sets forth numerous specific details to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art may make similar modifications without departing from the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0057] Except as shown in the operating examples or otherwise indicated, all numbers used in the specification and claims to express the amounts of ingredients, physicochemical properties, etc. are understood to be adjusted by the term "about" in all cases. Therefore, unless otherwise indicated, the numerical parameters listed in the above specification and the appended claims are approximate values, and those skilled in the art can appropriately change these approximate values ​​by utilizing the teachings disclosed herein to seek to obtain the desired properties. The use of numerical ranges expressed as endpoints includes all numbers within the range and any range within the range, for example, 1 to 5 includes 1, 1.1, 1.3, 1.5, 2, 2.75, 3, 3.80, 4 and 5, etc.

[0058] The following examples more particularly describe the present disclosure, and these examples are intended to be illustrative only, as various modifications and variations within the scope of the present disclosure will be apparent to those skilled in the art. Unless otherwise indicated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and used directly without further processing, and all instruments used in the examples are commercially available.

[0059] Preparation Example 1

[0060] Extraction and identification of dandelion exosomes

[0061] Material Selection: Dandelion plants grown in a pollution-free environment with ample sunlight and fertile soil were selected. Healthy leaves in their active growth phase (5 weeks old) were selected as dandelion materials. During this phase, leaf cell metabolism is active, and dandelion exosome production is high and activity is stable.

[0062] Extraction steps:

[0063] ① Pretreatment: Rinse the harvested dandelion leaves four times with deionized water to remove surface impurities, then soak them in 75% ethanol for four minutes for disinfection. Rinse three times with sterile deionized water to thoroughly remove any residual ethanol. Cut the disinfected leaves into approximately 0.5 cm × 0.5 cm pieces and place them in a homogenizer precooled to 4°C containing 0.1 M phosphate buffer (PBS, pH = 7.4) and 1% protease inhibitors. The mass-to-volume ratio of leaves to buffer was 1:5.

[0064] ② Homogenization and Centrifugation: Homogenize the leaves at 12,000 rpm for 4 minutes at 7°C to thoroughly break them up. Transfer the homogenate to a centrifuge tube and centrifuge at 3,000 g for 10 minutes at 4°C to remove large cell debris and tissue residue, retaining the supernatant. Next, transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000 g for 70 minutes at 4°C to pellet the dandelion exosomes at the bottom of the tube. Carefully discard the supernatant and resuspend the pellet in an appropriate amount of pre-chilled water.

[0065] ③ Purification: The resuspension was filtered through a 0.22 μm filter membrane to further remove impurities, and then ultracentrifuged (100,000 g, 4°C, 70 min). Finally, the purified dandelion exosomes were resuspended in water.

[0066] Identification method:

[0067] Particle size and zeta potential: Dynamic light scattering (DLS) was used to measure the particle size and zeta potential of dandelion exosomes. The average particle size of dandelion exosomes was 130 nm, and the zeta potential was -28 mV.

[0068] Transmission electron microscopy (TEM) observation: Take an appropriate amount of dandelion exosome suspension and drop it on a copper grid. After negative staining with 2% phosphotungstic acid, observe under a transmission electron microscope. Dandelion exosomes are spherical structures ( Figure 1 ).

[0069] Preparation Example 2

[0070] Microneedle mold preparation:

[0071] Model Design: Using professional 3D modeling software (SolidWorks), a microneedle mold model was designed based on the treatment requirements for diabetic foot ulcers. The microneedle length was set to 1200 μm, the tip angle to 25°, the inter-needle spacing to 1500 μm, and the substrate thickness to 800 μm.

[0072] 3D Printing and Post-Processing: The designed model was imported into a 3D printer (Formlabs Form3 light-curing 3D printer) and printed using a biocompatible light-curing resin material. The print layer thickness was set to 30 μm, the exposure time per layer was 15 seconds, and the print speed was moderate to ensure printing accuracy and quality. After printing, the mold was ultrasonically cleaned in isopropyl alcohol for 20 minutes to remove any uncured resin on the surface. The mold was then placed in a UV curing chamber for a secondary cure of 50 minutes to enhance its mechanical properties. Finally, the mold surface was silanized to reduce adhesion between the microneedle patch and the mold, facilitating subsequent demolding.

[0073] Example 1

[0074] Preparation of a dandelion exosome microneedle patch:

[0075] S1. Weigh 2 g of sodium alginate and 0.5 g of polyethylene glycol, slowly add them to 25 mL of water, and stir at 280 rpm in a 55°C constant temperature water bath for 35 minutes until they are completely dissolved to form a uniform and transparent sodium alginate-PEG solution.

[0076] S2. Slowly add 2 mL of the dandelion exosome suspension extracted and identified in Preparation Example 1 to the sodium alginate-PEG solution prepared in Step S1. Gently stir for 10 minutes to evenly disperse the dandelion exosomes. Then, slowly add calcium chloride solution dropwise while stirring to promote the formation of the "egg-box" structure. Simultaneously, add transglutaminase to a concentration of 1 U / mL to obtain a microneedle matrix.

[0077] Among them, the solvent in the dandelion exosome suspension is 0.1M PBS, and the concentration of dandelion exosomes is 3 mg / mL.

[0078] The steps for preparing the calcium chloride solution are as follows: weigh 0.6 g of calcium chloride, add it to 12 mL of deionized water, and stir to dissolve it, that is, the calcium chloride solution.

[0079] S3. Preparation of dandelion exosome microneedle patch:

[0080] S31, Perfusion: Slowly pour the microneedle matrix from step S2 into the microneedle mold using a micropipette, adding dropwise from one end of the mold to ensure that the solution completely fills the microneedle pores and avoids bubbles. The perfusion process is performed in a clean bench to ensure a sterile environment.

[0081] S32. Preliminary curing: Place the mold filled with the microneedle matrix in a refrigerator at 4°C for 45 minutes to obtain the microneedle body.

[0082] S33. Prepare a protective layer: Remove the microneedle body from the microneedle mold, then drip the composite solution into the mold. Place the microneedle body back into the mold, creating a uniform layer of composite solution on the microneedle body. The resulting microneedle mold is then incubated in a 37°C incubator for 2 hours to achieve secondary curing. Transglutaminase catalyzes the cross-linking of lysine and glutamine residues in the gelatin, further strengthening the mechanical strength of the microneedle.

[0083] The composite solution was prepared as follows: 1g of gelatin was weighed, 10mL of water was added, and the mixture was stirred in a 60°C water bath at 250rpm for 30 minutes to dissolve. After cooling to 40°C, the MMP-9 sensitive peptide was added and stirred until the concentration of the MMP-9 sensitive peptide was adjusted to 10U / mL to prepare the composite solution.

[0084] S34, Drying and demoulding: Transfer the mold to a vacuum drying oven and set the vacuum degree to 10-3m The microneedles were dried at 25°C for 18 hours to remove moisture from the microneedles and further solidify them. After drying, the molded microneedle patch was carefully peeled off from the mold to obtain the dandelion exosome microneedle patch.

[0085] Example 2

[0086] Preparation of a dandelion exosome microneedle patch:

[0087] S1. Weigh 1.5 g of sodium alginate and 0.26 g of polyethylene glycol, slowly add them to 25 mL of water, and stir at 360 rpm in a constant temperature water bath at 45°C for 60 minutes until they are completely dissolved to form a uniform and transparent sodium alginate-PEG solution.

[0088] S2. Slowly add 2 mL of the dandelion exosome suspension extracted and identified in Preparation Example 1 to the sodium alginate-PEG solution prepared in Step S1. Gently stir for 5 minutes to evenly disperse the dandelion exosomes. Then, slowly add calcium chloride solution dropwise while stirring to promote the formation of the "egg-box" structure. Simultaneously, add transglutaminase to a concentration of 0.6 U / mL to obtain a microneedle matrix.

[0089] Among them, the solvent in the dandelion exosome suspension is 0.1M PBS, and the concentration of dandelion exosomes is 2 mg / mL.

[0090] The steps for preparing the calcium chloride solution are as follows: weigh 0.4 g of calcium chloride, add it to 12 mL of deionized water, and stir to dissolve it, that is, the calcium chloride solution.

[0091] S3. Preparation of dandelion exosome microneedle patch:

[0092] S31, Perfusion: Slowly pour the microneedle matrix from step S2 into the microneedle mold using a micropipette, adding dropwise from one end of the mold to ensure that the solution completely fills the microneedle pores and avoids bubbles. The perfusion process is performed in a clean bench to ensure a sterile environment.

[0093] S32. Preliminary curing: Place the mold filled with the microneedle matrix in a refrigerator at 4°C for 45 minutes to obtain the microneedle body.

[0094] S33. Prepare a protective layer: remove the microneedle body from the microneedle mold, then add the composite solution into the microneedle mold, and then place the microneedle body in the microneedle mold, so that a uniform layer of composite solution is covered on the surface of the microneedle body; then place the obtained microneedle mold in a 37°C incubator and incubate for 2 hours to achieve secondary curing.

[0095] The composite solution was prepared as follows: 1g of gelatin was weighed, 10mL of water was added, and stirring was continued in a 50°C water bath at 200rpm for 35 minutes to dissolve the gelatin solution. After cooling to 40°C, the MMP-9 sensitive peptide was added and stirred until the concentration of the MMP-9 sensitive peptide was adjusted to 5U / mL to prepare the composite solution.

[0096] S34, Drying and demoulding: Transfer the mold to a vacuum drying oven and set the vacuum degree to 10-3 m The microneedles were dried at 25°C for 18 hours to remove moisture from the microneedles and further solidify them. After drying, the molded microneedle patch was carefully peeled off from the mold to obtain the dandelion exosome microneedle patch.

[0097] Example 3

[0098] Preparation of a dandelion exosome microneedle patch:

[0099] S1. Weigh 2.6 g of sodium alginate and 0.7 g of polyethylene glycol, slowly add them to 25 mL of water, and stir at 230 rpm in a constant temperature water bath at 60°C for 30 minutes until they are completely dissolved to form a uniform and transparent sodium alginate-PEG solution.

[0100] S2. Slowly add 7 mL of the dandelion exosome suspension extracted and identified in Preparation Example 1 to the sodium alginate-PEG solution prepared in Step S1. Gently stir for 15 minutes to evenly disperse the dandelion exosomes. Then, slowly add calcium chloride solution dropwise while stirring to promote the formation of the "egg-box" structure. Simultaneously, add transglutaminase to a concentration of 1.5 U / mL to obtain a microneedle matrix.

[0101] Among them, the solvent in the dandelion exosome suspension is 0.1M PBS, and the concentration of dandelion exosomes is 2 mg / mL.

[0102] The steps for preparing the calcium chloride solution are as follows: weigh 0.9 g of calcium chloride, add it to 12 mL of deionized water, and stir to dissolve it, thus obtaining the calcium chloride solution.

[0103] S3. Preparation of dandelion exosome microneedle patch:

[0104] S31, Perfusion: Slowly pour the microneedle matrix from step S2 into the microneedle mold using a micropipette, adding dropwise from one end of the mold to ensure that the solution completely fills the microneedle pores and avoids bubbles. The perfusion process is performed in a clean bench to ensure a sterile environment.

[0105] S32. Preliminary curing: Place the mold filled with the microneedle matrix in a refrigerator at 4°C for 45 minutes to obtain the microneedle body.

[0106] S33, preparation of protective layer: the microneedle body is taken out of the microneedle mold, then the composite solution is added dropwise into the microneedle mold, and then the microneedle body is placed in the microneedle mold, that is, a layer of uniform composite solution is covered on the surface of the microneedle body; the obtained microneedle mold is placed in a 37℃ incubator for 3 hours to realize secondary curing.

[0107] The preparation of the composite solution is as follows: 1 g of gelatin is weighed, 10 mL of water is added, and the mixture is stirred and dissolved in a 60℃ constant temperature water bath, the stirring speed is 250 rpm, and the stirring is continued for 30 minutes to prepare a gelatin solution. After cooling to 40℃, the MMP-9 sensitive peptide segment is added and stirred uniformly, and the MMP-9 sensitive peptide segment is adjusted to 15 U / mL to prepare the composite solution.

[0108] S34, drying and demolding: the mold is transferred to a vacuum drying oven, the vacuum degree is set to 10-3 m bar, the temperature is 25℃, and the drying is carried out for 18 hours to remove the water in the microneedle and further cure the microneedle. After drying, the formed microneedle patch is carefully peeled off from the mold to obtain the dandelion exosome microneedle patch.

[0109] Comparative Example 1

[0110] The steps for preparing the microneedle patch in Comparative Example 1 and Example 2 are basically the same, the main difference is that in step S2, no dandelion exosome is added, i.e. a blank microneedle patch is prepared.

[0111] Comparative Example 2

[0112] The steps for preparing the microneedle patch in Comparative Example 2 and Example 2 are basically the same, the main difference is that in step S2, no transglutaminase is added, i.e. a microneedle patch without enzyme crosslinking is prepared.

[0113] Comparative Example 3

[0114] The steps for preparing the microneedle patch in Comparative Example 3 and Example 2 are basically the same, the main difference is that in step S3, no MMP-9 sensitive peptide segment is added to the composite solution.

[0115] Experimental Example 1

[0116] Verification experiment of antioxidant synergistic effect of dandelion exosome microneedle patch.

[0117] Preparation of dandelion extract solution:

[0118] ① Pretreatment: Rinse the harvested dandelion leaves four times with deionized water to remove surface impurities, then soak them in 75% ethanol for four minutes for disinfection. Rinse three times with sterile deionized water to thoroughly remove any residual ethanol. Cut the disinfected leaves into approximately 0.5 cm × 0.5 cm pieces and place them in a homogenizer precooled to 4°C containing 0.1 M phosphate buffer (PBS, pH = 7.4) and 1% protease inhibitors. The mass-to-volume ratio of leaves to buffer was 1:5.

[0119] ② Homogenization and Centrifugation: Homogenize the leaves at 12,000 rpm for 4 minutes at 7°C to fully break them up. Transfer the homogenate to a centrifuge tube and centrifuge at 500 rpm for 3 minutes at 4°C. After centrifugation, the protoplast pellet will be at the bottom of the tube. Carefully aspirate the supernatant. Freeze-dry the supernatant to obtain the dandelion extract.

[0120] Experimental methods:

[0121] (1) DPPH free radical scavenging experiment

[0122] The dandelion exosomes extracted in Preparation Example 1 were used to prepare dandelion exosome solutions with concentrations of 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, and 0.40 mg / mL, respectively. Dandelion extract solutions with concentrations of 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, 0.30 mg / mL, and 0.40 mg / mL were also prepared.

[0123] A 96-well plate was taken and different concentrations of dandelion exosome solutions, different concentrations of dandelion extract solutions, and vitamin C solutions with concentrations of 0.005 mg / mL, 0.10 mg / mL, 0.020 mg / mL, 0.030 mg / mL, 0.040 mg / mL, and 0.060 mg / mL were added as a positive control group, and anhydrous ethanol was used as a negative control group.

[0124] DPPH free radical solution was added to each well and mixed with the sample solution in equal volumes, resulting in a total volume of 200 μL.

[0125] The 96-well plate was placed in a dark environment for reaction for 30 minutes.

[0126] The absorbance of each well was measured using a microplate reader at a wavelength of 517 nm.

[0127] The absorbance measurement results are shown in Table 1.

[0128] Table 1

[0129]

[0130]

[0131] By calculating the clearance rate and drawing the curve, the IC50 value of the dandelion exosome group was 0.14±0.02 mg / mL, and the IC50 value of the dandelion extract group was 0.29±0.03 mg / mL, confirming that the antioxidant capacity of dandelion exosomes is twice that of dandelion extract.

[0132] (2) Cell antioxidant experiment

[0133] Human skin fibroblasts induced by H2O2 oxidative damage were selected and divided into five groups: blank group (no treatment), model group (only H2O2 was added), dandelion exosome low, medium and high concentration groups (0.1 mg / mL, 0.2 mg / mL and 0.3 mg / mL of dandelion exosome solution were added before H2O2), dandelion extract low, medium and high concentration groups (dandelion extract solution of the same concentration was added before H2O2 was added) and positive control group (N-acetylcysteine ​​solution was added before H2O2 was added).

[0134] After culturing for 24 hours, the cells were stained with the fluorescent probe DCFH-DA, and the fluorescence intensity was measured using a fluorescence spectrometer after incubation for 30 minutes. The detection results are shown in Table 2.

[0135] Table 2. The results of intracellular ROS level detection are as follows:

[0136] Group ROS fluorescence intensity Blank group 120±10 Model Group 480±20 Dandelion exosome low concentration group 320±15 Dandelion exosomes medium concentration group 240±12 Dandelion exosome high concentration group 180±10 Low concentration dandelion extract group 380±16 Dandelion extract medium concentration group 300±13 High concentration dandelion extract group 260±11 Positive control group 150±8

[0137] The test data in Table 2 show that compared with the model group, the dandelion exosome group showed a significant decrease in ROS levels in a concentration-dependent manner. Furthermore, the ROS fluorescence intensity levels in the low-, medium-, and high-concentration dandelion exosome groups were lower than those in the low-, medium-, and high-concentration dandelion extract groups, respectively, further confirming the superior antioxidant effect of the dandelion exosome group.

[0138] Experimental Example 2

[0139] Validation experiment of anti-inflammatory targeted delivery of dandelion exosome microneedle patch

[0140] Experimental methods:

[0141] (1) Cell experiments

[0142] RAW264.7 macrophages were cultured to the logarithmic growth phase and stimulated with LPS (1 μg / mL) for 6 hours to establish an inflammatory model.

[0143] The dandelion exosomes medium concentration group and the dandelion extract medium concentration group were labeled with fluorescent markers, respectively.

[0144] The labeled dandelion exosomes medium concentration group and dandelion extract medium concentration group were incubated with inflammatory model cells for 2 hours respectively.

[0145] Flow cytometry was used to detect the cellular uptake of fluorescent markers, and the binding rates of the medium-concentration group of dandelion exosomes and the medium-concentration group of dandelion extract with inflammatory cells were calculated. The results are shown in Table 3.

[0146] Table 3

[0147] Group Binding rate (%) Dandelion exosomes medium concentration group 68.7±3.2 Dandelion extract medium concentration group 11.8±2.5

[0148] From the test results in Table 3, it can be seen that the binding rate of the medium concentration group of dandelion exosomes to inflammatory cells is 5.8 times that of the medium concentration group of dandelion extract (68.7% / 11.8%≈5.8), indicating that dandelion exosomes have stronger targeting.

[0149] The cell supernatant was collected and the secretion of TNF-α was detected using a TNF-α detection kit. The test results are shown in Table 4.

[0150] Table 4

[0151] Group TNF-α secretion (pg / mL) Model Group 124.5±8.3 Dandelion exosomes medium concentration group 27.6±4.1 Dandelion extract medium concentration group 83.7±6.5

[0152] From the test results in Table 4, it can be seen that the secretion of TNF-α in the medium concentration group of dandelion exosomes was reduced by 78% compared with the model group ((124.5-27.6) / 124.5≈78%), while the reduction in the medium concentration group of dandelion extract was smaller.

[0153] (2) Animal experiments

[0154] Male SD rats weighing 250-300 g were selected to establish a diabetic foot ulcer model.

[0155] The rats were divided into a model group, a dandelion exosome microneedle patch group, and a dandelion extract microneedle patch control group (the preparation method of the dandelion extract microneedle patch was the same as that of the dandelion exosome microneedle patch).

[0156] The drugs were administered regularly, and wound tissue was obtained 14 days after administration.

[0157] The expression of TNF-α in wound tissue was detected by immunohistochemistry. The test results are shown in Table 5.

[0158] Table 5. Immunohistochemical test results of wound tissue

[0159] Group TNF-α expression (IOD value) Model Group 186.3±15.7 Dandelion exosomes medium concentration group 42.5±12.3 Dandelion extract medium concentration group 103.7±14.2

[0160] From the test results in Table 5, it can be seen that the expression level of TNF-α in the medium concentration group of dandelion exosomes was significantly lower than that in the model group and the medium concentration group of dandelion extract, indicating that it can effectively inhibit the inflammatory response.

[0161] Experimental Example 3

[0162] Mechanical strength test of the microneedle patches prepared in Examples 1-3 and Comparative Examples 1-3

[0163] (1) Experimental setup

[0164] The microneedle patch was fixed on the fixture of a dynamic mechanical analyzer (DMA).

[0165] The test temperature range was set to 20°C-60°C, and the heating rate was 2°C / min.

[0166] Test frequency: 1Hz.

[0167] Strain control mode: 0.5%.

[0168] (2) Mechanical properties test

[0169] The storage modulus (G') of each group of microneedle patches was measured and recorded.

[0170] Each group of tests was performed with at least five replicates. The test results are shown in Table 6.

[0171] Table 6. Storage modulus data

[0172] Group Storage modulus (G') (kPa) Example 1 4.15±0.21 Example 2 3.68±0.35 Example 3 4.47±0.10 Comparative Example 1 3.39±0.80 Comparative Example 2 2.23±0.12 Comparative Example 3 3.72±0.29

[0173] Experimental conclusion: Compared with Example 2, in Comparative Example 1, no dandelion exosomes were added, and the storage modulus of the obtained microneedle patch decreased slightly, indicating that dandelion exosomes have a certain effect on improving the mechanical strength of the microneedle patch; while the storage modulus of Comparative Example 2 is lower, indicating that the addition of glutamine transaminase can significantly improve the mechanical strength of the microneedle patch through enzymatic cross-linking; in Comparative Example 3, no MMP-9 sensitive peptide was used, which had little effect on the storage modulus of the obtained microneedle patch, indicating that gelatin plays a major reinforcing role in the protective layer.

[0174] The storage modulus of the microneedle patches prepared in Examples 1-3 was as high as 3.68-4.47 kPa, indicating that the obtained microneedles had strong puncture force and were able to penetrate thick and tough necrotic tissue.

[0175] Experimental Example 4

[0176] Dandelion exosome release: A Franz diffusion cell transdermal test method was used. The microneedle patches of Example 2 and Comparative Example 3 were applied to one side of the stratum corneum of isolated pig skin. At different time points (2 hours, 24 hours, 48 ​​hours, and 72 hours) after application, the release of dandelion exosomes was tracked by fluorescent labeling, and the release of dandelion exosomes was measured.

[0177] The results show that, using the microneedle patch prepared in Example 2, after 2 hours of application, dandelion exosomes can be rapidly released through the mesopores, with a release rate of 60%. After 72 hours of continuous application, the cumulative release of dandelion exosomes reaches 85%, that is, the construction of the mesopores can enable dandelion exosomes to be released continuously for a long time. However, using the microneedle patch prepared in Comparative Example 3, after 24 hours of application, the release rate of dandelion exosomes is only 16%, and after 72 hours of continuous application, the cumulative release of dandelion exosomes reaches 30%. The reason may be that the lack of MMP-9 sensitive peptide segment causes the pore structure in the microneedle patch to deteriorate, thereby affecting the release of dandelion exosomes. The above results show that the MMP-9 sensitive peptide segment can interact with gelatin to form pores. The mesoporous structure can accurately control the release rate of dandelion exosomes according to the situation of the inflammatory site, thereby improving the targetedness and effectiveness of the treatment.

[0178] Among them, some raw material purchasing manufacturers are shown in Table 7:

[0179] Table 7

[0180] name Purchase manufacturer Positive control vitamin C solution Aladdin Biochemical Technology Co., Ltd. Negative control anhydrous ethanol Aladdin Biochemical Technology Co., Ltd. N-Acetylcysteine ​​Solution Aladdin Biochemical Technology Co., Ltd. Fluorescent probe DCFH-DA Shanghai Biyuntian Biotechnology Co., Ltd. Dandelion extract Meiluo Technology Co., Ltd. TNF-α Detection Kit Shanghai Biyuntian Biotechnology Co., Ltd. Silver ion antibacterial functional dressing Changsha Hairun Biotechnology Co., Ltd.

[0181] The above embodiments are only for illustrating the technical concept and features of the present invention. Its purpose is to enable people familiar with this technology to understand the content of the present invention and implement it. It cannot be used to limit the scope of protection of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A dandelion exosome microneedle patch, characterized in that: The invention comprises a microneedle body and a protective layer. The raw materials for preparing the microneedle body include: dandelion exosomes with a concentration of 0.1-0.4 mg / mL, sodium alginate with a concentration of 40-72 mg / mL, polyethylene glycol with a concentration of 7-20 mg / mL, calcium chloride with a concentration of 10-25 mg / mL, and glutamine transaminase with a concentration of 0.5-1.5 U / mL.

2. The dandelion exosome microneedle patch according to claim 1, characterized in that The raw materials for preparing the composite solution for the protective layer include gelatin and MMP-9 sensitive peptide segments; in the composite solution, the concentration of the gelatin is 50-150 mg / mL, and the concentration of the MMP-9 sensitive peptide segment is 2-20 U / mL.

3. The dandelion exosome microneedle patch according to claim 2, characterized in that The preparation method of the composite solution is as follows: gelatin is added into water, stirred and dissolved at 50-70°C with a stirring speed of 200-300 rpm to prepare a gelatin solution, cooled to 35-45°C, and then the MMP-9 sensitive peptide segment is added and stirred evenly to prepare a composite solution.

4. The dandelion exosome microneedle patch according to claim 1, characterized in that The length of the microneedle body is 1100-1300 μm, the needle tip angle of the microneedle body is 20-30 degrees, the distance between adjacent microneedle bodies is 1400-1600 μm, and the thickness of the microneedle base is 700-900 μm.

5. The dandelion exosome microneedle patch according to claim 2, characterized in that The method for preparing the protective layer comprises: coating the composite solution on the microneedle body, and performing secondary curing to obtain the protective layer.

6. A method for preparing the dandelion exosome microneedle patch according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Add sodium alginate and polyethylene glycol into water and mix well to prepare a sodium alginate-PEG solution; S2. Add the dandelion exosome suspension to the sodium alginate-PEG solution, stir for 5-15 minutes, add calcium chloride solution and transglutaminase to prepare the microneedle matrix; S3. Pour the microneedle matrix into the microneedle mold, perform preliminary solidification, prepare a protective layer, dry and demold, and obtain a dandelion exosome microneedle patch.

7. The method for preparing the dandelion exosome microneedle patch according to claim 6, characterized in that: In step S1, the specific conditions of the mixing are: stirring at a speed of 200-400 rpm at 45-60° C. for 0.5-1.0 h.

8. The method for preparing the dandelion exosome microneedle patch according to claim 6, characterized in that: In step S2, the concentration of calcium chloride in the calcium chloride solution is 30-75 mg / mL, and the solvent is water.

9. The method for preparing the dandelion exosome microneedle patch according to claim 6, characterized in that: In step S3, the specific conditions for the preliminary curing are: storage at 2-5°C for 0.5-1.0h.

10. Use of the dandelion exosome microneedle patch according to any one of claims 1 to 5 or the dandelion exosome microneedle patch prepared by the preparation method of claims 6 to 9 in chronic wound repair.

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