A nano-composite system and intelligent controlled-release microneedle patch for treating vitiligo and a preparation method thereof

Abstract

The present application relates to the field of biological medicine, and more particularly to a nano-composite system for treating vitiligo, an intelligent controlled-release microneedle patch and a preparation method thereof, wherein the nano-composite system contains enzyme-responsive micelles and polyethylene glycol modified polydopamine nanoparticles; the enzyme-responsive micelles are formed by glyceryl monostearate encapsulating ginseng root-derived exosomes; and the polyethylene glycol modified polydopamine nanoparticles are formed by covalent connection of a polydopamine core and methoxy-polyethylene glycol-amine. Compared with the prior art, the nano-composite system of the present application can realize the spatiotemporal programmed release of drugs in response to the pathological microenvironment, wherein the polydopamine nanoparticles provide instant antioxidant effect, and the exosome micelles trigger release under the condition of overexpression of matrix metalloproteinase-9, thereby synergistically exerting anti-inflammatory and promoting pigment regeneration effects; animal experiments have proved that the patch can effectively reverse the white spots and realize the recoloring of hair and skin. The microneedle patch thus prepared can realize transdermal self-administration, is convenient and painless.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine and dermatosis treatment, and particularly relates to a nano-composite system for treating vitiligo and an intelligent controlled-release microneedle patch. BACKGROUND

[0002] Vitiligo is a chronic depigmenting disease characterized by progressive loss of skin melanocytes, with a global prevalence of about 1%-2%. This disease not only brings heavy psychological burden to patients, but also significantly increases the risk of skin cancer due to the decline of epidermal photoprotection. Current research suggests that the pathogenesis of vitiligo revolves around the pathological cycle of "oxidative stress-immune inflammation-melanocyte dysfunction". Among them, oxidative stress is the core starting link, and the patient's melanocytes have inherent defects, leading to excessive accumulation of reactive oxygen species (ROS), while key antioxidant enzymes such as catalase (CAT) and superoxide dismutase (SOD) are damaged, causing imbalance of the oxidation-antioxidant system. Persistent oxidative stress further triggers an immune inflammatory cascade through mechanisms such as promoting T lymphocyte activation and enhancing melanocyte immunogenicity, ultimately forming a step-by-step amplification of the melanocyte destruction cycle.

[0003] Current clinical mainstream treatment methods include topical calcineurin inhibitors, narrow-band ultraviolet B (NB-UVB) phototherapy, and surgical transplantation, etc. However, these methods have obvious limitations in achieving stable repigmentation: topical drugs have limited efficacy outside the face and neck, and may cause skin irritation; phototherapy has a long treatment period, low complete repigmentation rate, and high recurrence rate after drug withdrawal; surgical transplantation is limited by the Koebner phenomenon and the risk of trauma. These current situations highlight the difficulty of existing technology in simultaneously intervening in multiple interconnected aspects of the pathological process of vitiligo.

[0004] In recent years, plant-derived nanovesicles (pNVs) have become a promising drug delivery platform due to their inherent biocompatibility and biodegradability. In particular, ginseng-derived exosomes (G-Exos) have become an ideal candidate for restoring melanocyte function and immune homeostasis due to their excellent antioxidant and anti-inflammatory efficacy. At the same time, polydopamine nanoparticles (PDA) as a biomimetic substitute for melanin not only provide immediate optical compensation through pigment deposition, but also effectively scavenge ROS through their phenolic hydroxyl groups, synergistically enhancing antioxidant defenses.

[0005] However, the single preparation form has obvious defects: the exosomes are directly applied and are easily hindered by the skin barrier, and it is difficult to effectively enrich at the lesion site; lack of lesion targeting and controlled release ability, easy to cause drug waste; unable to realize the synergistic intervention of oxidative stress, inflammatory damage and pigment regeneration in space and time. The existing drug delivery systems such as CN202110203774.4, CN201810643146.6 and the like disclosed exosome controlled release hydrogel still face problems such as complex material preparation, difficult to administer by minimally invasive method, poor adhesion at the action site and the like.

[0006] Therefore, there is an urgent need in the art to develop a new delivery system that can penetrate the skin barrier, realize lesion targeting, and respond to pathological microenvironment for intelligent drug release, to simultaneously regulate multiple key links of vitiligo pathogenesis, and break through the limitations of existing treatment technologies. SUMMARY

[0007] Based on the above reasons, the present application provides a nano-composite system and an intelligent controlled-release microneedle patch for treating vitiligo. Specifically, the present application aims to construct an intelligent responsive drug delivery system by using soluble microneedle technology to encapsulate ginseng root-derived exosomes (G-Exos) and polydopamine-polyethylene glycol nanoparticles (PDA@PEG), so as to realize the multiple synergistic intervention of oxidative stress, inflammatory response and pigment regeneration disorder in the pathological microenvironment of vitiligo. The present application can meet the treatment scenarios of vitiligo lesions at different sites and stages. The purpose of the present application can be achieved by the following technical solutions:

[0008] The present application first provides a nano-composite system for treating vitiligo, characterized in that it contains enzyme-responsive micelles and polyethylene glycol-modified polydopamine nanoparticles; the enzyme-responsive micelles are formed by encapsulating ginseng root-derived exosomes with glyceryl monostearate (TM); and the polyethylene glycol-modified polydopamine nanoparticles are composed of a polydopamine core and methoxy-polyethylene glycol-amine through covalent connection.

[0009] In an embodiment of the present application, the average hydrodynamic diameter of the polyethylene glycol-modified polydopamine nanoparticles is 150-170 nm, and the Zeta potential is -15 mV to -25 mV.

[0010] In a preferred embodiment of the present application, the polyethylene glycol-modified polydopamine nanoparticles are prepared by covalent connection of dopamine basic oxidative polymerization and PEG-NH2.

[0011] The present application further provides a preparation method of the above-mentioned nano-composite system, characterized in that it comprises the following steps:

[0012] (a) Extraction of ginseng root-derived exosomes: ginseng root-derived exosomes are extracted and purified from ginseng root tissues by differential centrifugation and sucrose density gradient centrifugation;

[0013] (b) Preparation of enzyme-responsive micelles: a glyceryl monostearate aqueous solution is mixed with a ginseng root-derived exosome solution, ultrasonic emulsification and dialysis purification are performed, and the enzyme-responsive micelles are obtained;

[0014] (c) Preparation of polyethylene glycol-modified polydopamine nanoparticles: dopamine is polymerized under alkaline conditions to form polydopamine nanoparticles, and after purification, the polydopamine nanoparticles are mixed with a methoxy-polyethylene glycol-amine solution, ultrasonic-assisted reaction and dialysis purification are performed, and the polyethylene glycol-modified polydopamine nanoparticles are obtained.

[0015] Another aspect of the present application provides an intelligent controlled-release microneedle patch for treating vitiligo, characterized in that the nanocomposite system is loaded in the needle tip matrix of the patch.

[0016] In a preferred embodiment of the present application, the needle tip matrix material of the microneedle is made of a polymer. Suitable polymers include, but are not limited to, hyaluronic acid, polyvinyl alcohol, alginate, gelatin, chitosan and derivatives thereof, which are used alone or in combination in the present application, and methacrylated hyaluronic acid (HAMA) and polyvinyl alcohol (PVA) are more preferred.

[0017] Each microneedle is arranged on a base layer to form a microneedle patch. The base layer is also composed of a polymer, including but not limited to polyvinyl alcohol, sodium carboxymethyl cellulose, alginate and polyvinylpyrrolidone, etc., which are used alone or in combination in the present application, and polyvinyl alcohol is more preferred.

[0018] In an embodiment of the present application, the mass concentration of methacrylated hyaluronic acid in the needle tip matrix is 3% to 8%, and the mass concentration of polyvinyl alcohol is 0.5% to 2%.

[0019] In the microneedle patch of the present application, a plurality of microneedles are arranged in a regular pattern. According to some embodiments, the distance between each microneedle is equal. The microneedle shape includes but is not limited to conical and prismatic, and conical is more preferred. The needle height is 700 to 800 μm, and the needle base diameter is 450 to 550 μm.

[0020] In an embodiment of the present application, 80 to 120 microneedles are arranged on a base layer with an area of 1 cm 2 .

[0021] Another aspect of the present application provides a preparation method of the intelligent microneedle patch described above, characterized in that it comprises the following steps:

[0022] (a) Dissolve methacrylated hyaluronic acid, polyvinyl alcohol and a photoinitiator in water to form a base solution;

[0023] (b) mixing the above nanocomposite system with the base solution to obtain a tip matrix solution;

[0024] (c) injecting the tip matrix solution into a microneedle mold, after centrifugal filling, pre-drying, ultraviolet light cross-linking curing, and covering with a backing layer, and after demolding, the microneedle patch is obtained.

[0025] Another aspect of the present application provides a pharmaceutical composition, characterized in that it comprises a therapeutically effective amount of the above nanocomposite system and a pharmaceutically acceptable carrier.

[0026] Another aspect of the present application provides a kit containing the smart microneedle patch, which consists of a microneedle patch and physiological saline packaged separately.

[0027] Another aspect of the present application provides the use of the above nanocomposite system or the above smart microneedle patch in the preparation of a medicine for preventing and / or treating vitiligo.

[0028] Another aspect of the present application provides the use of the above nanocomposite system or the above smart microneedle patch in the preparation of a medical device or a cosmetic product for antioxidant and / or anti-inflammatory purposes.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The nanocomposite system of the present application realizes multiple intervention on oxidative stress, inflammatory response and pigment regeneration disorder in the pathological microenvironment of vitiligo through the synergistic effect of enzyme-responsive micelles and polydopamine nanoparticles.

[0031] The microneedle patch of the present application can effectively penetrate the stratum corneum of the skin to achieve efficient drug delivery, and at the same time, through the intelligent response characteristics, it can achieve controllable release of the drug at the lesion site.

[0032] The preparation method provided by the present application has simple process and mild conditions, and is suitable for large-scale production.

[0033] Animal experiments have confirmed that the microneedle patch provided by the present application can significantly improve the skin lesion symptoms of the vitiligo model, promote the repigmentation of hair and skin, and has good biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Reaction mechanism diagram of PDA@PEG synthesis process.

[0035] Figure 2 Schematic diagram of microneedle patch preparation and release mechanism.

[0036] Figure 3 TEM image of G-Exos.

[0037] Figure 4Hydrodynamic size and zeta potential distribution of G-Exos.

[0038] Figure 5 Particle size change curve of G-Exos during 8 weeks of storage at -80 °C.

[0039] Figure 6 TEM and SEM images of PDA@PEG nanoparticles.

[0040] Figure 7 Hydrodynamic size and zeta potential distribution of PDA@PEG.

[0041] Figure 8 Particle size change curve of PDA@PEG nanoparticles during 60 days of storage at 25 °C.

[0042] Figure 9 Protective effects of G-Exos and PDA@PEG on H2O2-induced HaCaT and PIG1 cell viability.

[0043] Figure 10 ROS scavenging effects of each treatment group on PIG1 cells.

[0044] Figure 11 Effects of each treatment group on SOD, CAT activities and MDA content in H2O2-damaged HaCaT cells.

[0045] Figure 12 Inhibitory effects of each treatment group on LPS-induced TNF-a and IL-6 secretion by HaCaT cells.

[0046] Figure 13 PIG1 cell uptake of PDA@PEG nanoparticles.

[0047] Figure 14 Ultrastructure observation of PDA@PEG nanoparticle distribution in PIG1 cells.

[0048] Figure 15 Promoting effects of G-Exos and PDA@PEG on PIG1 cell melanin synthesis.

[0049] Figure 16 TEM image of G-Exos@TM composite micelles.

[0050] Figure 17 Comparison of particle size and zeta potential of G-Exos, TM and G-Exos@TM composite micelles.

[0051] Figure 18Particle size change of G-Exos™ composite micelles during incubation in PBS with / without MMP-9 and G-Exos cumulative release profile.

[0052] Figure 19 SEM images of the composite microneedle patch.

[0053] Figure 20 Force-displacement curve of the composite microneedle.

[0054] Figure 21 H&E staining sections of mouse skin after treatment with the composite microneedle patch.

[0055] Figure 22 Optical microscope images of the swelling process of the composite microneedle patch in PBS (pH 7.4).

[0056] Figure 23 In vitro release profiles of PDA@PEG and G-Exos in the composite microneedle patch in PBS with / without MMP-9.

[0057] Figure 24 Cytotoxicity evaluation of the composite microneedle extract on PIG1 and HaCaT cells.

[0058] Figure 25 Macroscopic repigmentation photographs of the back of vitiligo model mice in each group after different treatment periods, vitiligo scores, and quantitative analysis of the repigmentation area on the back.

[0059] Figure 26 H&E staining sections and quantitative analysis of dermal thickness of the back skin tissue of mice in each group.

[0060] Figure 27 Masson-Fontana staining sections of the back skin tissue of mice in each group.

[0061] Figure 28 Confocal microscope images and quantitative analysis of ROS levels in the skin tissue of mice in each group.

[0062] Figure 29 Determination results of SOD, CAT activity, and MDA content in the skin tissue of mice in each group.

[0063] Figure 30 Immunohistochemical staining images and quantitative analysis of TNF-α expression in the skin tissue of mice in each group.

[0064] Figure 31 ELISA determination results of TNF-α and IL-6 concentrations in the homogenate of the skin tissue of mice in each group. DETAILED DESCRIPTION

[0065] In order to further understand the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the scope of protection of the present application.

[0066] Unless otherwise specified, the reagents involved in the embodiments of the present application are all commercially available products, which can be purchased through commercial channels.

[0067] Example 1: Extraction and purification of ginseng root-derived exosomes (G-Exos)

[0068] Step 1: Pre-treatment of tissue

[0069] Step 1.1: Fresh ginseng root tissue was taken and placed in pre-cooled 0.01 M phosphate buffer solution (PBS, pH 7.4), and gently stirred with a glass rod. The surface was washed repeatedly 3 times to completely remove the dirt and impurities on the surface. The excess liquid on the surface of the tissue was absorbed with sterile filter paper, and then the ginseng root tissue was cut into small pieces with a volume of 1-2 cm 3 .

[0070] Step 1.2: 10 g of the cut ginseng tissue was accurately weighed and placed in a 200 mL high-speed tissue homogenizer, and 30 mL of pre-cooled PBS buffer was added at a mass-volume ratio of 1:3.

[0071] Step 2: Tissue homogenization and coarse filtration

[0072] The mixture homogenization cup was placed in an ice water bath, and the high-speed tissue homogenizer was used at a speed of 15,000 rpm for 60 seconds each time. After each homogenization, the system was cooled for 30 seconds, and the process was repeated 3 times until the tissue was completely broken. The tissue homogenate was poured into a 200-mesh stainless steel sieve, and a glass rod was used to guide the collection of the filtrate. Further filtration was performed through four layers of sterile gauze to remove residual fibrous impurities, and the final filtrate was collected.

[0073] Step 3: Fractionated centrifugation

[0074] Step 3.1: The filtrate obtained in step 2 was divided into 50 mL ultracentrifuge tubes and centrifuged at 2,000 ×g relative centrifugal force for 20 minutes at 4°C using a high-speed refrigerated centrifuge.

[0075] Step 3.2: After the end of centrifugation, the supernatant was carefully pipetted up to avoid touching the pellet at the bottom of the tube, and the collected supernatant was labeled as "Supernatant A". Supernatant A was transferred to a new ultracentrifuge tube and centrifuged at 10,000 x g for 60 min at 4 °C.

[0076] Step 3.3: After the end of centrifugation, the supernatant was carefully pipetted up again to avoid touching the pellet at the bottom of the tube, and the collected supernatant was labeled as "Supernatant B".

[0077] Step 4: Ultracentrifugation for preliminary enrichment of exosomes

[0078] Supernatant B was filtered through a 0.45 μm hydrophilic polyether sulfone needle filter to remove possible remaining micrometer-sized vesicles and impurities. The filtered supernatant was transferred to an ultracentrifuge tube and centrifuged at 120,000 x g for 2 h at 4 °C using an ultracentrifuge. After the end of centrifugation, the supernatant was carefully decanted, and a thin, light yellow film of pellet was observed at the bottom of the tube, which was the crude exosome pellet.

[0079] Step 5: Purification by sucrose density gradient centrifugation

[0080] The pellet obtained in Step 4 was gently resuspended with 1 mL of pre-cooled PBS buffer, avoiding vigorous pipetting, to obtain a crude exosome suspension. In an ultracentrifuge tube, 3 mL of 60%, 45%, 30%, and 8% (w / v) sucrose solutions were sequentially added from bottom to top to form a discontinuous sucrose density gradient. The 1 mL crude exosome suspension obtained in Step 4 was carefully added to the top of the sucrose density gradient using a Pasteur pipette. The gradient tube was placed in the horizontal rotor of an ultracentrifuge and centrifuged at 120,000 x g for 90 min at 4 °C. After the end of centrifugation, the tube was carefully removed, and a cream to cream-yellow band was observed at the interface between the 30% and 45% sucrose solutions. Using a 1 mL sterile syringe with a needle, the band at the interface was carefully pipetted to collect a high-purity G-Exos solution.

[0081] Step 6: Ultracentrifugation for purification and storage

[0082] The collected solution containing G-Exos after sucrose density gradient centrifugation was transferred into a suitable ultracentrifuge tube. Using an ultracentrifuge, centrifuge at 120,000 x g for 70-90 minutes at 4 °C. Carefully discard the supernatant, at this time there should be a visible yellowish or milky white precipitate at the bottom of the tube. Add an appropriate amount of pre-cooled PBS buffer to the centrifuge tube containing the G-Exos precipitate, gently blow or repeatedly rinse the bottom of the tube with a pipette gun to ensure that the precipitate is fully and gently resuspended uniformly. The resuspended solution can be left to stand at 4 °C for 1 hour to help dissolve. The protein concentration of the G-Exos resuspension was determined using the BCA protein quantification kit, strictly following the instructions. Dilute the solution with PBS buffer to a final protein concentration of 2.0 mg / mL. The diluted G-Exos solution was aliquoted at 50 μL / tube and stored in a -80 °C ultra-low temperature refrigerator for long-term storage.

[0083] Example 2: Preparation of enzyme-responsive complex micelles (G-Exos@TM)

[0084] Accurately weigh 10.0 mg of TM into a 5 mL clean glass sample bottle, and use a pipette to accurately measure 1.0 mL of ultrapure water into the sample bottle. Place the sample bottle in a constant temperature water bath at 60 °C for heating, while using an ultrasonic cell disruptor for ultrasonic assisted dissolution. Set the ultrasonic power to 100 W, the working mode to work for 2 seconds and interval for 3 seconds, and continue processing until the TM is completely dissolved, the solution is clear and transparent, and a TM pre-dispersion is obtained, which is ready for use.

[0085] Step 2: Mixing and emulsification of exosomes and micelles

[0086] Step 2.1: Measure 1.0 mL of G-Exos prepared in Example 1, with a protein concentration of 2.0 mg / mL.

[0087] Step 2.2: Place the TM pre-dispersion on a magnetic stirrer, maintain the system temperature at 60 °C, and stir at a constant speed of 500 rpm.

[0088] Step 2.3: Using a microsyringe pump, slowly and uniformly add the G-Exos solution measured in step 2.1 to the continuously stirred TM pre-dispersion at a constant flow rate of 1.0 mL / min.

[0089] Step 2.4: After the addition is complete, continue to maintain the 60 °C water bath and 500 rpm stirring conditions for 30 minutes to allow the G-Exos and TM molecules to fully interact with each other.

[0090] Step 2.5: Quickly transfer the above mixed solution to an ice water bath and cool to 0-4 °C.

[0091] Step 2.6: The mixed solution was subjected to ultrasonic treatment in an ice bath using the same probe sonicator (power setting: 200 W, pulse mode: work for 2 seconds, interval for 3 seconds) for a total of 5 minutes to complete the emulsification and drug loading process, forming the primary complex micelles.

[0092] Step 3: Dialysis purification and product collection

[0093] Step 3.1: The primary complex micelle solution after ultrasonic treatment was transferred to a regenerated cellulose dialysis bag with a molecular weight cut-off of 3,500 Da, ensuring that the dialysis bag was tightly sealed at both ends. The dialysis bag was completely immersed in 2.0 L of phosphate buffered saline (PBS, 0.01 M, pH 7.4) in a 4°C cold room or refrigerator, and the dialysis solution was slowly stirred using a magnetic stirrer. The dialysis purification process lasted for 24 hours, during which the dialysis solution was completely replaced every 4 hours (2.0 L of PBS each time).

[0094] Step 3.2: After dialysis, the final solution was carefully collected from the dialysis bag, which was the purified enzyme-responsive complex micelles loaded with ginseng root-derived exosomes (G-Exos@TM) solution. The obtained G-Exos@TM solution was divided into brown sample bottles and stored in a 4°C environment away from light for future use.

[0095] Example 3: Synthesis method of polydopamine-polyethylene glycol nanoparticles (PDA@PEG)

[0096] Step 1: Synthesis of polydopamine nanoparticles

[0097] Step 1.1: Accurately weigh 360 mg of dopamine hydrochloride and place it in a 250 mL three-necked round-bottom flask. Add 180 mL of deionized water and gently shake to initially disperse it.

[0098] Step 1.2: Place the three-necked flask in a 50°C constant temperature water bath, install a mechanical stirrer, and stir at a constant speed of 800 rpm until the dopamine hydrochloride is completely dissolved, obtaining a colorless transparent solution.

[0099] Step 1.3: Use a constant flow pump or microsyringe pump to slowly and uniformly add 1.52 mL of 1 M sodium hydroxide solution to the above solution at a rate of 0.05 mL / min.

[0100] Step 1.4: After the addition is complete, maintain the 50°C water bath and 800 rpm stirring speed for 5 hours. During this process, it can be observed that the color of the reaction system changes from colorless to light brown, dark brown, and finally dark black, indicating that polydopamine nanoparticles have been formed.

[0101] Step 1.5: After the reaction is completed, transfer the reaction solution to a centrifuge tube, and centrifuge at 12,000 x g for 10 min at room temperature using a high-speed centrifuge. Carefully discard the supernatant and collect the black precipitate at the bottom.

[0102] Step 1.6: Add 50 mL of deionized water to the precipitate, and resuspend the precipitate completely using a vortex shaker for 1 min. Then, centrifuge again at 12,000 x g for 10 min, and discard the supernatant. Repeat this washing process three times to ensure that unreacted monomers and byproducts are completely removed.

[0103] Step 1.7: After the final washing, resuspend the polydopamine nanoparticle precipitate in 5 mL of deionized water to obtain a preliminary purified PDA nanoparticle dispersion, which is ready for use.

[0104] Step 2: PEGylation modification and final purification

[0105] Step 2.1: Accurately weigh 100 mg of methoxy-polyethylene glycol-amine, and add it to the 5 mL PDA nanoparticle dispersion prepared in Step 1.

[0106] Step 2.2: Place the mixed solution in an ice water bath, and use a probe sonicator to treat it for a total of 40 min under the following conditions: power 300 W, pulse mode of 2 s on / 3 s off, to promote the physical adsorption and initial combination of mPEG-NH2 and the PDA particle surface.

[0107] Step 2.3: After the ultrasonic treatment, remove the ice bath, and place the reaction solution on a magnetic stirrer at room temperature (25 ± 2°C) for 12 h of stirring at a speed of 400 rpm to allow the mPEG-NH2 to be covalently grafted to the PDA nanoparticle surface through a Michael addition or Schiff base reaction.

[0108] Step 2.4: Transfer the entire reaction solution to a regenerated cellulose dialysis bag with a molecular weight cut-off of 8-14 kDa.

[0109] Step 2.5: Place the dialysis bag in a 2 L beaker, and dialyze it using deionized water for a total of 48 h. During this period, replace the dialysate every 6 h, and use fresh 2 L of deionized water each time to ensure that unreacted mPEG-NH2 molecules are completely removed.

[0110] Step 2.6: After the dialysis is completed, collect the dark black solution in the dialysis bag to obtain the purified polydopamine-polyethylene glycol nanoparticle solution. This solution can be sterile filtered through a 0.22 μm microporous filter, and stored in a dark environment at 4°C for later use (see reaction schematic in Figure 1 ).

[0111] Example 4: Hyaluronic acid microneedle patch loaded with G-Exos@TM and PDA@PEG and its preparation method

[0112] Step 1: Preparation of the composite microneedle tip matrix solution

[0113] Step 1.1: Accurately weigh the following components into a 2 mL brown screw thread glass vial to avoid light-induced pre-crosslinking of the photoinitiator:

[0114] Methacrylated hyaluronic acid (HAMA): 50.0 mg; Polyvinyl alcohol (PVA, alcoholysis degree 87-89%, Mw 31,000-50,000): 10.0 mg; Photoinitiator LAP: 1.0 mg

[0115] Step 1.2: Accurately pipette 1.0 mL of deionized water into the above vial.

[0116] Step 1.3: Immediately screw the cap tightly and place it on a vortex shaker to oscillate at 2500 rpm for 3 minutes to initially wet and disperse the powders.

[0117] Step 1.4: Subsequently, transfer the vial to a 37°C constant temperature shaker and oscillate at 150 rpm for 20 minutes until all components are completely dissolved, obtaining a clear, transparent, and uniform microneedle base solution.

[0118] Step 1.5: Accurately pipette the following two active component solutions in sequence:

[0119] G-Exos@TM composite micellar solution prepared in Example 2, 100 μL (0.2 mg of G-Exos protein mass contained, loading amount).

[0120] PDA@PEG nanoparticle solution prepared in Example 3, 50 μL (1.0 mg of nanoparticle dry weight, loading amount).

[0121] Step 1.6: Using a 100-1000 μL adjustable pipette, slowly add the above two active component solutions to the microneedle base solution prepared in Step 1.4 in sequence.

[0122] Step 1.7: Using the same pipette, set the volume to 500 μL, slowly pipette the mixed solution 15 times, the operation should be gentle to ensure that the two active ingredients are uniformly dispersed in the matrix, while avoiding the introduction of air bubbles as much as possible.

[0123] Step 1.8: Finally, obtain a homogeneous, double-active ingredient-loaded microneedle tip matrix solution, which is placed in a light-tight container and stored at room temperature in the dark until use, and the subsequent molding steps should be completed within 1 hour.

[0124] Step 2: Molding and fabrication of microneedle patch

[0125] Step 2.1: Hydrophilic treatment of polydimethylsiloxane (PDMS) microneedle mold using an oxygen plasma treater. The mold specification is a 10 x 10 array of conical needles with a height of 750 pm, a base diameter of 500 pm, and a center-to-center spacing of 500 pm. The treatment parameters are set as follows: power 100 W, and treatment time 60 seconds.

[0126] Step 2.2: The prepared composite microneedle tip matrix solution from Step 1 is uniformly and continuously dropped onto the surface of the plasma-treated PDMS mold using a pipette, ensuring that the liquid completely covers and is slightly higher than all the tip cavities.

[0127] Step 2.3: The mold is placed smoothly into the rotor of a benchtop centrifuge, and centrifuged at a relative centrifugal force of 2,000 x g for 10 minutes at room temperature (25 ± 2°C), allowing the tip matrix solution to fill and fill each tip cavity under the driving force of centrifugation.

[0128] Step 2.4: Immediately after centrifugation, a disposable plastic spatula is used to scrape the mold surface at an angle of about 45 degrees, removing the excess solution on the surface and ensuring that only enough matrix solution remains in the tip cavities.

[0129] Step 2.5: The treated mold is moved horizontally into a 37°C forced air drying oven for 2 hours to allow the tip structure to be initially shaped and concentrated.

[0130] Step 2.6: After pre-drying, the mold is placed on the workbench of a 405 nm wavelength UV crosslinking instrument. The UV light intensity is adjusted to 15 mW / cm 2 , ensuring that the light path is perpendicular to the mold surface, and irradiating for 60 seconds to allow the HAMA molecules to undergo sufficient photo-crosslinking reaction to form a stable hydrogel network structure.

[0131] Step 2.7: Accurately weigh 4.0 g of polyvinyl alcohol (PVA, same specifications as above) powder and dissolve in 20 mL of deionized water to prepare a 20% (w / v) PVA aqueous solution. The solution is placed in a 60°C water bath and stirred at 200 rpm until completely dissolved and clear to obtain the microneedle backing layer solution.

[0132] Step 2.8: The PVA backing solution prepared in Step 2.7 is added to the mold base portion of the solidified tip structure, and the droplet volume is just enough to completely cover the base area (thickness of about 1-2 mm) without overflowing to the tip portion.

[0133] Step 2.9: The mold was again transferred to a 37 °C forced-air drying oven for continuous drying for 48 hours to completely volatilize the water in the backing layer, solidify to form a flexible support layer, and form a firm physical bond with the tip layer.

[0134] Step 2.10: After drying was completed, the microneedle patch was carefully and completely removed from the edge of the mold at one corner using flat tweezers. The patch was wiped with a piece of dust-free paper to remove any excess edges that might be present on the back of the patch.

[0135] Step 2.11: Each microneedle patch prepared was placed in an aluminum foil bag and vacuum-sealed. The final product was stored in a dry, dark, and cool (4 °C) environment (see Figure 2 for a schematic diagram of the synthesis and release).

[0136] Test Example 1: Structural characterization of ginseng root-derived exosomes (G-Exos)

[0137] 1. Extraction efficiency and morphological observation

[0138] To verify the efficiency of the extraction method described in the present application (see Example 1) and the morphology of the product obtained, the following experiments were performed:

[0139] Step 1.1: Calculation of extraction efficiency. 1.0 kg of fresh ginseng root was accurately weighed, and the procedure in Example 1 was strictly followed to obtain the G-Exos solution after purification. The protein concentration of the obtained G-Exos solution was determined using a BCA protein quantification kit according to the manufacturer's instructions. The average value of three independent repeated experiments was calculated to obtain the amount of G-Exos protein stably obtained from each kilogram of fresh ginseng root as 50.0 ± 3.5 mg.

[0140] Step 1.2: Transmission electron microscopy morphological observation. 10 μL of the above G-Exos solution (protein concentration of about 0.1 mg / mL) was added dropwise to a carbon-supported copper mesh and allowed to adsorb for 3 minutes. Excess liquid was absorbed from the edge with filter paper, and then 10 μL of 2% (w / v) phosphotungstic acid solution (pH 7.0) was added dropwise for negative staining. After 1 minute of staining, the sample was again dried with filter paper. The prepared sample was observed under a JEM-1400 Flash transmission electron microscope at an acceleration voltage of 80 kV. As shown in Figure 3 , the G-Exos observed in the field of view exhibited a typical exosome sac-like or cup-like morphology, with an intact structure and good dispersion throughout the field of view, with no obvious aggregates, indicating that the G-Exos sample prepared in the present application had high purity.

[0141] 2. Particle size distribution and stability analysis

[0142] To characterize the physical properties and storage stability of G-Exos, the following analyses were performed:

[0143] Step 2.1: Particle size and polydispersity index determination. The G-Exos solution was diluted with sterile PBS to an appropriate detection concentration (protein concentration of about 0.05 mg / mL). The determination was performed using a Malvern Zetasizer Nano ZS90 dynamic light scattering instrument, with the detection temperature set to 25°C and the equilibration time set to 60 seconds. The results showed that the G-Exos had a main distribution peak of 137.1 ± 11.4 nm (n = 3) in the hydrodynamic diameter. Figure 4 A). The polydispersity index thereof was 0.15 ± 0.03, which was less than 0.2, indicating that the sample particle size was unimodal and narrow, and the system was highly uniform.

[0144] Step 2.2: Zeta potential determination. Using the same instrument, the Zeta potential of the original concentration G-Exos solution was determined at 25°C using laser Doppler electrophoresis technology. The average Zeta potential value was -21.29 ± 0.51 mV (n = 3) Figure 4 B). The high absolute value of the negative charge (generally considered to be greater than 20 mV) confirmed that the G-Exos had good colloidal stability in the aqueous medium due to the electrostatic repulsion between particles, and was not prone to aggregation.

[0145] Step 2.3: Storage stability evaluation. The G-Exos solution prepared in the same batch was divided and stored in a -80°C ultra-low temperature refrigerator. After 2 months of storage, a sample was taken out and the particle size distribution was determined again according to the method of step 2.1. The results showed that the particle size peak of the sample after storage did not shift significantly Figure 5 . This result proved that the G-Exos prepared according to the method of the present application could be stored for a long time at -80°C while maintaining its colloidal stability.

[0146] Test Example 2: Synthesis and characterization of polyethylene glycol modified polydopamine nanoparticles (PDA@PEG)

[0147] 1. Synthesis of PDA@PEG nanoparticles

[0148] Step 1.1: Synthesis and purification. The polydopamine nanoparticle dispersion was successfully obtained by oxidative polymerization of dopamine hydrochloride under alkaline conditions and subsequent centrifugal purification, strictly according to the method described in Example 3.

[0149] Step 1.2: Surface modification. mPEG5000-NH2 was added to the PDA dispersion obtained in the previous step, and the PEG chains were stably covalently linked to the PDA core by a Schiff base / Michael addition reaction through ice bath ultrasonic assisted dispersion and continuous stirring at room temperature.

[0150] Step 1.3: Final purification. Unreacted mPEG-NH2 was removed by dialysis, and the purified PDA@PEG nanoparticle solution was finally obtained for subsequent characterization and biological evaluation.

[0151] 2. Characterization of the physical and chemical properties of PDA@PEG nanoparticles

[0152] Step 2.1: Morphology observation. 10 μL of PDA@PEG solution (diluted to an appropriate multiple) was dropped onto a carbon-supported copper mesh, and after natural air-drying, it was observed using a transmission electron microscope (TEM) (A) and a scanning electron microscope (SEM) (B). The images showed that the obtained nanoparticles were monodisperse spherical in shape. Figure 6 A) and scanning electron microscope (SEM) (B). The images showed that the obtained nanoparticles were monodisperse spherical in shape. Figure 6 B) and scanning electron microscope (SEM) (B). The images showed that the obtained nanoparticles were monodisperse spherical in shape.

[0153] Step 2.2: Particle size determination. By counting at least 200 particles in the transmission electron microscope images, the average diameter of PDA@PEG nanoparticles was measured to be 162 ± 4.3 nm (A). Figure 7 A).

[0154] Step 2.3: Zeta potential determination. The PDA@PEG solution was diluted to an appropriate concentration with deionized water, and a nanoparticle size and Zeta potential analyzer was used for determination. The results showed that its Zeta potential was -19.72 ± 0.51 mV (B). Figure 7 B).

[0155] Step 2.4: Stability evaluation. The PDA@PEG solution was stored at 25°C for 60 days, and during this period, its particle size was determined by dynamic light scattering method at regular intervals, and no significant aggregation or precipitation was observed (C), proving that it has good long-term stability. Figure 8 C).

[0156] Test Example 3: Evaluation of the synergistic therapeutic effect of G-Exos and PDA@PEG

[0157] 1. Evaluation of synergistic cell protection effect

[0158] Step 1.1: Cell culture. Human immortalized keratinocytes (HaCaT) were cultured in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, BI) and 1% penicillin / streptomycin (100 U / mL). Human immortalized melanocytes (PIG1) were cultured in Medium 254 medium (Gibco) supplemented with 5% fetal bovine serum, 1% penicillin / streptomycin, and 10% HMGS-2 (Gibco). All cells were cultured at 37°C under 5% CO2 conditions.

[0159] Step 1.2: Cell grouping treatment. HaCaT and PIG1 cells were cultured according to the method described in step 1.1 and seeded in 96-well plates at a density of 1 x 10 4 cells per well. After 24 hours of adhesion, the culture medium was replaced with one containing 0.8 mmol / L H2O2 to establish an oxidative stress model, and the following grouping interventions were immediately performed: 1) control group; 2) G-Exos group (10 pg / mL); 3) PDA@PEG group (50 pg / mL); 4) combined treatment group (10 pg / mL G-Exos + 50 pg / mL PDA@PEG).

[0160] Step 1.3: Cell viability detection. After 24 hours of intervention, cell viability was detected using the CCK-8 method. The results showed that, compared with the single treatment groups, the combined treatment of G-Exos and PDA@PEG could more significantly reverse the H2O2-induced toxicity of HaCaT ( Figure 9 A) and PIG1 cells ( Figure 9 B).

[0161] 2. Evaluation of synergistic antioxidant effect

[0162] Step 2.1: Intracellular reactive oxygen species (ROS) level detection. PIG1 cells were seeded at the same density and treated with fresh culture medium containing various group preparations for 4 hours (grouping step 1.2). After treatment, the culture medium was discarded, and serum-free medium containing 10 pM DCFH-DA probe was added and incubated at 37°C for 30 minutes. Then the cells were washed twice with PBS, and morphological observation was performed by fluorescence microscopy ( Figure 10 ). The results showed that G-Exos was a more effective intracellular ROS scavenger than PDA@PEG, and the combined treatment group showed the greatest signal attenuation, indicating a synergistic scavenging effect.

[0163] Step 2.2: Endogenous antioxidant system and oxidative damage detection. After pre-treatment of HaCaT cells with 0.8 mmol / L H2O2 for 4 hours, the culture medium was replaced with one containing different preparations and continued to be cultured for 24 hours (grouping step 1.2). The cells were collected, and the activities of superoxide dismutase (SOD) and catalase (CAT) and the content of malondialdehyde (MDA) were measured using commercial kits. The results ( Figure 11 ) showed that G-Exos could significantly restore SOD and CAT activities and reduce MDA levels (p < 0.01 compared with the PDA@PEG group), and this effect was further amplified in the combined group (p < 0.001).

[0164] 3. Evaluation of synergistic anti-inflammatory effect

[0165] Step 3.1: Inflammatory cytokine assay. HaCaT cells were seeded at appropriate density and stimulated with 0.1 ng / mL of lipopolysaccharide (LPS) for 24 hours to establish an inflammation model, while group intervention was performed (grouping step 1.2). After incubation, cell culture supernatant was collected and the concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined using ELISA kits. Results Figure 12 showed that G-Exos alone treatment could inhibit TNF-α and IL-6 secretion by 62% and 57%, respectively, while combination therapy further strengthened this inhibitory effect, indicating the presence of anti-inflammatory synergy.

[0166] 4. Cell uptake and ultrastructure analysis of PDA@PEG

[0167] Step 4.1: Confocal microscopy observation. PIG1 cells were seeded in 15 mm glass bottom confocal dishes and co-incubated with 50 μg / mL PDA@PEG for 24 hours. Subsequently, cells were fixed using 4% paraformaldehyde, and cytoskeletal fibrous actin was stained using rhodamine-phalloidin (red fluorescence), and cell nuclei were counterstained using DAPI (blue fluorescence). Brightfield and fluorescence channel overlay images Figure 13 showed that PDA@PEG (dark particles under brightfield) was present in large quantities in the cytoplasm, and the cell morphology remained intact.

[0168] Step 4.2: Transmission electron microscopy observation. PIG1 cells treated with PDA@PEG were prepared routinely: first fixed with 2.5% glutaraldehyde at 4°C overnight, then post-fixed with 1% osmium tetroxide. Subsequently, dehydrated through an ethanol-acetone gradient series, infiltrated with acetone-epoxy resin mixture, and finally heat polymerized (35°C to 80°C). Ultra-thin sections of 70-90 nm were prepared using an ultramicrotome (Leica EM UC7), and observed under a transmission electron microscope after staining with uranyl acetate and lead citrate. Results Figure 14 clearly showed the distribution of PDA@PEG nanoparticles in intracellular vesicles and cytoplasmic compartments, confirming their effective internalization.

[0169] 5. Synergistic promotion of melanin synthesis

[0170] Step 5.1: Quantitative analysis of melanin content. PIG1 cells were seeded in 6-well plates and cultured overnight. Afterward, they were grouped for intervention (grouping as in Step 1.2) for 24 hours. After treatment, the culture medium was discarded, cells were washed with PBS, and the cell pellet was collected. 100 μL of 1 M sodium hydroxide solution was added to the pellet, and the mixture was incubated in an 80°C water bath for 1 hour to fully dissolve the melanin. After cooling, the absorbance of the solution was measured at 490 nm using a microplate reader to quantify the melanin content. Results ( Figure 15 The results showed that, compared with the H2O2-damaged model group, PDA@PEG monotherapy significantly increased melanin synthesis (by 1.32 times, p < 0.05), while the G-Exos / PDA@PEG combination further increased melanin production by 1.41 times (p < 0.01).

[0171] Test Example 4: Preparation, Characterization, and Enzyme-Response Release Study of G-Exos@TM Composite Micelles

[0172] 1. Preparation of G-Exos@TM composite micelles

[0173] G-Exos@TM composite micelles were prepared strictly according to the method described in Example 2. In brief, a homogeneous G-Exos@TM composite micelle solution was obtained by mixing an aqueous solution of glyceryl monostearate (TM) with a G-Exos solution, followed by ice-bath ultrasonic emulsification and dialysis purification.

[0174] 2. Physicochemical characterization of G-Exos@TM composite micelles

[0175] Step 2.1: Morphological observation. 10 μL of G-Exos@TM solution was added to a copper grid on a carbon-supported membrane. After standing for 3 minutes to absorb the adsorption, excess liquid was blotted away with filter paper. Then, 2% phosphotungstic acid solution was added for negative staining for 1 minute. Under a transmission electron microscope, vesicle-like structures were clearly surrounded by a membrane. Figure 16 ), consistent with the micellar encapsulation structure.

[0176] Step 2.2: Particle size and Zeta potential analysis. An appropriate amount of G-Exos@TM composite micelle solution was diluted with PBS and then measured using a nanoparticle size and Zeta potential analyzer. The results showed that the hydrodynamic diameter of G-Exos@TM was 182.7 ± 19 nm. Figure 17 A), the diameter of unmodified G-Exos is 144.7 ± 15 nm ( Figure 17 B). The zeta potential of TM itself is -1.6 ± 0.2 mV, the zeta potential of unmodified G-Exos is -26.6 ± 2.2 mV, and the zeta potential of G-Exos@TM is -11.2 ± 1.2 mV.Figure 17 C).

[0177] 3. Enzymatic responsive release property evaluation

[0178] Step 3.1: In vitro release experiment design. Freshly prepared G-Exos@TM complexes were diluted with the following two media respectively:

[0179] Group A: PBS (pH 7.4); Group B: PBS (pH 7.4) containing 1 ng / mL MMP-9.

[0180] All samples were placed in a 37°C constant temperature shaker and continuously shaken at a speed of 600 rpm.

[0181] Step 3.2: Particle size change monitoring. Samples were taken from each group every day, and the change in the hydrodynamic diameter of the particles was monitored by dynamic light scattering. The results show that (Fig. 3) Figure 18 A): In the presence of MMP-9, the particle size returned to the size of native G-Exos after 7 days; while in PBS without MMP-9, the particle size only decreased slightly to about 170 nm.

[0182] Step 3.3: Exosome release quantitative analysis. At the designated time points (1st, 3rd, 7th day), samples were taken from each group, resuspended after centrifugation. Using the TINGO exosome ELISA kit, according to the instructions, the amount of released G-Exos was quantified by detecting the exposure of the exosome surface marker CD63. The results show that (Fig. 4) Figure 18 B): In PBS, only less than 7% of G-Exos was released from the complex within 7 days, in the presence of MMP-9, almost all G-Exos was released by the 7th day.

[0183] Test Example 5: Preparation, performance evaluation and in vitro release study of composite microneedle patches

[0184] 1. Preparation of composite microneedle patches

[0185] G-Exos@TM and PDA@PEG loaded composite microneedle patches were prepared according to the method described in Example 4. Briefly, G-Exos@TM complex micelles were blended with PDA@PEG nanoparticles in the HAMA precursor solution, after injection into the PDMS mold, they were filled by centrifugation, pre-dried, cross-linked by UV light, and finally a PVA backing layer was added and demolded to obtain a complete composite microneedle patch.

[0186] 2. Microneedle morphology and structure characterization

[0187] Step 2.1: Stereomicroscope observation. The overall morphology of HAMA / G-Exos@TM / PDA@PEG composite microneedles was observed using a stereomicroscope. The results showed that the needle array was arranged in order (Fig. 2A). Figure 19 A).

[0188] Step 2.2: Morphological observation. The dried microneedle patch was fixed on the sample stage and observed by scanning electron microscope after gold spraying treatment. The SEM images showed (Fig. 2B) that the microneedles were regular conical, with sharp tips and uniform size, with a needle height of 750 μm and a base diameter of 500 μm. Figure 19 B), the microneedles were regular conical, with sharp tips and uniform size, with a needle height of 750 μm and a base diameter of 500 μm.

[0189] 3. Mechanical properties and skin penetration ability evaluation

[0190] Step 3.1: Mechanical strength test. A displacement-force testing system equipped with a 50 kg load sensor was used. The microneedle tip was vertically fixed on a stainless steel platform, and the sensor was lowered from 1 cm above the tip at a speed of 0.1 mm / s. After contact, the speed was reduced to 0.01 mm / s, and the loading continued until the displacement reached 800 μm. The force-displacement curve was recorded. The results showed (Fig. 3A) that the breaking force of HAMA / G-Exos@TM / PDA@PEG microneedles was 0.13 N / needle, which was higher than the minimum force required for skin insertion. Figure 20

[0191] Step 3.2: In vitro skin penetration experiment. Full-thickness mouse skin was taken and fixed on the experimental platform after removing the subcutaneous fat. The microneedle patch was vertically placed on the skin surface, and a thumb pressure was applied for 10 minutes. After removal, the skin sample was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining. Histological sections confirmed that the microneedle channels completely penetrated the stratum corneum (Fig. 3B), indicating that the microneedles had good skin penetration ability. Figure 21

[0192] 4. Swelling and drug release behavior study

[0193] Step 4.1: Swelling behavior test. The microneedle patch was immersed in PBS (pH 7.4) at 37°C, and the morphological changes were observed by digital optical microscope at regular time intervals. The results showed (Fig. 4A) that the microneedles rapidly absorbed water and swelled, and the structure dissociated over time, with the morphology basically collapsing after 15 minutes. This swelling property is crucial for promoting the sustained transdermal release of drugs in the hydrogel matrix. Figure 22

[0194] Step 4.2: Drug release curve determination. The microneedle patch was placed in PBS containing 1 ng / mL MMP-9 and PBS without MMP-9, respectively, and gently shaken at 37°C. The release medium was collected at different time points, and the following method was used for quantitative analysis:

[0195] ​​​PDA@PEG nanoparticles: measure their characteristic absorbance at 280 nm

[0196] G-Exos: detect CD63 surface marker by TINGO exosome ELISA kit

[0197] Step 4.3: Release profile analysis. The release curve shows that PDA@PEG releases rapidly in both PBS and MMP-9 solution within 2 hours, showing a burst release mode (A); the release of G-Exos shows an enzyme-triggered property: in the presence of MMP-9, the cumulative release rates on day 3 and day 7 are 71.3% and 99.4%, respectively; while in PBS without MMP-9, the release rates are only 18.5% and 38.2% (B) within the same period. Figure 23 Figure 23

[0198] 5. Biocompatibility evaluation

[0199] Step 5.1: Cytotoxicity detection. The microneedle extract was co-cultured with P1G1 and HaCaT cells for 24 hours, and cell viability was detected by CCK-8 method. The results show no significant toxicity difference compared with the untreated control group (P > 0.05) (Fig. 5A). Figure 24

[0200] Test Example 6: Evaluation of the therapeutic effect of composite microneedle patches on a vitiligo animal model

[0201] 1. Establishment of animal model and experimental design

[0202] Step 1.1: Establishment of vitiligo model. 4-6 week old female C57BL / 6 mice were selected, and after intraperitoneal injection of 5% chloral hydrate (350 mg / kg) anesthesia, an external emulsion containing 33.3% vaseline, 50% 4-benzyloxyphenol and 16.7% tretinoin was evenly applied to the depilation area, once every other day, for 30 days, to establish a stable vitiligo model. This treatment induces selective hair follicle melanin loss while maintaining epidermal integrity, closely simulating human vitiligo pathology.

[0203] Step 1.2: Experimental grouping and treatment. The successfully modeled mice were randomly divided into 5 groups (n=5): ① model group; ② HAMA microneedle group (empty microneedle); ③ HAMA / G-Exos@TM microneedle group; ④ HAMA / PDA@PEG microneedle group; ⑤ HAMA / G-Exos@TM / PDA@PEG microneedle group. From the 30th day of modeling, the corresponding microneedle patches were placed in the depigmentation area, and replaced every 3 days for 3 weeks.

[0204] 2. Macroscopic evaluation of treatment effect

[0205] ​​​Step 2.1: Observation of the repigmentation process. Take weekly photos of the back to record changes in hair color removal. Results show ( Figure 25 A):

[0206] Week 1: The combined treatment group showed significant hair repigmentation in the application area, which was superior to other groups;

[0207] Week 2: The repigmentation advantage in the combined treatment group was further enhanced;

[0208] Week 3: The combined treatment group achieved near-complete repigmentation, while the microneedle group also induced approximately 20% repigmentation.

[0209] Step 2.2: Vitiligo scoring. Scoring is based on the percentage of depigmented area (0-5 points). Results show ( Figure 25 B) All groups had an initial score >4 (>50% back depigmentation); the control group maintained a score of about 4 throughout the course; the combined treatment group's score gradually improved: it dropped to about 2 in the second week and further dropped to 1 in the third week.

[0210] Step 2.3: Quantitative analysis of polychromatic area. ImageJ quantitative analysis shows ( Figure 25 C), at week 3, the repigmentation surface area in the combined treatment group was significantly increased compared with that in the model control group (p < 0.001).

[0211] 3. Histological analysis

[0212] Step 3.1: Sample processing. After the last treatment, skin from the back was taken, fixed in 4% formalin, embedded in paraffin, and sectioned.

[0213] Step 3.2: H&E staining analysis. The results show ( Figure 26 The combined treatment group showed a significant increase in hair follicle density, with the follicles mainly in the late anagen phase; the monotherapy group showed an increase in the number of hair follicles, but these follicles were mainly stationary in the early anagen phase. Dermal thickness measurements showed that the combined treatment achieved the maximum dermal thickness (p < 0.01).

[0214] Step 3.3: Masson-Fontana staining analysis. The results show ( Figure 27 All microneedling treatment groups showed enhanced melanin synthesis and hair bulb development within the hair follicles; the combined treatment group showed elongated lower halves and maximum melanin content in the hair follicles; the control group maintained a sparse hair follicle structure with very few melanin granules.

[0215] Test Example 7: Evaluation of the antioxidant and anti-inflammatory effects of composite microneedle patches in a vitiligo model

[0216] 1. Experimental Design and Sample Preparation

[0217] Step 1: The operation was performed strictly according to the animal model establishment method, grouping scheme and treatment procedure described in Test Example 6. Briefly, the vitiligo model mice were treated with the corresponding microneedle patches for three weeks after random grouping, and the skin tissue samples were collected 24 hours after the last treatment for subsequent analysis.

[0218] 2. Oxidative stress level evaluation

[0219] Step 2.1: Skin tissue active oxygen detection. The mouse back skin tissue was taken to prepare 8 μm frozen sections, which were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% BSA, then incubated with ROS-specific primary antibody (1:200) at 4°C overnight, incubated with Cy3-labeled secondary antibody in the dark for 1 hour, and observed by confocal microscopy after DAPI restaining. Quantitative analysis showed that the ROS fluorescence intensity of the combination therapy group was significantly lower than that of the model group (p < 0.01), indicating that it effectively alleviated the oxidative stress state in the vitiligo model. Figure 28

[0220] Step 2.2: Antioxidant index detection. The skin tissue was taken to prepare a homogenate, and the SOD activity, CAT activity and MDA content were measured using a commercial kit. The results showed that (Fig. 2B) Figure 29 ): The SOD / CAT activity of the vitiligo model group was significantly reduced, and the MDA level was increased; each treatment group could reverse this trend, and the combination therapy group had the most significant effect on restoring antioxidant capacity.

[0221] 3. Inflammatory factor expression analysis

[0222] Step 3.1: Immunohistochemical staining (Fig. 3A) Figure 30 ). The skin paraffin sections were deparaffinated, hydrated, and then antigen-repaired, followed by incubation with rabbit anti-TNF-α monoclonal antibody (1:200) at 4°C overnight, incubation with HRP-labeled secondary antibody at room temperature for 1 hour, color development with DAB, hematoxylin restaining, and quantitative analysis of TNF-α positive expression area by ImageJ software.

[0223] Step 3.2: ELISA determination (Fig. 3B) Figure 31 ). The skin tissue homogenate supernatant was taken, and the TNF-α concentration and IL-6 concentration were quantitatively detected according to the kit instructions.

[0224] The results showed that the G-Exos monotherapy group and the combination therapy group could significantly inhibit the expression of TNF-α and IL-6, and the effect of the combination therapy was better than that of the single treatment group.

[0225] The above describes the preferred embodiments of the present application, but it is not intended to limit the present application. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present application.​

Claims

1. A nanocomposite system for the treatment of vitiligo, characterized in that, The enzyme-responsive micelles are formed by glyceryl monostearate encapsulating ginseng root-derived exosomes; the polyethylene glycol-modified polydopamine nanoparticles are composed of a polydopamine core and methoxy-polyethylene glycol-amine through covalent connection; the average hydrodynamic diameter of the enzyme-responsive micelles is 150-250 nm, and the Zeta potential is-5 mV to-15 mV; the average hydrodynamic diameter of the polyethylene glycol-modified polydopamine nanoparticles is 150-170 nm, and the Zeta potential is-15 mV to-25 mV.

2. An intelligent microneedle patch for treating vitiligo, characterized by, The tip matrix of the microneedle patch is loaded with the nanocomposite system of claim 1.

3. The smart microneedle patch of claim 2, wherein, The tip of the microneedle patch is composed of a photocrosslinked hydrogel, and the matrix material of the photocrosslinked hydrogel comprises methacrylated hyaluronic acid and polyvinyl alcohol.

4. The smart microneedle patch of claim 2, wherein, The microneedle of the microneedle patch is conical, with a needle height of 700-800 μm and a needle base diameter of 450-550 μm.

5. A method of preparing the smart microneedle patch according to any one of claims 2-4, characterized by, The method comprises the following steps: (a) dissolving methacrylated hyaluronic acid, polyvinyl alcohol and a photoinitiator in water to form a base solution; (b) mixing the nanocomposite system of claim 1 with the base solution to obtain a tip matrix solution; (c) injecting the tip matrix solution into a microneedle mold, centrifugally filling, pre-drying, ultraviolet light crosslinking and curing, and covering with a backing layer, and then demolding to obtain the microneedle patch.

6. A pharmaceutical composition, characterized by, The nanocomposite system of claim 1 in a therapeutically effective amount and a pharmaceutically acceptable carrier.

7. Use of the nanocomposite system of claim 1 or the smart microneedle patch of any one of claims 2-4 in the preparation of a medicament for preventing and / or treating vitiligo.

8. Use of the nanocomposite system of claim 1 or the smart microneedle patch of any one of claims 2-4 in the preparation of a medical device for antioxidant and / or anti-inflammatory purposes.

Images