Use of a complex for the preparation of a medicament for the treatment of androgenetic alopecia

Abstract

The application discloses application of a compound in preparation of a medicine for treating androgen alopecia, and relates to the technical field of biological medicines. The foreskin mesenchymal stem cell extracellular small vesicle has water-soluble and fat-soluble double-soluble characteristics, can be absorbed through the skin, and is administered in a manner of intravenous administration / mist administration and the like. The foreskin mesenchymal stem cell extracellular small vesicle is used as a carrier to load curcumin, so that the absorption of the curcumin can be effectively improved, the pharmacological effect of the curcumin can be improved, and the targeting and anti-inflammatory efficiency can be improved. Moreover, the compound obtained by loading the foreskin mesenchymal stem cell extracellular small vesicle with curcumin can easily penetrate into the skin, directly acts on the hair follicles damaged by androgen, has a good effect of treating androgen alopecia, and can significantly promote hair regeneration.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a complex in the preparation of a drug for treating androgenetic alopecia. Background Technology

[0002] Androgenetic alopecia (AGA) is the most common type of hair loss. In men, it typically manifests as a receding hairline (M-shaped) and thinning hair on the crown (O-shaped), eventually leading to male pattern baldness or total baldness. In women, it often presents as gradual thinning and widening of hair on the crown and at the parting, with the hairline generally not receding significantly. The pathological mechanism mainly involves the hair follicles becoming overly sensitive to androgens (primarily dihydrotestosterone) in the body, causing the follicles to gradually miniaturize, shortening the growth phase, and ultimately resulting in the growth of fine, soft vellus hair before it falls out.

[0003] In related technologies, drugs such as finasteride and minoxidil are mainly used to treat androgenetic alopecia, but these have problems such as slow onset of action and poor efficacy. Hair transplantation can also be used to change appearance, but the internal environment and local microenvironment are not improved, the survival time of transplanted hair is short, and hair loss is likely to recur after a few years. Therefore, there is a need for a drug that can effectively treat androgenetic alopecia. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes the use of a complex in the preparation of a medicament for treating androgenetic alopecia.

[0005] The use of a complex according to a first aspect of the present invention in the preparation of a medicament for treating androgenetic alopecia, the complex comprising extracellular microvesicles loaded with curcumin; the extracellular microvesicles being derived from preputial mesenchymal stem cells.

[0006] The application of the present invention, according to embodiments thereof, has at least the following beneficial effects: Curcumin has poor water solubility, low intestinal absorption, and rapid metabolism (oral bioavailability <1%), and is not easily absorbed through the skin. Extracellular vesicles of foreskin mesenchymal stem cells can inhibit inflammation and fibrosis in hair follicle stem cells and surrounding tissues, promoting tissue repair and hair follicle stem cell regeneration. Compared to extracellular vesicles of umbilical cord mesenchymal stem cells, foreskin mesenchymal stem cell extracellular vesicles exhibit better targeting (significantly higher expression rate of targeted chemokine genes), anti-inflammatory properties (significantly higher levels of anti-inflammatory factors IL10 and TGF), and the ability to promote repair and regeneration, making them more effective for treating androgenetic alopecia.

[0007] Extracellular vesicles of preputial mesenchymal stem cells possess dual water- and lipid-soluble properties, enabling transdermal absorption. Using these extracellular vesicles as a carrier to load curcumin effectively improves curcumin absorption, enhances its pharmacological effects, and improves targeting and anti-inflammatory efficacy. Furthermore, the complex described in this embodiment readily penetrates the skin, directly acting on androgen-damaged hair follicles, demonstrating excellent therapeutic effects on androgenetic alopecia and significantly promoting hair regeneration.

[0008] According to some embodiments of the present invention, the particle size of the extracellular microvesicles is 30 nm-150 nm. For example, it can be 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm or 150 nm.

[0009] According to some embodiments of the present invention, the particle size of the composite is 40 nm to 200 nm. For example, it can be 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185 nm, 190 nm, 195 nm, or 200 nm.

[0010] According to some embodiments of the present invention, the curcumin content in the complex is 15 ng to 30 ng per 100 million extracellular vesicles. For example, it can be 15 ng, 16 ng, 17 ng, 18 ng, 19 ng, 20 ng, 21 ng, 22 ng, 23 ng, 24 ng, 25 ng, 26 ng, 27 ng, 28 ng, 29 ng, or 30 ng.

[0011] According to some embodiments of the present invention, a method for loading the extracellular microvesicles with curcumin includes at least one of electroporation, microinjection, sonication, and culturing foreskin mesenchymal stem cells in a curcumin-containing culture medium.

[0012] According to some embodiments of the present invention, the preparation method of the complex includes the following steps: The complex was obtained by culturing foreskin mesenchymal stem cells in a culture medium containing curcumin and separating the extracellular vesicles from the culture medium.

[0013] According to some embodiments of the present invention, the culture medium is an extracellular vesicle-free culture medium.

[0014] According to some embodiments of the present invention, the culture medium is a classic medium used to ensure the growth and / or survival of the foreskin mesenchymal stem cells. The culture medium includes, but is not limited to, DMEM, DMEM / F12, MEM, alpha-MEM (α-MEM), IMDM, or RPMI.

[0015] According to some embodiments of the present invention, the concentration of curcumin in the culture medium is 0.625 μM-10 μM. For example, it can be 0.625 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, or 10 μM. According to some embodiments of the present invention, the culture temperature is 36-38°C, preferably 37°C.

[0016] According to some embodiments of the present invention, the culture time is 24 h to 48 h. For example, it can be 24 h, 26 h, 28 h, 30 h, 32 h, 34 h, 36 h, 38 h, 40 h, 42 h, 44 h, 46 h, or 48 h.

[0017] According to some embodiments of the present invention, the culture is a 3D culture. This makes it easier to control product quality, eliminates the need for a series of cumbersome operations such as digestion, subculturing, and inoculation during 2D culture, significantly reducing time, space, and labor costs; and results in high yield, good functional uniformity, and high quality, making it suitable for large-scale industrial production.

[0018] According to some embodiments of the present invention, the 3D culture includes either 3D static culture or 3D dynamic culture.

[0019] According to some embodiments of the present invention, the 3D dynamic culture method includes at least one of stirring, perfusion, and rotation.

[0020] According to some embodiments of the present invention, the 3D culture includes either a supportless 3D culture or a supported 3D culture.

[0021] According to some embodiments of the present invention, the supportless 3D culture includes at least one of the hanging drop method, the low-adhesion spherical culture plate method, and the magnetic levitation method.

[0022] According to some embodiments of the present invention, the supported 3D culture includes using at least one of hydrogel and microcarrier as a support for 3D culture.

[0023] According to some embodiments of the present invention, the material of the microcarrier includes a biocompatible material.

[0024] According to some embodiments of the present invention, the material of the microcarrier includes at least one of natural polymer materials and synthetic polymer materials.

[0025] According to some embodiments of the present invention, the natural polymeric material includes at least one of gelatin, collagen, cellulose, chitin, alginate, and dextran.

[0026] According to some embodiments of the present invention, the synthetic polymer material includes poly(hydroxyethyl methacrylate) (PHEMA) and polylactic acid. At least one of glycolic acid copolymer (PLGA) and polylactide (PDLLA).

[0027] According to some embodiments of the present invention, the density of the microcarrier in the culture medium is 5 g / L-7 g / L. For example, it can be 5 g / L, 5.1 g / L, 5.2 g / L, 5.3 g / L, 5.4 g / L, 5.5 g / L, 5.6 g / L, 5.7 g / L, 5.8 g / L, 5.9 g / L, 6 g / L, 6.1 g / L, 6.2 g / L, 6.3 g / L, 6.4 g / L, 6.5 g / L, 6.6 g / L, 6.7 g / L, 6.8 g / L, 6.9 g / L, or 7 g / L.

[0028] According to some embodiments of the present invention, the methods for separating the extracellular vesicles include, but are not limited to, at least one of differential centrifugation, density gradient centrifugation, ultrafiltration, and size exclusion chromatography.

[0029] According to some embodiments of the present invention, the dosage form of the drug includes any one of tablets, capsules, solutions, aerosols, sprays, ointments, or films.

[0030] According to some embodiments of the present invention, the dosage form of the drug is suitable for topical application. The dosage form includes, but is not limited to, any one of solutions, aerosols, sprays, ointments, or films. This avoids the systemic side effects and poor patient compliance associated with long-term oral medication.

[0031] According to some embodiments of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0032] According to some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of diluents, dispersants, binders, fillers, thickeners, lubricants, pH adjusters, flavor maskers, colorants, antioxidants, or antibacterial agents.

[0033] According to some embodiments of the present invention, the medicament further includes other active ingredients for treating androgenetic alopecia. These other active ingredients for treating androgenetic alopecia are loaded in the cellular extracellular vesicles.

[0034] According to some embodiments of the present invention, the other effective ingredients for treating androgenetic alopecia include, but are not limited to, minoxidil or finasteride.

[0035] According to some embodiments of the present invention, the complex has at least one of the functions of A1) to A3): A1) Promotes hair regeneration and / or growth; A2) Increases the expression level of β-catenin in skin tissue; A3) Inhibits the expression level of secretory curl-related protein 2 in skin tissue.

[0036] According to some embodiments of the present invention, the curcumin is effective at concentrations of 1 μM to 10 μM. For example, it can be 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 4.5 μM, 5 μM, 5.5 μM, 6 μM, 6.5 μM, 7 μM, 7.5 μM, 8 μM, 8.5 μM, 9 μM, 9.5 μM, or 10 μM. Therefore, it has good anti-inflammatory and proliferative effects.

[0037] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0038] Figure 1 Comparison of the anti-inflammatory abilities of the complexes in Example 1 and Comparative Example 2; A: Qualitative analysis results; B: Quantitative analysis results.

[0039] Figure 2 Characterization results of the curcumin complex of preputial mesenchymal stem cells in Example 1 and the extracellular vesicles of preputial mesenchymal stem cells in Comparative Example 1; A: Nanoparticle tracking particle size analysis results; B: Transmission electron microscopy image (scale bar: 200 nm); C: Western blot detection results; D: Fluorescence spectrophotometer detection results.

[0040] Figure 3This is a schematic diagram of the preparation process of the extracellular vesicle curcumin complex of preputial mesenchymal stem cells in Example 1 and the exogenous extracellular vesicle curcumin complex of preputial mesenchymal stem cells in Comparative Example 3.

[0041] Figure 4 This is an evaluation of hair regeneration in a diabetic mouse wound model using the curcumin complex from extracellular microvesicles of prepuce mesenchymal stem cells (Extracellular microvesicles) as described in Example 1; A: Schematic diagram of hair regeneration induced by the curcumin complex from extracellular microvesicles of prepuce mesenchymal stem cells; B: Typical immunofluorescence staining images of β-actin and SFRP2 in skin tissue on postoperative day 12 in each group (blue: DAPI; red: β-actin; green: SFRP2; scale bar: 200 μm; n=3); C: Quantitative analysis of β-actin and SFRP2 expression.

[0042] Figure 5 Hair growth in volunteer 1 before and after drug treatment (left: before the first dose; right: after the last dose).

[0043] Figure 6 Hair growth in volunteer 2 before and after drug treatment (left: before the first dose; right: after the last dose).

[0044] Figure 7 Hair growth in volunteer 3 before and after drug treatment (left: before the first dose; right: after the last dose). Detailed Implementation

[0045] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0046] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0047] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0048] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0049] Example 1 This example provides a curcumin complex from extracellular microvesicles of preputial mesenchymal stem cells, and its preparation method is as follows: (1) The foreskin removed by circumcision was longitudinally sectioned to separate the epidermis and dermis. The dermal tissue homogenate was evenly seeded into culture flasks for primary culture and passaged to the 3rd generation to establish a mesenchymal stem cell bank (P3 generation FS-MSCs). The P3 generation FS-MSCs were cultured at a rate of 6000 cells / cm³. 2 The cells were seeded at a density of [insert density here] into dextran-based microcarrier culture medium and incubated overnight. The rotation speed was adjusted (~30 rpm) until the cells did not settle or aggregate. After 4-5 days of culture, when the confluence of FS-MSCs reached 80-90%, the culture medium was changed to curcumin DMEM / F12 medium, and the supernatant was collected after 48 h of incubation.

[0050] The culture medium for the dextran-based microcarriers was formulated as follows: 3 g / L Cytodex 3 dextran-based microcarriers (Cytodex 3 dextran-based microcarriers). TM 3. Cytiva Corporation, USA), with the remainder in DMEM / F12 medium.

[0051] The method for preparing curcumin DMEM / F12 medium is as follows: add 10 mM curcumin ethanol solution to DMEM / F12 medium until the final curcumin concentration is 4 μM.

[0052] (2) The collected supernatant was centrifuged at 4℃ and 300×g for 10 min; then centrifuged at 4℃ and 2000×g for 20 min to remove residual cell debris and larger organelles; the supernatant was then further purified using a 0.22 μm filter. The purified supernatant was initially concentrated and filtered using a 500 kD hollow fiber membrane to obtain a concentrated solution. Further separation was performed using a HiScreen Capto Core 700 gel column combined with an AKTA Pure 25 macromolecular separation and purification system: all chromatography buffers (95% ethanol, Sangon Biotech, China, catalog number A507050) and buffer solutions (PBS, BasalMedia, China, catalog number B320KJ) were filtered through a 0.22 µm membrane for later use. Afterwards, the pump was cleaned, and the buffer inlet tube was transferred to deionized water for rinsing, followed by transfer to 20% ethanol for preservation. The program was selected, and after automatic cleaning was completed, the HiScreen Capto Core 700 gel column (HiScreen Capto Core 700, Cytiva, catalog number 17548151) was installed, and the program was adjusted until the column was equilibrated. After preparation, Superloop sample concentrate was used to fix the sample volume, and the filtrate was collected. The filtrate was then concentrated by centrifugation using a 100 kD ultrafiltration tube to obtain the curcumin complex from extracellular microvesicles of foreskin mesenchymal stem cells.

[0053] Comparative Example 1 This example provides extracellular microvesicles of foreskin mesenchymal stem cells (FSMSCs-sEVs), which are prepared in a manner that is basically the same as in Example 1, except that the curcumin DMEM / F12 medium in step (2) is replaced with an equal amount of DMEM / F12 medium.

[0054] Comparative Example 2 This example provides a curcumin complex of extracellular microvesicles of umbilical cord mesenchymal stem cells, which is prepared in a manner that is basically the same as in Example 1, except that P3 generation FS-MSCs are replaced with an equal amount of umbilical cord mesenchymal stem cells.

[0055] Comparative Example 3 This example provides a curcumin complex derived from exogenous foreskin mesenchymal stem cells via extracellular vesicles, the preparation method of which is as follows: 1.19×10 11The FSMSCs-sEVs obtained in Comparative Example 1 were slowly added to curcumin DMEM / F12 medium (concentration 4 μM), mixed thoroughly, and incubated in a water bath at 37°C in the dark for 2 h. Unloaded free curcumin molecules were removed by ultrafiltration (ultrafiltration tube molecular weight 100 kDa) (3000×g, 10 min). The ultrafiltrate was resuspended in PBS buffer solution to obtain the exogenous foreskin mesenchymal stem cell extracellular vesicle curcumin complex.

[0056] Experimental Example 1 2 mL containing 2×10 5 Raw264.7 cells were seeded in DMEM medium in 6-well plates and incubated at 37°C for several hours until cell adhesion. The culture medium was then replaced with DMEM medium containing 500 ng / mL LPS, and the cells were incubated at 37°C for 24 h to stimulate the M1 phenotype. The culture medium was then replaced with DMEM medium containing 50 μg / mL of the complex from Example 1 or Comparative Example 2, and the cells were incubated at 37°C for 48 h. Cells were stained with PE-labeled anti-CD86 antibody (Invitrogen), collected, and resuspended in PBS. Intracellular fluorescence intensity was measured using a flow cytometer (BD FACSAria III).

[0057] The results are as follows Figure 1 As shown.

[0058] Compared with cells treated with the curcumin complex of extracellular microvesicles of umbilical cord mesenchymal stem cells, cells treated with the curcumin complex of extracellular microvesicles of foreskin mesenchymal stem cells showed lower CD86 signal (red fluorescence, M1 phenotype). This indicates that the curcumin complex of extracellular microvesicles of foreskin mesenchymal stem cells has better anti-inflammatory activity than the curcumin complex of extracellular microvesicles of umbilical cord mesenchymal stem cells.

[0059] Experiment Example 2 The curcumin complex from extracellular vesicles of preputial mesenchymal stem cells in Example 1 and the extracellular vesicles from preputial mesenchymal stem cells in Comparative Example 1 were analyzed for particle size concentration and morphology by nanoparticle tracking and transmission electron microscopy (TEM). Lysis buffer (Beyotime Biotechnology Co., Ltd., China, catalog number C3632-100ml) and the sample solution were mixed in an equal volume ratio, gently pipetted to mix, and lysed on ice for 10 min. After centrifugation at 12000×g for 5 min at 4°C, the supernatant was collected, and the expression of positive (CD9 and CD63) markers was detected by Western blotting (sample loading was normalized to total protein content). The curcumin loading was detected using a fluorescence spectrophotometer (Ex=425 nm, Em=460~700 nm).

[0060] The results are as follows Figure 2 As shown.

[0061] The average sizes of the extracellular vesicles of preputial mesenchymal stem cells (PCSFCs) and the curcumin complex of PCSFCs were 84.8 nm and 83.6 nm, respectively. Transmission electron microscopy revealed that both PCSFCs and the curcumin complex exhibited typical cup-shaped structures less than 100 nm in diameter. Western blot analysis showed enrichment of extracellular vesicle markers (CD9 and CD63) in the curcumin complex, further confirming the successful isolation of the extracellular vesicles. Compared to the PCSFCs, the curcumin complex showed a distinct curcumin fluorescence peak at an intrinsic emission wavelength of 530 nm, demonstrating successful curcumin loading.

[0062] Experimental Example 3 The concentrations of the extracellular microvesicle curcumin complex from preputial mesenchymal stem cells in the corresponding solutions were tested using nanoparticle tracking analysis for the curcumin complex from preputial mesenchymal stem cells in Example 1 and Comparative Example 3. The content of extracellular vesicles and curcumin complexes of preputial mesenchymal stem cells in each milliliter of supernatant was determined. The curcumin content loaded in the extracellular vesicles of preputial mesenchymal stem cells was detected by fluorescence spectrophotometry. The specific method was as follows: First, a series of concentration gradient solutions were prepared using curcumin standard in a DMEM / F12 system consistent with the sample. The fluorescence intensity was measured using a fluorescence spectrophotometer, and a standard curve was established to obtain a linear regression equation between fluorescence intensity and curcumin concentration (ng / mL). Subsequently, after removing free curcumin from the curcumin-loaded extracellular vesicle samples, extracellular vesicle lysis buffer was added, and the samples were lysed on ice for 30 min. The fluorescence value of the samples was measured under the same detection parameters. A blank control of unloaded extracellular vesicles was set up to obtain blank fluorescence values, and the blank was subtracted to obtain net fluorescence. The net fluorescence value was converted into curcumin concentration according to the standard curve and the dilution factor, thereby calculating the total mass of curcumin in the sample system. Simultaneously, the number of extracellular microvesicle particles used for detection was measured using nanoparticle tracking analysis (NTA), and the final loading amount was obtained.

[0063] Table 1

[0064] like Figure 3 As shown in Table 1, the endogenous drug delivery method eliminates the need for secondary impurity removal, yields higher concentrations of curcumin complex from extracellular microvesicles of foreskin mesenchymal stem cells, and is more suitable for large-scale production and transformation.

[0065] Experiment Example 4 Previous studies have confirmed a positive correlation between β-catenin activity and trichiogenicity in the Wnt signaling pathway, and β-actin is a biomarker for the initiation of the active cycle of hair follicle stem cells. SFRP2, as a negative regulator that inhibits the nuclear translocation of β-catenin, participates in the regulation of various cellular functions and biological processes.

[0066] BALB / c mice were acclimatized for one week. Fasting blood glucose was measured once before modeling. Modeling was initiated by a single intraperitoneal injection of streptozotocin (STZ) at a dose of 120 mg / kg (STZ was dissolved in sterile 0.1 M sodium citrate buffer (pH=4.5) before use to prepare a 1 wt% STZ solution, which was then filtered through a 0.22 μm microporous membrane for sterilization). During this period, mice had free access to food and water. Fasting blood glucose (FBG) was measured by tail blood collection after 3 days. A FBG ≥ 16.7 mmol / L was considered a successful diabetic mouse model. Blood glucose levels were monitored weekly for 2 weeks after successful modeling of diabetes.

[0067] Diabetic mice were fasted for 12 hours before modeling. During modeling, they were anesthetized by intraperitoneal injection of sodium pentobarbital (1%, 50 mg / kg, IP). The lumbosacral region was shaved, and the skin was disinfected with 75% alcohol swabs. Two full-thickness skin excision wounds, extending to the subcutaneous layer, were created in the surgical area using a 6 mm diameter circular punch, thus establishing a full-thickness skin excision model. Postoperatively, the wounds hemostatically stopped naturally without bandaging.

[0068] Mice were randomly divided into three groups: (1) control group, where sterile saline solution was applied evenly to the wound; (2) Example 1 group, where 10 mg of sterile saline solution was applied evenly to the wound. 8 One extracellular vesicle / each (for two wounds) is applied evenly to the wound surface. (3) Comparative group 1, 10 8 One extracellular vesicle per mouse (two wounds) was applied and spread evenly on the wound surface. After different experimental groups received appropriate treatments, each wound was covered with a sterile dressing. All mice were individually housed in clean cages, provided with clean drinking water and free access to food, and their wounds were kept dry to prevent infection. Twelve days post-surgery, all mice were sacrificed, and skin tissue around the wounds was collected. Immunofluorescence staining was performed on the skin tissue, and RNA was extracted. The expression levels of β-actin and SFRP2 were detected by PCR (using ACTB as an internal reference gene). -ΔΔCt The relative expression levels of β-actin and SFRP2 were calculated using the method (with three independent replicates).

[0069] like Figure 4As shown.

[0070] The curcumin complex in extracellular microvesicles of prepuce mesenchymal stem cells has the ability to promote hair regeneration. After treatment with the curcumin complex in extracellular microvesicles of prepuce mesenchymal stem cells, a large number of hair follicles showed enlargement of subcutaneous hair bulbs and formation of inner root sheaths, which are typical morphological characteristics of transformation to the anagen phase. On the 12th day after surgery, hair follicle stem cells in the wound area were effectively activated—manifested by a significant upregulation of β-catenin expression, while the expression of secretory curl-associated protein 2 (SFRP2) was inhibited.

[0071] In summary, the application of extracellular vesicles of prepuce mesenchymal stem cells and the curcumin complex of prepuce mesenchymal stem cells can promote hair regeneration by activating hair follicle stem cells. Among them, the prepuce mesenchymal stem cell extracellular vesicle / curcumin complex has a better effect on promoting hair regeneration.

[0072] Experimental Example 5 Inclusion criteria for volunteers included: diagnosis of AGA by a dermatologist based on clinical presentation and dermoscopy; age 18-50 years; alopecia classification conforming to Hamilton-Norwood classification II-V; relatively stable hair loss status and absence of acute scalp inflammation within the past 3 months; willingness to cooperate with follow-up and imaging during the trial and signing an informed consent form. Exclusion criteria included: coexisting cicatricial alopecia, alopecia areata, tinea capitis, or other scalp diseases; use of treatment products that may affect hair growth within the past 6 months (including but not limited to minoxidil, finasteride, microneedling growth factor injections, hair transplantation, etc.); allergy to curcumin-related components; and individuals deemed unsuitable for enrollment by the researchers. During the trial, volunteers were not allowed to use any other products that might affect the results, and were only permitted to use mild cleansing shampoos.

[0073] Volunteers underwent microneedling with roller-assisted percutaneous delivery: After cleaning and drying the scalp, trained operators used disposable sterile roller instruments to perform microneedling treatment on the hair loss area. The rolling directions were longitudinal, transverse, and diagonal. After roller treatment, the prepuce mesenchymal stem cell extracellular microvesicle curcumin complex from Example 1 was evenly applied to the treated area and gently massaged to promote absorption. The dosage per administration was 5 × 10⁻⁶. 8 The number of extracellular vesicles was assessed by standardized scalp localization photography after counting the number of uses (photographs taken before the first administration and after the last administration).

[0074] Volunteer 1 (male, 40 years old) experienced significant hair growth and increased density after three consecutive treatments (twice a week).

[0075] Volunteer 2 (male, 50 years old) experienced significant hair growth and increased density after two consecutive treatments (administered once a week).

[0076] Volunteer 3 (male, 35 years old) experienced significant hair growth and increased density after six consecutive treatments (once a week).

[0077] The embodiments of the present invention have been described in detail above with reference to the examples. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention.

Claims

1. The use of a complex in the preparation of a medicament for treating androgenetic alopecia, characterized in that, The complex comprises extracellular microvesicles loaded with curcumin; the extracellular microvesicles are derived from foreskin mesenchymal stem cells.

2. The application according to claim 1, characterized in that, The particle size of the complex is 40 nm-200 nm; and / or, the curcumin content in the complex is 15 ng-30 ng per 100 million extracellular vesicles.

3. The application according to claim 1, characterized in that, The preparation method of the complex includes the following steps: The complex was obtained by culturing foreskin mesenchymal stem cells in a culture medium containing curcumin and separating the extracellular vesicles from the culture medium.

4. The application according to claim 3, characterized in that, The culture is a 3D culture.

5. The application according to claim 4, characterized in that, The 3D culture includes either 3D culture without support or 3D culture with support.

6. The application according to claim 5, characterized in that, The supportless 3D culture includes at least one of the hanging drop method, the low-adhesion spherical culture plate method, and the magnetic levitation method; and / or, the supported 3D culture includes using at least one of the hydrogel and microcarrier as a support for 3D culture.

7. The application according to claim 6, characterized in that, The material of the microcarrier includes at least one of natural polymer materials and synthetic polymer materials; and / or, the density of the microcarrier in the culture medium is 5 g / L-7 g / L.

8. The application according to claim 7, characterized in that, The natural polymer material includes at least one of gelatin, collagen, cellulose, chitin, alginate, and dextran; and / or, the synthetic polymer material includes poly(hydroxyethyl methacrylate) and polylactic acid. At least one of glycolic acid copolymer and polylactide.

9. The application according to claim 1, characterized in that, The dosage form of the drug includes any one of tablets, capsules, solutions, aerosols, sprays, ointments, or films.

10. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

Images