A method for preparing leukocytes, use of extracellular small vesicles or complexes thereof

Abstract

The application discloses a leukocyte preparation method and application of extracellular small vesicles or complexes thereof, and relates to the technical field of biotechnology.The application finds that leukocyte extracellular small vesicles can effectively improve the damage resistance and proliferation capacity of hair follicle cells, promote hair regeneration, and can be used for treating alopecia.After the leukocyte extracellular small vesicles are loaded with curcumin and other effective components for treating alopecia, the treatment effect on alopecia can be greatly improved, and allergic reactions are not easily caused.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing leukocytes, the application of their extracellular vesicles or complexes. 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] Currently, the main drugs for treating androgenetic alopecia are finasteride and minoxidil, which have problems such as slow onset of action and poor efficacy. While hair transplantation can quickly change appearance, it does not improve the internal environment and local microenvironment, and the survival time of transplanted hair is relatively short, with hair loss recurring after a few years.

[0004] Therefore, there is a need for a new drug that can be used to treat androgenetic alopecia. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention proposes the use of extracellular microvesicles of leukocytes or complexes thereof in the preparation of products for the prevention and / or treatment of hair loss.

[0006] The present invention also provides a complex of extracellular microvesicles of leukocytes.

[0007] The present invention also provides a method for preparing the above-mentioned composite.

[0008] The present invention also provides a method for preparing leukocytes.

[0009] The use of extracellular microvesicles of leukocytes or complexes thereof according to a first aspect of the present invention in the preparation of products for the prevention and / or treatment of hair loss.

[0010] The application of the present invention, according to embodiments thereof, has at least the following beneficial effects: Extracellular vesicles of leukocytes can effectively enhance the damage resistance and proliferation ability of hair follicle cells, promote hair regeneration, and can be used to treat hair loss (especially androgenetic alopecia).

[0011] According to some embodiments of the present invention, the hair loss includes androgenetic alopecia.

[0012] According to some embodiments of the present invention, the product includes at least one of a medicine and a personal care product.

[0013] According to some embodiments of the present invention, the hair care product includes at least one of shampoo, conditioner, hair serum, and hair oil.

[0014] According to some embodiments of the present invention, the content of extracellular vesicles or complexes of leukocytes in the product is 1wt%-99wt%.

[0015] 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.

[0016] 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.

[0017] According to some embodiments of the present invention, the drug further includes pharmaceutically acceptable excipients.

[0018] 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.

[0019] According to some embodiments of the present invention, the product further includes other effective ingredients for preventing and / or treating hair loss. These other effective ingredients for preventing and / or treating hair loss are loaded in the cellular extracellular vesicles.

[0020] According to some embodiments of the present invention, the other effective ingredients for preventing and / or treating hair loss include, but are not limited to, minoxidil or finasteride.

[0021] According to some embodiments of the present invention, the complex uses the extracellular vesicles of leukocytes as a carrier to load effective ingredients for treating hair loss.

[0022] According to some embodiments of the present invention, the complex comprises extracellular microvesicles of leukocytes loaded with curcumin.

[0023] According to a second aspect of the present invention, a complex comprises extracellular microvesicles of leukocytes loaded with curcumin.

[0024] The composite according to embodiments of the present invention has at least the following beneficial effects: The complex described in this embodiment (extracellular vesicles of leukocytes loaded with curcumin) solves the problems of poor absorption and solubility of curcumin. It contains the functional components of complete leukocyte extract but does not contain allergens such as fragmented cell debris. It organically combines the functions of extracellular vesicles of leukocytes with those of curcumin, possessing the dual functions of both leukocyte extract and curcumin. This significantly enhances the anti-damage and proliferative capacity of hair follicle cells, effectively improving hair loss. Furthermore, the complex is completely encapsulated by a cell membrane, ensuring the activity of the active ingredients during storage. The intact cell membrane also reduces antigenicity, minimizing the likelihood of allergic reactions, and provides high permeability.

[0025] According to some embodiments of the present invention, the particle size of the extracellular microvesicles of leukocytes is 30 nm-200 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, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm.

[0026] According to some embodiments of the present invention, the particle size of the composite is 30 nm to 200 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, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm.

[0027] According to some embodiments of the present invention, the curcumin content in the complex is 20 ng to 100 ng per 100 million leukocyte extracellular vesicles. For example, the curcumin content in the complex can be 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng, 50 ng, 55 ng, 60 ng, 65 ng, 70 ng, 75 ng, 80 ng, 85 ng, 90 ng, 95 ng, or 100 ng.

[0028] According to some embodiments of the present invention, a method for loading the extracellular vesicles with curcumin includes at least one of electroporation, microinjection, sonication, and culturing leukocytes in a curcumin-containing culture medium.

[0029] The method for preparing the composite according to the second aspect embodiment of the third aspect of the present invention includes the following steps: The complex is obtained by culturing leukocytes in a culture medium containing curcumin and separating the extracellular vesicles from the culture medium.

[0030] The preparation method according to embodiments of the present invention has at least the following beneficial effects: The preparation method of the embodiment involves adding curcumin to a leukocyte culture system. The leukocytes absorb curcumin into the cells via endocytosis and then secrete the endocytic curcumin through extracellular vesicles via paracrine signaling.

[0031] According to some embodiments of the present invention, the culture medium is an extracellular vesicle-free culture medium.

[0032] 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 leukocytes.

[0033] 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.

[0034] According to some embodiments of the present invention, the culture temperature is 36-38°C, preferably 37°C.

[0035] 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.

[0036] According to some embodiments of the present invention, the culture includes at least one of 2D culture and 3D culture. This facilitates product quality control, 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.

[0037] According to some embodiments of the present invention, the 3D culture includes either 3D static culture or 3D dynamic culture.

[0038] According to some embodiments of the present invention, the 3D dynamic culture method includes at least one of stirring, perfusion, and rotation.

[0039] According to some embodiments of the present invention, the 3D culture includes either a supportless 3D culture or a supported 3D culture.

[0040] 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.

[0041] 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.

[0042] According to some embodiments of the present invention, the material of the microcarrier includes a biocompatible material.

[0043] 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.

[0044] According to some embodiments of the present invention, the natural polymeric material includes at least one of gelatin, collagen, cellulose, chitin, alginate, and dextran.

[0045] 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).

[0046] 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.

[0047] 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.

[0048] A method for preparing leukocytes according to a fourth aspect of the present invention includes the following steps: S1. Expose mesenchymal stem cells to the first culture medium, culture them, and obtain inflammation-permitted mesenchymal stem cells; The first culture medium includes phytohemagglutinin (PHA), interleukin-2 (IL-2), interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α). S2. Expose the inflammatory-permitted mesenchymal stem cells to a second culture medium and culture them to obtain preliminarily induced leukocytes; The second culture medium includes IL-7, IL-13, IL-33, WKYMVm hexapeptide, butyramide, and RORγt agonist 3; S3. Contact the preliminarily induced white blood cells with the third culture medium and culture them to obtain the white blood cells; The third culture medium includes IL-7, IL-13, IL-33, TNF-α, WKYMVm hexapeptide, butyramide, RORγt agonist 3, carcinoembryonic antigen CEA, cancer antigen 125, MAGE-A1, NY-ESO-1, HER2 / neu, and EGFR.

[0049] The preparation method according to embodiments of the present invention has at least the following beneficial effects: Following PHA and IL-2 activation of cells, INF-γ and TNF-α enhance the inflammation-permitting reprogramming system, thereby increasing the anti-inflammatory properties of mesenchymal stem cells (MSCs) and maintaining these properties in induced dendritic cells (DCs). A six-factor system comprising IL-7, IL-13, IL-33, WKYMVm hexapeptide, butyzamide, and RORγt agonist 3 significantly improves the efficiency of MSC induction into leukocytes. Furthermore, multiple types of tumor antigen peptides can stimulate the immune system to generate an immune response against tumor cells, thereby stimulating DC maturation.

[0050] Mesenchymal stem cells (MSCs) possess multi-directional differentiation characteristics. By utilizing a combination of cellular factors, MSCs can be induced into leukocytes (specifically, dendritic cells, or DCs). The innovative induction method employs a two-step approach: first, reprogramming them into inflammation-permitting MSCs, and then inducing them into DCs, which enhances the anti-inflammatory properties of DCs. The preparation method described in this example avoids violations of transfusion laws and ethical controversies, enabling the industrial-scale production of leukocytes with good quality uniformity.

[0051] According to some embodiments of the present invention, the first culture medium comprises: 1 μg / mL-10 μg / mL PHA, 500 U / mL-2000 U / mL IL-2, 5 ng / mL-15 ng / mL IFN-γ, and 10 ng / mL-20 ng / mL TNF-α. For example, the concentration of PHA can be 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, 7 μg / mL, 7.5 μg / mL, 8 μg / mL, 8.5 μg / mL, 9 μg / mL, 9.5 μg / mL, or 10 μg / mL; the concentration of IL-2 can be 500 U / mL, 600 U / mL, 700 U / mL, 800 U / mL, 900 U / mL, 1000 U / mL, 1100 U / mL, 1200 U / mL, 1300 U / mL, 1400 U / mL, 1500 U / mL, 1600 U / mL, 1700 U / mL, or 10 μg / mL. U / mL, 1800 U / mL, 1900 U / mL or 2000 U / mL; the concentration of IFN-γ can be 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, 8.5 ng / mL, 9ng / mL, 9.5 ng / mL, 10 ng / mL, 10.5 ng / mL, 11 ng / mL, 11.5 ng / mL, 12 ng / mL, 12.5 ng / mL, 13 ng / mL, 13.5 ng / mL, 14 ng / mL, 14.5 ng / mL or 15 ng / mL; the concentration of TNF-α can be 10 ng / mL, 10.5 ng / mL, 11 ng / mL, 11.5 ng / mL, 12 ng / mL, 12.5 ng / mL, 13 ng / mL, 13.5 ng / mL, 14ng / mL, 14.5 ng / mL, 15 ng / mL, 15.5 ng / mL, 16 ng / mL, 16.5 ng / mL, 17 ng / mL, 17.5 ng / mL, 18 ng / mL, 18.5 ng / mL, 19 ng / mL, 19.5 ng / mL or 20 ng / mL.

[0052] According to some embodiments of the present invention, the second culture medium comprises 50 ng / mL-100 ng / mL IL-7, 50 ng / mL-150 ng / mL IL-13, 50 ng / mL-150 ng / mL IL-33, 10 ng / mL-50 ng / mL WKYMVm hexapeptide, 2 ng / mL-10 ng / mL butyramide, and 1 ng / mL-5 ng / mL RORγt agonist 3.For example: The concentration of IL-7 can be 50 ng / mL、55 ng / mL、60 ng / mL、65 ng / mL、70 ng / mL、75 ng / mL、80 ng / mL、85 ng / mL、90ng / mL、95 ng / mL or 100 ng / mL; The concentration of IL-13 can be 50 ng / mL、55 ng / mL、60 ng / mL、65 ng / mL、70 ng / mL、75 ng / mL、80 ng / mL、85 ng / mL、90 ng / mL、95 ng / mL、100 ng / mL、105 ng / mL、110 ng / mL、115 ng / mL、120 ng / mL、125 ng / mL、130 ng / mL、135 ng / mL、140 ng / mL、145 ng / mL or 150 ng / mL ng / mL; the concentration of IL-33 can be 50 ng / mL、55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, 100 ng / mL, 105 ng / mL, 110 ng / mL, 115 ng / mL, 120 ng / mL, 125 ng / mL, 130 ng / mL, 135 ng / mL, 140 ng / mL, 145 ng / mL or 150 ng / mL; the concentration of the WKYMVm hexapeptide may be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL or 50 ng / mL; the concentration of the butyramide may be 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, 8.5 ng / mL, 9 ng / mL, 9.5 ng / mL or 10 ng / mL; the concentration of the RORγt agonist 3 may be 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL or 5 ng / mL.

[0053] According to some embodiments of the present invention, the third culture medium comprises 20 ng / mL-50 ng / mL IL-7, 20 ng / mL-50 ng / mL IL-13, 20 ng / mL-50 ng / mL IL-33, 10 ng / mL-30 ng / mL TNF-α, 5 ng / mL-10 ng / mL WKYMVm hexapeptide, 2 ng / mL-5 ng / mL butyramide, 1 ng / mL-3 ng / mL RORγt agonist 3, 50 ng / mL-100 ng / mL carcinoembryonic antigen CEA, 50 ng / mL-100 ng / mL cancer antigen 125, 50 ng / mL-100 ng / mL LMAGE-A1, 50 ng / mL-100 ng / mL NY-ESO-1, 50 ng / mL-100 ng / mL HER2 / neu, 50 ng / mL-100ng / mL EGFR. For example: the concentration of IL-7 can be 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, or 50 ng / mL; the concentration of IL-13 can be 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, or 50 ng / mL; the concentration of IL-33 can be 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 45 ng / mL, or 50 ng / mL; the concentration of TNF-α can be 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, or 30 ng / mL; and the concentration of WKYMVm hexapeptide can be 5 ng / mL, 5.5 ng / mL, 6 ng / mL, or 6.5 ng / mL. ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, 8.5 ng / mL, 9 ng / mL, 9.5 ng / mL or 10 ng / mL; the concentration of butanamide can be 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL or 5 ng / mL; the concentration of the RORγt agonist 3 can be 1 ng / mL, 1.2 ng / mL, 1.4 ng / mL, 1.6 ng / mL, 1.8 ng / mL, 2 ng / mL, 2.2 ng / mL, 2.4 ng / mL, 2.6 ng / mL, 2.8 ng / mL or 3 ng / mL.The concentration of carcinoembryonic antigen (CEA) can be 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. The concentration of cancer antigen 125 can be 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL, or 100 ng / mL. The concentration of MAGE-A1 can be 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL or 100 ng / mL. The concentration of NY-ESO-1 can be 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL or 100 ng / mL. The concentration of HER2 / neu can be 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95ng / mL or 100 ng / mL. The concentration of EGFR can be 50 ng / mL, 55 ng / mL, 60 ng / mL, 65 ng / mL, 70 ng / mL, 75 ng / mL, 80 ng / mL, 85 ng / mL, 90 ng / mL, 95 ng / mL or 100 ng / mL.

[0054] According to some embodiments of the present invention, the cell culture media in the first, second, and third culture media are substantially free of serum. The cell culture media include lymphocyte culture media.

[0055] According to some embodiments of the present invention, the cell culture medium includes at least one of the following: KBM 581 lymphocyte serum-free medium, GT-T551 H3 medium, 12-725F UltraCULTURE medium, PC-1™ medium, X-VIVO™ serum-free medium, UltraCHO™ medium, UltraMDCK™ medium, Pro293™CDM system, Insect-XPRESS™ medium, HL-1™ completely serum-free medium, UltraDOMA™ medium, CTS™ serum-free cell culture medium, LymGro™ lymphocyte serum-free medium, TexMACS medium, IMSF100 medium, and UltraDOMA-PF™ protein-free medium.

[0056] According to some embodiments of the present invention, the culture in S1, S2, and S3 of the preparation method is independently selected from 2D culture and 3D culture, respectively.

[0057] According to some embodiments of the present invention, the mesenchymal stem cells are derived from at least one tissue selected from bone marrow, periosteum, synovium, skeletal muscle, dental pulp, umbilical cord, umbilical cord blood, placenta, peripheral blood, fat, and foreskin.

[0058] According to some embodiments of the present invention, the culture temperature in steps S1, S2, and S3 of the preparation method is 36-38°C.

[0059] According to some embodiments of the present invention, the culture time in S1 of the preparation method is 2-6 days.

[0060] According to some embodiments of the present invention, the culture time in S2 of the preparation method is 7-10 days. Specifically, S2 includes mixing and culturing mesenchymal stem cells with a second culture medium for 2-4 days, then continuing to add an equal volume of the second culture medium and culturing, replacing the 45v / v%-55v / v% culture medium with an equal volume of the second culture medium every 2-4 days.

[0061] According to some embodiments of the present invention, the culture time in S3 of the preparation method is 2-5 days.

[0062] 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

[0063] Figure 1Characterization results of induced leukocyte (DC) extracellular vesicles in Comparative Example 1 and the induced leukocyte (DC) extracellular vesicle / curcumin complex in Example 2. A: Nanoparticle tracking size analysis results and transmission electron microscopy images (scale bar: 200 nm) of induced leukocyte (DC) extracellular vesicles in Comparative Example 1; B: Nanoparticle tracking size analysis results and transmission electron microscopy images (scale bar: 200 nm) of the induced leukocyte (DC) extracellular vesicle / curcumin complex in Example 2; C: Western blot detection results; D: Fluorescence spectrophotometer detection results; Figure 2 Example 2 illustrates the protective effect of the induced leukocyte (DC) extracellular microvesicle / curcumin complex against hair follicle cell damage; * indicates significant difference. p <0.05), **** indicates a highly significant difference ( p <0.0001).

[0064] Figure 3 The therapeutic effects of induced leukocyte (DC) extracellular vesicles in Comparative Example 1 and the induced leukocyte (DC) extracellular vesicle / curcumin complex in Example 2 on the AGA model are shown in the figures. A: Photographs of representative mice; B: Statistical results of new hair growth length.

[0065] Figure 4 The photos show the hair growth of volunteer 1 before and after drug treatment; from left to right, the photos are before hair growth and after ten treatments.

[0066] Figure 5 The photos show the hair growth of volunteer 2 before and after drug treatment; from left to right, the photos are before hair growth and after ten treatments.

[0067] Figure 6 The photos show the hair growth of volunteer 3 before and after drug treatment; from left to right, the photos are before hair growth, after one hair growth treatment, and after two hair growth treatments. Detailed Implementation

[0068] 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.

[0069] 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.

[0070] In the description of this invention, the use of terms such as first, second, third, etc., is only for the purpose of distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of technical features indicated, or implicitly indicating the order of the technical features indicated.

[0071] 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.

[0072] Example 1 This example provides a method for obtaining leukocytes (DCs) using a serum-free culture medium and a two-step reprogramming induction regulator, as follows: (1) Preparation of culture medium: Preparation method of the first-step inflammation-permitted reprogramming medium: Add the prescribed amounts of PHA (final concentration 5 μg / mL), IL-2 (final concentration 1000 U / mL), IFN-γ (final concentration 10 ng / mL), and TNF-α (final concentration 15 ng / mL) to 250 mL of Corning 581 medium. After complete dissolution (1 hour), filter sterilize using a 0.22 μm filter and store at -20℃ for later use.

[0073] DC basal induction medium is used to initially induce inflammatory permitting mesenchymal stem cells into DC cells. The preparation method is as follows: Add the prescribed amounts of IL-7 (final concentration 75 ng / mL), IL-13 (final concentration 100 ng / mL), IL-33 (final concentration 100 ng / mL), WKYMVm hexapeptide (sequence Trp-Lys-Tyr-Met-Val-Met-NH2; final concentration 30 ng / mL), Butyzamide (final concentration 6 ng / mL), and RORγt agonist 3 (final concentration 3 ng / mL) to 250 mL of Corning 581 medium. After complete dissolution (1 hour), filter sterilize using a 0.22 μm filter and store at -20℃ for later use.

[0074] DC enhancement induction medium is used to enhance the function of DC cells obtained through basal induction. Its preparation method is as follows: The formulated amounts of IL-7 (final concentration 35 ng / mL), IL-13 (final concentration 35 ng / mL), IL-33 (final concentration 35 ng / mL), TNF-α (20 ng / mL), WKYMVm hexapeptide (sequence Trp-Lys-Tyr-Met-Val-Met-NH2; final concentration 7.5 ng / mL), Butyzamide (final concentration 3.5 ng / mL), RORγt agonist 3 (final concentration 2 ng / mL), carcinoembryonic antigen CEA (final concentration 50 ng / mL), cancer antigen 125 (CA-125, final concentration 50 ng / mL), MAGE-A1 (final concentration 50 ng / mL), NY-ESO-1 (final concentration 50 ng / mL), and HER2 / neu (human epidermal growth factor receptor 2, final concentration 50 ng / mL) are added. After dissolving EGFR (epidermal growth factor receptor, final concentration 50 ng / mL) in 250 mL of Corning 581 medium for 1 hour, the medium was filtered through a 0.22 μm filter for sterilization and stored at -20°C for later use.

[0075] (2) DC culture: (2.1) First step: Add cytodex 1 microcarrier (Corning brand, dosage 0.65 g / L, the same below) to the spinner flask culture system, seed 150 foreskin mesenchymal stem cells, culture the foreskin mesenchymal stem cells in the first-step inflammation-permitted reprogramming medium, and continue to culture in a 37℃, 5% CO2 incubator. After 3 days, the foreskin mesenchymal stem cells will grow to completely cover the surface of the microcarrier, and inflammation-permitted mesenchymal stem cells will be obtained. (2.2) Second step: Day 0 is the day when the foreskin mesenchymal stem cells completely cover the surface of the microcarrier.

[0076] On day 0, 250 mL of DC basal induction medium was added to the 3D culture spinner flask. On the third day, two days later, add 250 mL of new DC basal induction medium to bring the total volume to 500 mL. On day 5, the specific steps are as follows: remove the roller bottle from the incubator, place it in the biosafety cabinet, let it stand for 15-20 minutes to wait for the microcarriers and cells to settle to the bottom, then carefully aspirate the upper culture medium with a 50 mL pipette, and replace the culture medium with 250 mL of DC enhancement induction medium; culture until day 8 to obtain mature DC cells.

[0077] Remove the rotating flask from the incubator and place it in a biosafety cabinet. Let it stand for 15-20 minutes to allow the microcarriers and cells to settle at the bottom. Then, carefully aspirate the upper culture medium using a 50 mL pipette. Transfer the remaining cells and microcarriers at the bottom to 3-5 50 mL centrifuge tubes. Add 10 mL of PBS-EDTA (pre-cooled at 4°C, EDTA concentration of 1 mM) to each centrifuge tube containing microcarriers and cells, and incubate at 4°C for 10-15 minutes. Continue to add 10 mL of pre-cooled physiological saline to the centrifuge tube, rinse vigorously 3-4 times, and collect the solution in a 50 mL centrifuge tube. Make up the volume to 45 mL with physiological saline, centrifuge at 1200 rpm for 10 minutes, and discard the supernatant. The cell pellet is the DC cells induced by 3D foreskin mesenchymal stem cells.

[0078] Flow cytometry was used to verify the dendritic cell markers on the surface of the collected cells. The test results are shown in Table 1.

[0079] Table 1

[0080] Example 2 This example provides a method for preparing induced extracellular vesicle / curcumin complexes of leukocytes (DCs), with the following steps: (1) DC cells were cultured in a 3D 500 mL fermenter. The DC cells obtained in Example 1 were cultured at a density of 6000 cells / cm³. 2 Seed the cells at the density of the bottom growth area into the dextran-based microcarrier culture medium (3 g / L microcarrier, the remainder is DMEM / F12 medium), incubate overnight, adjust the rotation speed (~30 rpm) until the cells do not settle and aggregate, and culture for 4-5 days; (2) Replace the DC cell culture medium with curcumin DMEM / F12 medium, incubate at 37°C and 5% CO2 for 48 h, and then collect the supernatant. During incubation, DC cells endocytose curcumin into the cells and then secrete curcumin into the cell culture supernatant through paracrine signaling. 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.

[0081] (3) The supernatant was centrifuged at 4℃ and 300×g for 10 min, and then centrifuged at 4℃ and 2000×g for 20 min to remove residual cell debris and larger organelles. Then, the supernatant was further purified using a 0.22 μm filter. The purified supernatant was initially concentrated and filtered using a 500 kD hollow fiber membrane, and then further separated using a HiScreen CaptoCore 700 gel column combined with an AKTA Pure 25 macromolecular separation and purification system: all chromatography solvents (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 CaptoCore 700 gel column was installed, and the program was adjusted until the column equilibrated. After preparation, the supernatant obtained before Superloop loading was used to fix the sample collection volume, and the filtrate was collected after completion.

[0082] High-concentration 3D engineered extracellular vesicles / curcumin complexes of induced leukocytes were obtained by centrifugation using a 100 kD ultrafiltration tube.

[0083] Comparative Example 1 This example provides a method for preparing extracellular microvesicles of induced leukocytes (DCs). The steps are basically the same as those in Example 2, except that the curcumin DMEM / F12 medium in step (2) is replaced with an equal volume of DMEM / F12 medium.

[0084] Detection Example 1 (1) The particle size concentration and morphology of the nanoparticles (leukocyte extracellular vesicles / curcumin complex in Example 2) were analyzed by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), respectively. The extracted nanoparticles were characterized to meet the international standards for extracellular vesicles by Western blotting and fluorescence activated cell detection of characteristic proteins.

[0085] Add lysis buffer (Beyotime C3632) and sEVs samples at a 1:1 volume ratio, gently mix by pipetting, and lyse on ice for 10 min. Centrifuge at 12000×g for 5 min at 4℃, collect the supernatant, and determine the curcumin concentration using a fluorescence spectrophotometer. Set the excitation wavelength to 425 nm and detect the emission intensity from 460 to 700 nm to characterize whether the extracellular vesicle / curcumin complex of leukocytes was successfully loaded with curcumin.

[0086] The results are as follows Figure 1 As shown.

[0087] Nanoparticle tracking analysis showed that the average sizes of the induced leukocyte (DC) extracellular vesicles in Comparative Example 1 and the induced leukocyte (DC) extracellular vesicle / curcumin complex in Example 2 were 89.2 nm and 91.8 nm, respectively.

[0088] The morphology of nanoparticles under transmission electron microscopy showed that both the extracellular vesicles of induced leukocytes (DCs) and the curcumin complex of extracellular vesicles of induced leukocytes (DCs) had typical cup-shaped structures of less than 200 nm.

[0089] Western blot results showed that the curcumin complex of induced leukocyte (DC) extracellular microvesicles exhibited enrichment of extracellular microvesicle markers (Alix, CD63, TSG101, CD81, and CD9), but was negative for Calnexin markers, further validating the successful isolation of the induced leukocyte (DC) extracellular microvesicle / curcumin complex.

[0090] Compared to induced leukocyte (DC) extracellular vesicles, the DC / curcumin complex exhibited a distinct curcumin fluorescence peak at an emission wavelength of 530 nm, demonstrating successful curcumin loading. (Per 1.5 × 10⁻⁶) 9 Each extracellular vesicle carries 1 μg of curcumin.

[0091] Detection Example 2 The therapeutic effect of inducing extracellular microvesicles / curcumin complex in leukocyte (DC) cells on androgen (DHT) damage model in dermal papilla cells. Well-grown 3rd-5th generation (P3-P5) human dermal papilla cells (HDPCs) were used, at a concentration of 5 × 10⁻⁶ cells / cells. 3 Cells were seeded at a density of 100 μL per well in 96-well plates. After 24 hours of culture and cell adhesion, the supernatant was discarded, and complete medium containing 10 μmol / L dihydrotestosterone (DHT) was added for intervention, with 4 replicates per group. DHT was pre-dissolved in anhydrous ethanol to prepare a stock solution (ensuring the final ethanol concentration in the medium was below 0.1% before use), and a control group containing an equal volume of solvent was also included. (Drug (5 × 10⁻⁶)) 7 After treating each well with extracellular vesicles of leukocytes (DCs) or extracellular vesicles of leukocytes (DCs) / curcumin complex for 24 or 48 hours, 10 μL of CCK-8 solution was added to each well, and incubation continued for 2 hours. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability (%) was calculated as (OD value of experimental group / OD value of control group) × 100%.

[0092] The results are as follows Figure 2 As shown.

[0093] The cell survival rate of dermal papilla cells after DHT-induced damage was 60%; the cell survival rate of dermal papilla cells after intervention with simple induction of extracellular microvesicles of leukocytes (DCs) was 80%; and the cell survival rate of dermal papilla cells after intervention with induction of extracellular microvesicles of leukocytes (DCs) / curcumin complex was 110%; there were significant differences among the three groups.

[0094] Detection Example 3 Male C57BL / 6 mice aged 6–8 weeks and weighing 20–24 g were selected. After 7 days of acclimatization, on day 1 of the experiment, a 5 cm × 3 cm area of ​​hair was removed from the back of all mice. The animals were then randomly divided into five groups: a normal control group (Control group), a model group, a positive control group (minoxidil group), a model + induced leukocyte (DC) extracellular vesicle group, and a model + induced leukocyte (DC) extracellular vesicle / curcumin complex group. During the modeling period, the model group, positive control group (minoxidil group), model + induced leukocyte (DC) extracellular vesicle group, and model + induced leukocyte (DC) extracellular vesicle / curcumin complex group received intraperitoneal injections of 0.1 mL of testosterone propionate (dissolved in physiological saline, dosage 1 mg / day) daily. The normal control group received an equal volume of physiological saline solution intraperitoneally. This treatment was continued for 28 days. Additionally, the positive control group received a 2% minoxidil solution (100 μL / mouse / day) applied topically to the hair-removed area 30 minutes after each modeling administration. The model + induced leukocyte (DC) extracellular vesicle group and the model + induced leukocyte (DC) extracellular vesicle / curcumin complex group received subcutaneous injections of either the induced leukocyte (DC) extracellular vesicle from Example 1 or the induced leukocyte (DC) extracellular vesicle / curcumin complex from Example 2 at six points in the hair-removed area on the first day and 14th day of modeling administration. Each mouse received a total of 10 mg of the induced leukocyte (DC) extracellular vesicle from Example 1 or the induced leukocyte (DC) extracellular vesicle / curcumin complex from Example 2. 10 Nanoparticle concentration.

[0095] Photos were taken on days 1, 7, 14, 21, and 28 after hair removal to record hair growth in mice. On days 14, 21, and 28, hairs were randomly plucked from the shaved back area of ​​the mice. Ten hairs were plucked from each mouse, and the length of the hairs was measured using calipers. The results are expressed as mean ± SD.

[0096] like Figure 3 As shown.

[0097] On day 21, the hair lengths of the control group, model group, model + induced dendritic cell (DC) extracellular vesicle group, model + induced dendritic cell (DC) extracellular vesicle / curcumin complex group, and minoxidil group were 5.563, 2.533, 3.850, 4.033, and 3.055 mm, respectively. There were statistically significant differences between the treatment groups and the model group. The hair lengths of the induced dendritic cell (DC) derived extracellular vesicles and their complex with curcumin were significantly higher than those in the model group. This indicates that the treatment drugs have hair regeneration-promoting activity.

[0098] On day 28, the hair lengths in the control group, model group, model + induced leukocyte (DC) extracellular vesicle group, model + induced leukocyte (DC) extracellular vesicle / curcumin complex group, and minoxidil group were 8.518, 4.010, 5.945, 6.685, and 5.153 mm, respectively. The model + induced leukocyte (DC) extracellular vesicle / curcumin complex group showed a statistically significant difference compared to the minoxidil group. This indicates that the induced leukocyte (DC) extracellular vesicle / curcumin complex is more effective than traditional minoxidil.

[0099] In summary, inducing extracellular vesicles in leukocytes (DCs), especially after binding with curcumin, has shown good therapeutic potential, providing new experimental evidence for the treatment of AGA.

[0100] Detection Example 4 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.

[0101] 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. Rolling directions were longitudinal, transverse, and diagonal. After roller treatment, the induced leukocyte (DC) extracellular vesicle / curcumin complex from Example 1 was evenly applied to the treated area and gently massaged to promote absorption. The dosage was 10 mg / dL based on the number of vesicles per administration. 9The evaluation method is to count the number of times it is used and then perform standardized scalp positioning photography.

[0102] Volunteer 1 (male, 35 years old) was given the drug once a week. After 10 treatments, his hair became noticeably thicker and new hair grew.

[0103] Volunteer 2 (male, 40 years old) was given the medication twice a week. After two weeks of use, microscopic examination of the hair on the top of the head showed that the hair was significantly thicker and longer.

[0104] Volunteer 3 (male, 36 years old) was given the drug twice a week. After two treatments, the amount of hair on the top of his head increased significantly.

[0105] 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. Application of extracellular microvesicles of leukocytes or complexes thereof in the preparation of products for the prevention and / or treatment of hair loss.

2. The application according to claim 1, characterized in that, The product includes at least one of the following: medicines and personal care products.

3. A composite, characterized in that, The complex comprises extracellular vesicles of leukocytes loaded with curcumin.

4. The composite according to claim 3, characterized in that, Methods for loading the extracellular vesicles with curcumin include at least one of electroporation, microinjection, sonication, and culturing leukocytes in a curcumin-containing culture medium.

5. The method for preparing the composite according to claim 3 or 4, characterized in that, Includes the following steps: The complex is obtained by culturing leukocytes in a culture medium containing curcumin and separating the extracellular vesicles from the culture medium.

6. The preparation method according to claim 5, characterized in that, The concentration of curcumin in the culture medium was 0.625 μM-10 μM.

7. A method for preparing leukocytes, characterized in that, Includes the following steps: S1. Expose mesenchymal stem cells to the first culture medium, culture them, and obtain inflammation-permitted mesenchymal stem cells; The first culture medium includes phytohemagglutinin, IL-2, INF-γ, and TNF-α; S2. Expose the inflammatory-permitted mesenchymal stem cells to a second culture medium and culture them to obtain preliminarily induced leukocytes; The second culture medium includes IL-7, IL-13, IL-33, WKYMVm hexapeptide, butyramide, and RORγt agonist 3; S3. Contact the preliminarily induced white blood cells with the third culture medium and culture them to obtain the white blood cells; The third culture medium includes IL-7, IL-13, IL-33, TNF-α, WKYMVm hexapeptide, butyramide, RORγt agonist 3, carcinoembryonic antigen CEA, cancer antigen 125, MAGE-A1, NY-ESO-1, HER2 / neu, and EGFR.

8. The preparation method according to claim 7, characterized in that, The first culture medium comprises: 1 μg / mL-10 μg / mL PHA, 500 U / mL-2000 U / mL IL-2, 5 ng / mL-15 ng / mL IFN-γ, and 10 ng / mL-20 ng / mL TNF-α; And / or, the second culture medium comprises 50 ng / mL-100 ng / mL IL-7, 50 ng / mL-150 ng / mL IL-13, 50 ng / mL-150 ng / mL IL-33, 10 ng / mL-50 ng / mL WKYMVm hexapeptide, 2 ng / mL-10 ng / mL butyramide, and 1 ng / mL-5 ng / mL RORγt agonist 3; And / or, the third culture medium comprises 20 ng / mL-50 ng / mL IL-7, 20 ng / mL-50 ng / mL IL-13, 20 ng / mL-50 ng / mL IL-33, 10 ng / mL-30 ng / mL TNF-α, 5 ng / mL-10 ng / mL WKYMVm hexapeptide, 2 ng / mL-5 ng / mL butyramide, 1 ng / mL-3 ng / mL RORγt agonist 3, 50 ng / mL-100 ng / mL carcinoembryonic antigen CEA, 50 ng / mL-100 ng / mL cancer antigen 125, 50 ng / mL-100 ng / mL MAGE-A1, 50 ng / mL-100 ng / mL NY-ESO-1, 50 ng / mL-100 ng / mL HER2 / neu, 50 ng / mL-100 ng / mL IL-7, 20 ng / mL-50 ng / mL IL-13, 20 ng / mL-50 ng / mL IL-33, 10 ng / mL-30 ng / mL TNF-α, 5 ng / mL-10 ng / mL WKYMVm hexapeptide, 2 ng / mL-5 ng / mL butyramide, 1 ng / mL-3 ng / mL RORγt agonist 3, 50 ng / mL-100 ng / mL carcinoembryonic antigen CEA, 50 ng / mL-100 ng / mL carcinoembryonic antigen 125, 50 ng / mL-100 ng / mL MAGE-A1, 50 ng / mL-100 ng / mL NY-ESO-1, 50 ng / mL-100 ng ng / mL EGFR.

9. The preparation method according to claim 7, characterized in that, The cell culture media in the first culture medium, the second culture medium, and the third culture medium each independently include at least one of the following: KBM 581 lymphocyte serum-free medium, GT-T551 H3 medium, 12-725F UltraCULTURE medium, PC-1™ medium, X-VIVO™ serum-free medium, UltraCHO™ medium, UltraMDCK™ medium, Pro293™CDM system, Insect-XPRESS™ medium, HL-1™ completely serum-free medium, UltraDOMA™ medium, CTS™ serum-free cell medium, LymGro™ lymphocyte serum-free medium, TexMACS medium, IMSF100 medium, and UltraDOMA-PF™ protein-free medium.

10. The preparation method according to claim 7, characterized in that, The mesenchymal stem cells are derived from at least one of the following tissues: bone marrow, periosteum, synovium, skeletal muscle, dental pulp, umbilical cord, umbilical blood, placenta, peripheral blood, fat, and foreskin.

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