A composition for inducing hair follicle regeneration and promoting hair growth using exosomes, its preparation method and application.

Abstract

This invention proposes a composition for inducing hair follicle regeneration and promoting hair growth using exosomes, its preparation method, and its application, belonging to the field of pharmaceutical technology. Cell activating factors and activating particles are added to serum-free MSC culture medium to culture human umbilical cord mesenchymal stem cells. After cell separation, exosomes and the culture medium are collected. The exosomes are embedded in liposomes, and the surface is modified to obtain ε-polylysine-modified exosome liposomes. Polygonum multiflorum, Platycladus orientalis leaves, and tea are added to the culture medium for fermentation. The mixture is filtered, the filtrate is evaporated and crystallized, filtered again, and reacted with the ε-polylysine-modified exosome liposomes to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes. This composition has good solubility, can target and deliver exosomes and fermentation products, significantly improves adhesion rate, inhibits bacterial biofilm formation, has anti-inflammatory effects, and provides long-acting, slow-release drugs. It has a good effect on promoting hair follicle regeneration and hair growth, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth, its preparation method, and its application. Background Technology

[0002] With the fast pace of modern life and increasing environmental degradation, more and more people are experiencing varying degrees of sub-health issues due to high levels of stress, overtime work, late nights, and smog. These issues include increased scalp oil secretion, scalp inflammation, hair follicle atrophy, and hair loss. Hair loss has become a growing concern for modern people, especially young people. Currently, the main solutions for preventing and treating hair loss are medications and surgery. Drug treatments, primarily finasteride and minoxidil, often have serious side effects and are prone to causing drug dependence; once discontinued, hair loss worsens. Surgical treatments are expensive, invasive, and require a recovery period. Therefore, developing natural hair loss prevention and hair regrowth ingredients to address hair loss is of great significance.

[0003] Patent document CN112704688A discloses a hair growth injection solution based on autologous adipose-derived stem cell exosomes. The injection solution comprises autologous adipose-derived stem cell exosomes and water for injection. Its preparation method includes the following steps: Step 1, selection of autologous fat; Step 2, preparation of adipose-derived stem cells; Step 3, preparation of adipose-derived stem cell exosomes; Step 4, preparation of the hair growth injection solution containing adipose-derived stem cell exosomes. Patent document CN110339338A discloses a hair growth agent comprising adipose-derived mesenchymal stem cell conditioned medium, fibroblast exosomes, mannitol, trehalose, and dextran 40. The adipose-derived mesenchymal stem cells are cultured using a medium containing 20-100 mJ / cm³. 2 UVB irradiation doses cause adipose mesenchymal stem cells to secrete a variety of bioactive factors, which can stimulate the hair papilla and activate hair follicle stem cells, achieving hair growth effects from the inside out.

[0004] However, we know that exosome activity varies depending on the culture environment. The cell type and state that secrete exosomes are also important factors affecting exosome function. Therefore, developing an exosome composition with high hair follicle regeneration activity for inducing hair follicle regeneration and promoting hair growth is of great significance. Summary of the Invention

[0005] The purpose of this invention is to propose a composition for inducing hair follicle regeneration and promoting hair growth using exosomes, its preparation method, and its application. It has good solubility, can target and deliver exosomes and fermentation products, significantly improves adhesion rate, inhibits bacterial biofilm formation, has good anti-inflammatory effect, and provides long-acting, slow-release drug. It has a good effect on promoting hair follicle regeneration and hair growth, and has broad application prospects.

[0006] The technical solution of this invention is implemented as follows:

[0007] This invention provides a method for preparing a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth. Cell activating factors and activating particles are added to serum-free MSC culture medium to culture human umbilical cord mesenchymal stem cells. After cell separation, the culture medium is centrifuged to collect exosomes and the culture medium after culture. The exosomes are embedded in liposomes, and their surfaces are coupled with ε-polylysine to obtain ε-polylysine-modified exosome liposomes. Polygonum multiflorum, Platycladus orientalis leaves, and tea are added to the culture medium for fermentation. The mixture is filtered, and the filtrate is evaporated and crystallized. After filtration, the filtrate is filtered again to obtain a cocrystal of the fermentation product. This cocrystal is reacted with the ε-polylysine-modified exosome liposomes to obtain a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth.

[0008] As a further improvement to the present invention, the following steps are included:

[0009] S1. Preparation of culture medium: Cell activating factors and activating particles were added to MSC serum-free culture medium to prepare the culture medium;

[0010] S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment until the cells reached 90% confluence. The cells were digested, activated particles were separated by magnetism, centrifuged, and the supernatant was discarded. The cells were resuspended in the new medium prepared in step S1 and seeded into the new medium for continued expansion culture under the original conditions.

[0011] S3. Isolation of exosomes: After the cells are amplified to 90% confluence in step S2, the culture medium is discarded, the cells are washed, and the exosome collection medium is added to the solid cells. The cells are then cultured again, filtered through a syringe filter, centrifuged at low temperature at the first speed, and the supernatant is collected. The cells are then centrifuged at low temperature at the second speed, and the supernatant is collected. The cells are then centrifuged at low temperature at the third speed, and the supernatant is collected. The centrifuged precipitate is collected. The supernatant from the last centrifugation is retained and resuspended in PBS buffer to prepare exosome buffer.

[0012] S4. Preparation of exosomes and liposomes: Cholesterol, phosphatidylserine, and lecithin were dissolved in dichloromethane-ethanol solution, exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated, and the mixture was freeze-dried to obtain exosomes and liposomes.

[0013] S5. Natural molecular modification: ε-polylysine was dissolved in water, EDC and NHS were added, the mixture was stirred and activated, exosomes and liposomes were added, the mixture was stirred and reacted, and then freeze-dried to obtain ε-polylysine-modified exosomes and liposomes.

[0014] S6. Preparation of fermentation product cocrystal: Wash, dry and crush Polygonum multiflorum, Platycladus orientalis leaves and tea leaves, add the supernatant of the last centrifugation in step S3, inoculate with fermentation bacteria, ferment and culture, filter, evaporate the filtrate to crystallize, filter again to obtain fermentation product cocrystal.

[0015] S7. Preparation of a composition for inducing hair follicle regeneration and promoting hair growth using exosomes: Fermentation product cocrystals and ε-polylysine-modified exosome liposomes were added to water, stirred and mixed, and freeze-dried to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

[0016] As a further improvement of the present invention, the cell activating factor in step S1 includes tumor necrosis factor α and interleukin-1β in a mass ratio of 3-5:2. The amount of the cell activating factor added is 0.2-0.5 mg / L, and the amount of the activating particles added is 1-2 g / L. The preparation method of the activating particles is as follows: magnetic Fe3O4 particles are added to N,N-dimethylformamide, zirconium tetrachloride is added, and the reaction is carried out with hydrothermal stirring. 2-hydroxyterephthalic acid is added, and the reaction is continued with heat preservation. The product is separated by magnets, washed, dried, added to ethanol, aluminum isopropoxide is added, water and alkali are added, the reaction is stirred, separated by magnets, washed, and dried to obtain the activating particles.

[0017] As a further improvement of the present invention, the mass ratio of the magnetic Fe3O4 particles, zirconium tetrachloride, 2-hydroxyterephthalic acid, aluminum isopropoxide, water and alkali is 10:3-3.5:2.4-2.8:2-4:3-5:0.5-1, the alkali is NaOH or KOH, the temperature of the hydrothermal stirring reaction is 120-130℃, the time is 1-3h, the time for continuing the reaction at the temperature is 1-3h, and the time for stirring the reaction is 3-5h.

[0018] As a further improvement of the present invention, the conditions for low oxygen, external magnetic field, infrared light irradiation, and microwave treatment in step S2 are as follows: oxygen content 1-3 v / v%, external magnetic field strength 0.2-0.4T, infrared light irradiation wavelength 700-900nm, microwave power 300-500W, and treatment time 12-20h; the inoculation into the new culture medium is carried out at a passage ratio of 1:4-6; the exosome collection culture medium in step S3 is αMEM medium + 1wt% recombinant human insulin + 1wt% lysine + 1wt% glutamine, and the culture conditions are as follows. The culture was carried out in a CO2 incubator with a CO2 concentration of 5v / v% for 36-48 hours. The filter membrane of the needle filter had a pore size of 0.22μm. The low temperature was 2-4℃. The first rotation speed was 250-350g for 5-15 minutes, the second rotation speed was 1800-2200g for 5-15 minutes, the third rotation speed was 8000-12000g for 20-30 minutes, and the fourth rotation speed was 100000-120000g for 1.5-2.5 hours. The pH value of the PBS buffer was 7.2-7.7.

[0019] As a further improvement of the present invention, the mass ratio of cholesterol, phosphatidylserine and lecithin in step S4 is 5-10:3-5:1-3, the volume ratio of dichloromethane-ethanol solution is 10-15:3-5, the temperature of constant temperature stirring is 20-25℃ and the time is 20-30 min, and the power of ultrasonic treatment is 1000-1200W and the time is 20-30 min.

[0020] As a further improvement of the present invention, the mass ratio of ε-polylysine, EDC, NHS and exosome liposomes in step S5 is 3-5:1-2:1-2:7-10, the stirring activation time is 20-40 min, and the stirring reaction time is 12-15 h.

[0021] As a further improvement of the present invention, the mass ratio of Polygonum multiflorum, Platycladus orientalis leaves, tea leaves, and the supernatant from the last centrifugation in step S3 is 3-5:2-5:1-3:50-70, and the fermentation bacteria are a seed culture of selenium-enriched yeast and Bacillus subtilis, with a bacterial count of 10. 8 -10 9 The inoculum concentration is cfu / mL, and the inoculum size is 1-2 v / v% and 2-3 v / v%. The fermentation conditions are 20-25℃, 100-200 r / min, fermentation culture for 36-48 h, and the volatilization time is 5-7 d. In step S7, the mass ratio of the fermentation product cocrystal and ε-polylysine modified exosome liposomes is 10:2-3.

[0022] The present invention further protects a composition prepared by the above-described method that utilizes exosomes to induce hair follicle regeneration and promote hair growth.

[0023] This invention further protects the use of the above-mentioned composition for inducing hair follicle regeneration and promoting hair growth using exosomes in the preparation of topical medicines for promoting hair growth and preventing hair loss.

[0024] The present invention has the following beneficial effects:

[0025] Exosomes derived from human umbilical cord mesenchymal stem cells (HUC-MSCs) are important bioactive components involved in paracrine function. Their contents are highly similar to those of HUC-MSCs, and they can perform similar physiological functions. They play a crucial role in the repair of damaged tissues. They can be taken up by follicular papillary cells, which accelerate cell proliferation, increase cell nucleus size, clarify nucleoli, and increase organelles such as mitochondria. Components such as miR-181a-5p carried by exosomes can activate the Akt and Wnt / β-catenin signaling pathways, prompting telogen follicles to enter the anagen phase, thereby stimulating the proliferation of follicular papillary cells.

[0026] Adding cell activating factors, including tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), to the culture medium has a dual role in regulating stem cell exosome secretion and enhancing its germinal function. TNF-α and IL-1β activate the NF-κB pathway by binding to stem cell surface receptors (such as TNFR1 and IL-1R), promoting the expression of exosome biogenesis-related genes (such as Rab27a and TSG101), thus significantly increasing the amount of exosome secretion. At the same time, the addition of cell activating factors enables the exosomes secreted by stem cells to carry neutralizing cytokines of TNF-α or IL-1β (such as IL-1Ra), which can locally regulate the inflammatory microenvironment, avoid hair follicle degeneration caused by excessive inflammation, and thus have a better function in promoting germinal growth.

[0027] The additional activating particles added to the culture medium have a magnetic iron oxide core and are coated with UiO-66-OH and alumina. These particles vibrate and generate heat under low-frequency magnetic field and infrared light irradiation, which can promote the physiological activity of stem cells under suitable conditions, significantly increase the secretion and physiological activity of exosomes. At the same time, under hypoxic conditions, the HIF-1α pathway of stem cells is activated, and the content of VEGF and HGF in exosomes is significantly increased. It can also significantly increase the yield of exosomes and the content of active ingredients that promote hair regeneration. The hypoxic environment simulates the physiological hypoxia state, which can activate the stress response pathway of cells and prompt mesenchymal stem cells to secrete more exosomes rich in growth factors.

[0028] This invention improves the loading capacity of highly active exosomes by encapsulating them in liposomes, significantly enhancing transdermal absorption while protecting them from enzymatic degradation. Furthermore, the liposomes are modified by reacting ε-polylysine with phosphatidylserine, allowing for targeted delivery to the negatively charged surface of the hair follicle via the positive charge on their surface. This targeted delivery system specifically delivers exosomes to the hair follicle. Modifying the exosome surface or encapsulating them in targeted nanocarriers enables more precise action of exosomes on hair follicle cells, improving the therapeutic effect of the composition, reducing drug distribution and potential side effects in non-target tissues, significantly increasing exosome adhesion, inhibiting bacterial biofilm formation, exhibiting good anti-inflammatory effects, and prolonging scalp retention time, thus achieving a long-acting, slow-release effect and extending the duration of action.

[0029] This invention addresses the issue of waste culture medium containing a large amount of active substances after separating cells and exosomes from the culture medium following stem cell culture. Directly discarding this medium would result in resource waste and require additional wastewater treatment. Therefore, by adding substances such as Polygonum multiflorum, Platycladus orientalis leaves, and tea leaves to the culture medium and then fermenting it, the invention promotes the production of abundant active components in plant cells, greatly improving the hair growth and follicle development effects of the resulting fermented cocrystal. Furthermore, it also achieves the resource utilization of the waste culture medium.

[0030] This invention modifies exosomal liposomes with ε-polylysine loaded onto fermentation cocrystals, which greatly improves the solubility of the product. The product is delivered to hair follicles via liposomes, inhibiting collagen hardening around the hair follicles and promoting the proliferation of hair papilla cells, with significant effects.

[0031] The composition prepared by this invention, which utilizes exosomes to induce hair follicle regeneration and promote hair growth, has good solubility, can target and deliver exosomes and fermentation products, significantly improves adhesion rate, inhibits bacterial biofilm formation, has good anti-inflammatory effect, and provides long-acting, slow-release drug, thus having a good effect on promoting hair follicle regeneration and hair growth, and has broad application prospects. Detailed Implementation

[0032] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Magnetic Fe3O4 particles with an average particle size of 1-2 μm. Human umbilical cord mesenchymal stem cells were provided by Pronosei, 1×10 6cells / T25 cell culture flask. NHS, N-hydroxysuccinimide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0034] Preparation Example 1: Preparation of Activated Particles

[0035] The method is as follows: 1g of magnetic Fe3O4 particles were added to 100mL of N,N-dimethylformamide, and 0.3g of zirconium tetrachloride was added. The mixture was hydrothermally stirred at 120℃ for 1h. Then, 0.248g of 2-hydroxyterephthalic acid was added, and the mixture was kept at this temperature for another 1h. The mixture was separated by magnets, washed, and dried. The product was then added to 20mL of ethanol, along with 0.2g of aluminum isopropoxide, 0.3mL of water, and 0.05g of NaOH. The mixture was stirred for 3h, separated by magnets, washed, and dried to obtain activated particles.

[0036] Preparation Example 2: Preparation of Activated Particles

[0037] The method is as follows: 1g of magnetic Fe3O4 particles were added to 100mL of N,N-dimethylformamide, and 0.35g of zirconium tetrachloride were added. The mixture was hydrothermally stirred at 130℃ for 3h. Then, 0.28g of 2-hydroxyterephthalic acid was added, and the mixture was kept at this temperature for another 3h. The mixture was separated by magnets, washed, and dried. The product was then added to 20mL of ethanol, along with 0.4g of aluminum isopropoxide, 0.5mL of water, and 0.1g of KOH. The mixture was stirred for 5h, separated by magnets, washed, and dried to obtain activated particles.

[0038] Preparation Example 3: Preparation of Activated Particles

[0039] The method is as follows: 1g of magnetic Fe3O4 particles were added to 100mL of N,N-dimethylformamide, and 0.33g of zirconium tetrachloride was added. The mixture was hydrothermally stirred at 125℃ for 2h, and then 0.26g of 2-hydroxyterephthalic acid was added. The mixture was kept at this temperature for another 2h. The particles were separated by magnets, washed, and dried. The product was then added to 20mL of ethanol, along with 0.3g of aluminum isopropoxide, 0.4mL of water, and 0.07g of KOH. The mixture was stirred for 4h, and then separated by magnets, washed, and dried to obtain activated particles.

[0040] Comparative Preparation Example 1

[0041] The difference compared to Preparation Example 3 is that aluminum isopropoxide was not added.

[0042] The method is as follows: 1g of magnetic Fe3O4 particles were added to 100mL of N,N-dimethylformamide, 0.33g of zirconium tetrachloride was added, and the mixture was hydrothermally stirred at 125℃ for 2h. Then, 0.26g of 2-hydroxyterephthalic acid was added, and the mixture was kept at this temperature for another 2h. The mixture was separated by magnets, the product was washed, and dried to obtain activated particles.

[0043] Comparative Preparation Example 2

[0044] The difference from Preparation Example 3 is that zirconium tetrachloride and 2-hydroxyterephthalic acid were not added.

[0045] The method is as follows: 1g of magnetic Fe3O4 particles were added to 20mL of ethanol, 0.3g of aluminum isopropoxide, 0.4mL of water and 0.07g of KOH were added, and the mixture was stirred and reacted for 4h. The magnets were separated, washed and dried to obtain activated particles.

[0046] Comparative preparation example 3

[0047] The difference compared to Preparation Example 3 is that no magnetic Fe3O4 particles were added.

[0048] The method is as follows: Add 0.33 g of zirconium tetrachloride to 100 mL of N,N-dimethylformamide, and stir hydrothermally at 125 °C for 2 h. Add 0.26 g of 2-hydroxyterephthalic acid, and continue to react at this temperature for 2 h. Separate the product with a magnet, wash and dry it. Add 0.3 g of aluminum isopropoxide, 0.4 mL of water and 0.07 g of KOH to 20 mL of ethanol, stir and react for 4 h. Separate the product with a magnet, wash and dry it to obtain activated particles.

[0049] Example 1

[0050] This embodiment provides a method for preparing a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth, comprising the following steps:

[0051] S1. Preparation of culture medium: Cell activating factors and activating particles were added to MSC serum-free culture medium to prepare the culture medium;

[0052] The cell activating factor includes tumor necrosis factor α and interleukin-1β in a mass ratio of 3:2. The amount of the cell activating factor added is 0.2 mg / L, and the amount of the activating particles prepared in Preparation Example 1 added is 1 g / L.

[0053] S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment. The oxygen content was 1v / v%, the external magnetic field strength was 0.2T, the wavelength of the near-infrared light was 808nm, the microwave power was 300W, and the treatment time was 12h. The cells were cultured until they reached 90% confluence. The cells were digested, the activation particles were separated by magnetism, centrifuged, the supernatant was discarded, and the cells were resuspended in the new medium prepared in step S1. The cells were seeded into the new medium at a passage ratio of 1:4 and the culture was expanded under the original conditions.

[0054] S3. Exosome isolation: After amplification to 90% confluence in step S2, the culture medium was discarded, the cells were washed, and the solid cells were added to exosome collection medium (αMEM medium + 1 wt% recombinant human insulin + 1 wt% lysine + 1 wt% glutamine). The cells were cultured in a CO2 incubator at 5 v / v% CO2 concentration for 36 h, filtered through a 0.22 μm needle filter, centrifuged at 250 g for 5 min at 2 °C, and the supernatant was collected. The cells were then centrifuged at 1800 g for 5 min at 2 °C, and the supernatant was collected again. The cells were then centrifuged at 8000 g for 20 min at 2 °C, and the supernatant was collected again. The cells were then centrifuged at 100000 g for 1.5 h at 2 °C, and the centrifuged precipitate was collected. The supernatant from the last centrifugation was retained and resuspended in PBS buffer with a pH of 7.2 to a concentration of 30 μg / mL to prepare exosome buffer.

[0055] S4. Preparation of exosome liposomes: 0.5g cholesterol, 0.3g phosphatidylserine and 0.1g lecithin were dissolved in 50mL dichloromethane-ethanol solution, 100mL exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated at 1000W for 20min, and then freeze-dried to obtain exosome liposomes.

[0056] The volume ratio of the dichloromethane-ethanol solution is 10:3;

[0057] S5. Natural molecular modification: 0.3g of ε-polylysine was dissolved in 100mL of water, 0.1g of EDC and 0.1g of NHS were added, and the mixture was stirred and activated for 20min. Then, 0.7g of exosome liposomes were added, and the mixture was stirred and reacted for 12h. The mixture was then freeze-dried to obtain ε-polylysine-modified exosome liposomes.

[0058] S6. Preparation of fermentation product eutectics: Wash, dry, and pulverize 3g of Polygonum multiflorum, 2g of Platycladus orientalis leaves, and 1g of tea leaves. Add 50g of the supernatant from the last centrifugation in step S3. Inoculate with selenium-enriched yeast and Bacillus subtilis seed culture. The bacterial count of the seed culture is 10. 8 -10 9 The inoculum was 1 v / v% and 2 v / v%, at 20 °C, 100 r / min, and fermented for 36 h. After filtration, the filtrate was evaporated for 5 days to crystallize. After filtration, the fermentation product co-crystal was obtained.

[0059] S7. Preparation of a composition for inducing hair follicle regeneration and promoting hair growth using exosomes: 10g of fermentation product cocrystal and 2g of ε-polylysine-modified exosome liposomes were added to 100mL of water, stirred and mixed for 30min, and then freeze-dried to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

[0060] Example 2

[0061] This embodiment provides a method for preparing a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth, comprising the following steps:

[0062] S1. Preparation of culture medium: Cell activating factors and activating particles were added to MSC serum-free culture medium to prepare the culture medium;

[0063] The cell activating factor includes tumor necrosis factor α and interleukin-1β in a mass ratio of 5:2. The amount of the cell activating factor added is 0.5 mg / L, and the amount of the activating particles obtained in Preparation Example 2 added is 2 g / L.

[0064] S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment. The oxygen content was 3v / v%, the external magnetic field strength was 0.4T, the wavelength of the near-infrared light was 808nm, the microwave power was 500W, and the treatment time was 20h. The cells were cultured until they reached 90% confluence. The cells were digested, the activation particles were separated by magnetism, centrifuged, the supernatant was discarded, and the cells were resuspended in the new medium prepared in step S1. The cells were seeded into the new medium at a passage ratio of 1:6 and the culture was expanded under the original conditions.

[0065] S3. Exosome isolation: After amplification to 90% confluence in step S2, the culture medium was discarded, the cells were washed, and the solid cells were added to exosome collection medium (αMEM medium + 1 wt% recombinant human insulin + 1 wt% lysine + 1 wt% glutamine). The cells were cultured in a CO2 incubator at 5 v / v% CO2 concentration for 48 h, filtered through a 0.22 μm needle filter, centrifuged at 350 g for 15 min at 4 °C, and the supernatant was collected. The cells were then centrifuged at 2200 g for 15 min at 4 °C, and the supernatant was collected. The cells were then centrifuged at 12000 g for 30 min at 4 °C, and the supernatant was collected. The cells were then centrifuged at 120000 g for 2.5 h at 4 °C, and the centrifuged precipitate was collected. The supernatant from the last centrifugation was retained and resuspended in PBS buffer with a pH of 7.7 to a concentration of 70 μg / mL to prepare exosome buffer.

[0066] S4. Preparation of exosome liposomes: 1g cholesterol, 0.5g phosphatidylserine and 0.3g lecithin were dissolved in 50mL dichloromethane-ethanol solution, 100mL exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated at 1200W for 30min, and then freeze-dried to obtain exosome liposomes.

[0067] The volume ratio of the dichloromethane-ethanol solution is 15:5;

[0068] S5. Natural molecular modification: Dissolve 0.5g of ε-polylysine in 100mL of water, add 0.2g of EDC and 0.2g of NHS, stir and activate for 40min, add 1g of exosome liposomes, stir and react for 15h, freeze dry to obtain ε-polylysine modified exosome liposomes;

[0069] S6. Preparation of fermentation product eutectics: Wash, dry, and pulverize 5g of Polygonum multiflorum, 5g of Platycladus orientalis leaves, and 3g of tea leaves. Add 70g of the supernatant from the last centrifugation in step S3. Inoculate with selenium-enriched yeast and Bacillus subtilis seed culture. The bacterial count of the seed culture is 10. 8 -10 9 The inoculum was 2 v / v% and 3 v / v%, and the fermentation was carried out at 25°C and 200 r / min for 48 h. After filtration, the filtrate was evaporated for 7 days to crystallize. After filtration, the fermentation product co-crystal was obtained.

[0070] S7. Preparation of a composition for inducing hair follicle regeneration and promoting hair growth using exosomes: 10g of fermentation product cocrystal and 3g of ε-polylysine-modified exosome liposomes were added to 100mL of water, stirred and mixed for 30min, and then freeze-dried to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

[0071] Example 3

[0072] This embodiment provides a method for preparing a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth, comprising the following steps:

[0073] S1. Preparation of culture medium: Cell activating factors and activating particles were added to MSC serum-free culture medium to prepare the culture medium;

[0074] The cell activating factor includes tumor necrosis factor α and interleukin-1β in a mass ratio of 4:2. The amount of the cell activating factor added is 0.35 mg / L, and the amount of the activating particles prepared in Preparation Example 3 is 1.5 g / L.

[0075] S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment. The oxygen content was 2v / v%, the external magnetic field strength was 0.3T, the wavelength of the near-infrared light was 808nm, the microwave power was 400W, and the treatment time was 16h. The cells were cultured until they reached 90% confluence. The cells were digested, the activation particles were separated by magnetism, centrifuged, the supernatant was discarded, and the cells were resuspended in the new medium prepared in step S1. The cells were seeded into the new medium at a passage ratio of 1:5 and the culture was expanded under the original conditions.

[0076] S3. Exosome isolation: After amplification to 90% confluence in step S2, the culture medium was discarded, the cells were washed, and the solid cells were added to exosome collection medium (αMEM medium + 1 wt% recombinant human insulin + 1 wt% lysine + 1 wt% glutamine). The cells were cultured in a CO2 incubator at 5 v / v% CO2 concentration for 42 h, filtered through a 0.22 μm needle filter, centrifuged at 3°C ​​and 300 g for 10 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 2000 g for 10 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 10000 g for 25 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 110000 g for 2 h, and the centrifuged precipitate was collected. The supernatant from the last centrifugation was retained and resuspended in PBS buffer with a pH of 7.4 to a concentration of 50 μg / mL to prepare exosome buffer.

[0077] S4. Preparation of exosome liposomes: 0.7g cholesterol, 0.4g phosphatidylserine and 0.2g lecithin were dissolved in 50mL dichloromethane-ethanol solution, 100mL exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated at 1100W for 25min, and then freeze-dried to obtain exosome liposomes.

[0078] The volume ratio of the dichloromethane-ethanol solution is 12:4;

[0079] S5. Natural molecular modification: 0.4g of ε-polylysine was dissolved in 100mL of water, 0.15g of EDC and 0.15g of NHS were added, and the mixture was stirred and activated for 30min. Then, 0.85g of exosome liposomes were added, and the mixture was stirred and reacted for 13h. The mixture was then freeze-dried to obtain ε-polylysine-modified exosome liposomes.

[0080] S6. Preparation of fermentation product eutectics: Wash, dry, and pulverize 4g of Polygonum multiflorum, 3.5g of Platycladus orientalis leaves, and 2g of tea leaves. Add 60g of the supernatant from the last centrifugation in step S3. Inoculate with selenium-enriched yeast and Bacillus subtilis seed culture. The bacterial count of the seed culture is 10. 8 -10 9 The inoculum was 1.5 v / v% and 2.5 v / v%, and the fermentation was carried out at 22℃ and 150 r / min for 42 h. The mixture was filtered, and the filtrate was evaporated for 6 days to crystallize. The filtrate was then filtered again to obtain the cocrystal of the fermentation product.

[0081] S7. Preparation of a composition for inducing hair follicle regeneration and promoting hair growth using exosomes: 10g of fermentation product cocrystal and 2.5g of ε-polylysine modified exosome liposomes were added to 100mL of water, stirred and mixed for 30min, and then freeze-dried to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

[0082] Comparative Example 1

[0083] The difference from Example 3 is that the activating particles were prepared by Comparative Preparation Example 1.

[0084] Comparative Example 2

[0085] The difference from Example 3 is that the activating particles were prepared by Comparative Preparation Example 2.

[0086] Comparative Example 3

[0087] The difference from Example 3 is that the activating particles were prepared by Comparative Preparation Example 3.

[0088] Comparative Example 4

[0089] The difference compared to Example 3 is that no activating particles were added.

[0090] Specifically as follows:

[0091] S1. Preparation of culture medium: Cell activating factors were added to serum-free MSC culture medium to prepare the culture medium;

[0092] The cell activating factor includes tumor necrosis factor α and interleukin-1β in a mass ratio of 4:2, and the amount of the cell activating factor added is 0.35 mg / L.

[0093] Comparative Example 5

[0094] The difference compared to Example 3 is that no cell activating factor was added.

[0095] Specifically as follows:

[0096] S1. Preparation of culture medium: Activation particles were added to MSC serum-free culture medium to prepare the culture medium;

[0097] The amount of activating particles prepared in Preparation Example 3 was 1.5 g / L.

[0098] Comparative Example 6

[0099] The difference from Example 3 is that step S5 was not performed.

[0100] Specifically as follows:

[0101] S1. Preparation of culture medium: Cell activating factors and activating particles were added to MSC serum-free culture medium to prepare the culture medium;

[0102] The cell activating factor includes tumor necrosis factor α and interleukin-1β in a mass ratio of 4:2. The amount of the cell activating factor added is 0.35 mg / L, and the amount of the activating particles prepared in Preparation Example 3 is 1.5 g / L.

[0103] S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment. The oxygen content was 2v / v%, the external magnetic field strength was 0.3T, the wavelength of the near-infrared light was 808nm, the microwave power was 400W, and the treatment time was 16h. The cells were cultured until they reached 90% confluence. The cells were digested, the activation particles were separated by magnetism, centrifuged, the supernatant was discarded, and the cells were resuspended in the new medium prepared in step S1. The cells were seeded into the new medium at a passage ratio of 1:5 and the culture was expanded under the original conditions.

[0104] S3. Exosome isolation: After amplification to 90% confluence in step S2, the culture medium was discarded, the cells were washed, and the solid cells were added to exosome collection medium (αMEM medium + 1 wt% recombinant human insulin + 1 wt% lysine + 1 wt% glutamine). The cells were cultured in a CO2 incubator at 5 v / v% CO2 concentration for 42 h, filtered through a 0.22 μm needle filter, centrifuged at 3°C ​​and 300 g for 10 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 2000 g for 10 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 10000 g for 25 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 110000 g for 2 h, and the centrifuged precipitate was collected. The supernatant from the last centrifugation was retained and resuspended in PBS buffer with a pH of 7.4 to a concentration of 50 μg / mL to prepare exosome buffer.

[0105] S4. Preparation of exosome liposomes: 0.7g cholesterol, 0.4g phosphatidylserine and 0.2g lecithin were dissolved in 50mL dichloromethane-ethanol solution, 100mL exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated at 1100W for 25min, and then freeze-dried to obtain exosome liposomes.

[0106] The volume ratio of the dichloromethane-ethanol solution is 12:4;

[0107] S5. Preparation of fermentation product eutectics: Wash, dry, and pulverize 4g of Polygonum multiflorum, 3.5g of Platycladus orientalis leaves, and 2g of tea leaves. Add 60g of the supernatant from the last centrifugation in step S3. Inoculate with selenium-enriched yeast and Bacillus subtilis seed culture. The bacterial count of the seed culture is 10. 8 -10 9 The inoculum was 1.5 v / v% and 2.5 v / v%, and the fermentation was carried out at 22℃ and 150 r / min for 42 h. The mixture was filtered, and the filtrate was evaporated for 6 days to crystallize. The filtrate was then filtered again to obtain the cocrystal of the fermentation product.

[0108] S6. Preparation of a composition for inducing hair follicle regeneration and promoting hair growth using exosomes: 10g of fermentation product cocrystal and 2.5g of exosome liposomes were added to 100mL of water, stirred and mixed for 30min, and then freeze-dried to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

[0109] Comparative Example 7

[0110] The difference from Example 3 is that steps S6 and S7 were not performed.

[0111] Specifically as follows:

[0112] S1. Preparation of culture medium: Cell activating factors and activating particles were added to MSC serum-free culture medium to prepare the culture medium;

[0113] The cell activating factor includes tumor necrosis factor α and interleukin-1β in a mass ratio of 4:2. The amount of the cell activating factor added is 0.35 mg / L, and the amount of the activating particles prepared in Preparation Example 3 is 1.5 g / L.

[0114] S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment. The oxygen content was 2v / v%, the external magnetic field strength was 0.3T, the wavelength of the near-infrared light was 808nm, the microwave power was 400W, and the treatment time was 16h. The cells were cultured until they reached 90% confluence. The cells were digested, the activation particles were separated by magnetism, centrifuged, the supernatant was discarded, and the cells were resuspended in the new medium prepared in step S1. The cells were seeded into the new medium at a passage ratio of 1:5 and the culture was expanded under the original conditions.

[0115] S3. Exosome isolation: After amplification to 90% confluence in step S2, the culture medium was discarded, the cells were washed, and the solid cells were added to exosome collection medium (αMEM medium + 1 wt% recombinant human insulin + 1 wt% lysine + 1 wt% glutamine). The cells were cultured in a CO2 incubator at 5 v / v% CO2 concentration for 42 h, filtered through a 0.22 μm needle filter, centrifuged at 3°C ​​and 300 g for 10 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 2000 g for 10 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 10000 g for 25 min, and the supernatant was collected. The cells were then centrifuged at 3°C ​​and 110000 g for 2 h, and the centrifuged precipitate was collected. The supernatant from the last centrifugation was retained and resuspended in PBS buffer with a pH of 7.4 to a concentration of 50 μg / mL to prepare exosome buffer.

[0116] S4. Preparation of exosome liposomes: 0.7g cholesterol, 0.4g phosphatidylserine and 0.2g lecithin were dissolved in 50mL dichloromethane-ethanol solution, 100mL exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated at 1100W for 25min, and then freeze-dried to obtain exosome liposomes.

[0117] The volume ratio of the dichloromethane-ethanol solution is 12:4;

[0118] S5. Natural molecular modification: Dissolve 0.4g of ε-polylysine in 100mL of water, add 0.15g of EDC and 0.15g of NHS, stir and activate for 30min, add 0.85g of exosome liposomes, stir and react for 13h, freeze dry to obtain ε-polylysine modified exosome liposomes, which is the composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

[0119] Test Example 1

[0120] C57BL / 6J mice were randomly divided into a control group, a positive control group, Example 1-3 groups, and Comparative Examples 1-7 groups, with 10 mice in each group. The hair on the backs of the C57BL / 6J mice, covering an area of ​​approximately 2cm × 3cm, was shaved off with a razor. Then, depilatory cream was applied according to the instructions to remove any remaining hair. The mice were confirmed to be in the resting phase of hair growth (the skin was pink) to establish a C57BL / 6J mouse alopecia model. After successful modeling, 0.2mL of sample solution (prepared by adding the corresponding exosome-induced hair follicle regeneration and hair growth-promoting composition to deionized water to a concentration of 1mg / mL) was applied to the hair-removed area in Example 1-3 groups and Comparative Examples 1-7 groups. The positive control group was treated with an equal volume of minoxidil solution. The control group was treated with an equal volume of deionized water, and this treatment was repeated for 7 consecutive days.

[0121] After the experiment, C57BL / 6J mice were photographed and their hair growth was scored. The scoring criteria were as follows: no hair growth, flesh-colored skin in the hair-free area was 0 points, gray skin in the hair-free area was 1 point, black skin in the hair-free area was 2 points, and black skin in the hair-free area with a few hairs growing out was 3 points. The average score was taken.

[0122] Skin from the hairless area on the back of C57BL / 6J mice was taken, and circular skin patches were taken from the same location in the hairless area of ​​each mouse using a 20mm punch. All body hair on the skin patches was scraped off with a scalpel, weighed on an analytical balance, and the mean weight of hair from each group of C57BL / 6J mice was calculated.

[0123] The results are shown in Table 1.

[0124] Table 1

[0125] Group Hair growth score <![CDATA[Average hair mass of mice (mg / cm 2 )]]> control group 0.10±0.09 0.24±0.07 Positive control group 0.32±0.49* 0.78±0.27* Example 1 1.31±0.39* 2.95±0.57* Example 2 1.32±0.33* 2.99±0.48* Example 3 1.35±0.31* 3.04±0.51* Comparative Example 1 1.21±0.44 2.45±0.65 Comparative Example 2 1.19±0.41 2.40±0.59 Comparative Example 3 1.15±0.45 2.34±0.62 Comparative Example 4 0.92±0.47 2.02±0.60 Comparative Example 5 1.09±0.53 2.26±0.54 Comparative Example 6 1.01±0.49 2.12±0.55 Comparative Example 7 0.72±0.51 1.63±0.57

[0126] Note: * indicates P<0.05 compared to the control group.

[0127] As can be seen from the table above, the compositions for inducing hair follicle regeneration and promoting hair growth using exosomes prepared in Examples 1-3 of this invention have a good effect on promoting hair growth.

[0128] After the experiment, tissue samples were extracted from each group of mice, stained with hematoxylin and eosin (HE), and the growth status of hair follicles was observed. The number of hair follicles that entered the growth phase was counted and recorded, and the results are shown in Table 2.

[0129] Table 2

[0130] Group Number of mice whose hair follicles entered the growth phase (number of mice) Number of hair follicles control group 1 7 Positive control group 4 22 Example 1 10 65 Example 2 10 67 Example 3 10 70 Comparative Example 1 8 52 Comparative Example 2 8 49 Comparative Example 3 8 46 Comparative Example 4 6 37 Comparative Example 5 7 41 Comparative Example 6 7 43 Comparative Example 7 6 36

[0131] As can be seen from the table above, the compositions for inducing hair follicle regeneration and promoting hair growth using exosomes prepared in Examples 1-3 of the present invention have a good effect on inducing hair follicle regeneration.

[0132] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a composition that utilizes exosomes to induce hair follicle regeneration and promote hair growth, characterized in that, Cell activating factors and activating particles were added to serum-free MSC culture medium for culturing human umbilical cord mesenchymal stem cells. After cell isolation, exosomes and culture medium were collected by centrifugation. Exosomes were embedded in liposomes, and their surfaces were modified with ε-polylysine to obtain ε-polylysine-modified exosome liposomes. Polygonum multiflorum, Platycladus orientalis leaves, and tea leaves were added to the culture medium for fermentation. The mixture was filtered, and the filtrate was evaporated and crystallized. After filtration, the fermentation product cocrystal was obtained and reacted with the ε-polylysine-modified exosome liposomes to prepare a composition for inducing hair follicle regeneration and promoting hair growth using exosomes. The mass ratio of Polygonum multiflorum, Platycladus orientalis leaves, and tea leaves was 3-5:2-5:1-3. The cell activating factors were tumor necrosis factor α and interleukin-1β. The ratio is 3-5:2; the amount of cell activating factor added is 0.2-0.5 mg / L, and the amount of activating particles added is 1-2 g / L; the preparation method of the activating particles is as follows: magnetic Fe3O4 particles are added to N,N-dimethylformamide, zirconium tetrachloride is added, hydrothermal stirring reaction is carried out, 2-hydroxyterephthalic acid is added, the reaction is continued at a constant temperature, magnetic separation is performed, the product is washed, dried, added to ethanol, aluminum isopropoxide is added, water and alkali are added, stirring reaction is carried out, magnetic separation is performed, washing is performed, drying is performed to obtain activating particles; the mass ratio of magnetic Fe3O4 particles, zirconium tetrachloride, 2-hydroxyterephthalic acid, aluminum isopropoxide, water and alkali is 10:3-3.5:2.4-2.8:2-4:3-5:0.5-1.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of culture medium: Cell activating factors and activating particles were added to serum-free MSC culture medium to prepare the culture medium; S2. Stem cell culture: Primary human umbilical cord mesenchymal stem cells were resuscitated in serum-free MSC medium and transferred to a culture dish. The medium was then replaced with the medium prepared in step S1. The cells were cultured under hypoxia, external magnetic field, near-infrared light irradiation, and microwave treatment until the cells reached 90% confluence. The cells were digested, activated particles were separated by magnetism, centrifuged, and the supernatant was discarded. The cells were resuspended in the new medium prepared in step S1 and seeded into a new medium for continued expansion culture under the original conditions. S3. Isolation of exosomes: After the cells are amplified to 90% confluence in step S2, the culture medium is discarded, the cells are washed, and the exosome collection medium is added to the solid cells. The cells are then cultured again, filtered through a syringe filter, centrifuged at low temperature at the first speed, and the supernatant is collected. The cells are then centrifuged at low temperature at the second speed, and the supernatant is collected. The cells are then centrifuged at low temperature at the third speed, and the supernatant is collected. The centrifuged precipitate is collected. The supernatant from the last centrifugation is retained and resuspended in PBS buffer to prepare exosome buffer. S4. Preparation of exosomes and liposomes: Cholesterol, phosphatidylserine, and lecithin were dissolved in dichloromethane-ethanol solution, exosome buffer was added dropwise, the mixture was stirred at a constant temperature, the organic solvent was removed by vacuum evaporation, the mixture was sonicated, and the mixture was freeze-dried to obtain exosomes and liposomes. S5. Natural molecular modification: ε-polylysine was dissolved in water, EDC and NHS were added, the mixture was stirred and activated, exosomes and liposomes were added, the mixture was stirred and reacted, and then freeze-dried to obtain ε-polylysine-modified exosomes and liposomes; S6. Preparation of fermentation product cocrystal: Wash, dry and crush Polygonum multiflorum, Platycladus orientalis leaves and tea leaves, add the supernatant of the last centrifugation in step S3, inoculate with fermentation bacteria, ferment and culture, filter, evaporate the filtrate to crystallize, filter again to obtain fermentation product cocrystal; S7. Preparation of a composition for inducing hair follicle regeneration and promoting hair growth using exosomes: Fermentation product cocrystals and ε-polylysine-modified exosome liposomes were added to water, stirred and mixed, and freeze-dried to obtain a composition for inducing hair follicle regeneration and promoting hair growth using exosomes.

3. The preparation method according to claim 2, characterized in that, The alkali is NaOH or KOH, the temperature of the hydrothermal stirring reaction is 120-130℃, the time is 1-3h, the time of the continued heat preservation reaction is 1-3h, and the time of the stirring reaction is 3-5h.

4. The preparation method according to claim 2, characterized in that, The conditions for low oxygen, external magnetic field, infrared light irradiation, and microwave treatment in step S2 are as follows: oxygen content 1-3 v / v%, external magnetic field strength 0.2-0.4T, infrared light irradiation wavelength 700-900nm, microwave power 300-500W, and treatment time 12-20h. The exosomes are inoculated into a new culture medium at a passage ratio of 1:4-6. The exosome collection medium in step S3 is αMEM medium + 1wt% recombinant human insulin + 1wt% lysine + 1wt% glutamine, and the culture conditions are 5 v / v. The culture is carried out in a CO2 incubator with a CO2 concentration of % for 36-48 hours. The filter membrane of the needle filter has a pore size of 0.22 μm. The low temperature is 2-4℃. The first rotation speed is 250-350g for 5-15 min, the second rotation speed is 1800-2200g for 5-15 min, the third rotation speed is 8000-12000g for 20-30 min, and the fourth rotation speed is 100000-120000g for 1.5-2.5 hours. The pH value of the PBS buffer is 7.2-7.

7.

5. The preparation method according to claim 2, characterized in that, In step S4, the mass ratio of cholesterol, phosphatidylserine, and lecithin is 5-10:3-5:1-3, the volume ratio of the dichloromethane-ethanol solution is 10-15:3-5, the constant temperature stirring temperature is 20-25℃, the time is 20-30 min, and the ultrasonic treatment power is 1000-1200W for 20-30 min.

6. The preparation method according to claim 2, characterized in that, In step S5, the mass ratio of ε-polylysine, EDC, NHS and exosome liposomes is 3-5:1-2:1-2:7-10, the stirring activation time is 20-40 min, and the stirring reaction time is 12-15 h.

7. The preparation method according to claim 2, characterized in that, The mass ratio of Polygonum multiflorum, Platycladus orientalis leaves, tea leaves, and the supernatant from the last centrifugation in step S3 is 3-5:2-5:1-3:50-70. The fermentation bacteria are a seed culture of selenium-enriched yeast and Bacillus subtilis, and the bacterial count of the seed culture is 10. 8 -10 9 The inoculum concentration is cfu / mL, and the inoculum size is 1-2 v / v% and 2-3 v / v%. The fermentation conditions are 20-25℃, 100-200 r / min, fermentation culture for 36-48 h, and the volatilization time is 5-7 d. In step S7, the mass ratio of the fermentation product cocrystal and ε-polylysine modified exosome liposomes is 10:2-3.

8. A composition for inducing hair follicle regeneration and promoting hair growth by means of exosomes, prepared by any one of claims 1-7.

9. The use of the composition as described in claim 8, which utilizes exosomes to induce hair follicle regeneration and promote hair growth, in the preparation of topical medicines for promoting hair growth and preventing hair loss.