An external vesicle composition for promoting hair growth and its preparation method

Abstract

This invention relates to an extracellular vesicle composition for promoting hair growth and its preparation method, belonging to the field of regenerative medicine technology. It comprises the following active ingredients: an extracellular vesicle active ingredient derived from mesenchymal stem cells; cell-active factors selected from one or more of insulin-like growth factor, fibroblast growth factor, and vascular endothelial growth factor; an amino acid component selected from one or more of arginine, lysine, histidine, and proline; and a polypeptide component selected from one or two of hexapeptides and dipeptides. This extracellular vesicle composition for promoting hair growth and its preparation method, by culturing mesenchymal stem cells in a simulated microgravity environment, effectively simulates the three-dimensional growth environment in vivo, significantly promoting the proliferation and metabolic activity of stem cells, increasing the secretion of extracellular vesicles by 3 to 5 times compared to traditional two-dimensional culture; simultaneously, the microgravity-induced upregulation mechanism of key proteins further enhances the therapeutic efficacy of the extracellular vesicles.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine technology, specifically to an external vesicle composition for promoting hair growth and its preparation method, which is particularly suitable for the treatment and care of hair health problems such as androgenetic alopecia, alopecia areata, and various scalp environment imbalances, including thinning hair, receding hairline, and dormant hair follicles. Background Technology

[0002] Hair loss is a common problem affecting hundreds of millions of people worldwide, with androgenetic alopecia being the most prevalent, affecting approximately 50% of adult men and a significant proportion of women. Currently, commonly used clinical treatments for hair loss mainly include: topical minoxidil, oral finasteride, low-energy laser therapy, and hair follicle transplantation surgery. While minoxidil, an FDA-approved hair growth stimulant, has some efficacy, it suffers from slow onset of action, requires long-term continuous use, and rebound hair loss after discontinuation. Finasteride, on the other hand, is often disregarded by patients due to side effects such as sexual dysfunction.

[0003] In recent years, extracellular vesicles, as natural nanoscale messengers secreted by cells, have attracted widespread attention due to their ability to precisely deliver functional molecules between cells. Previous studies have shown that extracellular vesicles derived from mesenchymal stem cells can effectively promote fibroblast proliferation and migration by delivering growth factors and signaling molecules, demonstrating potential in tissue repair and hair regeneration. However, current technologies still have the following limitations: First, the activity and purity of the extravesicles are insufficient. Under traditional two-dimensional culture conditions, the yield and activity of extravesicles secreted by stem cells are low, and the extraction and purification process is crude, making it difficult to obtain high concentrations and high purity of active extravesicles.

[0004] Secondly, there is a lack of precise targeting of the hair follicle microenvironment. Most existing external vesicle products are not specifically optimized for the activation of hair follicle stem cells, making it difficult to effectively activate dormant hair follicles and improve the hair follicle microenvironment.

[0005] Third, the formulas are too simple and lack synergistic effects. Most existing products are based on single external vesicle components and are not scientifically compounded with active ingredients such as cell-active factors, amino acids, and peptides, making it difficult to exert the synergistic effect of multiple components in activating hair follicles.

[0006] Fourth, there is a lack of large-scale, standardized preparation processes. Existing methods for preparing exovesicles are mostly still in the laboratory stage, making it difficult to achieve stable scale-up from the laboratory to industrial production, resulting in large quality differences between batches of products.

[0007] Therefore, there is an urgent need for a hair growth promoting composition that is highly active, highly pure, can precisely target the hair follicle microenvironment, and has the capacity for large-scale production, in order to solve the problems of insufficient activity and purity of outer vesicles, lack of hair follicle targeting, single formulation, and difficulty in large-scale production in the existing technology. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an external vesicle composition for promoting hair growth and its preparation method. It has the advantages of high activity and yield of external vesicles, high purity, multi-component synergistic targeting of hair follicles, and significant hair growth promotion effect. It solves the problems of insufficient activity and yield, low purity, and lack of synergistic effect of existing external vesicle products.

[0009] To achieve the above objectives, the present invention provides the following technical solution: An external vesicle composition for promoting hair growth comprises the following active ingredients: The active ingredient of the extracellular vesicle is derived from the extracellular vesicles of mesenchymal stem cells, which are obtained by separating and extracting the extracellular vesicles after being cultured and expanded under a simulated microgravity environment. Cellular activity factors are selected from one or more of insulin-like growth factor, fibroblast growth factor, and vascular endothelial growth factor. The amino acid components are selected from one or more of arginine, lysine, histidine, and proline; The polypeptide component is selected from one or both of hexapeptides and dipeptides; And pharmaceutically or cosmetically acceptable excipients.

[0010] Furthermore, the simulated microgravity environment is achieved through a rotating cell culture system or a three-dimensional rotating bioreactor, with a culture time of 24–72 hours.

[0011] Furthermore, the extracellular vesicles have a particle size of 30–200 nm and a concentration of 1 × 10⁻⁶. 9 ~1×10 12 Particles / mL.

[0012] Furthermore, the extracellular vesicles are extracted and purified using a combination of tangential flow filtration and chromatography techniques, achieving a purity of ≥95%.

[0013] Furthermore, the mass ratio of each active ingredient in the composition is as follows: 1-10 parts extracellular vesicles, 0.01-1 parts cell-active factors, 0.1-5 parts amino acids, and 0.01-1 parts polypeptides.

[0014] Furthermore, the composition is a topical preparation, and the dosage form is selected from one of the following: serum, spray, gel, or microneedle patch.

[0015] The present invention also provides a method for preparing an external vesicle composition for promoting hair growth, comprising the following steps: S1. Stem cell expansion culture: Mesenchymal stem cells are seeded into a culture container containing serum-free culture medium and placed in a simulated microgravity bioreactor for expansion culture; S2, Extracellular vesicle secretion induction: Add cell activating factors to the culture system and continue culturing for 12-48 hours to induce mesenchymal stem cells to secrete extracellular vesicles; S3. Extracellular vesicle separation and extraction: Collect the culture supernatant, and successively remove cell debris by low-speed centrifugation, concentrate by tangential flow filtration, and purify by chromatography to obtain extracellular vesicles; S4. Combination of active ingredients: The extracellular vesicles obtained in S3 are mixed with cell-active factors, amino acids, and peptides in a certain proportion, excipients are added, and homogenization is performed to obtain an extracellular vesicle composition.

[0016] Furthermore, the cell activating factor in S2 is selected from one or both of tumor necrosis factor-α and interleukin-1α.

[0017] Furthermore, in S3, the low-speed centrifugation speed is 300-500×g, and the time is 5-15 minutes; the membrane pore size of the tangential flow filtration is 0.1-0.22μm; and the chromatography technique is size exclusion chromatography or ion exchange chromatography.

[0018] Furthermore, the rotation speed of the simulated microgravity environment in S1 is 10-20 rpm, the culture temperature is 37°C, and the CO2 concentration is 5%.

[0019] Compared with the prior art, the present invention provides an external vesicle composition for promoting hair growth and a method for preparing the same, which has the following beneficial effects: 1. The external vesicle composition for promoting hair growth and its preparation method effectively simulate the three-dimensional growth environment in vivo by culturing mesenchymal stem cells in a simulated microgravity environment, significantly promoting the proliferation and metabolic activity of stem cells, and increasing the secretion of external vesicles by 3 to 5 times compared with traditional two-dimensional culture; at the same time, the microgravity-induced upregulation mechanism of key proteins further enhances the therapeutic efficacy of external vesicles; the combined application of tangential flow filtration and chromatography technology enables the purity of external vesicles to reach more than 95%, taking into account both recovery rate and purity.

[0020] 2. This external vesicle composition for promoting hair growth and its preparation method involve scientifically combining highly active external vesicles with cell-active factors such as IGF-1, bFGF, and VEGF, amino acids such as arginine, and hexapeptides / dipeptides to form a four-fold synergistic mechanism of "vesicle targeted delivery—factor activation signal—amino acid nutrition—peptide structural support." The external vesicles, acting as natural nanocarriers, precisely deliver signaling molecules to hair follicle stem cells, activating dormant hair follicles. Cell-active factors promote hair follicle cell proliferation and angiogenesis; amino acids provide basic nutrition for hair growth; and peptides improve scalp microcirculation and barrier function.

[0021] 3. The external vesicle composition for promoting hair growth and its preparation method establish a complete process flow from stem cell expansion, external vesicle induction and secretion, separation and purification to active ingredient compounding. The parameters of each step are clear and controllable, with good batch reproducibility and potential for large-scale scale-up. The production in a sterile environment throughout the process ensures the safety and quality stability of the product, providing a feasible path for the industrial application of external vesicle technology in the field of hair health. Attached Figure Description

[0022] Figure 1 This is a process flow diagram of a method for preparing an external vesicle composition for promoting hair growth according to the present invention.

[0023] Figure 2 This is a comparative diagram showing the hair growth promoting effect of an external vesicle composition for promoting hair growth according to the present invention. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0025] Please see Figures 1 to 2 .

[0026] Example 1: Preparation of the exovesicle composition S1: Stem cell expansion culture Human umbilical cord mesenchymal stem cells (P3-P5 passage) were collected at a concentration of 1×10⁻⁶. 5 Cells / mL were seeded into culture vessels containing serum-free medium (such as MSC serum-free medium). The culture vessels were placed in a simulated microgravity rotating cell culture system (RCCS) with a rotation speed of 10–20 rpm, a culture temperature of 37°C, and a CO2 concentration of 5%, and cultured continuously for 48 hours. Fresh medium was replaced every 24 hours during the culture process.

[0027] S2: Extravesicle-induced secretion Cell activating factors—tumor necrosis factor-α (TNF-α, final concentration 10–50 ng / mL) and interleukin-1α (IL-1α, final concentration 5–20 ng / mL)—were added to the culture system, and the cells were cultured for another 24 hours under simulated microgravity to induce mesenchymal stem cells to secrete extracellular vesicles.

[0028] S3: Exovesicle separation and extraction Collect the culture supernatant and process it sequentially as follows: (1) Low-speed centrifugation: Centrifuge at 300×g for 10 minutes to remove cell debris and dead cells; (2) High-speed centrifugation: Centrifuge at 10,000×g for 30 minutes to remove cell debris; (3) Tangential flow filtration (TFF): Hollow fiber membrane modules with a pore size of 0.22 μm are used for concentration and washing to remove small molecule impurities and collect the retentate; (4) Chromatographic purification: The retentate was purified by size exclusion chromatography column and the elution peaks of the outer vesicles were collected; (5) Concentration: The purified vesicle solution is concentrated to the target concentration by ultrafiltration.

[0029] Through the above steps, high-purity extracellular vesicles were obtained. Nanoparticle tracking analysis (NTA) revealed that the extracellular vesicle particle size was 50–150 nm, with an average particle size of approximately 100 nm; the concentration was 5 × 10⁻⁶. 10 Particles / mL; purity (detected by Western Blot for external vesicle markers CD9, CD63, and CD81) ≥95%.

[0030] S4: Complex of active ingredients Weigh each component according to the following parts by mass: Extracellular vesicles (prepared in Example 1): 5 portions; IGF-1: 0.05 parts; bFGF: 0.03 parts; VEGF: 0.02 parts; Arginine: 0.5 parts; Lysine: 0.3 parts; Histidine: 0.2 parts; Acetyl Hexapeptide-8: 0.05 parts; Dipeptide-2: 0.03 parts; Excipients: 2 parts glycerin, 3 parts 1,3-butanediol, 0.1 parts sodium hyaluronate, 0.3 parts p-hydroxyacetophenone, and deionized water to 100 parts.

[0031] The above components are mixed under sterile conditions, homogenized under high pressure (800-1000 bar, 3-5 cycles), filtered through a 0.22 μm filter membrane for sterilization, and then filled into sterile bottles to obtain the external vesicle hair growth essence.

[0032] Example 2: Effects of different microgravity culture times on vesicle yield Following the preparation method of Example 1, the microgravity culture time was set to 24 hours, 48 ​​hours and 72 hours respectively to investigate the effect on the yield of external vesicles.

[0033] The effects of different microgravity culture times on vesicle yield are shown in Table 1.

[0034] Table 1

[0035] The results showed that culturing for 48 hours yielded the best external vesicle yield and particle size uniformity.

[0036] Example 3: Effects of different activating factors on exovesicle function Following the preparation method of Example 1, TNF-α, IL-1α, and a combination of both were used as activating factors.

[0037] The effects of different activating factors on the function of exovesicles are shown in Table 2.

[0038] Table 2

[0039] The results showed that the combined use of TNF-α and IL-1α could significantly increase the yield of external vesicles and their activity in promoting the proliferation of hair follicle stem cells.

[0040] Example 4: Effect of different compounding ratios on hair growth Referring to the compounding method in Example 1, different compounding ratios (mass ratios) of external vesicles and active factors were set to conduct hair follicle organ culture experiments.

[0041] The effects of different compounding ratios on hair growth are shown in Table 3.

[0042] Table 3

[0043] The results showed that group B (external vesicles:active factors:amino acids:peptides = 5:0.1:1:0.08) had the best hair growth promoting effect.

[0044] Example 5: In vitro hair follicle activation experiment of the composition Human hair follicle stem cells were cultured in vitro, and the composition prepared in Example 1 (experimental group), an equal amount of external vesicles (control group 1), an equal amount of active factor mixture (control group 2), and blank culture medium (blank group) were added respectively. After 7 days of culture, the proliferation activity and differentiation marker expression of hair follicle stem cells were detected.

[0045] Experimental results: Experimental group: The proliferation rate of hair follicle stem cells increased by 320% compared with the control group, and the expression of hair follicle differentiation markers (KRT15, ​​LEF1) was significantly upregulated; Control group 1: Proliferation rate increased by 180%, and the expression of differentiation markers was moderately upregulated; Control group 2: Proliferation rate increased by 120%, but there was no significant change in the expression of differentiation markers; Blank group: baseline proliferation level.

[0046] The results showed that the composition of the present invention significantly promoted the proliferation and differentiation of hair follicle stem cells through the synergistic effect of external vesicles with active factors, amino acids and peptides.

[0047] Example 6: Preliminary clinical application effects of the composition Thirty subjects with androgenetic alopecia (20 males and 10 females, aged 25-55 years) were selected and randomly divided into an experimental group (using the composition prepared in Example 1) and a control group (using the matrix control). The composition was used once in the morning and once in the evening for 90 consecutive days.

[0048] Evaluation indicators: Hair density (number of hair follicles per unit area) Hairline position changes Number of new hairs Subjects' self-rated satisfaction The results are shown in Table 4.

[0049] Table 4

[0050] In the experimental group, most subjects observed new hair growth along the hairline and a significant reduction in hair loss after 30 days of use, with stronger hair roots. After 90 days of use, the amount of hair on the top of the head increased significantly, and hair density improved markedly.

[0051] Comparative Example 1: Traditional two-dimensional culture of exovesicles Mesenchymal stem cells were cultured using a traditional two-dimensional adherent culture method (non-microgravity environment), and the remaining steps were the same as in Example 1.

[0052] Results: The yield of external vesicles was only 22% of that in Example 1, and the expression level of hair growth-related miRNAs (such as miR-218-5p) in the external vesicles was significantly lower than that in Example 1. At the same concentration, its effect on promoting the proliferation of hair follicle stem cells was only 45% of that in Example 1.

[0053] Comparative Example 2: Unpurified exovesicles The preparation method of Example 1 is followed, but the chromatography purification step is omitted, and only tangential flow filtration is used.

[0054] Results: The purity of the outer vesicles was 68%, containing a large amount of extraneous proteins and cell debris. In the hair follicle organ culture experiment, its effect on promoting hair growth was only 55% of that in Example 1, and some samples showed a slight inflammatory reaction (caused by impurities).

[0055] Comparative Example 3: Compositions without active factors and peptides Only the exovesicles prepared in Example 1 were used, without the addition of cell-active factors, amino acids and peptides, and all other conditions were the same.

[0056] Results: During 90 days of clinical application, the increase in hair density was only 12.3 hairs / cm². 2 This is far lower than the 28.6 roots / cm in Example 1. 2 This indicates that the combination of active factors and peptides has a significant synergistic effect on hair growth.

[0057] Comparative Example 4: Minoxidil Control Group Thirty subjects with androgenetic alopecia were treated for 90 days using commercially available 5% minoxidil solution under the same conditions.

[0058] Result: Hair density increased to 18.4 hairs / cm² 2 This is lower than the 28.6 roots / cm in Example 1. 2 The incidence of adverse reactions (scalp itching, erythema) was 15%, higher than the 3% in Example 1. This indicates that the composition of the present invention is superior to minoxidil in promoting hair growth and has a higher safety profile.

[0059] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0060] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An external vesicle composition for promoting hair growth, characterized in that, Includes the following active ingredients: The active ingredient of the extracellular vesicle is derived from the extracellular vesicles of mesenchymal stem cells, which are obtained by separating and extracting the extracellular vesicles after being cultured and expanded under a simulated microgravity environment. Cellular activity factors are selected from one or more of insulin-like growth factor, fibroblast growth factor, and vascular endothelial growth factor. The amino acid components are selected from one or more of arginine, lysine, histidine, and proline; The polypeptide component is selected from one or both of hexapeptides and dipeptides; And pharmaceutically or cosmetically acceptable excipients.

2. The external vesicle composition for promoting hair growth according to claim 1, characterized in that, The simulated microgravity environment is achieved through a rotating cell culture system or a three-dimensional rotating bioreactor, with a culture time of 24–72 hours.

3. The external vesicle composition for promoting hair growth according to claim 1, characterized in that, The extracellular vesicles have a particle size of 30–200 nm and a concentration of 1 × 10⁻⁶. 9 ~1×10 12 Particles / mL.

4. The external vesicle composition for promoting hair growth according to claim 1, characterized in that, The extracellular vesicles were extracted and purified using a combination of tangential flow filtration and chromatography techniques, achieving a purity of ≥95%.

5. The external vesicle composition for promoting hair growth according to claim 1, characterized in that, The mass ratio of each active ingredient in the composition is as follows: 1-10 parts extracellular vesicles, 0.01-1 parts cell-active factors, 0.1-5 parts amino acids, and 0.01-1 parts polypeptides.

6. The external vesicle composition for promoting hair growth according to claim 1, characterized in that, The composition is a topical preparation, and the dosage form is selected from one of the following: serum, spray, gel, or microneedle patch.

7. A method for preparing an external vesicle composition for promoting hair growth, comprising preparing the external vesicle composition for promoting hair growth as described in any one of claims 1-6, characterized in that, Includes the following steps: S1. Stem cell expansion culture: Mesenchymal stem cells are seeded into a culture container containing serum-free culture medium and placed in a simulated microgravity bioreactor for expansion culture; S2, Extracellular vesicle secretion induction: Add cell activating factors to the culture system and continue culturing for 12-48 hours to induce mesenchymal stem cells to secrete extracellular vesicles; S3. Extracellular vesicle separation and extraction: Collect the culture supernatant, and successively remove cell debris by low-speed centrifugation, concentrate by tangential flow filtration, and purify by chromatography to obtain extracellular vesicles; S4. Combination of active ingredients: The extracellular vesicles obtained in S3 are mixed with cell-active factors, amino acids, and peptides in a certain proportion, excipients are added, and homogenization is performed to obtain an extracellular vesicle composition.

8. A method for preparing an external vesicle composition for promoting hair growth according to claim 7, characterized in that, The cell activating factor in S2 is selected from one or both of tumor necrosis factor-α and interleukin-1α.

9. A method for preparing an external vesicle composition for promoting hair growth according to claim 7, characterized in that, In S3, the low-speed centrifugation speed is 300-500×g and the time is 5-15 minutes; the membrane pore size of the tangential flow filtration is 0.1-0.22μm; and the chromatography technique is size exclusion chromatography or ion exchange chromatography.

10. A method for preparing an external vesicle composition for promoting hair growth according to claim 7, characterized in that, The simulated microgravity environment in S1 has a rotation speed of 10-20 rpm, a culture temperature of 37°C, and a CO2 concentration of 5%.

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