Composition for treating alopecia and preparation method and application thereof

Abstract

The invention discloses a composition for treating alopecia and a preparation method and application thereof.The composition comprises exosome, a WNT activator and a BMP signal inhibitor, the mass molar ratio of the exosome to the WNT activator to the BMP signal inhibitor in the composition is 150-250 [mu] g: 6 nmol: 2.5 nmol, the composition can be obtained through direct mixing, alopecia regeneration treatment is conducted through local smearing, and the composition is free of side effects and free of toxic and side effects. The back hair covering area of the mouse alopecia area within 14-18 days can be close to 100%, and the hair regeneration effect is obviously improved in hair regeneration of physical alopecia, male alopecia and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a composition for treating hair loss, its preparation method, and its application. Background Technology

[0002] Exosomes are biological nanoscale spherical lipid bilayer vesicle structures with a diameter of 30-120 nm secreted by cells. Their main components include nucleic acids, proteins, and lipids. As a medium for intercellular communication and material exchange, exosomes have both physiological and pathological functions.

[0003] Patent application 202211189283.X first proposed the application of exosomes in promoting hair regeneration. It discloses the use of growth factor SCF contained in exosomes to promote the growth of stem cells and melanocytes, TGFβ3 to transform inflammatory cells into anti-inflammatory cells, VEGF and MIP-1 to stimulate angiogenesis and scalp cell formation, and FGF in combination with other growth factors to affect various cells. Hair follows a cycle of growth, rest, shedding, and regeneration. Hair loss is a condition mainly caused by the interruption of the body's hair growth cycle. While it generally does not threaten life, it has a significant impact on an individual's mood and quality of life. Currently, there is no cure for hair loss. Treatment mainly involves hair follicle transplantation and drug therapy, including oral and topical medications, primarily steroids and corticosteroids. However, these drugs generally have drawbacks due to adverse reactions. The aforementioned patent application provides a non-invasive and side-effect-free hair regrowth technology based on umbilical cord mesenchymal stem cell exosomes, which can promote hair growth by being directly applied to the skin. However, this approach has drawbacks such as complex exosome culture and limited hair regeneration promotion effect. Providing a hair loss treatment method that can improve hair regeneration effect and can be widely promoted is the key to solving the above problems. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a composition for treating hair loss, its preparation method and application, which, by combining exosomes, WNT activators and BMP signaling inhibitors, has a significantly improved hair regeneration effect in both physical hair loss and androgenetic alopecia.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] On the one hand, a composition for treating hair loss is provided, comprising exosomes, WNT activators, and BMP signaling inhibitors.

[0007] In another aspect, a method for preparing the above-mentioned composition for treating hair loss is provided, comprising directly mixing the exosomes, WNT activator and BMP signaling inhibitor.

[0008] On the other hand, there is an application of the above-mentioned composition for treating hair loss in the treatment of hair loss, including applying the medication to the hair loss area by means of topical application.

[0009] Compared with the prior art, the present invention has the following advantages:

[0010] 1. This invention creatively provides a hair regeneration strategy based on the combination of WNT activator and BMP signal inhibitor, which works synergistically with exosomes to achieve nearly 100% back hair coverage in 14-18 days. It has a significant improvement in hair regeneration effect in both physical hair loss and androgenetic alopecia.

[0011] 2. The composition of the present invention contains only exosomes, WNT activators and BMP signaling inhibitors. The composition for treating hair loss can be prepared by dissolving the above composition in a buffer solution.

[0012] 3. In the pharmaceutical composition of the present invention, the exosomes are derived from mesenchymal stem cells obtained by 3D microcarrier suspension culture technology. They have significantly increased particle density and miRNAs closely related to the regeneration and repair process. When used in combination with the WNT activator CHIR99021 and the BMP signaling inhibitor DM3189, they achieve a highly effective treatment effect for hair loss.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the therapeutic effect of the drug composition in Example 1 on hair loss in a physical hair loss model;

[0015] Figure 2 This is a schematic diagram showing the therapeutic effect of the pharmaceutical composition of Example 1 on hair loss in a male pattern baldness model;

[0016] Figure 3 A pathological staining image of hair follicles during the hair loss treatment process in a male pattern baldness model;

[0017] Figure 4 A bar chart showing the number of hair follicles during the hair loss treatment process in a male pattern baldness model;

[0018] Figure 5 Transmission electron microscopy images of exosomes (3D-Exos) from mesenchymal stem cells obtained by 3D microcarrier suspension culture technology in Example 1 and exosomes (2D-Exos) in Comparative Example 1.

[0019] Figure 6 This is a schematic diagram showing the particle size distribution and particle concentration test results of exosomes (2D-Exos) in Comparative Example 1.

[0020] Figure 7 This is a schematic diagram showing the particle size distribution and particle concentration test results of exosomes (3D-Exos) from mesenchymal stem cells obtained by 3D microcarrier suspension culture technology in Example 1.

[0021] Figure 8 This is a volcano plot showing the difference in miRNA abundance between exosomes (3D-Exos) from mesenchymal stem cells obtained by 3D microcarrier suspension culture technology in Example 1 and exosomes (2D-Exos) in Comparative Example 1.

[0022] Figure 9 This is a heatmap showing the cluster analysis of miRNA abundance in exosomes (3D-Exos) of mesenchymal stem cells obtained from 3D microcarrier suspension culture technology in Example 1 and exosomes (2D-Exos) in Comparative Example 1.

[0023] Figure 10 Scatter plot of highly expressed miRNA target gene pathway enrichment in exosomes (3D-Exos) of mesenchymal stem cells obtained from 3D microcarrier suspension culture technology in Example 1 and exosomes (2D-Exos) in Comparative Example 1. Detailed Implementation

[0024] The technical solution will now be clearly and completely described with reference to the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0025] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0026] In the following description, the terms “including,” “containing,” “having,” and “containing” are open-ended terms, meaning that they include but are not limited to.

[0027] Those skilled in the art should understand that, in the following description of the embodiments of this application, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0028] Those skilled in the art will understand that the numerical ranges in the embodiments of this application should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this application. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, the technical / scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While this application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this application. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] The technical principle adopted in this invention is based on the synergistic effect of exosomes, WNT activation pathway and BMP inhibition pathway to form a synergistic mechanism of "exosome microenvironment regulation + signal pathway activation", which fully activates hair follicle stem cells and promotes the growth rate and growth area of ​​hair.

[0031] On the one hand, a composition for treating hair loss is provided, comprising exosomes, WNT activators, and BMP signaling inhibitors.

[0032] The Wnt signaling pathway is a complex network of proteins that plays a major role in embryonic development and normal physiological processes in adult animals, participating in various cellular life activities. During their research, the inventors discovered that when the WNT activation pathway and the BMP inhibition pathway are combined with exosomes, hair follicle stem cells can be fully activated, promoting hair growth rate and increasing hair growth area.

[0033] In some embodiments, the molar ratio of the exosomes, WNT activator, and BMP signaling inhibitor is 150-250 μg: 6 nmol: 2.5 nmol.

[0034] In some embodiments, the exosomes are derived from mesenchymal stem cells obtained using 3D microcarrier suspension culture technology.

[0035] In some embodiments, the WNT activator is CHIR99021 and the BMP signaling inhibitor is DM3189.

[0036] In a further preferred embodiment, the WNT activator in the pharmaceutical composition of the present invention is CHIR99021, and the BMP signaling inhibitor is DM3189. The combination of the two with exosomes can fully activate hair follicle stem cells.

[0037] In some embodiments, the method for preparing the exosomes includes: culturing mesenchymal stem cells using 3D microcarrier suspension culture technology to obtain a cell culture supernatant, and then obtaining the exosomes by tangential flow ultrafiltration. In some embodiments, the microcarrier is a dextran microcarrier, and the seeding density of the mesenchymal stem cells on the microcarrier is 10-1. 4 cells / cm 2 The concentration of the microcarrier in the culture medium is 3 g / L, and the stirring speed in the suspension culture is 40 rpm.

[0038] In some preferred embodiments, the exosomes are derived from mesenchymal stem cells cultured using 3D microcarrier suspension culture technology and then obtained by tangential flow ultrafiltration (3D-Exos). Although 3D-Exos exosomes have a size similar to those obtained from 2D normoxic stem cell culture, their particle concentration is significantly higher than that of 2D exosomes, being 6.6 times that of 2D exosomes. Furthermore, miRNA sequencing results show that 3D-Exos exosomes contain significantly increased abundance of miRNAs closely related to promoting hair regeneration, such as miR-25-3p, miR-23a-3p, and miR-93-5p.

[0039] In another aspect, a method for preparing the above-mentioned composition for treating hair loss is provided, comprising directly mixing the exosomes, WNT activator and BMP signaling inhibitor in a buffer solution to obtain a composition system, wherein the concentration of exosomes in the composition system is 0.3~0.5 μg / μL.

[0040] On the other hand, there is an application of the above-mentioned composition for treating hair loss in the preparation of a product for treating hair loss, the application including applying the medicine to the hair loss area by means of topical application.

[0041] Prior to this application, a series of experiments were conducted. Some of the experimental results are listed below to provide a more detailed description of the invention. The following is a detailed description in conjunction with the embodiments.

[0042] Example 1

[0043] This embodiment provides a composition for treating hair loss, comprising exosomes, a WNT activator, and a BMP signaling inhibitor. The exosomes are mesenchymal stem cells obtained using 3D microcarrier suspension culture technology. Specifically:

[0044] Raw material source: Human umbilical cord mesenchymal stem cells, P3 generation;

[0045] P3 generation human umbilical cord mesenchymal stem cells were used at 10 4 cells / cm 2The microcarriers were seeded into 500 mL double-sided wall roller bottles at a density of 3 g / L. The roller bottles were placed on a magnetic stirrer and transferred to a 37℃ 5% CO2 incubator for stirring and culture at 40 rpm. The 500 mL double-sided wall roller bottles were from Corning Biotechnology. The microcarriers were dextran microcarriers with a particle size of 200 μm, model Cytodex1, purchased from Cytiva. The culture medium was serum-free human mesenchymal stem cell culture medium SC2013-G, purchased from Tianjin Haoyang.

[0046] After culturing for 7 days, the supernatant was collected and filtered sequentially through 0.45 μm and 0.22 μm filters to obtain the filtrate.

[0047] The filtrate was concentrated by tangential flow ultrafiltration through a 300 kD hollow fiber membrane column with a concentration ratio of 1:20 to obtain the concentrated system.

[0048] The concentrated system was replaced with physiological saline to obtain an exosome suspension;

[0049] The exosome suspension was sterilized by filtration through a 0.22 μm filter to obtain exosomes;

[0050] This embodiment also provides a method for preparing the above-mentioned composition for treating hair loss, comprising: adding 200 μg of the above-mentioned exosomes, CHIR99021 (WNT activator), and DM3189 (BMP signaling inhibitor) to 500 μL PBS buffer, mixing them evenly to obtain the composition system; wherein the concentration of CHIR99021 in the composition system is 12 μM and the concentration of DM3189 is 5 μM.

[0051] Comparative Example 1

[0052] This comparative study investigated the effect of exosomes on the performance of the composition for treating hair loss. It was the same as in Example 1, except that the exosomes in the composition were exosomes cultured under 2D conditions. Specifically, P3 human umbilical cord mesenchymal stem cells were cultured in T175 cell culture flasks with an oxygen concentration of 20%. The culture flasks were placed in a 37°C 5% CO2 incubator. After 3 days, the cell culture supernatant was collected and the exosomes were extracted.

[0053] Performance Evaluation

[0054] Mouse hair loss models were established by treating mice with hair removal cream. 500 μL of the composition system from Example 1 was evenly applied to the hair-removing areas of the mice, and they were cultured normally for the predetermined time. The results were as follows: Figure 1As shown, half of the mice using the composition system of the present invention (3D-EV+C+D) had full hair growth by day 14, and the hair growth area and growth rate were significantly better than those of the control group or the group that only used exosomes.

[0055] Mice were subcutaneously injected with testosterone propionate (5 mg / kg) daily in the back for 4 weeks. Afterward, the back hair was completely removed with a depilatory cream to obtain a dihydrotestosterone-induced male pattern baldness model. 500 μL of the composition system from Example 1 was evenly applied to the bald area of ​​the mice, and they were cultured normally for the predetermined time. The results were as follows... Figure 2 As shown, on day 18, the back hair coverage of mice using the composition system of this invention (3DEV+C+D) was close to 100%, significantly higher than that of the control group. The number of hair follicles was determined by pathological staining ( Figure 3 ) and hair follicle count bar chart ( Figure 4 It was confirmed that the number of hair follicles in mice using the composition of the present invention was significantly higher than that in other groups, indicating that the drug composition of exosomes, WNT activators and BMP signaling inhibitors of the present invention works synergistically to improve the quality of hair follicle regeneration.

[0056] Figure 5 The images show transmission electron microscopy (TEM) images of exosomes (3D-Exos) obtained from mesenchymal stem cells using the 3D microcarrier suspension culture technique in Example 1 and exosomes (2D-Exos) in Comparative Example 1. The sample preparation method was uranium acetate staining, in which the exosomes to be tested were fixed on a copper grid for staining. As can be seen, a clear double-layered membrane structure can be observed in both the exosomes (3D-Exos) obtained from mesenchymal stem cells using the 3D microcarrier suspension culture technique in Example 1 and the exosomes (2D-Exos) in Comparative Example 1.

[0057] The particle size distribution test results of exosomes (3D-Exos) from mesenchymal stem cells obtained by 3D microcarrier suspension culture technology in Example 1 and exosomes (2D-Exos) in Comparative Example 1 are as follows: Figure 6 and Figure 7 As shown, where Figure 6 The results show the particle size distribution of exosomes (2D-Exos) from Comparative Example 1. Figure 7The particle size distribution test results for exosomes (3D-Exos) obtained from mesenchymal stem cells using 3D microcarrier suspension culture technology in Example 1 are shown. The results were obtained using a nano-Courte particle size analyzer (resistance-inducing pulse method, RPS) manufactured by Shenzhen Rockchip Technology Co., Ltd. It can be seen that the particle sizes of exosomes (3D-Exos) obtained from mesenchymal stem cells using 3D microcarrier suspension culture technology in Example 1 and exosomes (2D-Exos) in Comparative Example 1 are both between 30 and 150 nm (average particle size of 3D-Exos is 71 nm, and average particle size of 2D-Exos is 69 nm), which meets the size requirements of MSC-exos. The particle concentration test results show that the particle concentration of exosomes (3D-Exos) obtained from mesenchymal stem cells using 3D microcarrier suspension culture technology in Example 1 is significantly higher than that of the comparative example (2D-Exos). The particle concentration of 3D-Exos is 2.76E+10 particles / mL, and the particle concentration of 2D-Exos is 4.17E+09 particles / mL.

[0058] The differences in miRNA content between exosomes (3D-Exos) obtained from mesenchymal stem cells in Example 1 (3D-Exos) and exosomes (2D-Exos) in Comparative Example 1 were analyzed based on miRNA sequencing. Figure 8 Volcano plot showing the difference in miRNA expression levels between the two. Figure 9 A heatmap of cluster analysis showing the difference in miRNA expression levels between the two groups. Figure 10 A scatter plot showing the enrichment of target gene signaling pathways for the two differentially expressed miRNAs. Combined with... Figures 8-10 It is evident that, compared to 2D-Exos, 3D-Exos contains 20 upregulated and 10 downregulated miRNAs. Some miRNAs with potential hair regeneration-promoting effects, such as miR-25-3p, miR-23a-3p, and miR-93-5p, are upregulated in 3D-Exos. These miRNAs mainly achieve this by targeting and inhibiting "hair follicle growth inhibitory factors" or activating "hair follicle stem cell proliferation / growth phase maintenance pathways." The target genes of the aforementioned upregulated miRNAs are mainly enriched in a series of signaling pathways such as PI3K / Akt, mTOR, HIF-1, and AMPK, which promote hair follicle cell activation, regulate cell proliferation and autophagy, promote angiogenesis, and regulate inflammation. Based on the hair regrowth promoting effect of the 3D-Exos corresponding composition, it can be seen that 2D culture affects the differentiation potential and self-renewal capacity of stem cells. This invention simulates the 3D microenvironment in vivo by 3D microcarrier culture, which allows the differentiation process of stem cells to fully interact with surrounding cells and matrix, resulting in exosomes with higher functionality and bioactivity. 3D-Exos, in combination with WNT activator and BMP signal inhibitor, fully activates hair follicle stem cells, promoting the growth rate and growth area of ​​hair.

Claims

1. A composition for treating hair loss, characterized in that, It includes exosomes, a WNT activator, and a BMP signaling inhibitor; the mass molar ratio of the exosomes, WNT activator, and BMP signaling inhibitor is 150~250 μg: 6 nmol: 2.5 nmol; the WNT activator is CHIR99021, and the BMP signaling inhibitor is DM3189.

2. The composition for treating hair loss according to claim 1, characterized in that, The exosomes are derived from mesenchymal stem cells obtained using 3D microcarrier suspension culture technology.

3. The composition for treating hair loss according to claim 2, characterized in that, The method for preparing the exosomes includes: culturing mesenchymal stem cells using 3D microcarrier suspension culture technology to obtain cell culture supernatant, and then obtaining the exosomes by tangential flow ultrafiltration.

4. The composition for treating hair loss according to claim 3, characterized in that, In the 3D microcarrier suspension culture technology, the microcarrier is a dextran microcarrier.

5. The composition for treating hair loss according to claim 4, characterized in that, The mesenchymal stem cells were seeded at a density of 10 on the microcarrier. 4 cells / cm 2 The concentration of the microcarrier in the culture medium is 3 g / L, and the stirring speed in the suspension culture is 40 rpm.

6. The composition for treating hair loss according to claim 3, characterized in that, The tangential flow ultrafiltration is performed using a 300 kD hollow fiber membrane column.

7. A method for preparing the composition for treating hair loss as described in claim 1, characterized in that, include: The exosomes, WNT activator, and BMP signaling inhibitor were directly mixed in a buffer solution to obtain the composition system.

8. The method according to claim 7, characterized in that, In the composition system, the concentration of exosomes is 0.3~0.5 μg / μL.

9. The use of the composition for treating hair loss as described in claim 1 in the preparation of a product for treating hair loss, characterized in that, The application includes applying medication using a topical application method.

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