Preparation method of high-activity exosome hair growth promoter

Abstract

The invention discloses a preparation method of a high-activity exosome hair growth promoter, which comprises the following steps: providing purified exosome, and mixing the purified exosome with hair growth active ingredients in a PBS (Phosphate Buffer Solution) with the pH value of 7.0-7.6, adding hydroxypropyl-beta-cyclodextrin with the final concentration of 0.5%-2% (w / v) and trehalose with the final concentration of 5%-10% (w / v), mildly incubating for 1-2 hours under the conditions that the temperature is 35-37 DEG C and the speed is 50-100 rpm, purifying, and freeze-drying to obtain a solid product. According to the invention, through the synergistic effect of the specific membrane permeability enhancer and the membrane stabilization protective agent, the efficient loading of the hair growth active component and the natural biological activity of the exosome are considered, the process is mild and controllable, the industrial amplification is easy, and the solid preparation solves the problem of inconvenient storage and transportation, and has strong practicability.

Description

Technical Field

[0001] This invention relates to the fields of biomedical technology and cosmetic technology, specifically to a method for preparing a highly active exosome hair growth promoter. Background Technology

[0002] Exosomes are extracellular vesicles, approximately 30-150 nanometers in diameter, actively secreted by cells. They carry important functional molecules such as proteins, lipids, and nucleic acids (e.g., miRNA, mRNA). As a natural drug delivery carrier, exosomes possess outstanding advantages such as good biocompatibility, low immunogenicity, excellent targeting, and the ability to cross biological barriers (e.g., the stratum corneum of the skin), showing great application potential in the field of hair regeneration.

[0003] To enhance the activation and repair effects of exosomes on hair follicles, specific hair growth active ingredients need to be loaded into the exosomes. However, existing loading technologies have significant drawbacks. For example, Chinese patent CN113894523A discloses a "method for preparing exosomes loaded with miRNA by electroporation," which uses a high-voltage electric field to form micropores on the exosome membrane to achieve loading. However, the high-intensity electric field can cause leakage of exosome contents and denaturation of membrane proteins. The published data shows that the exosome membrane integrity is only 65-70%, and the loss of biological activity exceeds 30%. Chinese patent CN112721568A discloses a "simple incubation method for preparing exosomes loaded with plant extracts." This method is gentle but relies on passive diffusion, and the loading efficiency is only 10-15%, making it difficult to achieve an effective therapeutic concentration. In addition, although the ultrasonic method (such as CN114013879A) can improve the loading efficiency, the ultrasonic shear force can cause exosomes to rupture and aggregate, significantly reducing uniformity and functional stability. Therefore, a core technological contradiction has long existed in this field: how to achieve high load efficiency while maximizing the preservation of the natural structure and biological activity of exosomes. Existing technologies have not provided an effective solution, which has become the main bottleneck for the commercialization of exosome-based hair growth products. Summary of the Invention

[0004] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for preparing a highly active exosome hair growth promoter. This method must meet the following requirements: mild and controllable process, high loading efficiency, maximum retention of exosome biological activity, and solve the problem of inconvenient storage and transportation of exosome preparations.

[0005] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a highly active exosome hair growth promoter, comprising the following preparation steps: S1. Provides purified exosomes; S2. Mix the exosomes described in step / 1 with the hair growth active ingredient in PBS buffer with a pH of 7.0-7.6 to form a mixture; S3. Add a membrane permeability enhancer and a membrane stabilizer to the mixture and incubate under mild conditions to achieve loading of the hair growth active ingredient into the exosome; the membrane permeability enhancer is hydroxypropyl-β-cyclodextrin, and its final concentration in the mixture is 0.1%-5% (w / v); the membrane stabilizer is trehalose, and its final concentration in the mixture is 1%-15% (w / v); the mild conditions are: at a temperature of 30℃-40℃, with slow stirring at a speed of 50rpm-150rpm, incubating for 0.5 hours-4 hours; S4. The loaded mixture is purified to remove unloaded components, thereby obtaining the highly active exosome hair growth promoter.

[0006] Preferably, the final concentration of hydroxypropyl-β-cyclodextrin in the mixture in S3 is 0.5%-2% (w / v).

[0007] Preferably, the final concentration of trehalose in the mixture in step S3 is 5%-10% (w / v).

[0008] Preferably, the mild conditions are: at a temperature of 35℃-37℃, with slow stirring at a speed of 50rpm-100rpm, incubated for 1-2 hours.

[0009] Preferably, the exosomes are derived from any one or more of human mesenchymal stem cells, adipose stem cells, or plant cells, and the exosomes are purified by size exclusion chromatography or ultracentrifugation.

[0010] Preferably, the hair growth active ingredient is any one or more of microRNA, small molecule polypeptide, or plant extract; the plant extract is selected from one or more of Polygonum multiflorum extract, Platycladus orientalis leaf extract, or rosemary extract.

[0011] Preferably, the purification is performed using ultrafiltration centrifugation or tangential flow filtration.

[0012] Preferably, after step S4, a formulation step S5 is further included: mixing the purified highly active exosome hair growth promoter with a lyophilization protectant and freeze-drying it to obtain a solid formulation.

[0013] (III) Beneficial Effects This invention provides a method for preparing a highly active exosome-based hair growth promoter. It has the following beneficial effects: 1. This invention utilizes the synergistic effect of hydroxypropyl-β-cyclodextrin and trehalose to achieve efficient loading of germinogenic active ingredients into exosomes without relying on severe physical conditions such as high-voltage electric fields and ultrasonic shear forces. At the same time, it can effectively maintain the integrity of the natural membrane structure of exosomes, avoid leakage of contents and denaturation of membrane proteins, and fundamentally solve the problem of the incompatibility between loading efficiency and activity maintenance in the prior art.

[0014] 2. The entire loading process of this invention is carried out in a mild environment close to physiological conditions. Key parameters such as temperature, reagent concentration, and incubation time are easy to control precisely, which can preserve the inherent targeted delivery and bioregulatory functions of exosomes to the greatest extent. The process is stable and reliable, the operation steps are simple, no complex special equipment is required, and it is easy to scale up industrial production.

[0015] 3. The final product of this invention is freeze-dried into a solid powder, which greatly improves the storage stability of the product. It can be stored for a long time without deep freezing conditions, effectively reducing storage and transportation costs. The product has excellent resolubility and can be quickly dissolved in aqueous systems, which facilitates its subsequent development into various practical dosage forms such as serums, gels, and soluble microneedles to meet the application needs of different scenarios. Detailed Implementation

[0016] 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.

[0017] Example 1: This invention provides a method for preparing a highly active exosome hair growth promoter, comprising the following preparation steps: S1. Provides purified exosomes; S2. Mix the exosomes from step / 1 with the hair growth active ingredient in PBS buffer with a pH of 7.0-7.6 to form a mixture; S3. Add a membrane permeability enhancer and a membrane stabilizer to the mixture and incubate under mild conditions to achieve loading of the hair growth active ingredient into the exosome. The membrane permeability enhancer is hydroxypropyl-β-cyclodextrin, with a final concentration of 0.1%-5% (w / v) in the mixture. The membrane stabilizer is trehalose, with a final concentration of 1%-15% (w / v) in the mixture. The mild conditions are: incubation at 30℃-40℃ with slow stirring at 50rpm-150rpm for 0.5 hours-4 hours. S4. Purify the loaded mixture to remove unloaded components and obtain a highly active exosome hair growth promoter.

[0018] The final concentration of hydroxypropyl-β-cyclodextrin in S3 in the mixture is 0.5%-2% (w / v).

[0019] The final concentration of trehalose in the mixture in S3 is 5%-10% (w / v).

[0020] The mild conditions are: at a temperature of 35℃-37℃, slowly stir at a speed of 50rpm-100rpm, and incubate for 1-2 hours.

[0021] Exosomes are derived from any one or more of human mesenchymal stem cells, adipose stem cells, or plant cells. Exosomes are purified by size exclusion chromatography or ultracentrifugation.

[0022] The active ingredient for hair growth is any one or more of microRNA, small molecule polypeptide or plant extract; the plant extract is selected from one or more of Polygonum multiflorum extract, Platycladus orientalis leaf extract or rosemary extract.

[0023] Purification was performed using ultrafiltration centrifugation or tangential flow filtration.

[0024] Following S4 is the formulation step S5: the purified highly active exosome hair growth promoter is mixed with a lyophilization protectant and then freeze-dried to obtain a solid formulation.

[0025] Example 2: (1) The exosome purification steps were the same as in Example 1, and the exosome suspension concentration was 1.1 × 10¹. 0 particles / mL; (2) Construction of the loading system: Take 1 mL of exosome suspension, add 60 μg of palmitoyl tripeptide-1 (final concentration 60 μg / mL), and mix well; (3) Mild active loading: Add 100 μL of 20% (w / v) hydroxypropyl-β-cyclodextrin aqueous solution (final concentration 2.0% (w / v)) and 200 μL of 50% (w / v) trehalose aqueous solution (final concentration 10.0% (w / v)), and incubate at 36℃ and 100 rpm for 2 h on a shaker; (4) The purification and preparation steps are the same as in Example 1, and exosome powder loaded with hair growth peptides is obtained.

[0026] Comparative Example 1: (1) The exosome purification steps were the same as in Example 1, and the exosome suspension concentration was 1.2 × 10¹. 0 particles / mL; (2) Take 1 mL of exosome suspension, add 50 μg miRNA-218-5p mimic, mix well and transfer to a 0.4 cm electroporation cup; (3) Electroporation instrument parameter settings: voltage 0.4kV, capacitance 125μF, resistance 200Ω, for electroporation treatment; (4) After treatment, incubate at 37°C for 30 min to restore the exosome membrane structure; (5) The purification and formulation steps are the same as in Example 1, and the exosome powder loaded by electroporation is obtained.

[0027] Comparative Example 2: (1) The exosome purification steps were the same as in Example 1, and the exosome suspension concentration was 1.2 × 10¹. 0 particles / mL; (2) Take 1 mL of exosome suspension, add 50 μg miRNA-218-5p mimic, and incubate in a 37℃ constant temperature water bath for 1.5 h (without HP-β-CD and trehalose, and without stirring). (3) The purification and formulation steps are the same as in Example 1, and the exosome powder loaded by simple incubation method is obtained.

[0028] Experimental example: 1. Sample pretreatment The lyophilized powders from Examples 1-2 and Comparative Examples 1-2 were reconstituted with 1 mL of PBS buffer (pH 7.4). The concentration of exosomes after reconstitution was detected by NTA. The concentration of all samples was adjusted to 1.0 × 10¹. 0 particles / mL, for later use.

[0029] 2. Comparison of detection methods and procedures (1) Load efficiency detection miRNA loading efficiency (Example 1, Comparative Examples 1-2): Take 200 μL of each reconstituted sample and extract total RNA using an exosome RNA extraction kit (Qiagen). Detect RNA purity using Nanodrop (A260 / A280 = 1.8-2.0). According to the qRT-PCR kit instructions, U6 was used as the internal reference gene to perform reverse transcription (42℃ for 30 min, 85℃ for 5 min) and PCR amplification (95℃ for 30 s, 40 cycles: 95℃ for 5 s, 60℃ for 30 s). Use 2 - The relative content of miRNA-218-5p in exosomes was calculated using the ΔΔCt method, and the loading efficiency was calculated as (miRNA content in exosomes / total initial amount of added miRNA) × 100%.

[0030] Hair growth peptide loading efficiency (Example 2): Take 200 μL of the reconstituted sample, add 200 μL of acetonitrile, vortex for 5 min, centrifuge at 10000×g for 5 min, and take the supernatant; HPLC detection conditions: C18 column (4.6×150mm), mobile phase acetonitrile-water (containing 0.1% trifluoroacetic acid, 50:50, v / v), flow rate 0.8mL / min, detection wavelength 220nm, column temperature 25℃; A standard curve was plotted using palmitoyl tripeptide-1 standard, and the peptide content in exosomes was calculated. Loading efficiency = (peptide content in exosomes / total initial peptide amount) × 100%.

[0031] (2) Exosome membrane integrity detection: Take 100 μL of each reconstituted sample, add 1 μL of DiO fluorescent dye (final concentration 5 μmol / L, DiO specifically labeled lipid bilayer), and incubate at 37°C in the dark for 30 min. Wash twice with PBS buffer, centrifuge at 10000×g for 5 min, and resuspend in 100μL PBS; Nanoflow cytometry detection: excitation wavelength 488nm, emission wavelength 510nm, counting 10,000 particles, recording the number of fluorescent positive particles (membrane-intact exosomes). Membrane integrity retention rate = (number of fluorescent positive particles / total number of particles) × 100%.

[0032] (3) Bioactivity detection: HDP cells in the logarithmic growth phase were seeded at 5×10³ cells / well in a 96-well plate, with 100μL of culture medium added to each well. The plates were then incubated at 37℃ and 5%CO2 for 24h. Discard the old culture medium and add culture medium containing each reconstituted sample (final exosome concentration 1×10⁻⁶). 8 (particles / mL), set up a blank control group (culture medium only), and 3 replicates per group; After culturing for another 48 hours, add 10 μL of LCK-8 reagent to each well and incubate at 37°C for 2 hours. The absorbance (OD value) at 450 nm was measured using an ELISA reader. The cell proliferation rate was calculated as (OD value of experimental group - OD value of blank control group) / (OD value of blank control group) × 100%.

[0033] 4. Experimental Results and Analysis All experimental data were statistically analyzed using SPSS 22.0 software. Quantitative data were expressed as mean ± standard deviation (x ± s). The t-test was used for comparisons between groups, and p < 0.05 was considered statistically significant. The results are shown in the table below: In summary, the loading efficiency of Examples 1 and 2 both reached over 68%, with Examples 2 (peptide loading) reaching as high as 70.5%, which is comparable to the electroporation method (Comparative Example 1: 65.8%) and significantly higher than the simple incubation method (Comparative Example 2: 13.5%), proving that the mild loading process of the present invention can achieve high-efficiency loading.

[0034] The exosome membrane integrity retention rates in Examples 1 and 2 both exceeded 92%, which is much higher than that of the electroporation method (68.4%) and close to that of the simple incubation method (94.1%). This indicates that the process of the present invention causes minimal damage to the exosome structure and can preserve its natural structure to the maximum extent.

[0035] Examples 1 and 2 showed significantly better growth-promoting effects on hair follicle cells than Comparative Examples 1 and 2. The cell proliferation rate of Example 2 reached 71.2%, while the electroporation method resulted in decreased biological activity (42.3%) due to membrane structure damage, and the simple incubation method had a weaker growth-promoting effect (35.6%) due to low loading efficiency. This fully demonstrates that the exosome hair growth promoter prepared in this invention has both high loading efficiency and high biological activity.

[0036] 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. A method for preparing a high activity exosome hair growth promoter, characterized by, The preparation process comprises the following steps: S1, providing purified exosomes; S2, mixing the exosomes of step S1 with a hair growth active ingredient in a PBS buffer with a pH value of 7.0-7.6 to form a mixture; S3, adding a membrane permeability enhancer and a membrane stability protective agent to the mixture and incubating under mild conditions to achieve loading of the hair growth active ingredient into the exosomes; the membrane permeability enhancer is hydroxypropyl-β-cyclodextrin, and the final concentration thereof in the mixture is 0.1%-5%(w / v); the membrane stability protective agent is trehalose, and the final concentration thereof in the mixture is 1%-15%(w / v); the mild conditions are: slow stirring at a speed of 50rpm-150rpm at a temperature of 30°C-40°C for 0.5 hours-4 hours; S4, purifying the loaded mixture to remove components not loaded to obtain the high-activity exosome hair growth promoter.

2. The method for preparing a highly active exosome hair growth promoter according to claim 1, characterized in that: In S3, the final concentration of hydroxypropyl-β-cyclodextrin in the mixture is 0.5%-2%(w / v).

3. The method of claim 1, wherein the method is for preparing a high-activity exosome hair growth promoter. In S3, the final concentration of trehalose in the mixture is 5%-10%(w / v).

4. The method of claim 1, wherein the method is for preparing a high-activity exosome hair growth promoter. The mild conditions are: slow stirring at a speed of 50rpm-100rpm at a temperature of 35°C-37°C for 1 hour-2 hours.

5. The method of claim 1, wherein the method is for preparing a high-activity exosome hair growth promoter. The exosomes are derived from any one or more of human mesenchymal stem cells, adipose stem cells or plant cells, and the exosomes are purified by size exclusion chromatography or ultracentrifugation.

6. The method of claim 1, wherein the method is for preparing a high-activity exosome hair growth promoter. The hair growth active ingredient is any one or more of microRNA, small molecule polypeptide or plant extract; the plant extract is selected from one or more of extract of Radix Polygoni Multiflori, extract of Biota orientalis or extract of Rosmarinus officinalis.

7. The method of claim 1, wherein the method is for preparing a high-activity exosome hair growth promoter. The purification is performed by ultrafiltration centrifugation or tangential flow filtration.

8. The method of claim 1, wherein the method is for preparing a high-activity exosome hair growth promoter. After S4, a preparation step S5 is further included: mixing the purified high-activity exosome hair growth promoter with a freeze-drying protective agent, and freeze-drying to obtain a solid preparation.

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