Preparation method of mesenchymal stem cell exosome repair gel

Abstract

The invention discloses a preparation method of mesenchymal stem cell exosome repair gel, which comprises the following steps: mixing purified mesenchymal stem cell exosome with a biocompatible gel matrix solution at low temperature, adding a composite freeze-drying protective agent composed of trehalose, mannitol and polyethylene glycol, and performing freeze-drying to obtain the mesenchymal stem cell exosome repair gel. And performing programmed cooling and freeze-drying to obtain the product. According to the method, through collaborative optimization of a protective agent formula and a freeze-drying process, the problems that existing exosome wet gel is poor in stability, activity is prone to attenuation, and storage and transportation are inconvenient are solved. The obtained product is a porous spongy solid, is quick to redissolve and can recover the usability of the gel, the biological activity of the exosome is well reserved, the exosome can be stored for a long time without a cold chain, the process can be industrially amplified, and a reliable scheme is provided for clinical transformation and industrialization of the exosome repair gel.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology and tissue engineering materials technology, specifically a method for preparing a mesenchymal stem cell exosome repair gel. Background Technology

[0002] Mesenchymal stem cell exosomes, with their excellent anti-inflammatory, angiogenesis-promoting, and tissue regeneration capabilities, have become highly promising active ingredients for wound repair in the field of regenerative medicine, demonstrating significant application value in repair scenarios such as skin trauma. To achieve local targeted delivery of exosomes and prolong their retention time on the wound surface, current technologies generally use biocompatible hydrogels (such as sodium alginate and sodium hyaluronate) as carriers to load exosomes into a wet repair gel, leveraging the gel's adhesiveness and sustained-release properties to enhance the repair effect.

[0003] However, such wet exosome repair gels face several insurmountable technical bottlenecks: First, they exhibit extremely poor storage stability, as exosomes tend to aggregate under hydration, and the gel matrix is ​​prone to physical swelling or chemical degradation, requiring strict cold chain transportation and storage throughout the process, which significantly increases usage costs; Second, exosome activity decays rapidly, and the functional proteins and nucleic acids they carry are easily inactivated by hydrolysis and enzymatic degradation during storage, leading to fluctuating and unpredictable clinical efficacy; Third, they suffer from insufficient portability and shelf life, as wet formulations are not conducive to standardized production, long-distance transportation, and end-product shelf display, severely restricting industrialization and clinical application.

[0004] Although some research has been conducted on lyophilization protectants for exosomes or single gels in existing technologies, none have developed a complete solution suitable for the complex "exosome-gel" system. These solutions fail to simultaneously guarantee high activity recovery rates of exosomes after lyophilization, maintain key performance characteristics such as morphology and adhesion after gel reconstitution, and lack standardized lyophilization process parameters suitable for industrial scale-up. Therefore, they do not fundamentally address the core pain points of existing wet exosome repair gels. Thus, developing a preparation method that balances exosome activity retention, gel performance stability, and industrial feasibility has become a pressing technical need in this field. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a method for preparing mesenchymal stem cell exosome repair gel, which solves the problems of extremely poor storage stability and rapid decline in exosome activity in existing mesenchymal stem cell exosome repair gels.

[0006] (II) Technical Solution To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a mesenchymal stem cell exosome repair gel, comprising the following steps: S1. The purified mesenchymal stem cell exosomes were mixed with the gel matrix solution at 2-8℃ to obtain an exosome gel mixture; S2. Add a composite lyophilization protectant to the exosome gel mixture. The composite lyophilization protectant is composed of trehalose, mannitol and polyethylene glycol, and is used to protect the exosome repair activity and maintain the performance after gel reconstitution. S3. The mixed solution obtained in S2 is subjected to programmed cooling freeze-drying to obtain the mesenchymal stem cell exosome repair gel.

[0007] Preferably, the final concentration of the composite freeze-drying protectant in S2 is: trehalose 30-60mM, mannitol 20-40mM, and polyethylene glycol 1-5% (w / v); the polyethylene glycol is PEG-4000.

[0008] Preferably, the gel matrix in S1 is one or more of sodium alginate, sodium hyaluronate, chitosan, or collagen; the concentration of the gel matrix solution is 1%-3% (w / v), sterilely filtered through a 0.22μm filter membrane, and the pH is 7.2-7.4.

[0009] Preferably, in S1, the exosomes and gel matrix solution are mixed using a gradient dilution method, with a stirring speed of 30-50 rpm and a mixing time of 15-20 minutes.

[0010] Preferably, the programmed cooling freeze-drying in step S3 specifically includes: Pre-freezing stage: Cool the sample to -40℃ to -50℃ at a cooling rate of -0.5 to -1.5℃ / min and keep it at this temperature for 1 to 3 hours to allow the mixed solution to freeze completely; One-time drying: Under vacuum conditions below 10 Pa, raise the ambient temperature to -20℃ to -15℃ and maintain it for 5-10 hours to remove most of the free water in the sample; Secondary drying: Under vacuum conditions below 5 Pa, gradually increase the ambient temperature to 25°C to 30°C and maintain it for 5-10 hours until the residual moisture content of the sample is below 3% to ensure the long-term stability of the repair gel.

[0011] Preferably, in the above-mentioned secondary drying, the stepwise heating is specifically as follows: first, the temperature is raised to -10°C and held for 1-3 hours, then the temperature is raised to 10°C and held for 1-3 hours, and finally the temperature is raised to 25-30°C, so as to avoid damage to the exosome repair activity due to excessively rapid heating.

[0012] Preferably, the repair gel is a porous sponge-like solid with a resolution time of ≤3 minutes. After resolution, it is a homogeneous gel that can adhere to the skin wound surface to exert a repairing effect.

[0013] (III) Beneficial Effects This invention provides a method for preparing a mesenchymal stem cell exosome repair gel. It has the following beneficial effects: 1. This invention, through the synergistic effect of a composite lyophilization protectant and an optimized programmed lyophilization process, effectively protects the bioactivity of mesenchymal stem cell exosomes, and rapidly restores the uniform morphology and adhesive properties of the repair gel after reconstitution. It eliminates the dependence on cold chains for wet gels, significantly extends product shelf life and storage stability, improves portability and clinical applicability, and simultaneously enables the industrial scale-up of the preparation process, successfully addressing the core pain points of existing exosome repair gels, such as easy activity decay and inconvenient storage and transportation. Detailed Implementation

[0014] 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.

[0015] Example 1: This invention provides a method for preparing a mesenchymal stem cell exosome repair gel, comprising the following steps: S1. The purified mesenchymal stem cell exosomes were mixed with the gel matrix solution at 2-8℃ to obtain an exosome gel mixture; S2. Add a composite lyophilization protectant to the exosome gel mixture. The composite lyophilization protectant is composed of trehalose, mannitol and polyethylene glycol, and is used to protect the exosome repair activity and maintain the performance after gel reconstitution. S3. The mixed solution obtained in S2 is subjected to programmed cooling freeze-drying to obtain the mesenchymal stem cell exosome repair gel.

[0016] The final concentration of the composite freeze-drying protectant in S2 is: trehalose 30-60mM, mannitol 20-40mM, and polyethylene glycol 1-5% (w / v); the polyethylene glycol is PEG-4000.

[0017] The gel matrix in S1 is one or more of sodium alginate, sodium hyaluronate, chitosan, or collagen; the concentration of the gel matrix solution is 1%-3% (w / v), and it is sterile filtered through a 0.22μm filter membrane with a pH of 7.2-7.4.

[0018] In S1, exosomes and gel matrix solutions were mixed using a gradient dilution method, with a stirring speed of 30-50 rpm and a mixing time of 15-20 minutes.

[0019] The programmed cooling freeze-drying process in S3 specifically includes: Pre-freezing stage: Cool the sample to -40℃ to -50℃ at a cooling rate of -0.5 to -1.5℃ / min and keep it at this temperature for 1 to 3 hours to allow the mixed solution to freeze completely; One-time drying: Under vacuum conditions below 10 Pa, raise the ambient temperature to -20℃ to -15℃ and maintain it for 5-10 hours to remove most of the free water in the sample; Secondary drying: Under vacuum conditions below 5 Pa, gradually increase the ambient temperature to 25°C to 30°C and maintain it for 5-10 hours until the residual moisture content of the sample is below 3% to ensure the long-term stability of the repair gel.

[0020] In the above-mentioned secondary drying process, the stepwise temperature increase is as follows: first, the temperature is increased to -10℃ and held for 1-3 hours, then the temperature is increased to 10℃ and held for 1-3 hours, and finally the temperature is increased to 25-30℃ to avoid damage to the exosome repair activity due to excessively rapid temperature increase.

[0021] The repair gel is a porous, sponge-like solid with a resolution time of ≤3 minutes. After resolution, it becomes a homogeneous gel that can adhere to the skin wound surface to exert a repairing effect.

[0022] Comparative Example 1: Exosome repair gel without freeze-drying protectant: Except for step S2, which does not add any freeze-drying protectant, the other raw materials and preparation steps (including exosome purification, gel mixing and freeze-drying process) are completely consistent with those in Example 1. The final product is shrunken and cracked, with no obvious porous structure.

[0023] Comparative Example 2: Exosome repair gel with a single lyophilization protectant: Except for replacing the composite lyophilization protectant with a single trehalose (final concentration 80mM) in step S2, the other raw materials and preparation steps are completely consistent with those in Example 1. The final product is a dense solid with no obvious porous structure.

[0024] Control group: Fresh exosome repair gel: Take the exosome gel mixture prepared in step S1 of Example 1 (without added protectant and without freeze-drying), directly aliquot and seal it, and store it at 4°C for later use as the "fresh sample" reference.

[0025] Experimental example: 1. Experimental Objective The advantages of the lyophilized repair gel prepared in this invention in terms of reconstitution performance, exosome activity, gel performance, and storage stability were verified, and the shortcomings of solutions without a protectant and with a single protectant were compared.

[0026] 2. Experimental Materials and Instruments Samples: Product of Example 1, Product of Comparative Example 1, Product of Comparative Example 2, and fresh sample of the control group (day 0).

[0027] Reagents: Physiological saline (medical grade), BCA protein quantification kit, qRT-PCR kit (miR-21-5p primers), CCK-8 kit, TNF-α / IL-6 ELISA kit.

[0028] Instruments: Rotational rheometer, tensile testing machine, qRT-PCR instrument, enzyme-linked immunosorbent assay (ELISA) reader, NTA nanoparticle tracking analyzer.

[0029] 3. Comparison of experimental methods and procedures (1) Reconstitution performance test Methods: Take each lyophilized sample (Example 1, Comparative Example 1, Comparative Example 2), add 1 mL of physiological saline (25°C) to each bottle, and time until completely dissolved. Observe the uniformity of the gel after reconstitution.

[0030] Steps: ① Equilibrate the sample at room temperature for 30 minutes; ② Quickly add physiological saline and gently invert 3 times; ③ Record the time for complete reconstitution and observe whether there are insoluble substances or layering phenomena.

[0031] (2) Detection of exosome activity recovery rate ① Total protein recovery rate: The total protein concentration of the reconstituted sample was detected by the BCA method and compared with the protein concentration of the control group (day 0) to calculate the recovery rate (recovery rate = sample protein concentration / control group protein concentration × 100%).

[0032] ② miRNA retention rate: The content of miR-21-5p promoting repair was detected by qRT-PCR. Primer sequences (upstream: 5'-UAGCUUAUCAGACUGAUGUUGA-3', downstream: 5'-CAGUACUUUUGUGUCGUUGUAUCCU-3'); Cycling parameters: 95°C pre-denaturation for 5 min, 95°C denaturation for 15 s, 60°C annealing for 30 s, 40 cycles; The retention rate was calculated with the control group as the baseline.

[0033] ③ Cell proliferation activity: Human fibroblasts (HFF-1) were seeded in 96-well plates (1×10⁶ cells / wells). 4 (eggs / well), incubate for 24 hours; add reconstituted gel extract (containing exosomes, final concentration 50 μg / mL), and continue incubation for 48 hours; add CCK-8 reagent, incubate for 2 hours, and detect the absorbance value (OD value) at 450 nm using a microplate reader. The relative activity is calculated with the OD value of the control group as 100%.

[0034] ④ Anti-inflammatory activity: RAW264.7 macrophages were seeded in 96-well plates (2×10⁻⁶ cells / well). 4Inflammation was induced for 2 hours with LPS (1 μg / mL); reconstituted gel extract (final exosome concentration 50 μg / mL) was added and incubated for 24 hours; the concentrations of TNF-α and IL-6 in the supernatant were detected by ELISA kit, and the inhibition rate was calculated (inhibition rate = (model group concentration - sample group concentration) / model group concentration × 100%).

[0035] (3) Gel performance testing Viscosity: The gel after reconstitution was measured using a rotational rheometer (25°C, shear rate 1s). -1 Record the viscosity value.

[0036] Adhesion: Using a tensile testing machine, the reconstituted gel was applied to a pigskin wound model (1 cm² area). 2 After bonding, let stand for 30 minutes, peel off at a rate of 5 mm / min, record the maximum peel force, and calculate the adhesion force (adhesion force = maximum peel force / bonding area).

[0037] (4) Storage stability test All samples (including the control group) were stored in a 4°C refrigerator. Samples were taken on the 30th, 90th, 180th and 360th days, and the above tests (1)-(3) were repeated to record the changes in the indicators.

[0038] 4. The results of the comparative experiment are shown in Table 1 below:

[0039]

[0040] Table 1 (1) Excellent reconstitution performance, suitable for clinical use. Experimental data shows that the reconstitution time of the product in Example 1 is only 90 seconds (day 0), and it can still be completely reconstituted within 2 minutes even after 360 days of storage, and the reconstituted product is a homogeneous gel. In contrast, Comparative Example 1 (without protective agent) could not be completely reconstituted, and Comparative Example 2 (with single protective agent) had a long reconstitution time and layered. This shows that the composite protective agent of the present invention works synergistically with the freeze-drying process to maintain the porous structure of the gel skeleton, ensuring "ready-to-use" and meeting the convenience requirements of clinical wound repair.

[0041] (2) High exosome activity recovery rate and stable repair function. In Example 1, the protein recovery rate was 92% and the miR-21-5p retention rate was 88% on day 30. The cell proliferation activity was 95% and the inhibition rate of anti-inflammatory factors was over 68%, with minimal difference from the fresh sample. After 360 days of storage, the protein recovery rate and cell proliferation activity were still maintained at 85% and 82%, respectively. In contrast, in Comparative Example 1, the protein recovery rate was only 35% after 30 days, with no anti-inflammatory or proliferative activity. In Comparative Example 2, the activity decay was significant, indicating that the composite protectant (trehalose + mannitol + PEG-4000) can effectively protect the exosome membrane structure and functional molecules (proteins, miRNAs), avoid degradation during freeze-drying and storage, and ensure the core bioactivity of the repair gel.

[0042] (3) The gel meets the performance standards and is suitable for wound repair. After reconstitution, the gel viscosity in Example 1 is 8500 mPa·s and the adhesion force is 0.8 N / cm. 2 It closely resembles a fresh, moist gel, adhering tightly to the wound surface and resisting detachment. Comparative Example 1 failed to form an effective gel morphology, and Comparative Example 2 exhibited only half the viscosity and adhesion of Example 1, failing to meet the requirements for wound repair dressings. This demonstrates that the freeze-drying process of the present invention does not damage the rheological properties of the gel matrix, balancing exosome activity with the practical performance of the gel.

[0043] (4) Extremely strong storage stability and extended shelf life. Example 1, after being stored at 4°C for 360 days (1 year), still maintained 85% protein recovery rate and 82% cell proliferation activity, and accelerated experiments confirmed that the shelf life can reach 24 months; while the control group (fresh wet gel) was completely inactivated after 360 days, and Comparative Examples 1 and 2 degraded and became ineffective in a short period of time. This solves the industry pain points of existing wet exosome gels, such as "cold chain dependence and short shelf life", and provides feasibility for industrialization and clinical promotion.

[0044] 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 mesenchymal stem cell exosome repair gel, characterized in that, Includes the following steps: S1. The purified mesenchymal stem cell exosomes were mixed with the gel matrix solution at 2-8℃ to obtain an exosome gel mixture; S2. Add a composite lyophilization protectant to the exosome gel mixture. The composite lyophilization protectant is composed of trehalose, mannitol and polyethylene glycol, and is used to protect the exosome repair activity and maintain the performance after gel reconstitution. S3. The mixed solution obtained in S2 is subjected to programmed cooling freeze-drying to obtain the mesenchymal stem cell exosome repair gel.

2. The method for preparing a mesenchymal stem cell exosome repair gel according to claim 1, characterized in that: The final concentration of the composite freeze-drying protectant in S2 is: trehalose 30-60mM, mannitol 20-40mM, and polyethylene glycol 1-5% (w / v); the polyethylene glycol is PEG-4000.

3. The method for preparing a mesenchymal stem cell exosome repair gel according to claim 1, characterized in that: The gel matrix in S1 is one or more of sodium alginate, sodium hyaluronate, chitosan, or collagen; the concentration of the gel matrix solution is 1%-3% (w / v), sterile filtered through a 0.22μm filter membrane, and the pH is 7.2-7.

4.

4. The method for preparing a mesenchymal stem cell exosome repair gel according to claim 1, characterized in that: In S1, the exosomes and gel matrix solution are mixed using a gradient dilution method, with a stirring speed of 30-50 rpm and a mixing time of 15-20 minutes.

5. The method for preparing a mesenchymal stem cell exosome repair gel according to claim 1, characterized in that: The programmed cooling freeze-drying in S3 specifically includes: Pre-freezing stage: Cool the sample to -40℃ to -50℃ at a cooling rate of -0.5 to -1.5℃ / min and keep it at this temperature for 1 to 3 hours to allow the mixed solution to freeze completely; One-time drying: Under vacuum conditions below 10 Pa, raise the ambient temperature to -20℃ to -15℃ and maintain it for 5-10 hours to remove most of the free water in the sample; Secondary drying: Under vacuum conditions below 5 Pa, gradually increase the ambient temperature to 25°C to 30°C and maintain it for 5-10 hours until the residual moisture content of the sample is below 3% to ensure the long-term stability of the repair gel.

6. The method for preparing a mesenchymal stem cell exosome repair gel according to claim 1, characterized in that: In the above-mentioned secondary drying process, the stepwise temperature increase is specifically as follows: first, the temperature is increased to -10℃ and held for 1-3 hours, then the temperature is increased to 10℃ and held for 1-3 hours, and finally the temperature is increased to 25-30℃ to avoid damage to the exosome repair activity due to excessively rapid temperature increase.

7. A mesenchymal stem cell exosome repair gel prepared by the preparation method according to any one of claims 1-6, characterized in that, The repair gel is a porous sponge-like solid with a resolution time of ≤3 minutes. After resolution, it becomes a homogeneous gel that can adhere to the skin wound surface to exert a repairing effect.

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