A composite freeze-dried product of collagen and exosomes, its preparation method and application

Abstract

This invention discloses a composite freeze-dried product of collagen and exosomes, its preparation method, and its applications, belonging to the field of biomedical materials technology. This invention optimizes the composite ratio of collagen and exosomes and the freeze-drying process parameters. By precisely controlling the pre-freezing cooling rate, sublimation drying, and desorption drying temperatures and times, it effectively avoids the problems of loose structure and uneven pores in collagen during freeze-drying, while reducing the damage to the exosome vesicle structure, achieving simultaneous and efficient preservation of the bioactivity of both. Experiments show that the freeze-dried product prepared by this invention exhibits good collagen structural integrity and an exosome bioactivity retention rate ≥85%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a composite freeze-dried product of collagen and exosomes, its preparation method and application. Background Technology

[0002] Collagen is the most abundant structural protein in the human body, widely found in tissues such as skin, bone, and cartilage. It possesses excellent biocompatibility, biodegradability, and cell adhesion, making it widely applicable in wound repair, tissue engineering, and cosmetics. Exosomes are nanoscale lipid bilayer vesicles secreted by cells, containing bioactive substances such as proteins, nucleic acids, and lipids. They can participate in intercellular communication, promote cell proliferation, differentiation, and damage repair, showing great potential for applications in regenerative medicine and skin care.

[0003] However, while exosomes possess high biological activity, they are also unstable and prone to degradation and denaturation at room temperature, leading to loss of biological activity and severely limiting their storage, transportation, and application. Currently, freeze-drying is a common method for preserving bioactive substances. By using low-temperature freezing and vacuum sublimation drying to remove moisture from the system, the structure and function of bioactive substances can be preserved to a certain extent.

[0004] However, existing technologies for the combined freeze-drying of collagen and exosomes still have many shortcomings: on the one hand, when collagen is freeze-dried alone, problems such as uneven pore size, loose structure, and poor rehydration are easily encountered, affecting its mechanical properties and application effects; on the other hand, exosomes are easily affected by factors such as low temperature and osmotic pressure changes during freeze-drying, leading to damage to their vesicle structure, reduced biological activity, or even inactivation; in addition, in existing combined freeze-drying methods, the compatibility between collagen and exosomes is poor, and stratification and aggregation are easily observed after freeze-drying, which cannot fully exert the synergistic effect of the two.

[0005] For example, the invention patent ZL202311078309.8, entitled "A Freeze-drying Method for Collagen Sponges," discloses a freeze-drying method for collagen sponges. This method mainly targets the freeze-drying of a single collagen system and does not involve the combined freeze-drying of exosomes, thus failing to solve the problem of preserving the activity of exosomes during the freeze-drying process. The invention patent 202310688610.4, entitled "A Collagen-Conjugated Exosome, Exosome Freeze-Dried Powder, and Preparation Method," discloses a collagen-conjugated exosome freeze-dried powder and its preparation method. Its focus is on the targeted modification of exosomes, and the freeze-drying process is relatively simple. It does not optimize the compound ratio of collagen and exosomes or the freeze-drying parameters, making it difficult to achieve simultaneous and efficient preservation of the bioactivity of both. Furthermore, the freeze-drying protectants commonly used in existing technologies are mostly single-component, unable to simultaneously meet the protection needs of both collagen and exosomes, further exacerbating the loss of their activity.

[0006] Therefore, developing a freeze-drying method that can simultaneously preserve the structural integrity of collagen and the biological activity of exosomes, and has good composite effect, good rehydration, and stable storage has become an urgent technical problem to be solved in this field. Summary of the Invention

[0007] Therefore, the purpose of this invention is to provide a composite freeze-dried product of collagen and exosomes and its preparation method, which can effectively solve the problems of loose structure and poor rehydration of collagen after freeze-drying, and easy loss of activity and poor compatibility of exosomes during freeze-drying. It achieves simultaneous and efficient preservation of the structural integrity of collagen and the biological activity of exosomes. At the same time, the freeze-dried product prepared by this method is stable in storage and has good rehydration, which can give full play to the synergistic effect of collagen and exosomes and has broad application prospects.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a composite lyophilized product of collagen and exosomes, wherein the lyophilized product is a porous and loose solid, and the lyophilized product is composed of collagen, exosomes and a composite lyophilization protectant; the composite lyophilization protectant is composed of trehalose, manganese sulfate, mannitol and proline; the porosity of the lyophilized product is 70%-85%, and the pore size distribution is 50-200 μm; after rehydration, the bioactivity retention rate of exosomes is ≥85%, and the triple helix structure integrity of collagen is ≥90%.

[0009] Secondly, the present invention provides a method for preparing the above-mentioned collagen and exosome composite lyophilized product, comprising the following steps: S1: Cool the composite mother liquor to below -40℃ at a rate of 1-2℃ / min, and keep it at that temperature for 2-24 hours to complete the pre-freezing; S2: The pre-frozen composite mother liquor was heated to -25°C to -15°C at a rate of 0.5-1°C / min under a vacuum of less than 20 Pa, and kept at this temperature for 8-36 hours for sublimation drying; then heated to 30°C to 40°C at a rate of 1-1.5°C / min and kept at this temperature for 3-12 hours for desorption drying; the composite freeze-dried product of collagen and exosomes was obtained. The concentration of exosomes in the composite mother liquor was 1×10⁻⁶. 9 -5×10 13 The concentration of particles / mL, the amount of compound lyophilization protectant added is 5%-15% of the total mass of compound mother liquor, the concentration of buffer solution is 0.01-0.02 mol / L, and the mass concentration of collagen solution is 0.5%-5%.

[0010] Sublimation drying removes free water from the system; analytical drying removes bound water from the system.

[0011] Based on the above technical solution, the preparation process of the composite mother liquor is further as follows: Collagen was dissolved in a buffer solution and stirred until completely dissolved to obtain a collagen solution. Exosomes and a compound lyophilization protectant were added to the collagen solution, stirred evenly, and the pH of the system was adjusted to 7.2-7.4. The solution was then allowed to stand in a water bath to degas, resulting in a compound mother liquor. The buffer solution was a phosphate buffer solution, and the compound lyophilization protectant was composed of trehalose, manganese sulfate, mannitol, and proline in a mass ratio of 5-8:0.2-0.5:2-4:1-2.

[0012] The pH was adjusted to 7.2–7.4 for the following reasons: 1) This pH range prevents collagen from denaturing, unwinding, or agglomerating during freeze-drying, ensuring a uniform structure and intact pores in the freeze-dried product; 2) The vesicle structure, membrane proteins, and internal bioactive substances of active exosomes exhibit the highest stability in a near-neutral physiological environment of pH 7.2–7.4, reducing damage to their structure and activity during freeze-drying; 3) This pH range allows collagen and exosomes to maintain good compatibility, resulting in a uniform and stable system without stratification, precipitation, or aggregation, which facilitates the smooth progress of subsequent freeze-drying processes; 4) Matching the pH of the human physiological environment allows the final product to be directly applied in the biomedical field after reconstitution, resulting in higher safety and compatibility.

[0013] Based on the above technical solution, further step S3 is included: after the desorption and drying are completed, inert gas is introduced into the vacuum environment and the freeze-dried product is sealed and packaged.

[0014] Based on the above technical solution, the composite mother liquor is further placed in a freeze-drying container and then placed in a pre-freezing device for cooling; step S2 is performed using a vacuum freeze dryer.

[0015] Based on the above technical solution, the collagen is derived from human, animal, cell fermentation, or microbial fermentation; the exosomes are derived from human, animal, plant, or cell culture.

[0016] Based on the above technical solution, the stirring speed is 100-500 r / min, the stirring time is 30-60 min, the standing degassing time is 15-30 min, and the degassing temperature is 4-8℃.

[0017] Based on the above technical solution, the freeze-drying container is a sterile freeze-drying bottle or freeze-drying tray, and the filling volume is 1 / 3 to 1 / 2 of the container volume.

[0018] The filling volume is 1 / 3 to 1 / 2 of the container volume to avoid liquid overflow during the freeze-drying process.

[0019] Based on the above technical solution, the temperature of the storage environment for the freeze-dried product is further specified as 2°C to 8°C.

[0020] Thirdly, the present invention provides the application of the above-mentioned composite lyophilized collagen and exosome in the preparation of biomedical products for wound repair, tissue engineering or skin treatment and care.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention optimizes the composite ratio of collagen and exosomes and the freeze-drying process parameters. By precisely controlling the pre-freezing cooling rate, sublimation drying, and desorption drying temperatures and times, it effectively avoids the problems of loose structure and uneven pores in collagen during freeze-drying, while reducing the damage to exosome vesicle structures, achieving simultaneous and efficient preservation of the bioactivity of both. A pre-freezing cooling rate of 1-2℃ / min is the optimal range for balancing collagen structure and exosome activity—too fast a rate forms small ice crystals that damage exosomes, while too slow a rate forms large ice crystals that damage the porous structure of collagen, which is not a conventional choice in this field. The controlled temperature rise during the sublimation and desorption stages avoids the collapse of collagen sponges and exosome inactivation caused by sudden temperature changes, which cannot be achieved by the existing direct heating and one-step freeze-drying process. Experiments show that the freeze-dried product prepared by this invention has good collagen structural integrity and an exosome bioactivity retention rate of ≥85%.

[0022] 2. This invention employs a composite freeze-drying protectant composed of trehalose, manganese sulfate, mannitol, and proline in a specific ratio. Trehalose forms a protective film on the surface of biomolecules, reducing the damage to their structure caused by low temperatures; manganese sulfate stabilizes the lipid bilayer structure of exosomes, improving their activity and stability; mannitol regulates the osmotic pressure of the system, preventing exosomes from rupturing due to changes in osmotic pressure; and proline protects the triple helix structure of collagen, preventing its denaturation and degradation. Trehalose and proline work synergistically, with the former replacing water molecules to bind to collagen peptide bonds, and the latter stabilizing the collagen triple helix structure through hydrogen bonds, achieving dual protection of collagen structure. Manganese sulfate and mannitol work synergistically, with the former stabilizing the phospholipid bilayer of exosomes through divalent cations, and the latter regulating the osmotic pressure of the system, preventing vesicle rupture caused by ice crystal formation, achieving dual protection of exosome activity. The four components, combined in a specific ratio, exhibit no antagonism and simultaneously address the protection needs of both collagen and exosomes, achieving a synergistic effect greater than the sum of its parts (1+1>2), which is unattainable by existing single / binary protectants. The four components work synergistically to more comprehensively protect the activity of collagen and exosomes, further improving the quality of freeze-dried products.

[0023] 3. The composite lyophilized product prepared by this invention is a porous and loose solid with high porosity, uniform pore size distribution, and good rehydration properties. It can be rapidly rehydrated within 1-10 minutes after the addition of buffer solution. After rehydration, the system is uniform and stable, without stratification or aggregation. It can fully exert the biocompatibility of collagen and the repair function of exosomes, with significant synergistic effects.

[0024] 4. The freeze-drying method of the present invention is simple to operate, operates under mild conditions, requires no complicated equipment, and is easy to scale up for industrial production; the freeze-dried product prepared has good stability and can be stored for more than 12 months at 2℃ to 8℃ without significant decrease in activity, which greatly reduces storage and transportation costs, expands its application range, and can be widely used in wound repair, tissue engineering, cosmetics, biomedicine and other fields. Detailed Implementation

[0025] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention. Example

[0026] A method for preparing a lyophilized compound of collagen and exosomes includes the following steps: S1 Preparation of the composite stock solution: Type I porcine skin collagen (95% purity) was dissolved in 0.01 mol / L phosphate buffer and stirred at 100 rpm for 30 min until completely dissolved, yielding a collagen solution with a mass concentration of 0.5%. Exosomes derived from human adipose-derived mesenchymal stem cells and a composite lyophilization protectant were added to the collagen solution, stirred thoroughly, and the pH was adjusted to 7.2. The solution was then degassed by standing in a 4℃ water bath for 15 min to obtain the composite stock solution. The concentration of exosomes in the composite stock solution was 1 × 10⁻⁶. 9 The particle / mL, the amount of compound freeze-drying protectant added is 5% of the total mass of the compound mother liquor, the compound freeze-drying protectant is composed of trehalose, manganese sulfate, mannitol and proline in a mass ratio of 5:0.2:2:1; S2 Pre-freezing treatment: Dispense the compound mother liquor into 2mL sterile lyophilized bottles, with a dispensing volume of 1mL, place them in a pre-freezing device, and lower the temperature to -40℃ at a cooling rate of 1℃ / min, keep warm for 2h to complete the pre-freezing; S3 Sublimation Drying: The pre-frozen composite mother liquor is transferred to a vacuum freeze dryer, the vacuum degree is adjusted to 10Pa, the shelf temperature is controlled to rise to -25℃ at a rate of 0.5℃ / min, and the temperature is kept for 8 hours for sublimation drying; S4 Desorption Drying: Keeping the vacuum level constant, the shelf temperature is increased to 30℃ at a rate of 1℃ / min, and kept at this temperature for 3 hours to perform desorption drying; S5 Sealed Preservation: After the desorption and drying process is completed, inert gas is introduced into the vacuum environment, the freeze-dried product is sealed and packaged, and stored in a low temperature environment of 2°C to obtain a collagen and exosome complex freeze-dried product.

[0027] The freeze-dried product prepared in this embodiment is a porous and loose solid with no shrinkage or cracking. The porosity is 70% and the pore size distribution is 50-150 μm. After rehydration, it completely dissolves within 1 minute. The system is uniform and does not aggregate. The exosome bioactivity retention rate is 85%, the collagen structural integrity is 92%, and there is no obvious denaturation. Example

[0028] A method for preparing a lyophilized compound of collagen and exosomes includes the following steps: S1 Preparation of the composite stock solution: 98% pure type III recombinant human collagen was dissolved in 0.015 mol / L phosphate buffer and stirred at 300 rpm for 45 min until completely dissolved, yielding a collagen solution with a mass concentration of 3.0%. Exosomes derived from human bone marrow mesenchymal stem cells and a composite lyophilization protectant were added to the collagen solution, stirred thoroughly, and the pH was adjusted to 7.3. The solution was then degassed by standing at 6°C for 22 min to obtain the composite stock solution. The concentration of exosomes in the composite stock solution was 3 × 10⁻⁶. 11The particle / mL, the amount of compound freeze-drying protectant added is 10% of the total mass of the compound mother liquor, the compound freeze-drying protectant is composed of trehalose, manganese sulfate, mannitol and proline in a mass ratio of 6.5:0.35:3:1.5; S2 Pre-freezing treatment: Dispense the compound mother liquor into sterile freeze-drying trays, filling the trays to 2 / 5 of their volume. Place the trays in a pre-freezing device and cool them to -45°C at a rate of 1.5°C / min. Keep them warm for 12 hours to complete the pre-freezing process. S3 Sublimation Drying: The pre-frozen composite mother liquor is transferred to a vacuum freeze dryer, the vacuum degree is adjusted to 15Pa, the shelf temperature is controlled to rise to -20℃ at a rate of 0.75℃ / min, and the temperature is kept for 24h to carry out sublimation drying; S4 Desorption Drying: Keeping the vacuum level constant, the shelf temperature is increased to 35℃ at a rate of 1.2℃ / min, and kept at this temperature for 8 hours for desorption drying; S5 Sealed Preservation: After the desorption and drying process is completed, inert gas is introduced into the vacuum environment, the freeze-dried product is sealed and packaged, and stored in a low temperature environment of 6°C to obtain a collagen and exosome complex freeze-dried product.

[0029] The freeze-dried product prepared in this embodiment is a porous and loose solid with no shrinkage or cracking. The porosity is 78% and the pore size distribution is 80-180 μm. After rehydration, it completely dissolves within 2 minutes. The system is uniform and free from aggregation. The exosome bioactivity retention rate is 88%, the collagen structural integrity is 95%, and the mechanical properties are good. Example

[0030] A method for preparing a lyophilized compound of collagen and exosomes includes the following steps: S1 Preparation of the composite stock solution: Type I bovine bone collagen (99% purity) was dissolved in 0.02 mol / L phosphate buffer and stirred at 500 rpm for 60 min until completely dissolved, yielding a collagen solution with a mass concentration of 5.0%. Exosomes derived from human umbilical cord mesenchymal stem cells and a composite lyophilization protectant were added to the collagen solution, stirred thoroughly, and the pH was adjusted to 7.4. The solution was then degassed by standing at 8°C for 30 min to obtain the composite stock solution. The concentration of exosomes in the composite stock solution was 5 × 10⁻⁶. 13 The particle / mL, the amount of compound freeze-drying protectant added is 15% of the total mass of the compound mother liquor, and the compound freeze-drying protectant is composed of trehalose, manganese sulfate, mannitol and proline in a mass ratio of 8:0.5:4:2; S2 Pre-freezing treatment: Dispense the compound mother liquor into 5mL sterile lyophilized bottles, with a dispensing volume of 2mL, place them in a pre-freezing device, and lower the temperature to -50℃ at a cooling rate of 2℃ / min, and keep them at this temperature for 24h to complete the pre-freezing. S3 Sublimation Drying: The pre-frozen composite mother liquor is transferred to a vacuum freeze dryer, the vacuum degree is adjusted to 20Pa, the shelf temperature is controlled to rise to -15℃ at a rate of 1℃ / min, and the temperature is kept for 36h for sublimation drying; S4 Desorption Drying: Keeping the vacuum level constant, the shelf temperature is increased to 40℃ at a rate of 1.5℃ / min and kept at this temperature for 12 hours for desorption drying; S5 Sealed Preservation: After the desorption and drying process is completed, inert gas is introduced into the vacuum environment, the freeze-dried product is sealed and packaged, and stored in a low-temperature environment of 8°C to obtain a collagen and exosome complex freeze-dried product.

[0031] The freeze-dried product prepared in this embodiment is a porous and loose solid with no shrinkage or cracking. The porosity is 85% and the pore size distribution is 100-200μm. After rehydration, it completely dissolves within 5 minutes. The system is uniform and free of agglomeration. The exosome bioactivity retention rate is 92%, and the collagen structural integrity is 97%. It has the best rehydration and mechanical properties.

[0032] Comparative Example 1 A method for preparing a composite freeze-dried product of collagen and exosomes, which differs from Example 2 in that: it is stored at 2°C for 12 months, while the other steps are the same.

[0033] The freeze-dried product prepared in this comparative example remained a porous and loose solid after being stored at 2°C for 12 months, without shrinkage or cracking. The porosity was 79%, and the pore size distribution was 80-180 μm. After rehydration, it completely dissolved within 2 minutes, the system was uniform and without agglomeration, the exosome bioactivity retention rate was 88%, the collagen structural integrity was 96%, and the mechanical properties were good.

[0034] Comparative Example 2 A method for preparing a composite lyophilized product of collagen and exosomes, which differs from Example 2 in that: no composite lyophilization protectant is added, while the other steps are the same.

[0035] The freeze-dried product prepared in this comparative example has a loose structure, uneven pores, and a porosity of only 52%. It takes more than 15 minutes to dissolve after rehydration, and a small amount of agglomeration occurs. The exosome bioactivity retention rate is only 60%, and the collagen structural integrity is only 75%, showing obvious denaturation.

[0036] Comparative Example 3 A method for preparing a composite freeze-dried product of collagen and exosomes, which differs from Example 2 in that the composite freeze-drying protectant is a single trehalose, while the other steps are the same.

[0037] The lyophilized product prepared in this comparative example had a porosity of 65%, and its dissolution rate after rehydration was slow, requiring 12 minutes to completely dissolve; the exosome bioactivity retention rate was 72%, and the collagen structural integrity was 82%.

[0038] Comparative Example 4 A method for preparing a composite freeze-dried product of collagen and exosomes, which differs from Example 2 in that: the pre-freezing cooling rate is 3℃ / min, the sublimation drying temperature is directly raised to -10℃, and the remaining steps are the same.

[0039] The freeze-dried product prepared in this comparative example showed obvious shrinkage and cracking, with a porosity of only 58% and uneven pore size distribution; the exosome bioactivity retention rate was 68%, the collagen was severely denatured, and the rehydration properties were extremely poor.

[0040] Comparative Example 5 A composite lyophilized product of collagen and exosomes was prepared according to ZL202311078309.8 "A method for lyophilizing collagen sponge", including the following steps: Type I porcine skin collagen (95% purity) and exosomes derived from human adipose-derived mesenchymal stem cells, used in Example 1, were dissolved in buffer salts to obtain collagen with a concentration of 0.5% and exosomes with a concentration of 1×10⁻⁶. 9 A mixed solution of particles / mL was prepared. The mixed solution was placed in a freeze dryer mold to a thickness of 5 mm. After spreading it evenly, it was repeatedly vacuumed three times at a vacuum degree of -35 Pa, with each vacuuming period lasting 60 min and the vacuuming temperature at 10 °C. After removing air bubbles from the solution, the freeze dryer was turned on to freeze-dry the pretreated mixed solution. The freeze-drying process included pre-freezing, sublimation drying, and desorption drying. After pre-freezing, a vacuum was applied, followed by sublimation drying and desorption drying under vacuum. The specific parameters were as follows: pre-freezing temperature of -35 °C, pre-freezing cooling rate of 1 °C / min, pre-freezing time of 3 h, sublimation drying temperature of -10 °C, sublimation drying stage duration of 15 h; vacuum degree of -30 Pa for sublimation drying under vacuum; desorption drying temperature of 30 °C, desorption drying time of 1 h; and total freeze-drying time of 27 h.

[0041] The freeze-dried product prepared in this comparative example is a porous and loose solid with slight shrinkage and cracking, and a porosity of 54%. It takes 16 minutes to dissolve after rehydration and a small amount of aggregation occurs. The exosome bioactivity retention rate is 42%, the collagen structural integrity is 82%, and the mechanical properties are good.

[0042] Comparative Example 6 A collagen-exosome composite lyophilized product was prepared according to the method described in document 202310688610.4, which includes the following steps: Type I porcine skin collagen (95% purity) and exosomes derived from human adipose-derived mesenchymal stem cells, as used in Example 1, were mixed with a lyophilization protectant at a ratio of 1:1:2 to obtain a collagen concentration of 0.5% and an exosome concentration of 1×10⁻⁶. 9 A mixed solution of particles / mL was prepared. The solution was then filtered through a 0.22 μm filter into a rapid-heat-conducting cup and immediately placed in liquid nitrogen in a sterile operating cabinet for 5–10 min to freeze. It was then rapidly transferred to a vacuum freeze dryer and freeze-dried for 24 h to obtain a lyophilized powder, which was stored at room temperature in a cool, dark place. The lyophilization protectant consisted of 35 g / L trehalose, 2.5 mM manganese sulfate, and 0.012 mol / L phosphate buffer, with a pH of 7.4.

[0043] The freeze-dried product prepared in this comparative example showed obvious shrinkage and cracking, with a porosity of only 50%; it required more than 20 minutes to dissolve after rehydration and exhibited aggregation; the exosome bioactivity retention rate was 69%, and the collagen structural integrity was 57%, showing obvious denaturation.

[0044] As can be seen from the above embodiments and comparative examples, the present invention, by optimizing the composition of the composite lyophilization protectant and the lyophilization process parameters, can effectively improve the quality of the collagen and exosome composite lyophilized product, achieving simultaneous and efficient preservation of the bioactivity of both, which has significant advantages over the prior art. The following summary table of experimental data further quantifies and verifies the technical effects of the present invention.

[0045] Table 1 below summarizes the key performance indicators of the lyophilized products prepared in Examples 1-3 and Comparative Examples 1-6. The detection methods are as follows: porosity was determined by mercury porosimetry; reconstitution time was measured using 0.015 mol / L phosphate buffer at room temperature; exosome bioactivity retention was determined by flow cytometry (with fresh exosome activity as 100%); collagen denaturation was determined by circular dichroism spectroscopy (retention of characteristic absorption peaks ≥90% indicates structural integrity). Each indicator was tested in triplicate.

[0046] Table 1: Summary Table of Experimental Data Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A lyophilized compound of collagen and exosomes, characterized in that, The lyophilized product is a porous, loose solid, composed of collagen, exosomes, and a composite lyophilization protectant. The composite lyophilization protectant is composed of trehalose, manganese sulfate, mannitol, and proline. The porosity of the lyophilized product is 70%-85%, and the pore size distribution is 50-200 μm. After rehydration, the bioactivity retention rate of exosomes is ≥85%, and the triple helix structure integrity of collagen is ≥90%.

2. The method for preparing the lyophilized compound of collagen and exosomes as described in claim 1, characterized in that, Includes the following steps: S1: Cool the composite mother liquor to below -40℃ at a cooling rate of 1-2℃ / min, and keep it at that temperature for 2-24 hours to complete the pre-freezing; S2: The pre-frozen composite mother liquor is heated to -25°C to -15°C at a rate of 0.5-1°C / min under a vacuum of less than 20Pa, and kept at this temperature for 8-36 hours for sublimation drying. The temperature was increased to 30°C to 40°C at a rate of 1-1.5°C / min, and held for 3-12 hours for desorption and drying; a composite lyophilized product of collagen and exosomes was obtained. The concentration of exosomes in the composite mother liquor was 1×10⁻⁶. 9 -5×10 13 The concentration of particles / mL, the amount of compound lyophilization protectant added is 5%-15% of the total mass of compound mother liquor, the concentration of buffer solution is 0.01-0.02 mol / L, and the mass concentration of collagen solution is 0.5%-5%.

3. The preparation method according to claim 2, characterized in that, The preparation process of the composite mother liquor is as follows: Collagen was dissolved in a buffer solution and stirred until completely dissolved to obtain a collagen solution. Exosomes and a compound lyophilization protectant were added to the collagen solution, stirred evenly, and the pH of the system was adjusted to 7.2-7.

4. The solution was then allowed to stand in a water bath to degas, resulting in a compound mother liquor. The buffer solution was a phosphate buffer solution, and the compound lyophilization protectant was composed of trehalose, manganese sulfate, mannitol, and proline in a mass ratio of 5-8:0.2-0.5:2-4:1-2.

4. The preparation method according to claim 2, characterized in that, It also includes step S3: After the desorption and drying are completed, inert gas is introduced into the vacuum environment and the freeze-dried product is sealed and packaged.

5. The preparation method according to claim 2, characterized in that, The composite mother liquor is placed in a freeze-drying container and then placed in a pre-freezing device for cooling; step S2 is performed using a vacuum freeze dryer.

6. The preparation method according to claim 2, characterized in that, The collagen is derived from human, animal, cell fermentation, or microbial fermentation; the exosomes are derived from human, animal, plant, or cell culture.

7. The preparation method according to claim 3, characterized in that, The stirring speed is 100-500 r / min, and the stirring time is 30-60 min; the standing degassing time is 15-30 min, and the degassing temperature is 4-8℃.

8. The preparation method according to claim 5, characterized in that, The freeze-drying container is a sterile freeze-drying bottle or freeze-drying tray, and the filling volume is 1 / 3 to 1 / 2 of the container volume.

9. The preparation method according to claim 2, characterized in that, The temperature of the freeze-dried product storage environment is 2°C to 8°C.

10. The use of the collagen and exosome lyophilized compound as described in claim 1 in the preparation of biomedical products for wound repair, tissue engineering or skin treatment care.