SIRT6 external vesicle freeze-drying protective agent, SIRT6 external vesicle freeze-dried powder, and preparation method and application of SIRT6 external vesicle freeze-dried powder

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses an SIRT6 outer vesicle freeze-drying protective agent, SIRT6 outer vesicle freeze-dried powder and a preparation method and application thereof. The SIRT6 external vesicle freeze-drying protective agent provided by the invention is prepared from the following components: 0.8 to 1.5 g / 100 mL of polyvinyl alcohol, 0.5 to 0.8 g / 100 mL of dextran, 5.5 to 6 g / 100 mL of trehalose, 2.5 to 4 g / 100 mL of mannitol, 0.35 to 0.4 g / 100 mL of vitamin C, 0.15 to 0.3 g / 100 mL of hydroxyectoine, 0.8 to 1.2 g / 100 mL of superoxide dismutase and a solvent which is a phosphate buffer solution. The invention further provides freeze-dried powder of the SIRT6 external vesicles and a preparation method of the freeze-dried powder. According to the SIRT6 outer vesicle freeze-drying protective agent, the structural damage of freeze-drying to the SIRT6 outer vesicle can be reduced, and the structural integrity and functionality of the SIRT6 outer vesicle are ensured.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to a SIRT6 exosome lyophilized protective agent, a SIRT6 exosome lyophilized powder, and a preparation method and application thereof. Background Art

[0002] In the biomedical field, extracellular vesicles, as an important intercellular communication tool, have great potential in the treatment and diagnosis of various diseases. In recent years, with the rapid development of biotechnology, the application scope of extracellular vesicles has been continuously expanded, especially in drug delivery systems, disease marker detection and cell therapy. However, extracellular vesicles face many challenges in practical application, especially maintaining their stability and activity during long-term storage and transportation has become a key issue that needs to be solved urgently.

[0003] To address these challenges, researchers often use a variety of methods to protect the structure and function of exosomes. Common methods include the use of various lyoprotectants, such as sugars (such as sucrose, glucose), polyols (such as glycerol, sorbitol), proteins (such as BSA), and other additives (such as surfactants). These lyoprotectants reduce the damage to exosomes caused by water removal during the freeze-drying process through physical and chemical effects, thereby improving their stability and activity.

[0004] SIRT6 exosomes are a type of multi-gene modified human umbilical cord mesenchymal exosomes derived from SIRT6 immortalized hUCMSCs. They have the characteristics of low immunogenicity, high stability, and the ability to naturally cross biological barriers, and have become the most attractive choice for the treatment of diseases such as OA. However, although the existing lyoprotectants used for SIRT6 exosomes can improve the stability of SIRT6 exosomes to a certain extent, they often cannot completely avoid the destruction of the SIRT6 exosome structure during the lyophilization process, resulting in impaired function.

[0005] Therefore, designing a more effective lyoprotectant to reduce the structural damage of SIRT6 extracellular vesicles during the lyophilization process and ensure their structural integrity and functionality is of great significance for the clinical application of SIRT6 extracellular vesicles. Summary of the invention

[0006] In order to reduce the structural damage of SIRT6 exosomes to SIRT6 exosomes during the freeze-drying process and ensure the structural integrity and functionality of SIRT6 exosomes after freeze-drying, the present application provides a SIRT6 exosome lyophilization protectant, SIRT6 exosome lyophilized powder, and a preparation method and application thereof.

[0007] In the first aspect, the SIRT6 exosome lyophilized protective agent provided in the present application adopts the following technical solution: A SIRT6 exosome lyophilized protective agent comprises the following components: polyvinyl alcohol 0.8-1.5 g / 100 mL, dextran 0.5-0.8 g / 100 mL, trehalose 5.5-6 g / 100 mL, mannitol 2.5-4 g / 100 mL, vitamin C 0.35-0.4 g / 100 mL, hydroxyectoine 0.15-0.3 g / 100 mL, superoxide dismutase 0.8-1.2 g / 100 mL, and the solvent is phosphate buffer.

[0008] The present application provides a SIRT6 exosome freeze-drying protective agent, which can significantly reduce the damage to the SIRT6 exosome structure during the freeze-drying process, thereby effectively maintaining its structural integrity and biological activity. Specifically, polyvinyl alcohol has good film-forming properties and stabilizing effects, and can form a protective film during the freeze-drying process to prevent the vesicles from rupturing. Dextran and trehalose, as non-reducing sugars, can form a stable glassy structure, reduce the formation of ice crystals when water evaporates, and thus reduce physical damage. Mannitol, as a filler, helps maintain the osmotic pressure balance inside and outside the cell and reduce the loss of substances inside the vesicle. Vitamin C has an antioxidant effect, can scavenge free radicals, and prevent oxidative damage; hydroxytetrahydropyrimidine can be used to stabilize the activity of the exosome, especially in the freeze-thaw cycle, can play a good role in protecting the exosome, thereby reducing the denaturation and damage of the exosome, so that the exosome always maintains its own structural integrity and activity. Superoxide dismutase can further scavenge excess free radicals and protect the biological molecules in the vesicle from oxidative damage. Through the combined action of the above-mentioned ingredients, this application achieves effective protection of the exosome structure, significantly reduces the loss rate during the freeze-drying process, improves the recovery rate of SIRT6 exosomes after freeze-drying and the stability of the biologically active substances they carry, extends the shelf life of the product, and lays a solid foundation for subsequent research and therapeutic applications.

[0009] Optionally, the SIRT6 extracellular vesicle lyoprotectant includes the following components: polyvinyl alcohol 1.0-1.2 g / 100 mL, dextran 0.5-0.8 g / 100 mL, trehalose 5.5-6 g / 100 mL, mannitol 2.5-4 g / 100 mL, vitamin C 0.35-0.4 g / 100 mL, hydroxytetrahydropyrimidine 0.2-0.25 g / 100 mL, superoxide dismutase 0.8-1.2 g / 100 mL, and the solvent is phosphate buffer.

[0010] In some embodiments, the content of polyvinyl alcohol can be 0.8-1.0 g / 100 mL, 0.8-1.2 g / 100 mL, 0.8-1.5 g / 100 mL, 1.0-1.2 g / 100 mL, 1.0-1.5 g / 100 mL, or 1.2-1.5 g / 100 mL.

[0011] In a specific embodiment, the content of polyvinyl alcohol can also be 0.8 g / 100 mL, 1.0 g / 100 mL, 1.2 g / 100 mL or 1.5 g / 100 mL.

[0012] In some embodiments, the content of hydroxytetrahydropyrimidine can be 0.15-0.2 g / 100 mL, 0.15-0.25 g / 100 mL, 0.15-0.3 g / 100 mL, 0.2-0.25 g / 100 mL, 0.2-0.3 g / 100 mL or 0.25-0.3 g / 100 mL.

[0013] In a specific embodiment, the content of hydroxyectoine can also be 0.15 g / 100 mL, 0.2 g / 100 mL, 0.25 g / 100 mL or 0.3 g / 100 mL.

[0014] Optionally, the SIRT6 extracellular vesicle lyoprotectant includes the following components: polyvinyl alcohol 10g / 100mL, dextran 6g / 100mL, trehalose 6g / 100mL, mannitol 3.5g / 100mL, vitamin C 4g / 100mL, hydroxytetrahydropyrimidine 2.5g / 100mL, superoxide dismutase 1.0g / 100mL, and the solvent is phosphate buffer.

[0015] Optionally, the concentration of the phosphate buffer is 0.01 M and the pH is 7.4.

[0016] In a second aspect, the present application provides a SIRT6 exosome lyophilized powder, comprising SIRT6 exosomes and the SIRT6 exosome lyophilized protectant according to any one of claims 1 to 4.

[0017] In the third aspect, the present application provides a method for preparing SIRT6 exosome lyophilized powder, comprising the following steps: adding a lyophilization protectant to the SIRT6 exosomes, and obtaining SIRT6 exosome lyophilized powder through pre-freezing, primary lyophilization and secondary lyophilization; the concentration of the SIRT6 exosomes in the lyophilization protectant is 5-10 g / 100 mL.

[0018] Optionally, the pre-freezing temperature is -10 to -15°C, and the time is 10-20 min; the primary freeze-drying temperature is -35 to -50°C, and the time is 30-60 min; the secondary freeze-drying temperature is -75 to -85°C, and the time is 1.5-2.5 h.

[0019] In a specific embodiment, the pre-freezing temperature may be -10°C or -15°C.

[0020] In a specific embodiment, the pre-freezing time can be 10 min or 20 min.

[0021] In some embodiments, the primary freezing temperature may be -35 to -40°C, -35 to -45°C, -35 to -50°C, -40 to -45°C, -40 to -50°C, or -45 to -50°C.

[0022] In a specific embodiment, the primary freezing temperature may also be -35°C, -40°C, -45°C or -50°C.

[0023] In some embodiments, the primary freezing time may be 30-40 min, 30-50 min, 30-60 min, 40-50 min, 40-60 min or 50-60 min.

[0024] In a specific embodiment, the time of the primary freezing can also be 30 min, 40 min, 50 min or 60 min.

[0025] In some embodiments, the secondary freezing temperature may be -75 to -80°C or -80 to -85°C.

[0026] In a specific embodiment, the secondary freezing temperature may also be -75°C, -80°C or -85°C.

[0027] In some embodiments, the secondary freezing time may be 1-1.5 h or 1.5-2.5 h.

[0028] In a specific embodiment, the secondary freezing time can also be 1 h, 1.5 h or 2.5 h.

[0029] In a fourth aspect, the present application provides a method for reconstitution of SIRT6 exosome lyophilized powder, comprising the following steps: adding physiological saline to the SIRT6 exosome lyophilized powder and resuspending it at a ratio of 0.05-0.1 mg / mL.

[0030] In summary, this application has the following beneficial effects: 1. The present application uses polyvinyl alcohol, trehalose, hydroxytetrahydropyrimidine, etc. to prepare a lyoprotectant, which can reduce the damage to the SIRT6 extracellular vesicle structure during the freeze-drying process, thereby effectively maintaining its structural integrity and biological activity.

[0031] 2. The present application further controls the addition amount of polyvinyl alcohol and hydroxyectoine within the following range: polyvinyl alcohol 1.0-1.2 g / 100 mL, hydroxyectoine 0.2-0.25 g / 100 mL, and the obtained SIRT6 extracellular vesicle freeze-dried protective agent has better use effect and better protection for SIRT6 extracellular vesicles. DETAILED DESCRIPTION

[0032] The present application provides a SIRT6 exosome lyophilized protective agent, comprising the following components: polyvinyl alcohol 0.8-1.5g / 100mL, dextran 0.5-0.8g / 100mL, trehalose 5.5-6g / 100mL, mannitol 2.5-4g / 100mL, vitamin C 0.35-0.4g / 100mL, hydroxytetrahydropyrimidine 0.15-0.3g / 100mL, superoxide dismutase 0.8-1.2g / 100mL, and the solvent is phosphate buffer. Further, the SIRT6 exosome freeze-dried protective agent includes the following components: polyvinyl alcohol 1.0-1.2g / 100mL, dextran 0.5-0.8g / 100mL, trehalose 5.5-6g / 100mL, mannitol 2.5-4g / 100mL, vitamin C 0.35-0.4g / 100mL, hydroxytetrahydropyrimidine 0.2-0.25g / 100mL, superoxide dismutase 0.8-1.2g / 100mL, and the solvent is phosphate buffer. The concentration of the phosphate buffer is 0.01M and the pH is 7.4.

[0033] The present application provides a SIRT6 exosome lyophilized powder, comprising SIRT6 exosomes and SIRT6 exosome lyophilized protective agent.

[0034] The preparation method of the above-mentioned SIRT6 exosome lyophilized powder comprises the following steps: adding 100 mL of lyophilization protectant to 15-30 mg of SIRT6 exosomes, first pre-freezing at -10 to -15°C for 10-20 minutes, and then freeze-drying once at -35 to -50°C for 30-60 minutes; then freeze-drying a second time at -75 to -85°C for 1.5 to 2.5 hours to obtain SIRT6 exosome lyophilized powder.

[0035] In this application, SIRT6 extracellular vesicles were extracted according to the method of Example 11 in CN118726486A; polyvinyl alcohol CAS No. 9002-89-5, purchased from Merck; dextran is dextran 70, CAS No. 58798-70-2; trehalose was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; mannitol CAS No. 87-78-5; vitamin C CAS No. 50-81-7; hydroxytetrahydropyrimidine CAS No. 165542-15-4; superoxide dismutase CAS No. 9054-89-1, purchased from Merck. The raw materials, reagents, solvents, etc. used in this application can all be obtained commercially.

[0036] The present application is further described in detail below in combination with preparation examples, embodiments, and performance testing experiments.

[0037] Preparation Example 1-4 Preparation Examples 1-4 each provide a SIRT6 extracellular vesicle freeze-dried protective agent.

[0038] The difference between the above preparation examples is that the amount of polyvinyl alcohol added to the SIRT6 extracellular vesicle freeze-drying protective agent is shown in Table 1 below.

[0039] The preparation method of the SIRT6 extracellular vesicle lyophilized protective agent provided in Preparation Example 1-4 includes the following steps: respectively weighing polyvinyl alcohol, 0.6 g of dextran, 6 g of trehalose, 3.5 g of mannitol, 0.4 g of vitamin C, 0.25 g of hydroxytetrahydropyrimidine, and 1.0 g of superoxide dismutase, dissolving the above components in 100 mL, 0.01 M, pH 7.4 phosphate buffer, mixing evenly, and obtaining the SIRT6 extracellular vesicle lyophilized protective agent.

[0040] Table 1 Addition amount of polyvinyl alcohol in the lyophilized protective agent of SIRT6 extracellular vesicles provided in Preparation Examples 1-4 Preparation Example 5-7 Preparation Examples 5-7 each provide a SIRT6 extracellular vesicle freeze-dried protective agent.

[0041] The difference between the above preparation examples is that the amount of hydroxyectoine added to the SIRT6 extracellular vesicle freeze-dried protective agent is shown in Table 2 below.

[0042] Table 2 Addition amount of hydroxyectoine in the freeze-dried protective agent of SIRT6 extracellular vesicles of Preparation Example 2 and Preparation Examples 5-7 Example Addition amount of hydroxytetrahydropyrimidine (g) 2 0.25 5 0.15 6 0.2 7 0.35 Comparative Preparation Example 1 Comparative Preparation Example 1 provides a SIRT6 extracellular vesicle freeze-dried protective agent.

[0043] The difference between the above preparation example and preparation example 2 is that the amount of polyvinyl alcohol added in the SIRT6 extracellular vesicle freeze-drying protective agent is 0.

[0044] Comparative Preparation Example 2 Comparative Preparation Example 2 provides a SIRT6 extracellular vesicle freeze-dried protective agent.

[0045] The difference between the above preparation example and preparation example 2 is that the amount of hydroxyectoine added in the SIRT6 extracellular vesicle freeze-drying protective agent is 0.

[0046] Comparative Preparation Example 3 Comparative Preparation Example 3 provides a SIRT6 extracellular vesicle freeze-dried protective agent.

[0047] The difference between the above preparation example and preparation example 2 is that the added amount of each component is: 2.0 g of polyvinyl alcohol, 1.0 g of dextran, 8 g of trehalose, 1.5 g of mannitol, 0.3 g of vitamin C, 0.35 g of hydroxyectoine, and 1.5 g of superoxide dismutase.

[0048] Examples 1-7 Examples 1-7 each provide a SIRT6 extracellular vesicle lyophilized powder.

[0049] The difference between the above embodiments is that the SIRT6 exosome lyophilized protective agent used in the SIRT6 exosome lyophilized powder is derived from Preparation Examples 1-7 respectively.

[0050] The preparation method of SIRT6 exosome lyophilized powder provided in Examples 1-7 includes the following steps: adding 100 mL of lyophilized protectant to 10 mg of SIRT6 exosomes, first pre-freezing at -10°C for 20 min, then freeze-drying once at -35°C for 60 min; then freeze-drying a second time at -75°C for 2.5 h to obtain SIRT6 exosome lyophilized powder.

[0051] Embodiment 8-13 Examples 8-13 each provide a SIRT6 extracellular vesicle lyophilized powder.

[0052] The difference between the above embodiment and embodiment 2 is that the freezing temperature and time in the preparation method of SIRT6 extracellular vesicle freeze-dried powder are as shown in Table 3 below Table 3 Freezing temperature and time in the preparation method of Example 2 and Examples 8-13 Comparative Examples 1-3 Comparative Examples 1-3 each provide a SIRT6 extracellular vesicle freeze-dried powder.

[0053] The difference between the above embodiments is that the SIRT6 exosome lyophilized protective agent used in the SIRT6 exosome lyophilized powder is derived from comparative preparation examples 1-3 respectively.

[0054] Comparative Example 4 Comparative Example 4 provides a SIRT6 extracellular vesicle freeze-dried powder.

[0055] The difference between the above embodiment and embodiment 2 is: the preparation method of SIRT6 extracellular vesicle freeze-dried powder.

[0056] The preparation method of SIRT6 exosome lyophilized powder provided in Comparative Example 4 comprises the following steps: adding 100 mL of lyophilization protectant to 10 mg of SIRT6 exosome vesicles, first pre-freezing at -10°C for 20 min, and then freeze-drying at -80°C for 2 h to obtain SIRT6 exosome lyophilized powder.

[0057] Comparative Example 5 Comparative Example 5 provides a SIRT6 extracellular vesicle freeze-dried powder.

[0058] The difference between the above embodiment and embodiment 2 is: the preparation method of SIRT6 extracellular vesicle freeze-dried powder.

[0059] The preparation method of SIRT6 extracellular vesicle freeze-dried powder provided in Comparative Example 5 includes the following steps: adding 100 mL of 0.01 M phosphate buffer with a pH of 7.4 to 10 mg of SIRT6 extracellular vesicles, first pre-freezing at -10°C for 20 min, and then freeze-drying once at -35°C for 60 min; then freeze-drying a second time at -75°C for 2.5 h to obtain SIRT6 extracellular vesicle freeze-dried powder.

[0060] Performance testing The effect of using SIRT6 extracellular vesicle lyophilization protective agent was investigated by detecting the relative expression level of SIRT6 before and after lyophilization of SIRT6 extracellular vesicles. The specific operation is as follows: Resuspend the SIRT6 extracellular vesicle lyophilized powder provided in Example 1-13 and Comparative Example 1-4 with physiological saline at a resuspension ratio of 0.08 mg / mL, and then detect the SIRT6 protein expression amount of SIRT6 extracellular vesicles in the reconstituted solution, and obtain the relative expression level of SIRT6 of SIRT6 extracellular vesicles before and after lyophilization. Note: The SIRT6 protein expression level of SIRT6 extracellular vesicles before lyophilization is recorded as 1. The results are shown in Table 4 below.

[0061] Table 4 Relative expression levels of SIRT6 after reconstitution of lyophilized powder of Examples 1-13 and Comparative Examples 1-5 According to the test results in Table 4, after the SIRT6 exosome freeze-dried powder provided in Examples 1-13 of the present application is re-dissolved, the relative expression level of SIRT6 in the SIRT6 exosomes is 0.98-0.99; while after the SIRT6 exosome freeze-dried powder provided in Comparative Examples 1-5 is re-dissolved, the relative expression level of SIRT6 in the SIRT6 exosomes is only 0.64-0.88. Therefore, it is shown that the use of the lyophilization protectant provided in Preparation Examples 1-7 of the present application as a protective agent for SIRT6 exosomes, and the use of pre-freezing and double freeze-drying processes to freeze-dry SIRT6 exosomes can ensure the structural integrity and biological activity of SIRT6 exosomes, and the clinical application of SIRT6 exosomes is of great significance.

[0062] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A SIRT6 exosome lyophilization protectant, characterized in that: The invention comprises the following components: polyvinyl alcohol 0.8-1.5 g / 100 mL, dextran 0.5-0.8 g / 100 mL, trehalose 5.5-6 g / 100 mL, mannitol 2.5-4 g / 100 mL, vitamin C 0.35-0.4 g / 100 mL, hydroxytetrahydropyrimidine 0.15-0.3 g / 100 mL, superoxide dismutase 0.8-1.2 g / 100 mL, and the solvent is phosphate buffer.

2. The SIRT6 exosome lyophilized protective agent according to claim 1, characterized in that The SIRT6 exosome lyophilized protective agent includes the following components: polyvinyl alcohol 1.0-1.2 g / 100 mL, dextran 0.5-0.8 g / 100 mL, trehalose 5.5-6 g / 100 mL, mannitol 2.5-4 g / 100 mL, vitamin C 0.35-0.4 g / 100 mL, hydroxytetrahydropyrimidine 0.2-0.25 g / 100 mL, superoxide dismutase 0.8-1.2 g / 100 mL, and the solvent is phosphate buffer.

3. The SIRT6 exosome lyophilized protective agent according to claim 1, characterized in that The SIRT6 extracellular vesicle freeze-dried protective agent includes the following components: polyvinyl alcohol 1.0g / 100mL, dextran 0.6g / 100mL, trehalose 6g / 100mL, mannitol 3.5g / 100mL, vitamin C 0.4g / 100mL, hydroxytetrahydropyrimidine 0.25g / 100mL, superoxide dismutase 1.0g / 100mL, and the solvent is phosphate buffer.

4. The SIRT6 exosome lyoprotectant according to any one of claims 1 to 3, characterized in that The concentration of the phosphate buffer is 0.01 M and the pH is 7.

4.

5. A SIRT6 exosome lyophilized powder, characterized in that: The method comprises SIRT6 exosomes and the SIRT6 exosome lyophilized protectant according to any one of claims 1 to 4.

6. The method for preparing the SIRT6 extracellular vesicle freeze-dried powder according to claim 5, characterized in that: The following steps are involved: A lyophilization protectant is added to the SIRT6 exosomes, and SIRT6 exosome lyophilized powder is obtained through pre-freezing, primary lyophilization and secondary lyophilization; the concentration of the SIRT6 exosomes in the lyophilization protectant is 5-10 g / 100 mL.

7. The method for preparing the SIRT6 extracellular vesicle freeze-dried powder according to claim 6, characterized in that: The pre-freezing temperature is -10~-15°C, and the time is 10-20min; the primary freeze-drying temperature is -35~-50°C, and the time is 30-60min; the secondary freeze-drying temperature is -75~-85°C, and the time is 1.5-2.5h.

8. A SIRT6 exosome injection, characterized in that: Including the SIRT6 extracellular vesicle freeze-dried powder as described in claim 5.

9. The method for preparing the SIRT6 extracellular vesicle injection according to claim 8, characterized in that: The following steps are involved: Physiological saline was added to the SIRT6 extracellular vesicle freeze-dried powder and resuspended at a ratio of 0.05-0.1 mg / mL.

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