Exosome freeze-drying protective agent capable of improving stability and application of exosome freeze-drying protective agent

Abstract

The invention belongs to the technical field of exosome freeze-drying protection, and particularly relates to an exosome freeze-drying protective agent capable of improving stability and application of the exosome freeze-drying protective agent. In order to overcome the defects that an existing exosome freeze-drying protective agent is complex in formula, the quality of a freeze-dried exosome is poor, the freeze-drying cost is high, and the long-term storage performance of the freeze-dried exosome cannot be guaranteed, the invention provides the exosome freeze-drying protective agent capable of improving the stability, and the exosome freeze-drying protective agent comprises trehalose or cane sugar and mannitol. Sucrose or trehalose is adopted, and mannitol is added as an excipient, so that the stability of the exosome can be improved. The protective agent disclosed by the invention is simple in formula component, low in cost, convenient to store and transport, and capable of being stored for a long time and keeping the exosome stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of exosome freeze-drying protection, and specifically relates to an exosome freeze-drying protective agent with improved stability and an application thereof. Background Art

[0002] Exosomes are a type of vesicles secreted by cells, containing a variety of nucleic acids, proteins, lipids, etc., and are responsible for the transport of cargo between cells. In the past decade, studies have been conducted in many fields of extracellular vesicles, including the role of extracellular vesicles in physiological and pathological processes, and their potential for clinical application in treatment and diagnosis. The application of exosomes is becoming more and more extensive, and how to store and transport them and how to ensure storage stability are extremely critical issues. However, at present, there are few studies on the storage conditions of exosomes, and there is still a lack of consensus on the storage conditions of exosomes. More comprehensive research is needed to systematically compare different storage methods over a long period of time.

[0003] Normally, exosomes can be stored at 4°C, -20°C and -80°C, but not at room temperature. When stored at 4°C, the particle concentration and RNA amount of exosomes decrease within 2-3 days; when stored at -20°C or -80°C, the particle concentration and RNA amount of exosomes also begin to decrease after a week of storage, which greatly limits the application of exosomes. At the same time, freezing and refrigeration conditions are not conducive to transportation. Therefore, it is urgent to develop a method to facilitate the transportation and storage of exosomes.

[0004] Freeze-drying technology is a common drying process that can achieve long-term stable preservation of bioactive substances. At present, some studies have used freeze-drying technology to store exosomes, but the existing freeze-dried exosomes have complex protective agents, poor quality of freeze-dried exosomes, high freeze-drying costs, and cannot guarantee the long-term storage performance of freeze-dried exosomes. Therefore, it is necessary to develop new and more effective exosome freeze-dried protective agents. Summary of the invention

[0005] The technical problem to be solved by the present invention is that the existing exosome lyophilization protectants have the defects of complex formula, poor quality of exosomes after lyophilization, high lyophilization cost, and failure to guarantee the long-term storage performance of exosomes after lyophilization.

[0006] The technical solution of the present invention to solve the above technical problems is: to provide an exosome freeze-drying protective agent with improved stability, comprising: trehalose or sucrose, and mannitol.

[0007] Among them, the above-mentioned exosome freeze-dried protective agent for improving stability includes: 4-10wt% trehalose or sucrose, 3-8wt% mannitol.

[0008] Among them, the above-mentioned exosome freeze-dried protective agent for improving stability includes: 8wt% trehalose and 5wt% mannitol.

[0009] Among them, the above-mentioned exosome freeze-dried protective agent for improving stability includes: 5wt% sucrose and 4wt% mannitol.

[0010] Among them, the above-mentioned exosome freeze-dried protective agent for improving stability also includes PBS or physiological saline.

[0011] Preferably, the amount of PBS or physiological saline added is based on the osmotic pressure reaching 280-320 mmol / L.

[0012] Among them, in the above-mentioned exosome lyophilization protective agent for improving stability, the exosomes are one of mesenchymal stem cell exosomes, 293 exosomes or engineered exosomes.

[0013] The present invention also provides a method for preparing an exosome freeze-dried protective agent with improved stability, comprising the following steps: Weigh trehalose or sucrose and mannitol, dissolve them in water to make the mass concentration of trehalose or sucrose reach 4-10wt%, and the mass concentration of mannitol reach 3-8wt%, stir and mix well to obtain an exosome freeze-dried protective agent with improved stability.

[0014] The beneficial effects of the present invention are: The present invention provides an exosome freeze-dried protective agent with improved stability, which can improve the stability of exosomes by using sucrose or trehalose and adding mannitol as an excipient. The protective agent of the present invention has simple formula ingredients, low cost, convenient storage and transportation, and can be stored for a long time and maintain the stability of exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Shown is the appearance of each sample after freeze-drying of the sample in Example 1; Figure 2 Shown is the appearance of each sample after freeze-drying of the sample in Example 2; Figure 3 Shown is the appearance of each sample after freeze-drying of the sample in Comparative Example 3; Figure 4 Shown is the appearance of each sample after freeze-drying of the sample of Comparative Example 4; Figure 5 Shown is the appearance of each sample after freeze-drying of the sample in Comparative Example 5; Figure 6 Shown is the appearance of each sample after freeze-drying of the sample 6 of Comparative Example; Figure 7 Shown are the electron microscopy results of 3M exosomes in Example 1 and Example 2 at -20°C. DETAILED DESCRIPTION

[0016] The present invention provides an exosome freeze-drying protective agent for improving stability, which comprises: trehalose or sucrose, mannitol, and water. Trehalose and sucrose are used as freeze-drying stabilizers and protective agents for exosomes, mannitol is an excipient for freeze-drying of exosomes, both of which play a protective role in the freeze-drying process of exosomes, and water plays a volume balancing role. Experimental verification shows that the ratio in the present invention can play a better protective role on exosomes.

[0017] The specific implementation modes of the present invention will be further explained below through examples, but it is not intended to limit the protection scope of the present invention to the scope described in the examples.

[0018] Example 1 Preparation of the Lyoprotectant of the Present Invention The freeze-drying protective agent is composed of 5% sucrose and 4% mannitol.

[0019] Example 2 Preparation of the Lyoprotectant of the Present Invention The freeze-drying protective agent is composed of: 8% trehalose and 5% mannitol.

[0020] Comparative Example 3 Preparation of other lyophilization protective agents The freeze-drying protective agent is composed of: 3% trehalose and 2% mannitol.

[0021] Comparative Example 4 Preparation of other lyophilization protective agents The freeze-drying protective agent comprises: 12% trehalose and 9% mannitol.

[0022] Comparative Example 5 Preparation of other lyophilization protective agents The freeze-drying protective agent is composed of: 15% trehalose and 10% mannitol.

[0023] Comparative Example 6 Preparation of other lyophilization protective agents The composition of the freeze-drying protective agent is: 5% trehalose.

[0024] The lyophilization protective agents of the embodiment and the comparative example were respectively used to freeze-dry the exosomes. The following performance tests were performed on the freeze-dried exosomes.

[0025] 1. Long-term stability testing of freeze-dried exosomes 1. Observe the freeze-dried appearance After the exosomes are freeze-dried, observe whether their overall appearance has shrunk, whether their surface position has dropped, whether there are convex spots on the surface, etc., and screen out the freeze-dried exosome prescriptions with better appearance.

[0026] 2. Long-term storage performance test at 25°C Freeze-dried exosomes with good appearance were selected and placed at 25°C for 30 days. Samples were taken and re-dissolved on 0 days, 11 days and 30 days to detect the exosome particle size, particle concentration, purity, CD9 positivity rate and CD63 positivity rate, and the stability of exosome freeze-dried powder at 25°C was observed.

[0027] 3. 2-8℃ long-term storage performance test Freeze-dried exosomes with good appearance were selected and stored at 2-8°C for 6 months. Samples were taken and re-dissolved in 0, 3, and 6 months to detect the exosome particle size, particle concentration, purity, CD9 positivity rate, and CD63 positivity rate, and the long-term stability of the exosome freeze-dried powder at 2-8°C was observed.

[0028] 4. -20℃ long-term storage performance test Freeze-dried exosomes with good appearance were selected and stored at -20°C for 6 months. Samples were taken and re-dissolved at 0, 3, and 6 months to detect the exosome particle size, particle concentration, purity, CD9 positivity rate, and CD63 positivity rate, and the long-term stability of exosome freeze-dried powder at -20°C was observed.

[0029] The stability performance results of Example 1-Comparative Example 6 are as follows Figure 1-6 shown.

[0030] Figure 1 The freeze-dried product has a good appearance after freeze-drying, with no drop in surface position, no shrinkage or cracks, no shrinkage at the bottom, and no convex spots on the surface.

[0031] Figure 2 The freeze-dried appearance of Example 2 shows that the freeze-dried product has a good appearance, with no drop in the surface position, no shrinkage as a whole, no cracks, no shrinkage at the bottom, and no convex spots on the surface.

[0032] Figure 3 This is the freeze-dried appearance of Comparative Example 3. After freeze-drying, the surface position of the product has dropped, it has shrunk severely, and the appearance is poor.

[0033] Figure 4 This is the freeze-dried appearance of Comparative Example 4. After freeze-drying, the surface position of the product did not drop, the bottom shrank severely, and the appearance was poor.

[0034] Figure 5 This is the freeze-dried appearance of Comparative Example 5. After freeze-drying, the surface position of the product did not drop, the bottom shrank severely, and the appearance was poor.

[0035] Figure 6 This is the freeze-dried appearance of Comparative Example 6. The surface position of the product did not drop after freeze-drying. There were cracks on the surface of 3 bottles, and there was no shrinkage at the bottom, and the appearance was poor.

[0036] It can be seen that the appearance of the freeze-dried products of Comparative Examples 3-6 was poor, so no subsequent exosome-related tests were performed. The freeze-dried products of Examples 1 and 2 had good appearance, and the following performance tests were performed on them.

[0037] 2. Stability testing of freeze-dried exosomes The samples of Examples 1 and 2 were respectively placed at 25°C for 30 days, and samples were taken at 0 days, 11 days and 30 days to detect the exosome particle size, particle concentration, purity, CD9 positivity rate and CD63 positivity rate; in addition, the samples of Examples 1 and 2 were respectively placed at 2-8°C and -20°C for 6 months, and samples were taken at 0 months, 3 months and 6 months to detect the exosome particle size, particle concentration, protein concentration, purity, CD9 positivity rate and CD63 positivity rate. The results are shown in Table 1 below.

[0038] Table 1 Stability of freeze-dried exosomes at 25℃ The results show that after the exosome samples were placed at 25°C for one month after freeze-drying, the exosome particle size, particle concentration, protein concentration, purity, CD9 positivity rate and CD63 positivity rate did not change significantly, that is, the stability of the exosome freeze-dried product was significantly improved.

[0039] 3. Stability of freeze-dried exosomes at 2-8℃ The samples of Examples 1 and 2 were placed at 2-8°C for 6 months, and samples were taken at 0M, 3M and 6M to detect the exosome particle size, particle concentration, purity, CD9 positive rate and CD63 positive rate; The experimental results are shown in Table 2.

[0040] Table 2 Stability of freeze-dried exosomes at 2-8℃ The results show that after the exosome samples were placed at 2-8°C for 6 months after freeze-drying, the exosome particle size, particle concentration, protein concentration, purity, CD9 positivity rate and CD63 positivity rate did not change significantly, that is, the stability of the exosome freeze-dried product was significantly improved.

[0041] 4. Stability of freeze-dried exosomes at -20℃ The exosomes in Example 1 and Example 2 were respectively collected at 0M, 3M and 6M for electron microscopy, particle size, particle concentration, protein concentration, purity, CD9 positivity rate and CD63 positivity rate.

[0042] The experimental results are as follows Figure 7 And as shown in Table 3.

[0043] Table 3 -20℃ freeze-dried exosome stability The results show that after the exosome samples were placed at 2-8°C for 6 months after freeze-drying, the exosome particle size, particle concentration, protein concentration, purity, CD9 positivity rate and CD63 positivity rate did not change significantly, that is, the stability of the exosome freeze-dried product was significantly improved.

[0044] The present invention provides an exosome lyophilization protective agent with a simple formula and high safety. From the perspective of freeze-dried prescription ingredients, the prescription designed by the present invention has simple ingredients and higher safety. From the perspective of cost, most of the current freeze-dried prescriptions use trehalose, which is relatively expensive. The prescription included in the present invention uses sucrose to replace trehalose, and compares the effects of sucrose and trehalose freeze-dried prescriptions, while maintaining the long-term storage stability of exosomes, reducing storage costs, which is conducive to the long-term storage of a large number of exosome samples.

Claims

1. Exosome freeze-dried protective agent to improve stability, It is characterized in that Composition includes: Trehalose or sucrose, mannitol.

2. The exosome freeze-dried protective agent for improving stability according to claim 1, It is characterized in that Composition includes: 4-10wt% trehalose or sucrose, 3-8wt% mannitol.

3. The exosome freeze-dried protective agent for improving stability according to claim 1, It is characterized in that Composition includes: 8wt% trehalose, 5wt% mannitol.

4. The exosome freeze-dried protective agent for improving stability according to claim 1, It is characterized in that Composition includes: 5wt% sucrose, 4wt% mannitol.

5. The exosome freeze-dried protective agent for improving stability according to claim 1, Features: PBS or saline is also included.

6. The exosome freeze-dried protective agent for improving stability according to claim 5, Features: The amount of PBS or saline added is based on the osmotic pressure reaching 280-320 mmol / L.

7. The exosome freeze-dried protective agent for improving stability according to claim 1, Features: The exosomes are one of mesenchymal stem cell exosomes, 293 exosomes or engineered exosomes.

8. A method for preparing the exosome freeze-dried protective agent with improved stability according to any one of claims 1 to 7, It is characterized in that The following steps are involved: Weigh trehalose or sucrose and mannitol, dissolve them in water to make the mass concentration of trehalose or sucrose reach 4-10wt%, and the mass concentration of mannitol reach 3-8wt%, stir and mix well to obtain an exosome freeze-dried protective agent with improved stability.

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