Exosome cryopreservation protective solution and preparation method thereof

Abstract

The application relates to an exosome low-temperature cryopreservation protective solution and a preparation method thereof. The exosome low-temperature cryopreservation protective solution comprises the following components in percentage by mass: 1-5wt% of bovine serum albumin, 0.1-0.5wt% of sialic acid and 89.5-97.9wt% of PBS buffer solution without Ca 2+ and Mg 2+ . The exosome low-temperature cryopreservation protective solution provided by the application has simple components, can long-term store exosomes at low temperature and does not affect the biological activity of the exosomes.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an exosome cryopreservation protective solution and a preparation method thereof. Background Art

[0002] Exosomes are extracellular vesicles with a diameter of 30 to 150 nm secreted by living cells that have the function of transmitting information. Exosomes have a double lipid membrane structure and can mediate intercellular signaling by delivering deoxyribonucleic acid (DNA), ribonucleic acid (RNA), lipids, and proteins to target cells.

[0003] Endothelial cell-derived exosomes are rich in microRNAs (miRNAs), cytokines, and growth factors. These exosomes can regulate angiogenesis and the biological functions of surrounding cancer cells, fibroblasts, neurons, and immune cells through these exosome contents. A growing body of research indicates that exosomes have important biological effects, including reducing apoptosis, alleviating inflammation, promoting angiogenesis, inhibiting fibrosis, and enhancing tissue repair potential. Furthermore, compared to living cells, exosomes have a more defined composition and are easier to control in terms of quality, offering promising clinical applications in the treatment of tissue injuries such as myocardial infarction and skin trauma.

[0004] Exosomes have low stability and are easily inactivated. To maintain their biological activity, they must be stored and transported at low temperatures. The loss of exosome biological activity often occurs during the storage and preservation stage. Studies have shown that exosomes stored at low temperatures will still experience structural damage such as enlargement of diameter, exosome rupture, inversion of the outer membrane, and reduction in biological activity such as degradation or inactivation of contents. The International Society for Extracellular Vesicles recommends that exosomes be stored in PBS buffer at -80°C. This method can maintain the biological activity of exosomes for 3-6 months. However, exosomes will still be damaged during storage, which will affect the recovery rate and biological activity of exosomes. The short shelf life also greatly limits the normal use of exosomes.

[0005] Current studies have found that relatively simple liquid environments are not conducive to the preservation of exosomes, while complex and acidic environments that simulate body fluids are more conducive to the long-term preservation of exosomes. In the absence of a protective agent, exosomes naturally aggregate, precipitate, fuse, and break down. Therefore, existing technologies typically preserve the activity of exosomes by adding cryoprotectants, such as culture medium or bovine serum. However, bovine serum can introduce potential pathogenic factors and cause inconsistent stability between exosome batches. There are also freeze-dried formulation methods designed for exosomes. While these methods can preserve exosome activity to a certain extent, the reagent preparation and operation are cumbersome, making them unsuitable for the application of exosome preparations. Gel preparations are another method for storing exosomes. While they can also preserve the integrity of exosomes, the preparation process requires a large amount of polymeric pharmaceutical excipients, which can seriously affect the biological properties and practical applications of exosomes.

[0006] Therefore, there is an urgent need for a cryopreservation protection reagent with simple ingredients, high biosafety, the ability to preserve exosomes for a long time at a lower temperature without affecting their biological activity. Summary of the Invention

[0007] To address the above problems, the present invention provides an exosome cryopreservation protective solution and a preparation method thereof. The exosome cryopreservation protective solution has simple ingredients and can preserve exosomes for a long time at low temperatures without affecting their biological activity.

[0008] In the first aspect, the present invention provides an exosome cryopreservation protection solution, wherein the exosome cryopreservation protection solution comprises, by mass percentage, 1-5 wt% bovine serum albumin, 1-5 wt% dextran 70, 0.1-0.5 wt% sialic acid, and no Ca 2+ Mg 2+ 89.5~97.9wt% of PBS buffer solution.

[0009] Preferably, the exosome cryopreservation protection solution comprises, by mass percentage, 2 wt% bovine serum albumin, 2 wt% dextran 70, 0.25 wt% sialic acid, and no Ca 2+ Mg 2+ PBS buffer solution 95.75wt%.

[0010] Preferably, the average molecular weight of the dextran 70 is 60,000 to 80,000.

[0011] Preferably, no Ca 2+ Mg 2+ The pH value of the PBS buffer solution is 6.4-8.8, preferably 6.8-8.8, more preferably 7.4.

[0012] In a second aspect, the present application provides a preparation method of the exosome cryopreservation solution, which comprises the following steps: dissolving dextran 70 in a PBS buffer solution without Ca 2+ , Mg 2+ to obtain a dextran 70-PBS colloidal solution, and then mixing the dextran 70-PBS colloidal solution with a sialic acid and a bovine serum albumin solution to obtain the exosome cryopreservation solution.

[0013] Preferably, the temperature for dissolving the dextran 70 in the PBS buffer solution without Ca 2+ , Mg 2+ is 50-60°C.

[0014] Preferably, the preparation method further comprises the operation of filtering the mixed solution formed after mixing the dextran 70-PBS colloidal solution with the sialic acid and the bovine serum albumin solution.

[0015] Preferably, the pore size of the filter membrane is 0.2-0.3 μm, preferably 0.22 μm.

[0016] Advantages

[0017] The exosome cryopreservation solution provided by the present application has a clear composition, and the exosomes added with the exosome cryopreservation solution provided by the present application can be stably stored at a low temperature of-20°C for 9-12 months, while maintaining the biological activity of the exosomes;

[0018] The exosome cryopreservation solution provided by the present application has simple components and higher biological safety and clinical application potential compared with the existing protection solution.

[0019] All components of the exosome cryopreservation solution provided by the present application can be produced industrially, are cheap and easy to obtain, and have a low-cost advantage. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 FIG. 1 is a comparison chart of Zeta potentials of dextran 70 and dextran 40-PBS colloidal solutions with different mass percentages;

[0021] Figure 2 FIG. 4 is an exosome particle size distribution and quantity diagram of brain microvascular endothelial cell-derived exosomes stored in the exosome cryopreservation solution prepared by the method of Example 1 of the present application within 12 months;

[0022] Figure 3 FIG. 5 is a diagram showing the particle size and morphological changes of brain microvascular endothelial cell-derived exosomes stored in the exosome cryopreservation solution prepared by the method of Example 1 of the present application within 12 months;

[0023] Figure 4 Schematic diagram of the comparison of the cell proliferation activity of exosomes after 12 months of storage in different exosome protective solutions. DETAILED DESCRIPTION

[0024] The present invention is further described below through embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0025] Exosomes are membrane vesicles released into the extracellular matrix after fusion of intracellular multivesicular bodies (MVBs) with the cell membrane. They are nanoscale lipid-encapsulated structures with a diameter of 30-150 nm, encapsulating substances such as proteins, mRNA, and microRNA. Exosomes naturally occur in body fluids, including blood, saliva, urine, and breast milk. Secreted exosomes enter these fluids, travel through the circulatory system, and reach other cells and tissues, exerting remote regulatory effects.

[0026] First, the present invention provides an exosome cryopreservation protection solution. The exosome cryopreservation protection solution comprises, by mass percentage, 1-5 wt% bovine serum albumin (BSA), 1-5 wt% dextran 70 (Dextran), 0.1-0.5 wt% sialic acid, and no Ca. 2+ Mg 2+ PBS buffer solution 89.5 ~ 97.9wt%; preferably, bovine serum albumin 2wt%, dextran 702wt%, sialic acid 0.25wt%, without Ca 2+ Mg 2+ PBS buffer solution 95.75wt%.

[0027] Exosomes are stored in a limited variety of cryopreservation solutions, and their preservation effects are poor. This can easily lead to structural damage, loss of biological activity, or degradation of contents during storage, affecting subsequent detection of exosomes. In light of this, the inventors of this application, through extensive experiments, have discovered that an exosome preservation solution containing PBS buffer, dextran 70, bovine serum albumin, and sialic acid can effectively protect exosomes, maintaining their morphological structure and biological activity for long periods at -20°C.

[0028] Adding bovine serum albumin (BSA) can increase the protein concentration in the exosome cryopreservation solution, thereby preventing protease hydrolysis of exosome membrane proteins. In some embodiments, the BSA content in the exosome cryopreservation solution can be controlled to 1-5 wt %. Excessively high BSA levels can lead to cytotoxicity and increase formulation costs, while low BSA levels can compromise protease inhibition.

[0029] Dextran is an inert, water-soluble polymer produced by the fermentation of sucrose by Leuconostoc mesenteroides. It has been widely used in the biomedical field as a natural, non-permeable cryoprotectant. It is currently widely used in clinical practice as an antithrombotic drug. Depending on the number of polymerized molecules, dextran can be primarily divided into: high-molecular-weight dextran (average molecular weight 100,000-200,000), medium-molecular-weight dextran 70 (average molecular weight 60,000-80,000), low-molecular-weight dextran 40 (average molecular weight 20,000-40,000), and small-molecule dextran (average molecular weight 10,000-20,000).

[0030] Dextran 70, as a non-permeable cryoprotectant, can form a protective colloidal film on the surface of exosomes under low temperature conditions, making the exosomes frost-resistant and avoiding structural damage caused by freezing and thawing during storage and use. In some embodiments, the content of dextran 70 (Dextran) in the exosome cryopreservation protective solution can be controlled to be 1-5wt%, preferably 2wt%, and the 2wt% dextran 70-PBS colloidal solution has the highest stability. If the content is too high, the stability of the colloidal solution will be reduced and cytotoxicity will be generated; if the content is too low, the density of the colloidal solution will be too low and it will be difficult to form a complete and stable protective film outside the exosomes.

[0031] Sialic acid is a general term for acylated neuraminic acid, a nine-carbon sugar, also known as N-acetylneuraminic acid. It is widely distributed in nature and has been confirmed to be ubiquitous in vertebrates and mammals, with the highest concentration in cerebrospinal fluid and mucus. It is an important component of glycoproteins, oligosaccharides, and glycolipids. Sialic acid can bind to membrane proteins on biological membranes, thereby forming a layer of polysaccharide-protein complex on the surface of the cell membrane. Therefore, it has the functions of glycoprotein protection, antibacterial and antiviral properties, and preventing cell membrane adhesion. The chemical structure of sialic acid N-acetylneuraminic acid used in the exosome cryopreservation protection solution provided by the present invention is as follows:

[0032]

[0033] Sialic acid usually exists in the form of oligomers. In the exosome cryopreservation protective solution, it can form a protective membrane by binding to the glycolipids on the exosome membrane. At the same time, sialic acid can ionize hydrogen ions, provide an acidic environment for the exosomes, and make the exosomes negatively charged, thereby avoiding collision and fusion between exosomes, and thus avoiding the loss of exosomes. In addition, sialic acid also has the function of protecting exosome membrane proteins and resisting virus recognition, thereby ensuring the biological activity of exosomes. In some embodiments, the content of sialic acid in the exosome cryopreservation protective solution can be controlled to be 0.1 to 0.5 wt%. Sialic acid can significantly affect the pH of the preparation. If the content is too high, the exosome cryopreservation protective agent will become over-acidified and produce cytotoxicity; if the content is too low, the protective effect of sialic acid on exosomes will be weakened or even disappear.

[0034] In some embodiments, no Ca 2+ Mg 2+ The pH value of the PBS buffer solution can be 6.4-8.8, preferably 6.8-8.8, and more preferably 7.4. In the principle of the present invention, sialic acid will be adsorbed on the surface of the exosome membrane through protein-protein interaction, causing the exosome to carry a negative charge and thus play a lubricating role, thereby preventing the natural aggregation or extrusion collision of the exosome, and Ca 2+ Mg 2 + The presence of may destroy the charge distribution on the exosome membrane and thus affect the protective effect.

[0035] The following is an exemplary description of the method for preparing the cryopreservation protective solution for exosomes provided by the present invention. The preparation method may include the following steps: heating the solution without Ca 2+ Mg 2+ The PBS buffer solution was heated to 95°C and then allowed to cool at room temperature until it was free of Ca 2+ Mg 2+ After cooling the PBS buffer solution to 50-60°C, dextran 70 is dissolved therein to obtain a dextran 70-PBS colloidal solution. After the colloidal solution cools to room temperature, the dextran 70-PBS colloidal solution is mixed with a solution of sialic acid and bovine serum albumin to obtain the exosome cryopreservation solution. The cryopreservation solution must be filtered through a 0.2-0.3 μm filter membrane before use.

[0036] PBS buffer solution heated at high temperature (such as 95°C) can effectively inactivate harmful substances such as proteases, nucleases, microorganisms, etc. in the preservation solution. When the PBS buffer solution is cooled to 50-60°C, it can quickly and effectively dissolve dextran 70 to form a dextran 70-PBS colloidal solution.

[0037] Dextran 70 is a high molecular weight compound that is not easily soluble in PBS buffer at room temperature. PBS buffer at 50-60°C can quickly dissolve dextran 70 and form a colloidal solution. Excessively high temperatures may cause dextran 70 to degrade; if the temperature is too low, dextran 70 will deposit at the bottom of the container and be difficult to dissolve quickly.

[0038] BSA and sialic acid are both protein compounds, which are easily denatured and lose their activity when exposed to high temperatures. Therefore, BSA and sialic acid need to be added to a dextran 70-PBS colloidal solution cooled to room temperature.

[0039] It should also be noted that the exosome cryopreservation solution needs to be filtered through a filter membrane before use. Preferably, the pore size of the filter membrane can be 0.2-0.3 μm, preferably 0.22 μm. A filter membrane with a pore size of 0.2-0.3 μm can effectively remove insoluble impurities and contaminants such as bacteria and fungi from the preparation. A pore size that is too large will prevent the removal of small particulate impurities, while a pore size that is too small will cause the filter membrane to clog.

[0040] The following examples are further listed to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all fall within the scope of protection of the present invention. The specific process parameters and the like in the following examples are only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description herein, and are not limited to the specific numerical values ​​exemplified below.

[0041] Unless otherwise specified, the present invention does not specify specific techniques or conditions, according to the conventional technical conditions in the field or according to the product instructions. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0042] Example 1

[0043] Add dextran 70 with a molecular weight of 60,000 to a solution preheated to 95°C and cooled to 50°C without Ca 2+ Mg 2+ PBS buffer solution to obtain dextran 70-PBS colloidal solution; after the colloidal solution is cooled to room temperature, bovine serum albumin and sialic acid are dissolved in the dextran 70-PBS colloidal solution to prepare exosome cryopreservation protection solution. The exosome cryopreservation protection solution comprises the following components by mass percentage: bovine serum albumin 2wt%, dextran 70 2wt%, sialic acid 0.25wt%, and no Ca 2+ Mg 2+95.75wt% of PBS buffer; the pH value of the obtained exosome cryopreservation protection solution is 6.44.

[0044] Comparative Example 1

[0045] This comparative example adopts the storage method recommended by the International Extracellular Vesicle Association, and the exosomes are stored in PBS buffer. The PBS buffer used in this comparative example has a pH value of 7.5 and does not contain Ca 2+ Mg 2+ PBS buffer.

[0046] Comparative Example 2

[0047] The preparation method of this comparative example refers to Example 1, with the main difference that no sialic acid is added; the components of the prepared exosome cryopreservation solution include, by mass percentage, 2 wt% bovine serum albumin, 704 wt% dextran, and no Ca 2+ Mg 2+ 94wt% PBS buffer; the pH value of the obtained exosome cryopreservation protection solution is 7.4.

[0048] Comparative Example 3

[0049] The preparation method of this comparative example refers to Example 1, with the main difference that: dextran 70 is not added; the components of the prepared exosome cryopreservation solution include: in terms of mass percentage, 2wt% bovine serum albumin, 0.25wt% sialic acid, and no Ca 2+ Mg 2+ 97.75wt% PBS buffer; the pH value of the obtained exosome cryopreservation protection solution is 6.5.

[0050] Comparative Example 4

[0051] The preparation method of this comparative example refers to Example 1, with the main difference that the proportion of dextran 70 is increased; the components of the prepared exosome cryopreservation solution include, by mass percentage, 2wt% bovine serum albumin, 6wt% dextran 70, 0.25wt% sialic acid, and no Ca 2+ Mg 2+ 91.75wt% of PBS buffer; the pH value of the obtained exosome cryopreservation protection solution is 6.32.

[0052] Comparative Example 5

[0053] The preparation method of this comparative example refers to Example 1, with the main difference being that dextran 40 is replaced by dextran 70; the components of the prepared exosome cryopreservation solution include, by mass percentage, 2 wt% bovine serum albumin, 0.2 wt% dextran 40, 0.25 wt% sialic acid, and no Ca 2+Mg 2+ 97.55wt% of PBS buffer; the pH value of the obtained exosome cryopreservation protection solution is 6.52.

[0054] Comparative Example 6

[0055] The preparation method of this comparative example refers to Example 1, with the main difference being that dextran 40 is replaced by dextran 70; the components of the prepared exosome cryopreservation solution include, by mass percentage, 2 wt% bovine serum albumin, 1 wt% dextran 40, 0.25 wt% sialic acid, and no Ca 2+ Mg 2+ 96.75wt% PBS buffer; the pH value of the obtained exosome cryopreservation protection solution is 6.48.

[0056] Next, the storage performance of the cryopreservation protective solutions in Comparative Examples 1-6 and Example 1 was studied using exosomes derived from brain microvascular endothelial cells.

[0057] (1) Screening of protective agents added to exosome cryopreservation protection solution.

[0058] According to the method of Example 1, 0.5 wt%, 1 wt%, 2 wt%, 4 wt%, 6 wt%, 8 wt%, 12 wt%, and 16 wt% dextran 70 and dextran 40-PBS colloidal solutions were prepared, and the zeta potential of the solutions was measured. Figure 1 As shown in the figure, it can be seen that the dextran 70-PBS colloidal solution is more stable than the dextran 40-PBS colloidal solution.

[0059] (2) Screening of the concentration of protective solution for exosome cryopreservation.

[0060] The exosome cryopreservation solutions prepared in Example 1 and Comparative Examples 4-6 were measured for zeta potential using a potentiometer. The specific experimental results are shown in Table 1 below. The results show that the exosome cryopreservation solution prepared with 1-5 wt% dextran 70 exhibited the highest stability.

[0061] Table 1 Effects of different exosome cryopreservation protection solution components on the cryopreservation stability of exosomes

[0062]

[0063]

[0064] (3) Extraction of exosomes from brain microvascular endothelial cells.

[0065] Human brain microvascular endothelial cells (HCMEC / D3) in the logarithmic growth phase were cultured and exosomes were extracted. HCMEC / D3 and serum-free culture medium were purchased from Suzhou Kemer Biotechnology Co., Ltd. The specific method is as follows: When the cell confluence reaches 60-70%, serum-free low-glucose DMEM medium is added and cultured for another 48 hours, after which the conditioned medium is collected. Exosomes are then isolated and extracted by ultracentrifugation or ultrafiltration: 1000 mL of cell culture medium is centrifuged at 300 g for 10 minutes to remove cells, and then at 3000 g for 20 minutes to further remove cell debris and impurities. The resulting supernatant is added with an appropriate amount of exosome extraction reagent according to the instructions of the exosome extraction kit, mixed by inversion, and incubated at 4°C for 12 hours. After incubation, the mixture is centrifuged at 10,000 g for 1 hour at 4°C. The supernatant is discarded, and the exosomes are contained in the pellet at the bottom of the tube. The exosomes are resuspended in 1 mL of pre-chilled PBS, filtered through a 0.22 μm disposable filter, and finally centrifuged at 110,000 g for 90 minutes to obtain endothelial cell-derived exosomes. The resulting exosomes are resuspended in different cryoprotectant solutions and stored at -20°C.

[0066] (4) Comparison of the number of exosomes derived from brain microvascular endothelial cells in different preservation methods.

[0067] The extracted exosomes were packaged and stored in different protective solutions of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1 respectively; the storage condition was -20°C. The starting number of exosomes used for the test was 2.81×10 10 The particles were divided and placed in different exosome cryopreservation solutions at -20°C for 3, 6, 9, and 12 months, and then the exosomes were identified. The specific test results are as follows: Figure 2 As shown in Table 2 below. The results show that the exosome cryopreservation protection solution prepared in Example 1 of the present invention can better protect the number of exosomes.

[0068] Table 2 Effects of different exosome cryopreservation solutions on the number of exosomes over time

[0069]

[0070] (5) Comparison of the particle size of exosomes derived from brain microvascular endothelial cells in different preservation methods.

[0071] The extracted exosomes were packaged and stored in different protective solutions of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Example 1; the storage condition was -20°C. The initial exosome particle size for the test was 126.3+ / -4.2nm. After packaging, they were placed in different exosome cryopreservation solutions and stored at -20°C for 3, 6, 9, and 12 months. The exosome NTA nanoparticle size was then identified. The specific test results are shown in the figure. Figure 2 、 3 As shown in Table 3 below. The results show that the exosome cryopreservation protection solution prepared in Example 1 of the present invention can better protect the exosome particle size and structure.

[0072] Table 3 Effects of different exosome cryopreservation protective solutions on exosome particle size over time

[0073]

[0074] (6) The effects of different exosome cryopreservation solutions on the biological activity of exosomes.

[0075] The starting exosome concentration used for the test was 2.81 × 10 10 After packaging, they were placed in different exosome cryopreservation solutions and stored at -20°C for 3, 6, 9, and 12 months, and then diluted to 1.0×10 10 The test solution was added into the vascular endothelial cell proliferation model to detect the biological activity of exosomes (CCK-8). The specific test results are as follows Figure 4 The results showed that the exosome cryopreservation protection solution prepared in Example 1 of the present invention can better protect the biological activity of exosomes and maintain the biological activity of exosomes in promoting cell proliferation (each solvent was used as a negative control to exclude the influence of the solvent formula itself).

[0076] Although the present invention has been described in detail through the above preferred embodiments, it should be understood that the above description is not intended to limit the present invention. After reading the above description, various modifications and substitutions of the present invention will become apparent to those skilled in the art. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A cryopreservation solution for exosomes, characterized in that: The exosome cryopreservation protection solution comprises, by mass percentage, 1-5 wt% bovine serum albumin, 1-5 wt% dextran 70, 0.1-0.5 wt% sialic acid, and no Ca 2+ Mg 2+ 89.5~97.9 wt% of PBS buffer solution.

2. The exosome cryopreservation protective solution according to claim 1, characterized in that The exosome cryopreservation protection solution comprises, by mass percentage, 2 wt% bovine serum albumin, 2 wt% dextran 70, 0.25 wt% sialic acid, and no Ca 2+ Mg 2+ 95.75 wt% of PBS buffer solution.

3. The exosome cryopreservation protective solution according to claim 1 or 2, characterized in that: The average molecular weight of the dextran 70 is 60,000 to 80,000.

4. The exosome cryopreservation protective solution according to claim 1 or 2, characterized in that: No calcium 2+ Mg 2+ The pH value of PBS buffer solution is 6.4-8.

8.

5. The exosome cryopreservation protective solution according to claim 4, characterized in that: No calcium 2+ Mg 2+ The pH value of PBS buffer solution is 6.8-8.

8.

6. The exosome cryopreservation protective solution according to claim 5, characterized in that No calcium 2+ Mg 2+ The pH value of PBS buffer solution is 7.

4.

7. A method for preparing the exosome cryopreservation protective solution according to claim 1, characterized in that: The preparation method comprises the following steps: dissolving dextran 70 in a solution containing no Ca 2+ Mg 2+ A dextran 70-PBS colloidal solution is obtained from a PBS buffer solution, and then the dextran 70-PBS colloidal solution is mixed with sialic acid and bovine serum albumin solution to obtain the exosome cryopreservation protection solution.

8. The preparation method according to claim 7, wherein Dextran 70 dissolved in Ca-free 2+ Mg 2+ The temperature of the PBS buffer solution is 50-60 °C.

9. The preparation method according to claim 7, characterized in that The preparation method further comprises the operation of filtering a mixed solution formed by mixing the dextran 70-PBS colloidal solution with the sialic acid and bovine serum albumin solution using a filter membrane.

10. The preparation method according to claim 9, wherein The pore size of the filter membrane is 0.2-0.3 μm.

11. The preparation method according to claim 10, characterized in that The pore size of the filter membrane is 0.22 μm.

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