Exosome preserving fluid

Abstract

The invention belongs to the technical field of biology, and particularly discloses an exosome preserving fluid, which is prepared from the following ingredients in parts by weight: 0.01 to 0.1 part of sodium hyaluronate, 0.1 to 1 part of threonine, 0.5 to 1.2 parts of chitosan, 1 to 4 parts of bovine serum albumin, 1.5 to 3.5 parts of phospholipid substances, 3 to 9 parts of polyhydroxy compounds, 2 to 10 parts of ellagic acid, 2 to 10 parts of tomato extracts, 5 to 15 parts of dimethyl sulfoxide and 64.1 to 66.99 parts of normal saline. The exosome preserving fluid provided by the invention can provide stable conditions for storing the exosome, so as to protect the structure of the exosome and biological information molecules or drugs carried by the exosome from biological, chemical or mechanical damage, effectively maintain the stability, activity and function of the exosome, and ensure that the exosome is not lost in the preservation process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an exosome preservation solution. Background Art

[0002] Exosomes are tiny membrane vesicles that can be secreted by most cells. Exosomes have important biological functions, including intercellular signaling, immune regulation, and material transport. In recent years, the application of exosomes in disease diagnosis, gene therapy, and drug delivery has made significant progress. However, exosomes are susceptible to oxidative damage, membrane structure destruction, protein degradation, and other problems during extraction, storage, and transportation, resulting in loss of function.

[0003] Currently, commonly used methods for preserving exosomes include freezing, low-temperature and refrigerated storage, but most of the existing preservation solutions cannot effectively solve problems such as oxidative damage, membrane rupture, precipitation and aggregation during the exosome preservation process.

[0004] In view of this, this application is filed. Summary of the invention

[0005] The present invention provides an exosome preservation solution, which can provide stable conditions for storing exosomes to protect the exosome structure and the bioinformatic molecules or drugs carried by the exosomes from biological, chemical or mechanical damage, effectively maintain the stability, activity and function of the exosomes, and ensure that they are not lost during the preservation process.

[0006] The present invention solves the technical problem by adopting the following technical solutions: An exosome preservation solution comprises the following components in parts by weight: 0.01-0.1 parts of sodium hyaluronate, 0.1-1 parts of threonine, 0.5-1.2 parts of chitosan, 1-4 parts of bovine serum albumin, 1.5-3.5 parts of phospholipid substances, 3-9 parts of polyhydroxy compounds, 2-10 parts of ellagic acid, 2-10 parts of tomato extracts, 5-15 parts of dimethyl sulfoxide, and 64.1-66.99 parts of normal saline.

[0007] The present invention creatively combines the above-mentioned raw materials to obtain an exosome preservation solution with excellent performance. The exosomes can provide stable conditions for storing exosomes to protect the exosome structure and the bioinformatic molecules or drugs carried by them from biological, chemical or mechanical damage, effectively maintain the stability, activity and function of the exosomes, and ensure that they are not lost during the storage process.

[0008] As a natural polyphenol compound, ellagic acid has strong antioxidant capacity. It can scavenge free radicals, reduce the damage of oxidative stress to the exosome membrane and internal components, and protect exosomes from oxidative degradation. Ellagic acid can also prevent the destruction of the exosome membrane by inhibiting peroxidation reactions and reducing lipid peroxidation. In addition, ellagic acid also has astringent properties and can bind to biological macromolecules such as proteins and polysaccharides, which may affect the stability and biological activity of exosomes.

[0009] Tomato extract is rich in antioxidants such as lycopene and flavonoids. Lycopene is a carotenoid with strong antioxidant effects that can effectively remove free radicals in the body and protect exosomes from oxidative damage. When synergistically acting with ellagic acid, lycopene further enhances resistance to oxidative stress and helps maintain the structure and function of exosomes.

[0010] The present invention creatively combines ellagic acid and tomato extract. Ellagic acid and tomato extract protect exosomes at multiple levels through different antioxidant mechanisms (such as free radical scavenging, lipid peroxidation inhibition, etc.), and their complementary effects enhance the antioxidant effect, prolong the stability and activity of exosomes, and improve the overall stability of exosomes, so that exosomes can avoid membrane rupture and leakage of contents during freezing, refrigeration or long-term storage. Ellagic acid combines with proteins or other biomacromolecules on the surface of exosomes to form a protective layer, which helps to stabilize the structure of exosomes. The components in tomato extract may also interact with exosomes in a similar way to further consolidate their structural stability.

[0011] As a preferred embodiment of the present invention, the composition comprises the following components in parts by weight: 0.04-0.08 parts of sodium hyaluronate, 0.2-0.6 parts of threonine, 0.6-1 parts of chitosan, 1.5-3 parts of bovine serum albumin, 1.8-3 parts of phospholipids, 4-8 parts of polyhydroxy compounds, 4-7 parts of ellagic acid, 5-9 parts of tomato extract, 8-12 parts of dimethyl sulfoxide, and 64.96-66.22 parts of normal saline.

[0012] As a preferred embodiment of the present invention, the following components are included in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of phospholipids, 5 parts of polyols, 6 parts of ellagic acid, 8 parts of tomato extract, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0013] As a preferred embodiment of the present invention, the polyhydroxy compound is glycerol glucoside.

[0014] As a preferred embodiment of the present invention, the phospholipid substance is soybean lecithin.

[0015] As a preferred embodiment of the present invention, the preparation method of the tomato extract is: (1) Wash and mash the tomatoes, add water to make pulp, and obtain a mixed liquid; (2) adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing ultrasound-assisted enzymolysis, centrifuging, filtering and drying to obtain a tomato extract.

[0016] As a preferred embodiment of the present invention, the mass ratio of tomato to water is 1:(4-10).

[0017] As a preferred embodiment of the present invention, the mass ratio of the mixed solution, pectinase, cellulase, β-glucanase, and mixed solution is (0.005~0.02):(0.005~0.02):(0.005~0.02):1.

[0018] As a preferred embodiment of the present invention, the ultrasonic power is 150~300W.

[0019] As a preferred embodiment of the present invention, the enzymatic hydrolysis temperature is 45-55° C., and the enzymatic hydrolysis time is 2-5 h.

[0020] Beneficial effects of the present invention: The exosome preservation solution of the present invention can provide stable conditions for storing exosomes to protect the exosome structure and the bioinformatic molecules or drugs carried by it from biological, chemical or mechanical damage, effectively maintain the stability, activity and function of the exosomes, and ensure that they are not lost during the preservation process. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0023] In the present invention, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0024] In the present invention, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0025] The reagents or instruments used in the present invention without indicating the manufacturer are all conventional products that can be obtained commercially. The raw materials used in the comparative examples and the raw materials used in the parallel experiments of the embodiments are the same commercially available products unless otherwise specified.

[0026] Example 1 An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 6 parts of ellagic acid, 8 parts of tomato extract, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0027] The concentration of the physiological saline is 0.9%.

[0028] The preparation method of the tomato extract is: (1) washing the tomatoes, crushing them with a tissue crusher, and adding water to prepare a pulp to obtain a mixed solution; the mass ratio of the tomatoes to the water is 1:8; (2) Adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing enzymolysis with the aid of 200 W ultrasound, centrifuging, filtering and drying to obtain a tomato extract; the mass ratio of the mixed solution, pectinase, cellulase, β-glucanase and the mixed solution is 0.01:0.01:0.01:1; the enzymolysis temperature is 48°C and the enzymolysis time is 4 hours.

[0029] Example 2 An exosome preservation solution comprises the following components in parts by weight: 0.04 parts of sodium hyaluronate, 0.6 parts of threonine, 0.6 parts of chitosan, 3 parts of bovine serum albumin, 1.8 parts of soybean lecithin, 8 parts of glycerol glucoside, 4 parts of ellagic acid, 9 parts of tomato extract, 8 parts of dimethyl sulfoxide, and 64.96 parts of normal saline.

[0030] The concentration of the physiological saline is 0.9%.

[0031] The preparation method of the tomato extract is: (1) washing the tomatoes, crushing them with a tissue crusher, and adding water to prepare a pulp to obtain a mixed solution; the mass ratio of the tomatoes to the water is 1:8; (2) Adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing enzymolysis with the aid of 200 W ultrasound, centrifuging, filtering and drying to obtain a tomato extract; the mass ratio of the mixed solution, pectinase, cellulase, β-glucanase and the mixed solution is 0.01:0.01:0.01:1; the enzymolysis temperature is 48°C and the enzymolysis time is 4 hours.

[0032] Example 3 An exosome preservation solution comprises the following components in parts by weight: 0.08 parts of sodium hyaluronate, 0.2 parts of threonine, 1 part of chitosan, 1.5 parts of bovine serum albumin, 3 parts of soybean lecithin, 4 parts of glycerol glucoside, 7 parts of ellagic acid, 5 parts of tomato extract, 12 parts of dimethyl sulfoxide, and 66.22 parts of normal saline.

[0033] The concentration of the physiological saline is 0.9%.

[0034] The preparation method of the tomato extract is: (1) washing the tomatoes, crushing them with a tissue crusher, and adding water to prepare a pulp to obtain a mixed solution; the mass ratio of the tomatoes to the water is 1:8; (2) Adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing enzymolysis with the aid of 200 W ultrasound, centrifuging, filtering and drying to obtain a tomato extract; the mass ratio of the mixed solution, pectinase, cellulase, β-glucanase and the mixed solution is 0.01:0.01:0.01:1; the enzymolysis temperature is 48°C and the enzymolysis time is 4 hours.

[0035] Example 4 An exosome preservation solution comprises the following components in parts by weight: 0.01 parts of sodium hyaluronate, 1 parts of threonine, 0.5 parts of chitosan, 4 parts of bovine serum albumin, 1.5 parts of soybean lecithin, 9 parts of glycerol glucoside, 2 parts of ellagic acid, 10 parts of tomato extract, 5 parts of dimethyl sulfoxide, and 66.99 parts of normal saline.

[0036] The concentration of the physiological saline is 0.9%.

[0037] The preparation method of the tomato extract is: (1) washing the tomatoes, crushing them with a tissue crusher, and adding water to prepare a pulp to obtain a mixed solution; the mass ratio of the tomatoes to the water is 1:8; (2) Adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing enzymolysis with the aid of 200 W ultrasound, centrifuging, filtering and drying to obtain a tomato extract; the mass ratio of the mixed solution, pectinase, cellulase, β-glucanase and the mixed solution is 0.01:0.01:0.01:1; the enzymolysis temperature is 48°C and the enzymolysis time is 4 hours.

[0038] Example 5 An exosome preservation solution comprises the following components in parts by weight: 0.1 parts of sodium hyaluronate, 0.1 parts of threonine, 1.2 parts of chitosan, 1 part of bovine serum albumin, 3.5 parts of soybean lecithin, 3 parts of glycerol glucoside, 10 parts of ellagic acid, 2 parts of tomato extract, 15 parts of dimethyl sulfoxide, and 64.1 parts of normal saline.

[0039] The concentration of the physiological saline is 0.9%.

[0040] The preparation method of the tomato extract is: (1) washing the tomatoes, crushing them with a tissue crusher, and adding water to prepare a pulp to obtain a mixed solution; the mass ratio of the tomatoes to the water is 1:8; (2) Adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing enzymolysis with the aid of 200 W ultrasound, centrifuging, filtering and drying to obtain a tomato extract; the mass ratio of the mixed solution, pectinase, cellulase, β-glucanase and the mixed solution is 0.01:0.01:0.01:1; the enzymolysis temperature is 48°C and the enzymolysis time is 4 hours.

[0041] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not contain ellagic acid, and the other contents are the same.

[0042] An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 8 parts of tomato extract, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0043] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not contain tomato extract, and the other contents are the same.

[0044] An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 6 parts of ellagic acid, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0045] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that an equal amount of tomato extract is used to replace ellagic acid in Comparative Example 3, and the other contents are the same.

[0046] An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 14 parts of tomato extract, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0047] Comparative Example 4 The difference between Comparative Example 4 and Example 1 is that an equal amount of ellagic acid is used to replace the tomato extract in Comparative Example 4, and the other contents are the same.

[0048] An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 14 parts of ellagic acid, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0049] Comparative Example 5 The difference between Comparative Example 5 and Example 1 is that Comparative Example 5 uses an equal amount of vitamin C to replace ellagic acid, and the other contents are the same.

[0050] An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 6 parts of vitamin C, 8 parts of tomato extract, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0051] Comparative Example 6 The difference between Comparative Example 6 and Example 1 is that Comparative Example 6 uses an equal amount of vitamin C to replace the tomato extract, and the other contents are the same.

[0052] An exosome preservation solution comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of soybean lecithin, 5 parts of glycerol glucoside, 6 parts of ellagic acid, 8 parts of vitamin C, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

[0053] Comparative Example 7 The difference between Comparative Example 7 and Example 1 is that the amount of each raw material is not within the scope of the present invention, and the others are the same.

[0054] An exosome preservation solution comprises the following components in parts by weight: 0.005 parts of sodium hyaluronate, 0.05 parts of threonine, 0.2 parts of chitosan, 0.5 parts of bovine serum albumin, 1 part of soybean lecithin, 2 parts of glycerol glucoside, 0.5 parts of ellagic acid, 11.5 parts of tomato extract, 20 parts of dimethyl sulfoxide, and 64.245 parts of normal saline.

[0055] Comparative Example 8 The difference between Comparative Example 8 and Example 1 is that the amount of each raw material is not within the scope of the present invention, and the others are the same.

[0056] An exosome preservation solution comprises the following components in parts by weight: 0.2 parts of sodium hyaluronate, 1.5 parts of threonine, 1.5 parts of chitosan, 6 parts of bovine serum albumin, 5 parts of soybean lecithin, 10 parts of glycerol glucoside, 11.5 parts of ellagic acid, 0.5 parts of tomato extract, 4 parts of dimethyl sulfoxide, and 59.8 parts of normal saline.

[0057] Test Case The particle size is 40nm and the concentration is 1 10 10 The mesenchymal stem cell exosomes were evenly mixed with the exosome preservation solution in a volume ratio of 3:7, refrigerated and stored at 4°C for 3 months, and the concentration and particle size of the preserved exosomes were detected.

[0058] Table 1

[0059] As can be seen from Table 1, the exosome preservation solution of the present invention can provide stable conditions for storing exosomes to protect the exosome structure and the bioinformatic molecules or drugs carried by it from biological, chemical or mechanical damage, effectively maintain the stability, activity and function of the exosomes, and ensure that they are not lost during the storage process.

[0060] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An exosome preservation solution, characterized in that: The invention comprises the following components in parts by weight: 0.01-0.1 parts of sodium hyaluronate, 0.1-1 parts of threonine, 0.5-1.2 parts of chitosan, 1-4 parts of bovine serum albumin, 1.5-3.5 parts of phospholipid substances, 3-9 parts of polyhydroxy compounds, 2-10 parts of ellagic acid, 2-10 parts of tomato extracts, 5-15 parts of dimethyl sulfoxide and 64.1-66.99 parts of normal saline.

2. The exosome preservation solution according to claim 1, characterized in that The invention comprises the following components in parts by weight: 0.04-0.08 parts of sodium hyaluronate, 0.2-0.6 parts of threonine, 0.6-1 parts of chitosan, 1.5-3 parts of bovine serum albumin, 1.8-3 parts of phospholipid substances, 4-8 parts of polyhydroxy compounds, 4-7 parts of ellagic acid, 5-9 parts of tomato extracts, 8-12 parts of dimethyl sulfoxide and 64.96-66.22 parts of normal saline.

3. The exosome preservation solution according to claim 1, characterized in that The composition comprises the following components in parts by weight: 0.05 parts of sodium hyaluronate, 0.5 parts of threonine, 0.8 parts of chitosan, 2 parts of bovine serum albumin, 2 parts of phospholipids, 5 parts of polyols, 6 parts of ellagic acid, 8 parts of tomato extract, 10 parts of dimethyl sulfoxide, and 65.65 parts of normal saline.

4. The exosome preservation solution according to claim 1, characterized in that The polyhydroxy compound is glycerol glucoside.

5. The exosome preservation solution according to claim 1, characterized in that The phospholipid substance is soybean lecithin.

6. The exosome preservation solution according to claim 1, characterized in that The preparation method of the tomato extract is: (1) Wash and mash the tomatoes, add water to make pulp, and obtain a mixed liquid; (2) adding the mixed solution, pectinase, cellulase and β-glucanase into the mixed solution, performing ultrasound-assisted enzymolysis, centrifuging, filtering and drying to obtain a tomato extract.

7. The exosome preservation solution according to claim 6, characterized in that The mass ratio of the tomato to water is 1:(4-10).

8. The exosome preservation solution according to claim 6, characterized in that The mass ratio of the mixed liquid, pectinase, cellulase, β-glucanase, and mixed liquid is (0.005~0.02):(0.005~0.02):(0.005~0.02):

1.

9. The exosome preservation solution according to claim 6, characterized in that The ultrasonic power is 150-300W.

10. The exosome preservation solution according to claim 6, characterized in that The enzymolysis temperature is 45-55° C., and the enzymolysis time is 2-5 hours.

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