Exosome separation method based on heterogeneous folded microspheres and application

Abstract

The invention discloses an exosome separation method based on heterogeneous folded microspheres and application of the exosome separation method. The exosome separation method comprises the following steps: S1, filling a separation column with hydrophilic-hydrophobic heterogeneous folded microspheres as a stationary phase, and balancing with a first eluent to obtain an exosome separation column; and S2, loading an exosome suspension to be separated and purified to the exosome separation column, eluting with a second eluent, and collecting the eluent, wherein the average particle size of the hydrophilic-hydrophobic heterogeneous wrinkled microspheres is 2 to 1200 [mu] m, and the diameter of wrinkles is 5 to 1000 nm; the second eluent contains 0-40% of methanol, ethanol or acetonitrile. The exosome separation method has the advantages of simplicity in operation, easiness in condition control, low cost, wide application range and the like, and can be used for high-purity and large-scale separation and purification of exosomes from different sources.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome separation, and in particular to an exosome separation method and application based on heterogeneous wrinkled microspheres. Background Art

[0002] Exosomes are extracellular vesicles (EVs) with a diameter of 40-160 nm that are actively secreted by cells. As part of normal physiology, they are widely involved in many physiological and pathological processes. Related studies have shown that the components of exosomes are related to immune response, viral pathogenicity, pregnancy, cardiovascular disease, central nervous system-related diseases and cancer progression, and have great clinical application potential in the early diagnosis of related diseases. In addition, exosomes have the advantages of low immunogenicity, low toxicity, long blood circulation time, and can cross the blood-brain barrier. They are an excellent class of drug carrier materials that can deliver short-chain RNA, antisense oligonucleotides, chemotherapeutic drugs and immunomodulators to target receptors. Therefore, exosomes have very important clinical application research value in both disease diagnosis and treatment research. However, exosome separation still has defects such as low purity and difficulty in large-scale separation, which greatly limits the clinical application of exosomes.

[0003] In the related technologies, many separation and purification technologies have been developed based on the density, size, surface charge and antigen-antibody characteristics of exosomes, such as ultracentrifugation, density gradient centrifugation, membrane filtration, polymer precipitation, microfluidic separation and size exclusion chromatography. Among them, ultracentrifugation and density gradient centrifugation developed based on exosome density, although the technology is becoming more mature and widely used, have problems such as expensive instruments, low purity, time-consuming and labor-intensive operation, and poor repeatability. The EXODUS exosome separation system developed based on membrane filtration is used for the separation and purification of exosomes from urine and saliva, but it is insufficient in processing exosomes with more complex sources. The polymer precipitation method uses size-exclusion polymers to "hijack" water molecules, thereby reducing the solubility of exosomes, and then sedimenting under low-speed centrifugation conditions, but this method has problems such as low purity and polymer contamination. Microfluidic separation is a microscale separation method based on the physical and biochemical properties of exosomes. It has high separation purity, but is only suitable for the separation and purification of a small amount of exosomes. Size exclusion chromatography usually uses materials including dextran, agarose, amide gel, modified silica gel, resin and polypropylene to separate and purify exosomes. Its operation is simple, but it is difficult to separate exosomes with high purity and on a large scale from exosome solutions with complex backgrounds.

[0004] Based on this, there is an urgent need to develop an exosome isolation method that is simple to operate, has a wide range of applications, and has a high recovery rate. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an exosome separation method and application based on heterogeneous wrinkled microspheres, which uses hydrophilic-hydrophobic heterogeneous wrinkled microspheres as a stationary phase to fix the exosomes, and then elutes them with an eluent containing a polar organic solvent (such as methanol, ethanol or acetonitrile) to obtain high-purity exosomes. The exosome separation method of the present invention has the advantages of simple operation, easy control of conditions, wide application range, and low cost, and can be used for high-purity and large-scale separation and purification of exosomes from different sources.

[0006] A first aspect of the present invention provides a method for isolating exosomes, comprising the following steps:

[0007] S1, filling the hydrophilic-hydrophobic heterogeneous wrinkled microspheres into a separation column as a stationary phase, and obtaining an exosome separation column after equilibration with a first eluent;

[0008] S2, loading the exosome suspension to be separated and purified onto the exosome separation column, then eluting with a second eluent, and collecting the eluent to obtain;

[0009] The average particle size of the hydrophilic-hydrophobic heterogeneous wrinkled microspheres is 2 to 1200 μm, and the diameter of the wrinkles is 5 to 1000 nm;

[0010] The second eluent contains 0-40% of an organic solvent, and the organic solvent includes at least one of methanol, ethanol, and acetonitrile.

[0011] The method for separating exosomes according to an embodiment of the present invention has at least the following beneficial effects:

[0012] The exosome separation method based on heterogeneous wrinkled microspheres of the present invention fixes the exosomes by using hydrophilic-hydrophobic heterogeneous wrinkled microspheres as the stationary phase, and then elutes them using an eluent containing a polar organic solvent (such as methanol, ethanol or acetonitrile) to obtain high-purity exosomes. The exosome separation method of the present invention has the advantages of simple operation, easy control of conditions, wide application range, and low cost, and can be used for high-purity and large-scale separation and purification of exosomes from different sources.

[0013] In some embodiments of the present invention, the preparation method of the hydrophilic-hydrophobic heterogeneous wrinkled microspheres comprises: mixing an oil phase solution with an aqueous phase solution, then introducing an inert gas, and reacting at 50 to 100° C. to obtain;

[0014] The oil phase solution is obtained by mixing a hydrophobic monomer, a swelling solvent and an initiator, and the water phase solution is obtained by mixing a water-soluble monomer, a stabilizer and a solvent.

[0015] In some embodiments of the present invention, the hydrophobic monomer includes at least one of styrene, divinylbenzene, vinylimidazole, vinylpyridine, ethyl methacrylate, vinyl chloride, methyl methacrylate, vinylthiazole, cyclohexyl vinyl ether, hexafluorobutyl acrylate, hexafluorobutyl methacrylate, vinyl n-butyl ether, octadecenoic acid, methacrylic anhydride, and glyceryl methacrylate.

[0016] In some preferred embodiments of the present invention, the hydrophobic monomer includes at least one of styrene, glyceryl methacrylate, and divinylbenzene.

[0017] In some embodiments of the present invention, the swelling solvent includes at least one of alkylbenzene, n-hexane, n-heptane, petroleum ether, ethyl acetate, cyclohexane, dichloromethane, chloroform, carbon tetrachloride, imidazole, pyridine, and tetrahydrofuran.

[0018] In some embodiments of the present invention, the alkylbenzene includes at least one of toluene, xylene, ethylbenzene and propylbenzene.

[0019] In some preferred embodiments of the present invention, the swelling solvent includes at least one of toluene, petroleum ether and chloroform.

[0020] In some embodiments of the present invention, the initiator includes at least one of azobisisobutyronitrile, benzoyl peroxide, and dimethyl azobisisobutyrate.

[0021] In some embodiments of the present invention, the mass volume ratio of the hydrophobic monomer, the swelling solvent and the initiator is 3-5 mL: 1-4 mL: 0.05-0.2 g.

[0022] In some embodiments of the present invention, the water-soluble monomer includes at least one of acrylic acid, vinylamide, vinylpyrrolidone, methacrylic acid, methyl methacrylate, pentaerythritol triacrylate, acrylamide, itaconic acid, sodium styrene sulfonate, ethylene urea, N-styryl urea, fumaric acid, maleic acid, and maleic anhydride.

[0023] In some preferred embodiments of the present invention, the water-soluble monomer includes at least one of acrylic acid, vinylamide, and sodium styrene sulfonate.

[0024] In some embodiments of the present invention, the stabilizer includes at least one of sodium octadecyl sulfonate, sodium octadecyl sulfate, hexadecyl trimethyl ammonium bromide, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, polydimethylsiloxane, polyethylene glycol ether, polypropylene ether, polyoxyethylene ethyl ether, polyethylene glycol sulfate, polyoxyethylene monostearate, isobutyl hydroxyphenyl ether, isopentyl dihydroxyphenyl ether, polyethylene glycol distearate, polyacrylamide, and polyvinyl pyrrolidone.

[0025] In some preferred embodiments of the present invention, the stabilizer is selected from at least one of sodium octadecyl sulfonate, sodium octadecyl sulfate, polyvinyl alcohol, polyacrylic acid, polyethylene glycol, and polyvinyl pyrrolidone.

[0026] In some embodiments of the invention, the solvent comprises water.

[0027] In some embodiments of the present invention, the mass volume ratio of the water-soluble monomer, the stabilizer and the solvent is 0.5-2 mL: 0.5-2 g: 75-150 mL.

[0028] In some embodiments of the present invention, the volume ratio of the aqueous phase solution to the oil phase solution is 2 to 100:1.

[0029] In some preferred embodiments of the present invention, the volume ratio of the aqueous phase solution to the oil phase solution is 10 to 50:1.

[0030] In some embodiments of the present invention, the hydrophilic-hydrophobic heterogeneous wrinkled microspheres include at least one of polystyrene-acrylic acid heterogeneous wrinkled microspheres, polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres, polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres, and polystyrene-polyacrylamide heterogeneous wrinkled microspheres.

[0031] In some embodiments of the present invention, the packing method includes wet column packing or dry column packing.

[0032] In some preferred embodiments of the present invention, the packing method is a wet column packing method, which includes dispersing the hydrophilic-hydrophobic heterogeneous wrinkled microspheres in a dispersion liquid and then packing them into the separation column as a stationary phase.

[0033] In some embodiments of the present invention, the dispersion liquid is an organic solvent with a concentration of 5% to 50%, and the organic solvent is selected from at least one of methanol, ethanol, and acetonitrile.

[0034] In some embodiments of the present invention, the separation column is selected from any one of a gravity separation type chromatographic separation column, a pump separation type chromatographic separation column, and a centrifugal separation type chromatographic separation column.

[0035] In some embodiments of the present invention, the first eluent contains 5-40% methanol, ethanol or acetonitrile.

[0036] In some embodiments of the present invention, the first eluent is selected from at least one of phosphate buffer, ammonium formate buffer, acetic acid / ammonium acetate buffer, disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, ammonium bicarbonate buffer, and Tris buffer.

[0037] In some preferred embodiments of the present invention, the first eluent is selected from any one of phosphate buffer, ammonium formate buffer, and acetic acid / ammonium acetate buffer.

[0038] In some embodiments of the present invention, the second eluent further comprises at least one buffer salt selected from the group consisting of phosphate, ammonium formate, acetic acid / ammonium acetate, disodium hydrogen phosphate / sodium dihydrogen phosphate, ammonium bicarbonate, and Tris.

[0039] In some embodiments of the present invention, the pH value of the first eluent and / or the second eluent is 3-12, and the concentration of the buffer salt is 0.2-200 mM.

[0040] In some embodiments of the present invention, the volume of the second eluent is 0.5 to 50 times the sample loading volume.

[0041] In some preferred embodiments of the present invention, the volume of the second eluent is 1 to 2 times the sample loading volume.

[0042] In some embodiments of the present invention, the elution method includes gravity elution, centrifugal elution or pump elution.

[0043] In some embodiments of the present invention, the gravity elution is suitable for purification of exosomes in 10 μL to 5 mL of biological fluid, and the volume of the second eluent is 1 to 50 times the sample volume.

[0044] In some embodiments of the present invention, the centrifugal elution is suitable for purification of exosomes in 5 μL to 25 mL of biological fluid; the volume of the second eluent is 0.5 to 5 times the sample volume.

[0045] In some embodiments of the present invention, the centrifugal elution is suitable for purification of exosomes in 10 μL to 25 mL of biological fluid; the volume of the second eluent is 2 to 5 times the sample volume.

[0046] In some embodiments of the present invention, the separation and purification relies on centrifugal force for elution, and the centrifugation conditions are 100 to 10,000×g and the time is 5 to 1,800 s.

[0047] In some embodiments of the present invention, the pump-in elution is suitable for continuous separation of exosomes in 50 mL to 1000 L of biological fluid. After the loading is completed, the second eluent is pumped in for elution, and the elution is monitored at 280 nm using an ultraviolet detector.

[0048] In some embodiments of the present invention, during the pump-in elution process, the pump-in flow rate of the second eluent is 0.1 to 200 mL / min.

[0049] In some preferred embodiments of the present invention, during the pump-in elution process, the pump-in flow rate of the second eluent is 0.1 to 100 mL / min.

[0050] In some more preferred embodiments of the present invention, during the pump-in elution process, the pump-in flow rate of the second eluent is 5 to 50 mL / min.

[0051] In some embodiments of the present invention, the exosome suspension to be separated and purified includes but is not limited to plant-derived berry fruits (strawberry, blueberry, mulberry, raspberry, kiwi, cactus fruit, grape, etc.), citrus fruits (mandarin orange, tangerine, orange, grapefruit, lemon, orange, etc.), pomelo fruits (apple, pear, hawthorn, loquat, peach, lychee, etc.), melon fruits (watermelon, cantaloupe, cantaloupe, papaya, sweet potato, etc.), root and stem Chinese medicinal materials (ginger, ginseng, Panax notoginseng, Panax notoginseng, Chuanxiong, Atractylodes macrocephala, Angelica sinensis, Polygonatum odoratum, lily, Gastrodia elata, etc.), body fluid sources (urine, blood, saliva, tears, cerebrospinal fluid, milk, etc.), cell sources (endothelial cells, immune cells, platelets, smooth muscle cells, mesenchymal stem cells, human renal epithelial cells, cardiomyocytes, cancer cells, etc.) and the like.

[0052] The second aspect of the present invention provides a recovery method of a separation column based on heterogeneous wrinkled microspheres, comprising separating the exosomes using the separation method of any one of the first aspects, and then adding an organic solution to the exosome separation column for elution.

[0053] In some embodiments of the present invention, the organic solution is selected from a methanol solution, an ethanol solution or an acetonitrile solution.

[0054] In some embodiments of the present invention, the concentration of the organic solution is 10% to 90%.

[0055] In some embodiments of the present invention, the amount of the organic solution added is 1 to 100 times the volume of the separation column.

[0056] Preferably, the amount of the organic solution added is 1 to 50 times the volume of the separation column.

[0057] The third aspect of the present invention provides the use of the exosome separation method as described in any one of the first aspect or the recovery method as described in the second aspect in the purification of exosomes.

[0058] The application according to the embodiment of the present invention has at least the following beneficial effects: the exosome separation method and separation column recovery method of the present invention are used to purify exosomes, which not only helps to improve the purity of exosomes and reduce the impurity content, but also has simple operation and low cost, and can be used for high-purity and large-scale separation and purification of exosomes from different sources.

[0059] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0061] Figure 1 This is a picture of the gravity separation type chromatographic separation column of the present invention;

[0062] Figure 2 Transmission electron microscopy images of kiwifruit-derived exosomes before and after separation and impurities eluted from the column in Example 1 of the present invention, scale: 500 nm;

[0063] Figure 3 Transmission electron microscopy images of kiwifruit-derived exosomes separated under different elution conditions of the present invention, scale: 500 nm;

[0064] Figure 4 Transmission electron microscopy images of human breast cancer cell exosomes before and after separation and impurities eluted from the column in Example 2 of the present invention, scale: 500 nm;

[0065] Figure 5 This is the result of exosome separation based on heterogeneous wrinkled microspheres of the present invention, scale: 500nm;

[0066] Figure 6 Transmission electron microscopy images of exosome separation under different elution conditions in Example 2 of the present invention, scale: 500 nm;

[0067] Figure 7 This is the result of the exosome recovery experiment of the present invention;

[0068] Figure 8 This is a picture of the pump-in separation type chromatographic separation column of the present invention;

[0069] Fig. 9 Transmission electron microscopy images of lemon-derived exosomes before and after separation and impurities eluted from the column in Example 3 of the present invention, scale: 500 nm;

[0070] Fig.10 This is a picture of the centrifugal separation type chromatographic separation column of the present invention;

[0071] Fig.11 Transmission electron microscopy images of mesenchymal stem cell exosomes before and after separation and impurities eluted from the column in Example 4 of the present invention, scale: 500 nm;

[0072] Fig.12 This is a picture of the 75mL pump-feed scale separation column system of the present invention;

[0073] Fig.13Transmission electron microscopy images of human breast cancer cell-derived exosomes before and after separation and impurities eluted from the column in Example 5 of the present invention, scale: 500 nm;

[0074] Fig.14 Transmission electron microscopy images of urine-derived exosomes before and after separation and impurities eluted from the column in Example 6 of the present invention, scale: 500 nm;

[0075] Fig.15 Transmission electron microscopy images of blood-derived exosomes before and after separation and impurities eluted from the column in Example 7 of the present invention, scale: 500 nm;

[0076] Fig.16 This is the exosome separation result based on size exclusion chromatography in Comparative Example 1 of the present invention, scale: 500 nm;

[0077] Fig.17 This is the exosome separation result based on ultracentrifugation method in comparative example 2 of the present invention, scale: 500 nm;

[0078] Fig.18 This is the exosome separation result based on the PEG precipitation method in Comparative Example 3 of the present invention, scale: 500 nm;

[0079] Fig.19 This is the exosome separation result based on the EXODUS method in Comparative Example 4 of the present invention, scale: 500 nm. DETAILED DESCRIPTION

[0080] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0081] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0082] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every 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 combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0083] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0084] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0085] In the description of the present invention, unless otherwise specified, the solvent in the "solution" is water, such as an ethanol solution is a mixture of ethanol and water.

[0086] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0087] Example 1

[0088] This embodiment provides an exosome separation method based on polystyrene-acrylic acid heterogeneous wrinkled microspheres, which uses kiwifruit exosome suspension as the separation raw material and a gravity separation type chromatographic separation column as the column for separation. The specific separation method is as follows.

[0089] (1) Preparation of kiwifruit exosome suspension:

[0090] The kiwifruit was washed and peeled, and the juice was squeezed by a juicer. During the juicing process, PBS precooled to 4°C was added to dilute the juice. The juice was centrifuged at 500g×10min, 2000g×20min, 5000g×30min, and 10000g×60min to remove the pulp residue. The supernatant was filtered through a filter to remove the suspended fruit hairs and seeds to obtain the kiwifruit exosome suspension.

[0091] (2) Exosome isolation from polystyrene-acrylic acid heterogeneous wrinkled microspheres:

[0092] The separation of exosomes based on polystyrene-acrylic acid heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0093] S1. Weigh 0.2 g of polystyrene-acrylic acid heterogeneous wrinkled microspheres (average particle size 100 μm) and disperse them in 70% ethanol solution to obtain a polystyrene-acrylic acid heterogeneous wrinkled microsphere dispersion, wherein the preparation method of the polystyrene-acrylic acid heterogeneous wrinkled microspheres is as follows:

[0094] 1.0g polyvinyl alcohol, 0.01g sodium octadecyl sulfonate and 1.5mL acrylic acid are dissolved in 100mL water to prepare an aqueous phase; 2.5mL styrene, 1.5mL divinylbenzene, 3.5mL chloroform and 0.06g azobisisobutyronitrile are mixed to prepare an oil phase; then the aqueous phase and the oil phase are mixed, stirred at 300rpm for 30min at 30°C to obtain suspended droplets, and then inert gas is introduced, and the temperature is raised to 70°C for polymerization for 12h, the reaction is stopped, and the polystyrene-acrylic acid heterogeneous wrinkled microspheres are obtained after washing with ethanol solution and drying. The average particle size of the polystyrene-acrylic acid heterogeneous wrinkled microspheres prepared by this method is tested to be 100±5μm, and the diameter of the wrinkles is about 25nm.

[0095] S2, by wet column packing method, the polystyrene-acrylic acid heterogeneous wrinkled microsphere dispersion is filled into a 1 mL gravity separation type chromatographic separation column with a sieve plate (such as Figure 1 As shown), deionized water is added to continuously increase the water content in the ethanol solution until it approaches 100%, and then a PBS buffer solution of pH 7.4 is used for elution to balance the separation column to obtain an exosome separation column for standby use;

[0096] S3. Take 50 μL of the above kiwifruit exosome suspension and load it into the above exosome separation column, then use 400 μL of PBS buffer (pH 7.4) containing 20% ​​ethanol for gravity elution, collect the eluate, and obtain the purified kiwifruit exosomes.

[0097] (3) Regeneration of exosome separation column:

[0098] After the exosomes are separated, they can be regenerated by elution with 1 mL of 70% ethanol solution for secondary use.

[0099] The kiwifruit exosomes before and after separation and purification were observed by electron microscopy. Figure 2 As shown, it has excellent separation and purification effect. Further, this example also tested the effect of gravity elution using 400 μL of phosphate buffer containing 75% ethanol. The results are as follows Figure 3As shown, the elution effect is not as good as the former, but it still shows a certain separation and purification effect. In addition, the recovery rate of exosomes recovered by the separation method of this embodiment was statistically analyzed, and the results showed that it reached 93%. It can be seen that the exosome separation method based on polystyrene-acrylic acid heterogeneous wrinkled microspheres of the present invention can efficiently separate, purify and recover exosomes.

[0100] Example 2

[0101] This embodiment provides a method for separating exosomes based on polystyrene-acrylic acid heterogeneous wrinkled microspheres, which uses human breast cancer cell exosomes as separation raw materials and a gravity separation type chromatographic separation column as a column for separation. The specific separation method is as follows.

[0102] (1) Preparation of human breast cancer cell exosome suspension:

[0103] The cell culture medium from which human breast adenocarcinoma cells were removed was centrifuged at 500g×10min, 2000g×20min, 5000g×30min, and 10000g×60min to remove cell debris and organelles, thereby obtaining a human breast adenocarcinoma cell exosome suspension.

[0104] (2) Exosome isolation from polystyrene-acrylic acid heterogeneous wrinkled microspheres:

[0105] The separation of exosomes based on polystyrene-acrylic acid heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0106] S1, weighing 0.2g of polystyrene-acrylic acid heterogeneous wrinkled microspheres (average particle size 100 μm) and dispersing them in 70% ethanol solution to obtain a polystyrene-acrylic acid heterogeneous wrinkled microsphere dispersion, wherein the preparation method of the polystyrene-acrylic acid heterogeneous wrinkled microspheres is referred to Example 1;

[0107] S2. The polystyrene-acrylic acid heterogeneous wrinkled microsphere dispersion is loaded into a 1 mL gravity separation type chromatographic separation column with a sieve plate by a wet column packing method, and deionized water is added to continuously increase the water content in the ethanol solution until it approaches 100%, and then 0.1 M PBS buffer (pH 7.4) is used for elution to balance the separation column to obtain an exosome separation column for standby use;

[0108] S3. Take 50 μL of the above human breast adenocarcinoma cell exosome suspension and load it into the above exosome separation column, then use 400 μL of PBS buffer (pH 7.4) containing 10% ethanol for gravity elution, collect the eluate, and obtain the purified human breast adenocarcinoma cell exosomes.

[0109] (3) Regeneration of exosome separation column:

[0110] After the exosomes are separated, they can be regenerated by elution with 1 mL of 70% ethanol solution for secondary use.

[0111] The exosomes of human breast cancer cells before and after separation and purification were observed and counted by electron microscopy. Figure 4 and Figure 5 As shown, it has excellent separation and purification effect. Further, this example also tested the effect of gravity elution using 400 μL PBS buffer (pH 7.4) containing 45% ethanol. The results are as follows Figure 6 As shown, the elution effect is not as good as the former, but it still shows a certain separation and purification effect. In addition, the recovery rate of exosomes recovered by the separation method of this embodiment was statistically analyzed, and the results are as follows Figure 7 As shown, it reaches 96%, which shows that the exosome separation method based on polystyrene-acrylic acid heterogeneous wrinkled microspheres of the present invention can efficiently separate, purify and recover exosomes.

[0112] Example 3

[0113] This embodiment provides an exosome separation method based on polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres, which uses lemon exosome suspension as the separation raw material and a pump-type separation chromatographic separation column as the column for separation. The specific separation method is as follows.

[0114] (1) Preparation of lemon exosome suspension:

[0115] The lemon was washed and peeled, and the juice was squeezed by a juicer. During the juicing process, PBS precooled to 4°C was added to dilute the juice. The juice was centrifuged at 500g×10min, 2000g×20min, 5000g×30min, and 10000g×60min to remove the pulp residue. The supernatant was filtered through a filter to remove the suspended fiber to obtain the lemon exosome suspension.

[0116] (2) Exosome isolation from polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres:

[0117] The separation of exosomes based on polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0118] S1. Weigh 2.0 g of polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres (average particle size 200 μm) and disperse them in a 60% methanol aqueous dispersion to obtain a polymethacrylate-polyacrylamide heterogeneous wrinkled microsphere dispersion. The preparation method of polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres is as follows:

[0119] 1.0g polyvinyl pyrrolidone, 0.05g sodium octadecyl sulfate and 0.75mL vinylamide are dissolved in 80mL water to prepare an aqueous phase; 2mL methacrylate glycerol, 2mL divinylbenzene, 0.5mL petroleum ether and 0.08g azobisisobutyronitrile are mixed to prepare an oil phase; then the aqueous phase and the oil phase are mixed, stirred at 200rpm for 10min at 40°C to obtain suspended droplets, and then inert gas is introduced, and the temperature is raised to 80°C for polymerization for 12h, the reaction is stopped, and the polymethacrylate glycerol-polyacrylamide heterogeneous wrinkled microspheres are obtained after washing with ethanol solution and drying. The polymethacrylate glycerol-polyacrylamide heterogeneous wrinkled microspheres prepared by the method are tested to have an average particle size of 200±15μm, and the diameter of the wrinkles is about 50nm.

[0120] S2, by wet column packing method, fill the above polymethacrylate-polyacrylamide heterogeneous wrinkled microsphere dispersion into 12mL and pump it into the separation type chromatographic separation column (such as Figure 8 As shown), deionized water is added to continuously increase the water content in the ethanol solution until it approaches 100%, and then 0.1 M ammonium formate buffer with a pH of 5.4 is used for elution to balance the separation column to obtain an exosome separation column for standby use;

[0121] S3. Take 500 μL of the above lemon exosome suspension and load it into the above exosome separation column, then elute with 800 μL of 0.1 M ammonium formate buffer (pH 5.4) containing 35% ethanol, collect the eluate, and obtain the purified lemon exosomes.

[0122] (3) Regeneration of exosome separation column:

[0123] After the exosomes are separated, they can be eluted and regenerated with 10 mL of 60% ethanol solution for secondary use.

[0124] The lemon exosomes before and after separation and purification were observed by electron microscopy. Fig. 9 As shown, it has excellent separation and purification effect.

[0125] Example 4

[0126] This embodiment provides an exosome separation method based on polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres, which uses mesenchymal stem cell exosomes as separation raw materials and a centrifugal separation type chromatographic separation column as a column for separation. The specific separation method is as follows.

[0127] (1) Preparation of mesenchymal stem cell exosome suspension:

[0128] The cell culture medium from which mesenchymal stem cells were removed was centrifuged at 500g×10min, 2000g×20min, 5000g×30min, and 10000g×60min to remove cell debris and organelles, and then a human mesenchymal stem cell exosome suspension was obtained.

[0129] (2) Exosome isolation from polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres:

[0130] The separation of exosomes based on polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0131] S1. Weigh 0.05 g of polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres (average particle size 5 μm) and disperse them in 50% ethanol solution to obtain a dispersion of polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres. The preparation method of polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres is as follows:

[0132] 0.80g polyethylene glycol, 0.015g sodium octadecyl sulfate and 1.5g sodium styrene sulfonate are dissolved in 120mL water to prepare an aqueous phase; 4mL divinylbenzene, 0.5mL toluene and 0.12g azobisisobutyronitrile are mixed to prepare an oil phase; then the aqueous phase and the oil phase are mixed, stirred at 600rpm for 60min at 40°C to obtain suspended droplets, and then inert gas is introduced, and the temperature is raised to 80°C for polymerization for 12h, the reaction is stopped, and the polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres are obtained after washing with ethanol solution and drying. The average particle size of the polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres prepared by the method is tested to be 5±0.5μm, and the diameter of the wrinkles is about 10nm.

[0133] S2, by wet column packing method, the above polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microsphere dispersion is filled into a 0.8 mL centrifugal separation type chromatographic separation column with a sieve plate (such as Fig.10 As shown), deionized water was added to continuously increase the water content in the ethanol solution until it was close to 100%, and then 0.1 M ammonium formate buffer (pH 6.4) was used for elution to balance the separation column to obtain an exosome separation column for standby use;

[0134] S3. Take 20 μL of the above-mentioned mesenchymal stem cell exosome suspension and load it into the above-mentioned exosome separation column, then add 40 μL of ammonium formate buffer (pH=6.4) containing 10% ethanol, centrifuge at 300 g for 30 s to elute, collect the eluate, and obtain the purified mesenchymal stem cell exosomes.

[0135] (3) Regeneration of exosome separation column:

[0136] After the exosomes are separated, they can be regenerated by centrifugation and elution with 5 mL of 70% ethanol solution for secondary use.

[0137] Electron microscopic observation of the mesenchymal stem cell exosomes before and after separation and purification was performed. Fig.11 As shown, it has excellent separation and purification effect.

[0138] Example 5

[0139] This embodiment provides an exosome separation method based on polystyrene-polyacrylamide heterogeneous wrinkled microspheres, which uses human breast cancer cell exosomes as separation raw materials and a pump-type separation chromatographic separation column as a column for separation. The specific separation method is as follows.

[0140] (1) Preparation of human breast cancer cell exosome suspension:

[0141] The preparation method of the human breast cancer cell exosome suspension in this example is described in reference to Example 2.

[0142] (2) Exosome isolation from polystyrene-polyacrylamide heterogeneous wrinkled microspheres:

[0143] The separation of exosomes based on polystyrene-polyacrylamide heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0144] S1. Weigh 50 g of polystyrene-polyacrylamide heterogeneous wrinkled microspheres (average particle size 100 μm) and disperse them in a 40% acetonitrile aqueous dispersion to obtain a polystyrene-polyacrylamide heterogeneous wrinkled microsphere dispersion. The preparation method of the polystyrene-polyacrylamide heterogeneous wrinkled microspheres is as follows:

[0145] 0.05 g of sodium octadecyl sulfate and 0.75 g of acrylamide were dissolved in 80 mL of water to prepare an aqueous phase; 2 mL of styrene, 2 mL of divinylbenzene, 0.5 mL of petroleum ether and 0.08 g of azobisisobutyronitrile were mixed to prepare an oil phase; the aqueous phase and the oil phase were then mixed, stirred at 300 rpm for 10 min at 40 ° C to obtain suspended droplets, and then an inert gas was introduced, and the temperature was raised to 70 ° C for polymerization for 12 h, the reaction was stopped, and the polystyrene-polyacrylamide heterogeneous wrinkled microspheres were obtained after washing with ethanol solution and drying. The average particle size of the polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres prepared by the method was tested to be 100±10 μm, and the diameter of the wrinkles was about 35 nm;

[0146] S2, by wet column packing method, the above polystyrene-polyacrylamide heterogeneous wrinkled microsphere dispersion is filled into a 75 mL pump-in separation type chromatographic separation column with a sieve plate (the simplified device diagram is as shown in FIG. Fig.12As shown), deionized water is added to continuously increase the water content in the dispersion until it approaches 100%, and then 0.1 M ammonium formate buffer at pH 6.4 is used for elution to balance the separation column to obtain an exosome separation column for standby use;

[0147] S3. Take 5000 μL of the above-mentioned human breast adenocarcinoma cell exosome suspension and continuously load it into the above-mentioned exosome separation column at a flow rate of 5 mL / min using a direct injection pump, and then use PBS buffer (pH 7.4) containing 10% ethanol to pump in for elution, collect the eluate at a volume of 1 mL, and then use an ultraviolet detector to measure the ultraviolet absorption of the eluate at 280 nm, collect the eluate with ultraviolet absorption, and obtain the purified human breast adenocarcinoma cell exosomes.

[0148] (3) Regeneration of exosome separation column:

[0149] After the exosomes are separated, they can be eluted and regenerated with 200 mL of 70% ethanol solution for secondary use.

[0150] The exosomes of human breast cancer cells before and after separation and purification were observed by electron microscopy. Fig.13 As shown, it has excellent separation and purification effect.

[0151] Example 6

[0152] This embodiment provides an exosome separation method based on polystyrene-polyacrylamide heterogeneous wrinkled microspheres. The difference between this embodiment and embodiment 5 is that the source of exosomes is different. This embodiment uses urine-derived exosome suspension as the separation raw material. The specific separation method is as follows.

[0153] (1) Preparation of urine-derived exosome suspension:

[0154] The urine sample was centrifuged at 10000 g for 30 min to remove impurities and obtain a urine exosome suspension.

[0155] (2) Exosome isolation from polystyrene-polyacrylamide heterogeneous wrinkled microspheres:

[0156] The separation of exosomes based on polystyrene-polyacrylamide heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0157] S1. Weigh 0.4 g of polystyrene-polyacrylamide heterogeneous wrinkled microspheres (average particle size 100 μm) and disperse them in a 40% acetonitrile aqueous dispersion to obtain a polystyrene-polyacrylamide heterogeneous wrinkled microsphere dispersion. The preparation method of the polystyrene-polyacrylamide heterogeneous wrinkled microspheres is as follows:

[0158] 0.05 g of sodium octadecyl sulfate and 0.75 g of acrylamide were dissolved in 80 mL of water to prepare an aqueous phase; 2 mL of styrene, 2 mL of divinylbenzene, 0.5 mL of petroleum ether and 0.08 g of azobisisobutyronitrile were mixed to prepare an oil phase; the aqueous phase and the oil phase were then mixed, stirred at 300 rpm for 10 min at 40 ° C to obtain suspended droplets, and then an inert gas was introduced, and the temperature was raised to 70 ° C for polymerization for 12 h, the reaction was stopped, and the polystyrene-polyacrylamide heterogeneous wrinkled microspheres were obtained after washing with ethanol solution and drying. The average particle size of the polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres prepared by this method was tested to be 100±10 μm, and the diameter of the wrinkles was about 35 nm.

[0159] S2. The polystyrene-acrylic acid heterogeneous wrinkled microsphere dispersion is loaded into a 1 mL gravity separation type chromatographic separation column with a sieve plate by a wet column packing method, and deionized water is added to increase the water content in the ethanol solution until it is close to 100%, and then PBS with pH 7.4 is used for elution to balance the separation column. The exosome separation column is obtained and set aside;

[0160] S3. Take 100 μL of the above urine-derived exosome suspension and load it into the above exosome separation column, then elute it with 1200 μL of PBS (pH 5.4) containing 20% ​​methanol, collect the eluate, and obtain the purified urine exosomes.

[0161] (3) Regeneration of exosome separation column:

[0162] After the exosomes are separated, they can be regenerated by elution with 2 mL of 70% ethanol solution for secondary use.

[0163] Electron microscopic observation of urine-derived exosomes before and after separation and purification was performed. Fig.14 As shown, it has excellent separation and purification effect.

[0164] Example 7

[0165] This embodiment provides an exosome separation method based on polystyrene-polyacrylamide heterogeneous wrinkled microspheres. The difference between this embodiment and embodiment 5 is that the source of exosomes is different. This embodiment uses blood-derived exosome suspension to separate the raw material. The specific separation method is as follows.

[0166] (1) Preparation of blood-derived exosome suspension:

[0167] The blood samples were centrifuged at 500g×10min, 2000g×20min, and 5000g×30min to remove cells, debris, and organelles to obtain a blood-derived exosome suspension.

[0168] (2) Exosome isolation from polystyrene-polyacrylamide heterogeneous wrinkled microspheres:

[0169] The separation of exosomes based on polystyrene-polyacrylamide heterogeneous wrinkled microspheres in this embodiment specifically includes the following steps:

[0170] S1. Weigh 2 g of polystyrene-polyacrylamide heterogeneous wrinkled microspheres (average particle size 100 μm) and disperse them in a 40% acetonitrile aqueous dispersion to obtain a polystyrene-polyacrylamide heterogeneous wrinkled microsphere dispersion. The preparation method of the polystyrene-polyacrylamide heterogeneous wrinkled microspheres refers to Example 5.

[0171] S2. The polystyrene-acrylic acid heterogeneous wrinkled microsphere dispersion is loaded into a 1 mL gravity separation type chromatographic separation column with a sieve plate by a wet column packing method, and deionized water is added to increase the water content in the ethanol solution until it is close to 100%, and then PBS with pH 7.4 is used for elution to balance the separation column. The exosome separation column is obtained and set aside;

[0172] S3. Take 20 μL of the above blood-derived exosome suspension and load it into the above exosome separation column, then elute it with 200 μL of PBS (pH 5.4) containing 20% ​​methanol, collect the eluate, and obtain the purified blood exosomes.

[0173] (3) Regeneration of exosome separation column:

[0174] After the exosomes are separated, they can be eluted and regenerated with 40 mL of 50% ethanol solution for secondary use.

[0175] Electron microscopic observation of the blood-derived exosomes before and after separation and purification was performed. Fig.15 As shown, it has excellent separation and purification effect.

[0176] Comparative Example 1: Size Exclusion Chromatography

[0177] In this comparative example, conventional size exclusion chromatography was used to separate and purify exosomes from a suspension of exosomes derived from human breast cancer cells. The preparation method of the suspension of exosomes derived from human breast cancer cells was referred to Example 2, and the specific separation steps were as follows:

[0178] First, the qEV column (purchased from IZON) was washed with 5 mL of phosphate buffered saline (PBS) according to the product instructions. After 500 μL of human breast cancer cell-derived exosome suspension was loaded onto the qEV column, it was washed with 10 mL of PBS, and the previous 3 mL of eluate was discarded. Then, the subsequent 1.5 mL of eluate containing exosomes was collected to obtain purified human breast cancer cell-derived exosomes.

[0179] The isolated and purified human breast cancer cell exosomes were observed by electron microscopy. Fig.16 As shown, it was shown that the exosome environment contained impurities and the separation and purification effect was poor.

[0180] Comparative Example 2: Ultracentrifugation

[0181] In this comparative example, ultracentrifugation was used to separate and purify exosomes from a suspension of exosomes derived from human breast cancer cells. The preparation method of the suspension of exosomes derived from human breast cancer cells was referred to Example 2, and the specific separation steps were as follows:

[0182] 500 μL of human breast cancer cell-derived exosome suspension was taken into a centrifuge tube, and then centrifuged at 120,000×g for 1 hour, the supernatant was removed, and the precipitate was collected and resuspended in an appropriate amount of PBS.

[0183] The isolated and purified human breast cancer cell exosomes were observed by electron microscopy. Fig.17 As shown, the purified exosome environment contains more impurities, and obvious impurity peaks can be detected, indicating that it is difficult to collect high-purity exosomes using ultracentrifugation.

[0184] Comparative Example 3: PEG precipitation method

[0185] In this comparative example, the PEG precipitation method was used to separate and purify the exosomes in the exosome suspension derived from human breast cancer cells. The preparation method of the exosome suspension derived from human breast cancer cells was referred to Example 2, and the specific separation steps were as follows:

[0186] 0.2 mL of PEG purification solution was added to 1 mL of human breast adenocarcinoma cell-derived exosome suspension, mixed well, incubated on a shaker at 4 °C for 14 h, and then centrifuged at 1500 × g for 30 min at 4 °C to remove the supernatant, and then centrifuged at 1500 × g for 5 min to remove the residual solution, and 50 μL of PBS was added to resuspend the exosome pellet.

[0187] The isolated and purified human breast cancer cell exosomes were observed by electron microscopy. Fig.18 As shown, the content of impurities in the purified exosome environment is improved compared with the ultracentrifugation method, but there are still relatively high impurity peaks.

[0188] Comparative Example 4: EXODUS Method

[0189] In this comparative example, the EXODUS method is used to separate and purify the exosomes in the exosome suspension derived from human breast adenocarcinoma cells, wherein the preparation method of the exosome suspension derived from human breast adenocarcinoma cells is referred to Example 2, and the specific purification process is entrusted to Shenzhen Huixin Biotechnology Co., Ltd. to use the EXODUS exosome separation system for purification, wherein the volume of the exosome suspension is 250mL, and the membrane pore size of the system is about 25nm. The exosome suspension is placed in a separation membrane, and impurities are removed and concentrated under ultrasound to obtain.

[0190] The isolated and purified human breast cancer cell exosomes were observed under electron microscopy. Fig.19 As shown, a large number of impurities can still be observed in the purified exosome environment, indicating that it is difficult to obtain high-purity exosomes using conventional exosome separation methods.

[0191] In summary, the present invention provides an exosome separation method based on heterogeneous wrinkled microspheres, which uses hydrophilic-hydrophobic heterogeneous wrinkled microspheres as a stationary phase to separate and purify exosomes. The method has the advantages of simple operation, easy control of conditions, wide application range, low cost, etc., and can be widely used in the high-purity and large-scale separation and purification of exosomes from different sources.

[0192] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for isolating exosomes, characterized in that: The following steps are involved: S1, filling the hydrophilic-hydrophobic heterogeneous wrinkled microspheres into a separation column as a stationary phase, and obtaining an exosome separation column after equilibration with a first eluent; S2, loading the exosome suspension to be separated and purified onto the exosome separation column, then eluting with a second eluent, and collecting the eluent to obtain; The average particle size of the hydrophilic-hydrophobic heterogeneous wrinkled microspheres is 2 to 1200 μm, and the diameter of the wrinkles is 5 to 1000 nm; The second eluent contains 0-40% of an organic solvent, and the organic solvent includes at least one of methanol, ethanol, and acetonitrile.

2. The separation method according to claim 1, characterized in that The preparation method of the hydrophilic-hydrophobic heterogeneous wrinkled microspheres comprises: mixing an oil phase solution with an aqueous phase solution, then introducing an inert gas, and reacting at 50-100° C. to obtain; The oil phase solution is obtained by mixing a hydrophobic monomer, a swelling solvent and an initiator, and the water phase solution is obtained by mixing a water-soluble monomer, a stabilizer and a solvent; Preferably, the hydrophobic monomer includes at least one of styrene, divinylbenzene, vinylimidazole, vinylpyridine, ethyl methacrylate, vinyl chloride, methyl methacrylate, vinylthiazole, cyclohexyl vinyl ether, hexafluorobutyl acrylate, hexafluorobutyl methacrylate, vinyl n-butyl ether, octadecenoic acid, methacrylic anhydride, and glyceryl methacrylate; and / or, the water-soluble monomer comprises at least one of acrylic acid, vinylamide, vinylpyrrolidone, methacrylic acid, methyl methacrylate, pentaerythritol triacrylate, acrylamide, itaconic acid, sodium styrene sulfonate, ethylene urea, N-styryl urea, fumaric acid, maleic acid, and maleic anhydride; Preferably, the volume ratio of the aqueous phase solution to the oil phase solution is 2 to 100:

1.

3. The separation method according to claim 1, characterized in that The hydrophilic-hydrophobic heterogeneous wrinkled microspheres include at least one of polystyrene-acrylic acid heterogeneous wrinkled microspheres, polymethacrylate-polyacrylamide heterogeneous wrinkled microspheres, polydivinylbenzene-sodium polystyrene sulfonate heterogeneous wrinkled microspheres, and polystyrene-polyacrylamide heterogeneous wrinkled microspheres.

4. The separation method according to claim 1, characterized in that The packing method includes wet column packing or dry column packing; Preferably, the packing method is a wet column packing method, which comprises dispersing the hydrophilic-hydrophobic heterogeneous wrinkled microspheres in a dispersion liquid, and then packing them into the separation column as a stationary phase; Preferably, the dispersion liquid is an organic solvent with a concentration of 5% to 50%, and the organic solvent is selected from at least one of methanol, ethanol and acetonitrile.

5. The separation method according to claim 4, characterized in that The separation column is selected from any one of a gravity separation type chromatographic separation column, a pump separation type chromatographic separation column, and a centrifugal separation type chromatographic separation column.

6. The separation method according to any one of claims 1 to 5, characterized in that: The first eluent is selected from at least one of phosphate buffer, ammonium formate buffer, acetic acid / ammonium acetate buffer, disodium hydrogen phosphate / sodium dihydrogen phosphate buffer, ammonium bicarbonate buffer, and Tris buffer.

7. The separation method according to claim 6, characterized in that The second eluent further comprises at least one buffer salt selected from the group consisting of phosphate, ammonium formate, acetic acid / ammonium acetate, disodium hydrogen phosphate / sodium dihydrogen phosphate, ammonium bicarbonate, and Tris; Preferably, the volume of the second eluent is 0.5 to 50 times the sample loading volume.

8. The separation method according to claim 6, characterized in that The elution method includes gravity elution, centrifugal elution or pump elution.

9. A recovery method for a separation column based on heterogeneous wrinkled microspheres, characterized in that: The method comprises separating the exosomes by the method for separating the exosomes according to any one of claims 1 to 8, and then adding an organic solution to the exosome separation column for elution; Preferably, the organic solution is selected from a methanol solution, an ethanol solution or an acetonitrile solution; Preferably, the concentration of the organic solution is 10% to 90%.

10. Use of the method for isolating exosomes according to any one of claims 1 to 8 or the method for recovering exosomes according to claim 9 in purifying exosomes.

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