PH-mediated plasma extracellular vesicle and lipoprotein separation method and application thereof

Abstract

The invention provides a method for separating pH-mediated plasma extracellular vesicles and lipoproteins and application of the pH-mediated plasma extracellular vesicles and lipoproteins, and belongs to the technical field of biology. Ciba blue functionalized magnetic beads are mixed with plasma in an alkaline pH environment, and the Ciba blue functionalized magnetic beads can selectively adsorb lipoprotein in the plasma and do not adsorb extracellular vesicles in the plasma. Extracellular vesicles without lipoprotein pollution can be obtained from the plasma treated by the Ciba blue functionalized magnetic beads through a titanium dioxide magnetic bead method. According to the separation method, the extracellular vesicles and the lipoproteins in the plasma can be accurately separated, and the separated lipoproteins and the separated extracellular vesicles are high in purity and basically free of cross contamination. Through small RNA sequencing analysis, it is found that the separated lipoprotein and extracellular vesicles respectively carry biological information expressing the physiological and pathological characteristics of the lipoprotein and the extracellular vesicles.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a method for separating plasma extracellular vesicles and lipoproteins mediated by pH and its application. Background Art

[0002] Extracellular vesicles (EVs) are a general term for membrane-bound vesicles released by cells. Among them, small extracellular vesicles (sEVs) with a diameter range less than 200 nm, especially exosomes (30 - 150 nm), have received a great deal of research attention in recent years. Lipoproteins (LPPs) are a class of single phospholipid layer spherical particles composed of a core of sterol esters and triglycerides and an outer shell of apolipoproteins, phospholipids, cholesterol, etc. The density and size of EVs and lipoproteins highly overlap, and their physicochemical properties are extremely similar. Moreover, both can be used as potential biomarkers for tumor diagnosis. Since there are 20 - 100 times more lipoproteins than EVs in the blood circulation and there is a lack of effective separation methods, a large amount of lipoproteins are often mixed into the plasma EVs obtained by researchers, which greatly affects the sensitivity and accuracy of EV detection methods. At the same time, the biological significance of lipoproteins in disease research is also ignored.

[0003] At present, there are many studies on purifying plasma heteroproteins using Cibacron Blue (CB). However, for the application research in the field of extracellular vesicles or exosomes, directly using CB or its microspheres to purify plasma will lead to the loss of extracellular vesicles or exosomes. Because in the plasma medium, CB will capture plasma exosomes or extracellular vesicles together in a neutral or physiological pH environment, resulting in the inaccurate separation of lipoproteins and vesicles in the plasma. Therefore, in order to accurately analyze the research of lipoproteins and extracellular vesicles in plasma in disease mechanisms and diagnosis, researchers need to develop a highly efficient, accurate and convenient method for separating plasma extracellular vesicles and lipoproteins. At present, there is no relevant report on the method for accurately separating plasma extracellular vesicles and lipoproteins from plasma using CB. Summary of the Invention

[0004] The present invention provides a method for separating plasma extracellular vesicles and lipoproteins mediated by pH and its application. In an alkaline pH environment, CB-functionalized magnetic beads (CBMB) will selectively remove lipoproteins in plasma without showing an adsorption effect on extracellular vesicles in plasma, and can accurately separate lipoproteins and extracellular vesicles in plasma, reducing losses.

[0005] To solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a method for separating plasma extracellular vesicles and lipoproteins mediated by pH. The plasma is diluted with a buffer solution having a pH of 8 to 12, and the diluted plasma is mixed with Cibacron Blue-functionalized magnetic beads. The Cibacron Blue-functionalized magnetic beads and the supernatant are separated, and the lipoproteins are separated from the Cibacron Blue-functionalized magnetic beads. The supernatant is mixed with titanium dioxide magnetic beads, the titanium dioxide magnetic beads are separated, and the extracellular vesicles are separated from the titanium dioxide magnetic beads.

[0007] Preferably, the buffer solution is one of MES buffer solution, PBS buffer solution, and carbonate buffer solution.

[0008] Preferably, the Cibacron Blue-functionalized magnetic beads and the titanium dioxide magnetic beads need to be washed with water 2 to 4 times respectively before use.

[0009] Preferably, the magnetic beads used in the preparation of the Cibacron Blue-functionalized magnetic beads include one or more of amino magnetic beads, carboxyl magnetic beads, silanol magnetic beads, and epoxy magnetic beads.

[0010] Preferably, the temperature for mixing the diluted plasma with the Cibacron Blue-functionalized magnetic beads is 2 to 6 °C, and the mixing time is 1 to 2 h.

[0011] Preferably, the supernatant plasma sample after treatment with the Cibacron Blue-functionalized magnetic beads needs to adjust the pH of the system to neutral with HCl solution and then capture it with titanium dioxide magnetic beads.

[0012] Preferably, the separated titanium dioxide magnetic beads are mixed with ammonia water to release the extracellular vesicles, and then through ultrafiltration, the ammonia water in the filtrate is replaced with PBS to obtain an extracellular vesicle PBS suspension.

[0013] Preferably, the temperature for mixing the titanium dioxide magnetic beads with ammonia water is 20 to 30 °C, and the mixing time is 10 to 15 min.

[0014] Preferably, the concentration of the ammonia water is 5 to 15%.

[0015] The present invention provides the application of the separation method in separating lipoproteins and extracellular vesicles in plasma.

[0016] The present invention provides the application of the separation method in separating high-density lipoproteins and extracellular vesicles in plasma.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention provides a method for separating plasma extracellular vesicles and lipoproteins mediated by pH. Under acidic and neutral conditions, CB-modified magnetic beads can simultaneously adsorb extracellular vesicles and lipoproteins in plasma. However, in an alkaline pH environment, CB-modified magnetic beads selectively adsorb lipoproteins in plasma and do not show adsorption to extracellular vesicles in plasma. Thus, the plasma treated with CB-modified magnetic beads contains extracellular vesicles but no lipoproteins, and extracellular vesicles free of lipoprotein contamination can be isolated by the titanium dioxide magnetic bead method. Compared with the scheme of directly purifying plasma with CB, the method of the present invention can reduce the loss of extracellular vesicles and obtain high-purity lipoproteins and extracellular vesicles.

[0019] Through small RNA sequencing analysis, the present invention found that the isolated lipoproteins and extracellular vesicles carry biological information expressing their respective physiological and pathological characteristics, which can be used for downstream precise analysis of the research on lipoproteins and extracellular vesicles in plasma in disease mechanisms and diagnosis. Description of the Drawings

[0020] Figure 1 Schematic diagram of the separation of plasma lipoproteins and small extracellular vesicles mediated by pH.

[0021] Figure 2 Characterization of CBMB. Among them, (A) Scanning electron microscope image and appearance of magnetic beads with Si-OH groups on the surface (upper left), and the corresponding elemental analysis is shown in the upper right table; (B) Scanning electron microscope image and appearance of CBMB prepared from Si-OH magnetic beads (lower left), and the corresponding elemental analysis is shown in the lower right table; the scale bar is 5 μm.

[0022] Figure 3 Immunoblot protein characterization of standards.

[0023] Figure 4 Adsorption test results of CBMB on EV standards. Among them, (A) Results of the change in size distribution of EV standards before and after treatment with CBMB under different pH environments; (B) Results of the change in the number of particles of EV standards before and after treatment with CBMB under different pH environments; (C) Results of the change in the average size of EV standards before and after treatment with CBMB under different pH environments.

[0024] Figure 5Analysis results of the size, quantity of isolated plasma EV particles, and proteins on CBMB and titanium dioxide magnetic beads. Among them, (A) Schematic diagram of the process of pre-treating plasma with CBMB and then isolating EV from the treated plasma using titanium dioxide magnetic beads; (B) Size distribution curve of EV particles isolated under different pH environments; (C) Average particle size of EV isolated under different pH environments; (D) Number of EV particles isolated under different pH environments; (E) Quantitative results of proteins captured on CBMB and titanium dioxide magnetic beads under different pH environments; (F) Analysis results of characteristic proteins by immunoblotting of proteins on CBMB and titanium dioxide magnetic beads under different pH environments.

[0025] Figure 6 Results of the effect of CBMB dosage on the purity of EV isolated by titanium dioxide magnetic beads.

[0026] Figure 7 Electron microscopy analysis of EV isolated by titanium dioxide magnetic beads to observe the effect of CBMB-treated plasma on the purity of EV isolated by titanium dioxide magnetic beads. Among them, (A) Scanning electron microscopy analysis. From left to right are the electron micrographs of titanium dioxide magnetic beads, titanium dioxide magnetic beads directly isolating plasma EV, and titanium dioxide magnetic beads isolating EV from CBMB-treated plasma. The arrow indicates the EV adsorbed on titanium dioxide, scale bar: 1μm; (B) Immunotransmission electron microscopy analysis. From left to right are the plasma EV directly isolated by titanium dioxide magnetic beads and the EV isolated by titanium dioxide magnetic beads from CBMB-treated plasma. The arrow indicates the lipoprotein co-isolated with EV. The immunogold antibody is anti-APOE, scale bar: 200nm.

[0027] Figure 8 Comparative analysis results of small RNAs carried by EV and lipoproteins. Among them, (A) Venn diagram showing the number of small RNAs carried by lipoproteins and EV; (B) Analysis of the biotypes of small RNAs carried by EV and lipoproteins; (C) Comparison of the biological process pathways enriched by EV and lipoproteins, showing the entries with p values less than 0.05.

[0028] Figure 9Detection and analysis results of products during the separation of extracellular vesicles and high-density lipoproteins in plasma. Among them, (A) Size distribution curve of EV particles isolated under different pH environments; (B) Number of EV particles isolated under different pH environments; (C) Size of EV particles isolated under different pH environments; (D) Transmission electron micrograph of the plasma EV and high-density lipoprotein mixture obtained by UC (left) and the transmission electron micrograph of the plasma EV and high-density lipoprotein mixture after CBMB sorting (pH 9.0) obtained by UC (right), arrows indicate high-density lipoproteins, scale bar: 200 nm; (E) Immunoblot analysis of proteins in the plasma EV and high-density lipoprotein mixture obtained by UC, high-density lipoproteins adsorbed by CBMB, and the remaining EV samples after CBMB sorting. Detailed implementation mode

[0029] The present invention provides a method for separating extracellular vesicles and lipoproteins in plasma mediated by pH. The plasma is diluted with a buffer solution with a pH of 8 to 12, filtered through a 0.22 μm filter (millipore), and then the diluted plasma is mixed with Cibacron Blue-functionalized magnetic beads. The Cibacron Blue-functionalized magnetic beads and the supernatant are separated, and lipoproteins are separated from the Cibacron Blue-functionalized magnetic beads; the supernatant is mixed with titanium dioxide magnetic beads, the titanium dioxide magnetic beads are separated, and extracellular vesicles are separated from the titanium dioxide magnetic beads. In the present invention, in acidic and neutral environments, Cibacron Blue-functionalized magnetic beads (CBMB) tend to simultaneously capture extracellular vesicles and lipoproteins in plasma; while in alkaline environments, CBMB only selectively adsorbs lipoproteins. The extracellular vesicles obtained by the separation method of the present invention are mainly small extracellular vesicles (sEV) with a diameter range less than 200 nm. The separation method of the present invention can selectively remove lipoproteins by regulating the pH of the buffer solution without affecting the separation of downstream extracellular vesicles, and the experimental principle is as Figure 1 shown.

[0030] In the present invention, the buffer solution is one of MES buffer solution, PBS buffer solution, and carbonate buffer solution, preferably carbonate buffer solution. The pH of the carbonate buffer solution is preferably 8 to 12. Setting the pH to alkaline in the present invention is beneficial for Cibacron Blue-functionalized magnetic beads to adsorb lipoproteins in plasma without adsorbing extracellular vesicles in plasma, and can accurately separate lipoproteins and extracellular vesicles in plasma.

[0031] In the present invention, the Cibacron Blue-functionalized magnetic beads and the titanium dioxide magnetic beads need to be washed with water 2 to 4 times respectively before use. After washing the Cibacron Blue-functionalized magnetic beads and the titanium dioxide magnetic beads in the present invention, the water is removed by magnetic separation, and the Cibacron Blue-functionalized magnetic beads and the titanium dioxide magnetic beads are collected respectively for standby.

[0032] In the present invention, the magnetic beads used in the preparation of Cibacron Blue-functionalized magnetic beads include one or more of amino magnetic beads, carboxyl magnetic beads, silanol magnetic beads, and epoxy magnetic beads, preferably silanol magnetic beads. In the present invention, the Cibacron Blue is immobilized on the silanol magnetic beads by reacting the chlorine (Cl) group on the Cibacron Blue molecule with the silanol (Si-OH) group on the silanol magnetic beads to obtain Cibacron Blue-functionalized magnetic beads (CBMB).

[0033] In the present invention, the diluted plasma is mixed with the Cibacron Blue-functionalized magnetic beads by rotation at 4 °C for 1 to 2 h, and then the supernatant is transferred to a clean centrifuge tube to separate the Cibacron Blue-functionalized magnetic beads. At this time, the Cibacron Blue-functionalized magnetic beads adsorb the lipoproteins in the plasma. In the present invention, the supernatant (the plasma treated with the Cibacron Blue-functionalized magnetic beads) is mixed with the washed titanium dioxide magnetic beads by rotation at 4 °C for 1 to 2 h, and the supernatant is removed by magnetic separation. Ammonia water is added to the titanium dioxide magnetic beads adsorbed with extracellular vesicles (the separated titanium dioxide magnetic beads), and the mixture is rotated at room temperature for 10 to 15 min. At this time, the extracellular vesicles are released into the ammonia water, and ultrafiltration is performed 3 to 5 times with a 3 kD ultrafiltration tube, and the ammonia water is replaced with PBS, and the solution is recovered to obtain a PBS solution containing extracellular vesicles. In the present invention, the supernatant plasma treated with the Cibacron Blue-functionalized magnetic beads is adjusted to neutral pH with an HCl solution and then captured with titanium dioxide magnetic beads.

[0034] The ammonia water concentration in the present invention is 5 to 15%, preferably 10%.

[0035] The present invention provides the application of the separation method in separating lipoproteins and extracellular vesicles in plasma. The separation method of the present invention can accurately separate lipoproteins and extracellular vesicles from plasma, and the obtained lipoproteins and extracellular vesicles have high purity and low loss.

[0036] The present invention also provides a method for separating extracellular vesicles and high-density lipoproteins in plasma, comprising the following steps: (1) Take 2 mL of human plasma (thawed on ice after taking out from -80°C), and dilute it to 12 mL with PBS to obtain diluted plasma; (2) Filter the diluted plasma through a 0.22-μm filter (millipore), and obtain a mixed precipitate of small EVs and high-density lipoproteins by ultracentrifugation (120,000 g, 90 min). Resuspend the precipitate with 200 μL of PBS; (3) Take 5 mg of CB-functionalized magnetic beads, wash them 3 times with water, and remove the water by magnetic separation; (4) Add 300 μL of carbonate buffer solution with pH 9.0 (Shanghai Macklin) to the CB-functionalized magnetic beads in step (3), then mix with the product in step (2), rotate and mix at 4°C for 1 h, and perform magnetic separation to obtain CB-functionalized magnetic beads adsorbed with high-density lipoproteins. Transfer the supernatant to a clean centrifuge tube; (5) Ultrafilter the plasma (the supernatant in step (4)) treated with CB-functionalized magnetic beads 3 times using a 3-kD ultrafiltration tube (millipore), resuspend it with PBS (pH 7.4, gibco), and recover a volume of 200 μL to separate EVs.

[0037] In the present invention, unless otherwise specified, all components or reagents are commercially available products well-known to those skilled in the art.

[0038] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0039] Example 1

[0040] 1. Preparation of CB-functionalized magnetic beads: Select silica hydroxyl magnetic beads (with a diameter of 1 μm) to prepare CB-functionalized magnetic beads. The steps are as follows:

[0041] (1) Dissolve 0.08 g of CB (Cibacron Blue 3G-A, Sigma) in 2 mL of pure water to obtain a CB aqueous solution;

[0042] (2) Dissolve 0.16 g of sodium carbonate in 2 mL of pure water to obtain a sodium carbonate aqueous solution;

[0043] (3) Mix the CB aqueous solution with 50 mg of silica hydroxyl magnetic beads (purchased from Dongna), and oscillate at 50°C with an oscillator at 200 rpm for 1 h;

[0044] (4) Add an aqueous solution of sodium carbonate to system (3), continue to shake for 4 h, and magnetically separate to discard the supernatant (the magnet attracts the magnetic beads from the outside of the tube, and the liquid is removed with a pipette). Wash the magnetic beads successively with water and 95% ethanol to obtain CB-functionalized magnetic beads. Then disperse the CB-functionalized magnetic beads in phosphate Tween buffer (PBST, pH 7.4) and store at 4 °C for later use.

[0045] 2. Scanning electron microscopy elemental analysis was performed on the CB-functionalized magnetic beads. It was found that a large amount of elements such as carbon, sodium, and sulfur were detected on the surface of the magnetic beads after CB modification, and the color of the magnetic beads changed from brown to dark blue, indicating that the CB molecules were successfully modified onto the surface of the magnetic beads ( Figure 2 ).

[0046] 3. Investigate the adsorption of CBMB to the EV standard. The test steps are as follows: To test the EV standard sample, mix 3 mg of CBMB with an EV standard sample containing approximately 10 10 EV particles, add an equal volume of buffer to obtain a mixture; incubate the mixture on a rotary mixer at 4 °C for 1 h; then track the changes in particle number and size by NTA analysis. The following buffers were used to achieve different pH values: pH 9.0 carbonate buffer (Shanghai Macklin, product number: C885534); PBS (1x) at pH 7.4 (gibco); MES buffer at pH 6.0 (Shanghai Macklin, product number M885671). Among them, the EV standard was obtained from urine extracellular vesicles obtained by ultracentrifugation. Urine EVs were obtained by the conventional method of ultracentrifugation in the current research. The characterization of the EV standard is shown in the figure (see below Figure 3 ). In addition, immunoblot protein characterization was also performed on high-density and low-density lipoprotein standards (Yeasen Biotech Co., Ltd. (Shanghai)). Figure 3 The results showed that the EVs isolated from urine contained the corresponding characterization proteins, which could be used to investigate the adsorption of CBMB.

[0047] Figure 4 The results found that CBMB had no effect on the size distribution of EVs in acidic, neutral, and alkaline environments ( Figure 4 A, Figure 4 C), but under acidic conditions, CBMB adsorbed to the EV standard, and approximately 40% of the EV standard was captured by CBMB ( Figure 4 B).

[0048] Example 2 Isolation and purification of extracellular vesicles and lipoproteins in plasma

[0049] The preparation of CB-functionalized magnetic beads was the same as in Example 1.

[0050] (1) Take 200 μL of human plasma (thawed on ice after being taken out from -80 °C), dilute it to 1 mL with pH 9.0 carbonate buffer (Shanghai Macklin, catalog number C885534), and filter it with a 0.22 μm filter (Millipore) to obtain diluted plasma;

[0051] (2) Take 5 mg of CB-functionalized magnetic beads (the CB-functionalized magnetic beads prepared in step 1 of this example), wash them 3 times with water, and remove the water by magnetic separation;

[0052] (3) Add the diluted plasma to the washed CB-functionalized magnetic beads, mix by rotation at 4 °C for 1 h, perform magnetic separation to obtain the CB-functionalized magnetic beads adsorbed with lipoproteins, and transfer the supernatant to a clean centrifuge tube;

[0053] (4) Take 5 mg of titanium dioxide magnetic beads (Nanjing Dongna), wash them 3 times with water, and remove the water by magnetic separation;

[0054] (5) Adjust the pH of the plasma treated with CB-functionalized magnetic beads (the supernatant in step (3)) to 7.4 with HCl (0.5 M), and add it to the washed titanium dioxide magnetic beads. Mix by rotation at 4 °C for 1 - 2 h, remove the supernatant by magnetic separation to obtain the titanium dioxide magnetic beads adsorbed with sEV;

[0055] (6) Add 150 μL of 10% ammonia water to the titanium dioxide magnetic beads adsorbed with sEV, mix by rotation at room temperature for 15 min, and sEV is released into the ammonia water;

[0056] (7) Ultrafilter the ammonia water containing sEV 4 times with a 3 kD ultrafiltration tube (Millipore), replace the ammonia water with PBS (pH 7.4, Gibco), recover a volume of 150 μL, and separate out sEV.

[0057] Example 3

[0058] The preparation of CB-functionalized magnetic beads is the same as that in Example 1.

[0059] The steps for separating and purifying plasma extracellular vesicles and lipoproteins are as follows:

[0060] (1) Take 200 μL of human plasma (thawed on ice after being taken out from -80 °C), dilute it to 1 mL with pH 9.5 carbonate buffer (Shanghai Macklin, catalog number C885536), and filter it with a 0.22 μm filter (Millipore) to obtain diluted plasma;

[0061] (2) Take 5 mg of CB-functionalized magnetic beads (the CB-functionalized magnetic beads prepared in step 1 of this example), wash them 3 times with water, and remove the water by magnetic separation;

[0062] (3) Add the diluted plasma to the washed CB-functionalized magnetic beads, mix by rotation at 4 °C for 1 h, magnetically separate to obtain the CB-functionalized magnetic beads adsorbed with lipoproteins, and transfer the supernatant to a clean centrifuge tube;

[0063] (4) Take 5 mg of titanium dioxide magnetic beads (Nanjing Dongna), wash them 3 times with water, and remove the water by magnetic separation;

[0064] (5) Adjust the pH of the plasma (the supernatant in step (3)) treated with CB-functionalized magnetic beads to 7.4 with HCl (0.5 M), and add it to the washed titanium dioxide magnetic beads. Mix by rotation at 4 °C for 2 h, magnetically separate to remove the supernatant to obtain the titanium dioxide magnetic beads adsorbed with sEV;

[0065] (6) Add 150 μL of 10% aqueous ammonia solution to the titanium dioxide magnetic beads adsorbed with sEV, mix by rotation at room temperature for 15 min, and sEV is released into the aqueous ammonia solution;

[0066] (7) Ultrafilter the aqueous ammonia containing sEV 4 times with a 3 kD ultrafiltration tube (millipore), replace the aqueous ammonia with PBS (pH 7.4, gibco), recover a volume of 100 μL, and separate out sEV.

[0067] Example 4

[0068] The preparation of CB-functionalized magnetic beads is the same as in Example 1.

[0069] The steps for separating and purifying plasma extracellular vesicles and lipoproteins are as follows:

[0070] (1) Take 200 μL of human plasma (frozen and thawed on ice after taking out from -80 °C), dilute it to 1 mL with pH 10 carbonate buffer (Shanghai Macklin, product number C885533) to obtain diluted plasma;

[0071] (2) Take 5 mg of CB-functionalized magnetic beads (the CB-functionalized magnetic beads prepared in step 1 of this example), wash them 3 times with water, and remove the water by magnetic separation;

[0072] (3) Add the diluted plasma to the washed CB-functionalized magnetic beads, mix by rotation at 4 °C for 1 h, magnetically separate to obtain the CB-functionalized magnetic beads adsorbed with lipoproteins, and transfer the supernatant to a clean centrifuge tube;

[0073] (4) Take 5 mg of titanium dioxide magnetic beads (Nanjing Dongna), wash them 3 times with water, and remove the water by magnetic separation;

[0074] (5) Adjust the pH of the plasma treated with CB-functionalized magnetic beads (the supernatant described in step (3)) to 7.4 with HCl (0.5 M), add it to the washed titanium dioxide magnetic beads, mix by rotation at 4 °C for 2 h, remove the supernatant by magnetic separation to obtain titanium dioxide magnetic beads adsorbed with sEV;

[0075] (6) Add 150 μL of 10% aqueous ammonia solution to the titanium dioxide magnetic beads adsorbed with sEV, mix by rotation at room temperature for 15 min, and sEV is released into the aqueous ammonia solution;

[0076] (7) Ultrafilter the ammonia water containing sEV 4 times with a 3 kD ultrafiltration tube (millipore), replace the ammonia water with PBS (pH 7.4, gibco), recover a volume of 150 μL, and separate out sEV.

[0077] Comparative Example 1

[0078] The preparation of CB-functionalized magnetic beads was the same as in Example 1.

[0079] The steps for separating and purifying plasma sEV and lipoproteins were different from those in Example 2 in that in step (1), it was diluted with a pH 6.0 MES buffer (Shanghai Macklin, product number M885671), and the remaining steps were the same.

[0080] Comparative Example 2

[0081] The preparation of CB-functionalized magnetic beads was the same as in Example 1.

[0082] The steps for separating and purifying sEV and lipoproteins in plasma were different from those in Example 2 in that in step (1), it was diluted with a pH 7.4 PBS buffer (gibco), and the remaining steps were the same.

[0083] Example 5

[0084] 1. Investigate the effect of plasma pretreatment by CBMB under different pH environments (pH 6.0 (Comparative Example 1), pH 7.4 (Comparative Example 2), pH 9.0 (Example 2)) on the separation of plasma EV by titanium dioxide.

[0085] Detect the particle size and particle number of small extracellular vesicles obtained in Example 2, Comparative Example 1, and Comparative Example 2 by a nanoparticle sizer (Nanosight NS300, Malvern Instruments Ltd). Perform immunoblotting analysis of characteristic proteins on the proteins on the CBMB obtained in step (3) and the titanium dioxide magnetic beads obtained in step (5) of Example 2, Comparative Example 1, and Comparative Example 2. Perform Nanodrop quantitative analysis on the proteins on the CBMB obtained in step (3) and the titanium dioxide magnetic beads CBMB obtained in step (5) of Example 1, Comparative Example 1, and Comparative Example 2. The results are asFigure 5 .

[0086] As can be seen from Figure 5 , the combination of CB magnetic beads and titanium dioxide magnetic beads can efficiently separate plasma EVs and lipoproteins ( Figure 5 A). Treating plasma with CB magnetic beads under different pH conditions does not affect the size distribution of vesicles captured by titanium dioxide magnetic beads in the next step ( Figure 5 B, Figure 5 C). However, since CBMB tends to capture both small extracellular vesicles and lipoproteins in plasma simultaneously in acidic and neutral environments, while in alkaline environments, CBMB only selectively adsorbs lipoproteins, the number of particles ( Figure 5 D) and the amount of protein ( Figure 5 E) captured by titanium dioxide magnetic beads from plasma treated with CBMB in alkaline environment are higher than those in acidic and neutral environments. Further, through immunoblotting, it was found that a large amount of EV protein markers (Alix and CD9) were characterized on the titanium dioxide magnetic beads from plasma treated with CBMB in alkaline environment, while lipoprotein markers (APOA, APOE) were enriched on the CB magnetic beads ( Figure 5 F).

[0087] 2. Effect of the dosage of CBMB-treated plasma on the purity of small extracellular vesicles separated by titanium dioxide magnetic beads

[0088] The separation and purification steps of small extracellular vesicles and lipoproteins in plasma are different from those in Example 2 in that the dosages of CBMB in step (2) are 0, 1 mg, 2 mg, 3 mg, and 4 mg respectively, and the other steps are the same. Immunoblotting analysis of characteristic proteins was performed on the titanium dioxide magnetic beads containing small extracellular vesicles obtained from the above experiments. The results are shown in Figure 6 .

[0089] The results show that as the amount of CBMB increases, the lipoprotein markers detectable on the titanium dioxide magnetic beads gradually decrease. When the dosage of CBMB reaches 4 mg, the lipoprotein marker (APOE) can no longer be detected on the titanium dioxide magnetic beads, while the concentration of the small extracellular vesicle protein marker Alix is very high ( Figure 6 ), indicating that a certain amount of CBMB can completely adsorb lipoproteins in plasma and separate small extracellular vesicles with higher purity.

[0090] 3. Electron microscopy analysis of titanium dioxide magnetic beads, small extracellular vesicles directly separated from plasma by titanium dioxide magnetic beads, and small extracellular vesicles separated from plasma treated with CBMB by titanium dioxide magnetic beads, to observe the effect of CBMB-treated plasma on the purity of small extracellular vesicles separated by titanium dioxide magnetic beads.

[0091] The steps for the isolation and purification of small extracellular vesicles from plasma using titanium dioxide magnetic beads are different from those in Example 2 in that the steps related to treating plasma with CBMB are removed, and the remaining steps are the same.

[0092] The steps for separating small extracellular vesicles from plasma treated with CBMB using titanium dioxide magnetic beads are the same as those in Example 2.

[0093] Through scanning electron microscopy analysis, it was found that after treating plasma with CBMB (pH 9.0), since lipoproteins in the plasma had been removed by CBMB, the purity of small extracellular vesicles captured by titanium dioxide magnetic beads from the CBMB-treated plasma was very high ( Figure 7 A). Subsequently, colloidal immunogold transmission electron microscopy was used to analyze the particles released from the titanium dioxide magnetic beads. It was found that without treating plasma with CBMB, a large number of lipoprotein structures were released from the titanium dioxide; while after treating plasma with CBMB at pH 9.0, pure small extracellular vesicle particles (cup-shaped structures) were released from the titanium dioxide ( Figure 7 B).

[0094] 4. Comparative analysis of small RNAs carried by small extracellular vesicles and lipoproteins

[0095] Take the CBMB that adsorbed lipoproteins obtained in step (3) of Example 2 and the titanium dioxide magnetic beads that adsorbed small extracellular vesicles obtained in step (5), and directly extract RNA on the magnetic beads, followed by small RNA sequencing analysis.

[0096] The steps for RNA extraction and sequencing analysis are as follows:

[0097] For the lipoprotein components enriched on CBMB and the EV components enriched on titanium dioxide magnetic beads, without releasing them, directly extract the RNA components encapsulated in the particles using the miRNeasy Mini Kit (Qiagen, 217004), and then use the NEBNext Multiplex Small RNA Library Preparation Kit (NEB, USA) to generate a small RNA sequencing library. RNA-seq is performed on the Illumina NovaSeq 6000 platform. The following databases are used to classify the types of small RNAs: RFAM for snRNA and snoRNA, and Ensembl for rRNA, tRNA, YRNA, repeats, exons, introns, etc. The DEGseq R package is used to identify differentially expressed genes (DEGs), with adjusted P < 0.05 and fold change > 2. The following databases: mirdb, mirtarbase, tarbase, and targetscan are used to predict the target genes of miRNAs for enrichment analysis (KEGG and GO).

[0098] The results showed that lipoproteins and small extracellular vesicles carried different numbers of small RNAs, and the common ones accounted for a relatively large proportion.Figure 8 A). Except for the known miRNA and YRNA categories, similar RNA ratios are shown on most RNA types on lipoproteins and small extracellular vesicles ( Figure 8 B). In addition, by performing differential analysis on the small RNAs carried by lipoproteins and small extracellular vesicles, it is found that the small RNAs in lipoproteins are closely related to lipid metabolism, and the small RNAs in extracellular vesicle particles are involved in vesicle localization, transport, establishment of vesicle localization, etc. Figure 8 C).

[0099] In summary, based on the pH value-mediated high-affinity separation system of CBMB, the present invention can efficiently separate lipoproteins and small extracellular vesicles from plasma, and these lipoproteins and small extracellular vesicles carry biological information expressing their respective physiological and pathological characteristics.

[0100] Example 6

[0101] The steps for separating extracellular vesicles and high-density lipoproteins in plasma are as follows:

[0102] (1) Take 2 mL of human plasma (frozen and thawed on ice after taking out from -80°C), and dilute it to 12 mL with PBS to obtain diluted plasma;

[0103] (2) After filtering the diluted plasma with a 0.22 μm filter (millipore), obtain a mixed precipitate of small EVs and high-density lipoproteins by ultracentrifugation (UC, 120,000 g, 90 min), and resuspend the precipitate with 200 μL of PBS;

[0104] (3) Take 4 mg of CB-functionalized magnetic beads (prepared in Example 1), wash them 3 times with water, and remove the water by magnetic separation;

[0105] (4) Add 300 μL of pH 9.0 carbonate buffer (Shanghai Macklin) to the CB-functionalized magnetic beads in step (3), then mix with the product in step (2), mix by rotation at 4°C for 1 h, perform magnetic separation to obtain CB-functionalized magnetic beads adsorbed with high-density lipoproteins, and transfer the supernatant to a clean centrifuge tube;

[0106] (5) Ultrafilter the plasma treated with CB-functionalized magnetic beads (the supernatant in step (4)) 3 times using a 3 kD ultrafiltration tube (millipore), resuspend it with PBS (pH 7.4, gibco), and recover a volume of 200 μL to isolate EVs.

[0107] The particle sizes and particle numbers of EVs in the mixed precipitate of EVs and high-density lipoproteins obtained by ultracentrifugation in step (2) of this example and the small extracellular vesicle particles obtained in step (5) were detected by a nanoparticle size analyzer (Nanosight NS300, Malvern Instruments Ltd). The morphologies of the mixed precipitate of small EVs and high-density lipoproteins obtained by ultracentrifugation in step (2) of this example and the small extracellular vesicles obtained in step (5) were analyzed by transmission electron microscopy. Immunoblot protein characterization was used to analyze the mixed precipitate of small EVs and high-density lipoproteins obtained by ultracentrifugation in step (2) of the example, the high-density lipoprotein CB-functionalized magnetic beads adsorbed in step (4), and the small extracellular vesicles obtained in step (5).

[0108] The results are shown in Figure 9 . It can be analyzed that since high-density lipoprotein is a particle smaller than EV, when the mixture of high-density and EV is treated with CBMB under alkaline conditions, compared with acidic and neutral buffers, more of the particles adsorbed by CBMB are small-sized particles, that is, high-density lipoprotein particles. It was found by transmission electron microscopy that both EVs and lipoprotein particles were present in the sample obtained by ultracentrifugation, while after treatment with CBMB, the lipoprotein particles had been removed by the magnetic beads. Further immunoblotting revealed that high-density lipoprotein was mainly distributed on the CBMB magnetic beads, while EVs were present in the treated sample, thus indicating that EVs and high-density lipoproteins were effectively separated.

[0109] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for separating pH-mediated plasma extracellular vesicles and lipoproteins, characterized in that, The plasma is diluted with a buffer solution having a pH of 8 to 12, the diluted plasma is mixed with Cibacron Blue-functionalized magnetic beads, the Cibacron Blue-functionalized magnetic beads and the supernatant are separated, and lipoproteins are separated from the Cibacron Blue-functionalized magnetic beads; the supernatant is mixed with titanium dioxide magnetic beads, the titanium dioxide magnetic beads are separated, and extracellular vesicles are separated from the titanium dioxide magnetic beads.

2. The separation method according to claim 1, characterized in that, The buffer solution is one of MES buffer solution, PBS buffer solution, and carbonate buffer solution.

3. The separation method according to claim 1, wherein Before use, the Cibacron Blue-functionalized magnetic beads and the titanium dioxide magnetic beads need to be washed 2 to 4 times with pure water respectively.

4. The separation method according to claim 1, wherein, The magnetic beads used in the preparation of the Cibacron Blue-functionalized magnetic beads include one or more of amino magnetic beads, carboxyl magnetic beads, silanol magnetic beads, and epoxy magnetic beads.

5. The separation method according to claim 1, wherein The temperature for mixing the diluted plasma with the Cibacron Blue-functionalized magnetic beads is 2 to 6 °C, and the mixing time is 1 to 2 h.

6. The separation method according to claim 1, wherein, The separated titanium dioxide magnetic beads are mixed with ammonia water to release extracellular vesicles, and then through ultrafiltration, the ammonia water in the filtrate is replaced with PBS to obtain an extracellular vesicle PBS suspension.

7. The separation method according to claim 6, characterized in that, The temperature of the mixing is 20 to 30 °C, and the mixing time is 10 to 15 min.

8. The separation method according to claim 6, wherein The concentration of the ammonia water is 5 to 15%.

9. Use of the separation method according to any one of claims 1 to 8 in separating plasma extracellular vesicles and lipoproteins.

10. Use of the separation method according to any one of claims 1 to 8 in separating plasma extracellular vesicles and high-density lipoproteins.

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