One-to-one fusion method of lipidosome and extracellular vesicles

Abstract

The invention discloses a one-to-one fusion method of liposome and extracellular vesicles, which comprises the following steps: dissolving cholesterol, phospholipid, cholesterol containing an acrylate or acrylamide structure and phospholipid containing an acrylate or acrylamide structure in a solvent, and then removing the solvent to obtain a precipitate, namely a product A; adding a phosphate buffer solution containing acrylate or acrylamide structure molecules into the product A, incubating and extruding to obtain a product B; co-incubating the product B and polyethylene glycol amine, and centrifuging to obtain a precipitate which is modified lipidosome; mixing the modified lipidosome solution, the ammonium persulfate solution, the molecular solution containing the acrylate or acrylamide structure and the extracellular vesicles, and performing co-incubation to realize one-to-one fusion of the lipidosome and the extracellular vesicles to obtain the lipidosome-extracellular vesicle compound. The method has the characteristics that the analysis cost of the unicellular vesicles is greatly reduced, and the comprehensiveness and accuracy of biological information detection are improved.

Description

Technical Field

[0001] The invention relates to an extracellular vesicle processing method, in particular to a one-to-one fusion method of liposome and extracellular vesicle. Background Art

[0002] Extracellular vesicles are bilayer lipid nanovesicles rich in a variety of biomolecules (including polysaccharides, proteins, and nucleic acids) released by cells to the extracellular space. In the early stages of cancer, cancer cells will secrete a large number of extracellular vesicles into the circulatory system. Therefore, extracellular vesicles are considered to be potential circulating biomarkers for early cancer screening. However, since other cells also release extracellular vesicles, extracellular vesicles in the circulatory system have high heterogeneity. In addition, extracellular vesicles secreted by the same cancer cells have significant differences in size and biomarker composition. Therefore, single extracellular vesicle analysis is the best means to identify cancer-related extracellular vesicles and analyze their biological information. The diameter of a single extracellular vesicle is small, generally 30-1000nm. At present, the available single extracellular vesicle analysis methods include nanoflow cytometry, super-resolution fluorescence microscopy, atomic force microscopy, and microfluidic-based digital detection technology. These methods require expensive equipment and high technical requirements for detection personnel, and most medical institutions in the country have not yet been equipped, which hinders the clinical application and promotion of single extracellular vesicle analysis methods in early cancer screening.

[0003] Fluorescence microscope and flow cytometer are two common analytical devices in clinical practice. These two analytical devices are low-cost and have low technical requirements for testers, but can only perform single-particle analysis on particles (such as cells) with a diameter of more than 500nm. Single extracellular vesicle analysis based on fluorescence microscope and flow cytometer is prone to lose a large amount of biological information in single extracellular vesicles, resulting in the problem of being unable to accurately and comprehensively analyze the biological information of single extracellular vesicles. Summary of the invention

[0004] The purpose of the present invention is to provide a one-to-one fusion method of liposomes and extracellular vesicles. The present invention has the characteristics of greatly reducing the cost of single extracellular vesicle analysis and improving the comprehensiveness and accuracy of biological information detection.

[0005] The technical solution of the present invention: a one-to-one fusion method of liposomes and extracellular vesicles, comprising the following steps:

[0006] S1. Preparation of modified liposomes:

[0007] a. Dissolve cholesterol, phospholipids, cholesterol containing acrylate or acrylamide structure, and phospholipids containing acrylate or acrylamide structure in a solvent, and then remove the solvent by rotary evaporation to obtain a precipitate of all cholesterol and phospholipids, which is product A;

[0008] b. Add phosphate buffer solution containing acrylate or acrylamide structural molecules to product A, incubate and extrude to obtain product B;

[0009] c. Incubate product B with polyethylene glycol amine and then centrifuge to obtain a precipitate which is a liposome with an inner surface modified with an acrylate or acrylamide chemical structure;

[0010] S2. Mix a liposome solution whose inner surface is modified with an acrylate or acrylamide chemical structure, an ammonium persulfate solution, a molecular solution containing an acrylate or acrylamide structure, and extracellular vesicles, and incubate them together to achieve a one-to-one fusion of liposomes and extracellular vesicles to obtain a liposome-extracellular vesicle complex.

[0011] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process a of step S1, the total concentration of cholesterol and cholesterol containing acrylate or acrylamide structure is 1.0 mg / mL-10.0 mg / mL, and the total concentration of phospholipids and phospholipids containing acrylate or acrylamide structure is 1.0 mg / mL-10.0 mg / mL; wherein the cholesterol containing acrylate or acrylamide structure accounts for 0%-50% of the total cholesterol mass, the phospholipids containing acrylate or acrylamide structure account for 0%-50% of the total phospholipid mass, and the cholesterol containing acrylate or acrylamide structure and the phospholipids containing acrylate or acrylamide structure are not both 0%.

[0012] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process a of step S1, a low boiling point solvent is used as the solvent, and the solvent includes any one of ethanol, dichloromethane or chloroform.

[0013] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process b of step S1, the solid-to-liquid ratio of product A to phosphate buffer solution is (8-12) mg: (2-5) ml.

[0014] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process b of step S1, the concentration of the acrylate or acrylamide structure molecules dissolved in the phosphate buffer solution is 0 mg / mL-5.0 mg / mL.

[0015] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process b of step S1, a porous membrane is used to extrude the incubated solution, and the pore size of the porous membrane is 400-1100 nm.

[0016] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process b of step S1, the incubation time is 5-60 minutes and the incubation temperature is 20-40°C.

[0017] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in the process c of step S1, the co-incubation time is 1-24 hours, and the incubation temperature is 20-40°C.

[0018] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, during the process c of step S1, the centrifugal force of the centrifugal treatment is greater than 5000g, and the centrifugation time is 5-15min.

[0019] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in step S2, the concentration of the liposome solution whose inner surface is modified with an acrylate or acrylamide chemical structure is 0.1 mg / mL-3.0 mg / mL, the concentration of the ammonium persulfate solution is 0.1 mg / mL-5.0 mg / mL, and the concentration of the molecular solution containing an acrylate or acrylamide structure is 0.1 mg / mL-5.0 mg / mL.

[0020] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in step S2, the pH value of the mixed solution is higher than 7.0.

[0021] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in step S2, the co-incubation time is 5-60 min, and the co-incubation temperature is 20-40°C.

[0022] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in step S2, the extracellular vesicles include extracellular vesicles in human body fluids or extracellular vesicles extracted from human tissues, and the human body fluids include any one of serum, plasma, urine, peritoneal cavity and tears.

[0023] In the aforementioned one-to-one fusion method of liposomes and extracellular vesicles, in step S2, the extracellular vesicles further include extracellular vesicles in various animal and plant tissues or in various animal and plant cell culture fluids.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention modifies liposomes to obtain liposomes with an inner surface modified with an acrylate or acrylamide chemical structure, and then fuses the liposomes with an inner surface modified with an acrylate or acrylamide chemical structure with extracellular vesicles in body fluids one-to-one to obtain a liposome-extracellular vesicle complex with a size expanded to the micron level, so as to facilitate fluorescent labeling of biological molecules such as proteins, nucleic acids or polysaccharides contained in the extracellular vesicles, so as to realize single extracellular vesicle analysis based on a fluorescence microscope or a flow cytometer, greatly reduce the cost of single extracellular vesicle analysis, and can detect the same biological information as a nano flow detector and a super-resolution fluorescence microscope. The biological information detection is comprehensive and accurate, and the feasibility of large-scale early cancer screening is improved.

[0026] Therefore, the present invention has the characteristics of greatly reducing the cost of single extracellular vesicle analysis and improving the comprehensiveness and accuracy of biological information detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of liposomes whose inner surfaces are modified with acrylate or acrylamide chemical structures.

[0028] Figure 2 Transmission electron microscopy images of liposomes, extracellular vesicles, and liposome-extracellular vesicle complexes.

[0029] Figure 3 This is a graph showing changes in fluorescence resonance energy transfer signals of three types of extracellular vesicle solutions, namely, liposomes modified with acrylate or acrylamide chemical structures on the inner surface, unmodified liposomes, and phosphate buffer solution as a function of incubation time.

[0030] Figure 4 This is a correlation analysis chart of the positive rate analysis results of specific proteins expressed by conventional flow cytometry and nano-flow cytometry, among which EpCAM + and EGFR + Indicates that extracellular vesicles contain EpCAM and EGFR, respectively.

[0031] Figure 5 This is a heat map of the analysis results of specific proteins expressed based on fluorescence microscopy and super-resolution fluorescence microscopy, among which EpCAM - and EGFR - Respectively, extracellular vesicles do not contain EPCAM and EGFR proteins.

[0032] Figure 6 This is a correlation analysis chart of the positive rate analysis results of specific miRNAs expressed by conventional flow cytometry and nano-flow cytometry; miR-21 + 、miR-126 + and miR-21 + / miR-126 + Extracellular vesicles containing miR-21 + 、miR-126 + and miR-21 + and miR-126 + .

[0033] Figure 7 This is the receiver operating characteristic curve for early breast cancer screening. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0035] The one-to-one fusion method of liposomes and extracellular vesicles comprises the following steps:

[0036] S1. Preparation of modified liposomes:

[0037] a. Dissolve cholesterol, phospholipids, cholesterol containing acrylate or acrylamide structure, and phospholipids containing acrylate or acrylamide structure in a solvent, and then remove the solvent by rotary evaporation to obtain a precipitate of all cholesterol and phospholipids, which is product A;

[0038] The total concentration of cholesterol and cholesterol containing acrylate or acrylamide structure is 1.0 mg / mL-10.0 mg / mL, and the total concentration of phospholipids and phospholipids containing acrylate or acrylamide structure is 1.0 mg / mL-10.0 mg / mL; wherein cholesterol containing acrylate or acrylamide structure accounts for 0%-50% of the total cholesterol mass, phospholipids containing acrylate or acrylamide structure accounts for 0%-50% of the total phospholipid mass, and cholesterol containing acrylate or acrylamide structure and phospholipids containing acrylate or acrylamide structure are not both 0%.

[0039] The solvent is a low boiling point solvent, and the solvent includes any one of ethanol, dichloromethane or chloroform.

[0040] b. Add 3 mL of phosphate buffer solution containing acrylate or acrylamide structural molecules to 10 mg of product A, incubate, and extrude the incubated solution using a porous membrane with a pore size of 400-1100 nm to obtain product B;

[0041] The solid-to-liquid ratio of product A to phosphate buffer solution is (8-12) mg: (2-5) ml.

[0042] The concentration of the acrylate or acrylamide structure molecules dissolved in the phosphate buffer solution is 0 mg / mL-5.0 mg / mL.

[0043] c. Incubate product B with polyethylene glycol amine for 1-24 hours at a temperature of 20-40°C, then centrifuge at a centrifugal force greater than 5000g for 5-15 minutes. The precipitate obtained is a liposome with an inner surface modified with an acrylate or acrylamide chemical structure, such as Figure 1 As shown;

[0044] S2. Mix 0.1 mg / mL-3.0 mg / mL of liposome solution whose inner surface is modified with acrylate or acrylamide chemical structure, 0.1 mg / mL-5.0 mg / mL of ammonium persulfate solution, 0.1 mg / mL-5.0 mg / mL of molecular solution containing acrylate or acrylamide structure and extracellular vesicles. The pH value of the mixed solution is higher than 7. Incubate them together. The incubation time is 5-60 minutes. The incubation temperature is 20-40°C to achieve one-to-one fusion of liposomes and extracellular vesicles to obtain a liposome-extracellular vesicle complex.

[0045] Extracellular vesicles include extracellular vesicles in human body fluids or extracellular vesicles extracted from human tissues. Human body fluids include any one of serum, plasma, urine, peritoneal cavity and tears.

[0046] Extracellular vesicles also include extracellular vesicles in various animal and plant tissues or in various animal and plant cell culture fluids.

[0047] Embodiment 1:

[0048] The one-to-one fusion method of liposomes and extracellular vesicles comprises the following steps:

[0049] S1. Preparation of modified liposomes:

[0050] a. Dissolve cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphocholine, cholesterol-polyethylene glycol 2000-acrylamide and 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol-acrylamide in ethanol, and then remove the ethanol by rotary evaporation to obtain a precipitate of all cholesterol and phospholipids, which is product A;

[0051] The total concentration of cholesterol and cholesterol-polyethylene glycol 2000-acrylamide is 5.0 mg / mL, and the total dosage is 7.5 mg. The total concentration of 1,2-distearoyl-sn-glycerol-3-phosphocholine and 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol 2000-acrylamide is 10.0 mg / mL, and the total dosage is 2.5 mg; among them, cholesterol-polyethylene glycol 2000-acrylamide accounts for 5% of the total cholesterol mass, and 1,2-distearoyl-3-phosphatidylethanolamine-polyethylene glycol 2000-acrylamide accounts for 10% of the total phospholipid mass.

[0052] b. Add 5 mL of phosphate buffer solution containing 1.0 mg / mL methylene bisacrylamide to 5 mg of product A, incubate for 30 min at a temperature of 37° C., and then extrude the incubated solution using a porous membrane with a pore size of 1000 nm to obtain product B;

[0053] c. Co-incubate product B with 0.01 mg / mL polyethylene glycol amine with a molecular weight of 2000 for 12 hours at a temperature of 37°C, and then centrifuge at a centrifugal force of 7000 g for 10 minutes to obtain a precipitate that is a liposome with an inner surface modified with acrylamide chemical structure;

[0054] S2. Mix a 0.3 mg / mL liposome solution whose inner surface is modified with an acrylamide chemical structure, a 1 mg / mL ammonium persulfate solution, a 1 mg / mL methylene bisacrylamide solution, and 50 μL of extracellular vesicles of the patient's plasma to be tested. The pH value of the mixed solution is 7.4. Incubate them together for 30 minutes at a temperature of 25°C to achieve one-to-one fusion of liposomes and plasma extracellular vesicles to obtain liposome-extracellular vesicle complex 1.

[0055] Embodiment 2:

[0056] The one-to-one fusion method of liposomes and extracellular vesicles comprises the following steps:

[0057] S1. Preparation of modified liposomes:

[0058] a. Dissolve cholesterol, 1,2-distearoyl-sn-glycerol-3-phosphocholine, cholesterol-polyethylene glycol 2000-methacrylate and 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine-polyethylene glycol 2000-acrylate in chloroform, and then remove the chloroform by rotary evaporation to obtain a precipitate of all cholesterol and phospholipids, which is product A;

[0059] The total concentration of cholesterol and cholesterol-polyethylene glycol 2000-methacrylate is 10.0 mg / mL, and the total concentration of 1,2-distearoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine-polyethylene glycol 2000-acrylate is 10.0 mg / mL; among them, cholesterol-polyethylene glycol 2000-methacrylate accounts for 25% of the total cholesterol mass, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine-polyethylene glycol 2000-acrylate accounts for 35% of the total phospholipid mass.

[0060] b. Add 3.0 mL of phosphate buffer solution containing 5.0 mg / mL polyethylene glycol diacrylate to product A, incubate for 30 min at a temperature of 30° C., and then extrude the incubated solution using a porous membrane with a pore size of 600 nm to obtain product B;

[0061] c. Co-incubate product B with 0.01 mg / mL polyethylene glycol amine with a molecular weight of 20,000 for 12 hours at a temperature of 20°C, and then centrifuge at a centrifugal force of 8,000 g for 15 minutes to obtain a precipitate that is a liposome with an acrylate chemical structure modified on the inner surface;

[0062] S2. Mix a 3.0 mg / mL liposome solution whose inner surface is modified with an acrylate chemical structure, a 5.0 mg / mL ammonium persulfate solution, a 5.0 mg / mL polyethylene glycol diacrylate solution, and 50 μL of extracellular vesicles from the patient's urine to be tested. The pH value of the mixed solution is 8. Incubate them together for 60 minutes at a temperature of 20°C to achieve one-to-one fusion of liposomes and urine extracellular vesicles to obtain liposome-extracellular vesicle complex 2.

[0063] Embodiment 3:

[0064] This example is basically the same as Example 1, except that the extracellular vesicles are extracellular vesicles secreted by HUVEC cells, and a liposome-extracellular vesicle complex 3 is obtained.

[0065] Embodiment 4:

[0066] This example is basically the same as Example 1, except that the extracellular vesicles are extracellular vesicles secreted by HL60 cells, and a liposome-extracellular vesicle complex 4 is obtained.

[0067] Embodiment 5:

[0068] This example is basically the same as Example 1, except that the extracellular vesicles are extracellular vesicles secreted by HCT116 cells, and a liposome-extracellular vesicle complex 5 is obtained.

[0069] Embodiment 6:

[0070] This example is basically the same as Example 1, except that the extracellular vesicles are extracellular vesicles secreted by MCF-7 cells, and a liposome-extracellular vesicle complex 6 is obtained.

[0071] Embodiment 7:

[0072] This example is basically the same as Example 1, except that the solvent is dichloromethane, the concentration of the acrylate or acrylamide structure molecules dissolved in the phosphate buffer solution is 0 mg / mL, the extracellular vesicles are extracellular vesicles in plant tissues, and a liposome-extracellular vesicle complex 7 is obtained.

[0073] Hydration particle size detection experiment: The hydrated particle size values ​​of the liposomes containing acrylamide chemical structure, the extracellular vesicles of MCF-7 cells, and the liposome-extracellular vesicle complex of MCF-7 cells after fusion in Example 6 were analyzed by transmission electron microscopy. The analysis results are shown in Figure 2 shown.

[0074] like Figure 2 As shown in the figure, transmission electron microscopy shows that the average hydrated particle size of the extracellular vesicles of MCF-7 cells before fusion is about 120nm, the average hydrated particle size of liposomes containing acrylate or acrylamide chemical structures is 957.1±13.5nm, and the average hydrated particle size of the liposome-extracellular vesicle complex of MCF-7 cells after fusion is 1148.9±52.0nm. This shows that after fusion treatment, the diameter of the liposome-extracellular vesicle complex increases, which is convenient for analysis by flow cytometry and fluorescence microscopy.

[0075] Fusion detection experiment: The extracellular vesicles in Example 6 labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindocyanine perchlorate and the extracellular vesicles in Example 6 labeled with 3,3-dioctadecyloxocarbocyanine perchlorate were mixed at the same concentration. The two perchlorate-labeled extracellular vesicle solutions were treated with the same amount and concentration of liposome solution with acrylamide chemical structure modified on the inner surface, liposome solution without any modification and phosphate buffer solution, and ammonium persulfate solution and methylenebisacrylamide solution were added during the treatment process according to the method of step S2 in Example 1 to achieve fusion of extracellular vesicles. Then the flow cytometer was used for analysis. The analysis results are shown in Figure 3 shown.

[0076] from Figure 3 It can be seen that during the co-incubation of the above solutions, the fluorescence resonance energy transfer effect of the extracellular vesicle solution treated with phosphate buffer solution did not increase. This is because the two perchlorates need to appear on the surface of the same liposome-extracellular vesicle mixture to cause the fluorescence resonance energy transfer effect, and there are no liposomes in the phosphate buffer solution, so the fusion of extracellular vesicles cannot be caused. The fluorescence resonance energy transfer effect of the extracellular vesicle solution treated with a liposome solution whose inner surface is modified with an acrylate or acrylamide chemical structure is equivalent to that of the extracellular vesicle solution treated with a phosphate buffer solution, which shows that no two or more extracellular vesicles fuse to the same liposome during the fusion process, reflecting the one-to-one fusion of liposomes and extracellular vesicles. The fluorescence resonance energy transfer effect of the extracellular vesicle sample treated with a liposome solution without any modification is rapidly enhanced, indicating that two or more extracellular vesicles fuse to the same liposome during the fusion process.

[0077] Membrane protein analysis and detection experiment: Mix the fluorescent aptamer sequence solution that can bind to EpCAM and EGFR proteins and the liposome-extracellular vesicle complex solution of Example 3-6, incubate at 25°C for 30 minutes, centrifuge for 10 minutes after incubation, and the centrifugal force is above 5000g. Take the precipitate after centrifugation and redisperse it in 100μL of phosphate buffer solution to obtain the labeled liposome-extracellular vesicle complex. The labeled liposome-extracellular vesicle complex was analyzed by conventional flow cytometer and fluorescence microscope respectively. At the same time, the extracellular vesicles of Example 3-6 were directly labeled with the fluorescent aptamer sequence that can bind to EpCAM and EGFR proteins, and the labeled extracellular vesicles were analyzed by nanoflow cytometer and super-resolution fluorescence microscope respectively.

[0078] The results of flow cytometry and nanoflow cytometry analysis are shown in Figure 4 As shown. Figure 4 It can be seen that the analysis results of flow cytometry and nanoflow cytometry are basically consistent, showing that the EpCAM and EGFR expressions of HUVEC cells, HL60 cells, HCT116 cells, and MCF-7 cells after fusion detected by flow cytometry are consistent with the EpCAM and EGFR expressions of different cells before fusion detected by nanoflow cytometry.

[0079] This indicates that the biological information of the extracellular vesicles in the liposome-extracellular vesicle complex is not lost, and flow cytometry detection can also detect the accurate biological information of the extracellular vesicles in the liposome-extracellular vesicle complex.

[0080] The results of fluorescence microscopy and super-resolution fluorescence microscopy analysis are shown in Figure 5 As shown. Figure 5 It can be seen that the analysis results of fluorescence microscopy and super-resolution fluorescence microscopy are basically consistent, showing that the expression of EPCAM and EGFR in different extracellular vesicles detected by fluorescence microscopy and super-resolution fluorescence microscopy before and after fusion is consistent.

[0081] This indicates that the biological information of extracellular vesicles in the liposome-extracellular vesicle complex is not lost, and fluorescence microscopy can also detect accurate biological information of extracellular vesicles in the liposome-extracellular vesicle complex.

[0082] miRNA analysis and detection experiment: The fluorescent beacons that dissolved the DNA hairpin structure were used to label the liposome-extracellular vesicle complexes of Example 3, Example 5 and Example 6, respectively, and then the labeled liposome-extracellular vesicle complexes were analyzed using a conventional flow cytometer.

[0083] At the same time, under the action of streptolysin, the RNA in the extracellular vesicles of Example 3, Example 5 and Example 6 was directly labeled with a fluorescent beacon with a DNA hairpin structure, and the labeled extracellular vesicles were analyzed using a nanoflow cytometer.

[0084] The results of flow cytometry and nanoflow cytometry analysis are shown in Figure 6 As shown. Figure 6 It can be seen that the analysis results of the flow cytometer are basically consistent with the analysis results of the nano-flow cytometer, indicating that the expression of miR-21 and miR-126 in different extracellular vesicles detected by the flow cytometer and the nano-flow cytometer before and after fusion is consistent.

[0085] This indicates that the biological information of extracellular vesicles in the liposome-extracellular vesicle complex is not lost, and flow cytometry can also detect accurate biological information of extracellular vesicles in the liposome-extracellular vesicle complex.

[0086] Cancer early screening test:

[0087] The liposome-extracellular vesicle complex 1 in Example 1 was labeled with a fluorescent aptamer sequence that can bind to CA125, HER2, PSMA and CA15-3 proteins, and the labeled liposome-extracellular vesicle complex was analyzed by flow cytometry. The analysis results are shown in Figure 7 shown.

[0088] from Figure 7 It can be seen that the area under the curve of the receiver operating characteristic curve can reach 0.934, therefore, it is shown that the liposome-extracellular vesicle complex can be used to distinguish between benign breast nodules and malignant breast nodules.

[0089] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. For those skilled in the art, the technical solutions described in the above embodiments can be modified, or some of the technical features therein can be replaced by equivalents; and all these modifications and replacements should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A one-to-one fusion method of liposomes and extracellular vesicles, characterized in that: The following steps are involved: S1. Preparation of modified liposomes: a. Dissolve cholesterol, phospholipids, cholesterol containing acrylate or acrylamide structure, and phospholipids containing acrylate or acrylamide structure in a solvent, and then remove the solvent by rotary evaporation to obtain a precipitate of all cholesterol and phospholipids, which is product A; b. Add phosphate buffer solution containing acrylate or acrylamide structural molecules to product A, incubate and extrude to obtain product B; c. Incubate product B with polyethylene glycol amine and then centrifuge to obtain a precipitate which is a liposome with an inner surface modified with an acrylate or acrylamide chemical structure; S2. Mix a liposome solution whose inner surface is modified with an acrylate or acrylamide chemical structure, an ammonium persulfate solution, a molecular solution containing an acrylate or acrylamide structure, and extracellular vesicles, and incubate them together to achieve a one-to-one fusion of liposomes and extracellular vesicles to obtain a liposome-extracellular vesicle complex.

2. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process a of step S1, the total concentration of cholesterol and cholesterol containing acrylate or acrylamide structure is 1.0 mg / mL-10.0 mg / mL, and the total concentration of phospholipids and phospholipids containing acrylate or acrylamide structure is 1.0 mg / mL-10.0 mg / mL; wherein the cholesterol containing acrylate or acrylamide structure accounts for 0%-50% of the total cholesterol mass, the phospholipids containing acrylate or acrylamide structure accounts for 0%-50% of the total phospholipid mass, and the cholesterol containing acrylate or acrylamide structure and the phospholipids containing acrylate or acrylamide structure are not both 0%.

3. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process a of step S1, the solvent is a low boiling point solvent, and the solvent includes any one of ethanol, dichloromethane or chloroform.

4. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process b of step S1, the solid-liquid ratio of product A to phosphate buffer solution is (8-12) mg: (2-5) ml.

5. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process b of step S1, the concentration of the molecules containing acrylate or acrylamide structure dissolved in the phosphate buffer solution is 0 mg / mL-5.0 mg / mL.

6. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process b of step S1, the incubated solution is extruded using a porous membrane, and the pore size of the porous membrane is 400-1100 nm.

7. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process b of step S1, the incubation time is 5-60 minutes, and the incubation temperature is 20-40°C.

8. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process c of step S1, the co-incubation time is 1-24 hours, and the incubation temperature is 20-40°C.

9. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In the process c of step S1, the centrifugal force of the centrifugal treatment is greater than 5000g, and the centrifugal time is 5-15min.

10. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In step S2, the concentration of the liposome solution whose inner surface is modified with an acrylate or acrylamide chemical structure is 0.1 mg / mL-3.0 mg / mL, the concentration of the ammonium persulfate solution is 0.1 mg / mL-5.0 mg / mL, and the concentration of the molecular solution containing an acrylate or acrylamide structure is 0.1 mg / mL-5.0 mg / mL.

11. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In step S2, the pH value of the mixed solution is higher than 7.

0.

12. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In step S2, the co-incubation time is 5-60 minutes, and the co-incubation temperature is 20-40°C.

13. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In step S2, the extracellular vesicles include extracellular vesicles in human body fluids or extracellular vesicles extracted from human tissues, and the human body fluids include any one of serum, plasma, urine, peritoneal cavity and tears.

14. The one-to-one fusion method of liposomes and extracellular vesicles according to claim 1, characterized in that: In step S2, the extracellular vesicles also include extracellular vesicles in various animal and plant tissues or extracellular vesicles in various animal and plant cell culture fluids.

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