阅读说明：本技术 一种循环细胞外囊泡原位标记和快速分离方法 (Circulating extracellular vesicle in-situ labeling and rapid separation method ) 是由 陈刚 余自力 刘海明 吴敏 赵怡芳 于 2021-07-16 设计创作，主要内容包括：本发明公开了一种循环细胞外囊泡原位标记和快速分离方法,将磷脂聚乙二醇或其衍生物修饰的物质注射到受试者的血液循环系统,磷脂聚乙二醇或其衍生物修饰的物质在游离状态下自行组装至循环细胞外囊泡的细胞膜磷脂双分子层上。该标记方法可应用在循环细胞外囊泡代谢动力学监测和从活体中快速分离出循环细胞外囊泡等方面,将有助于循环细胞外囊泡的基础研究和临床应用。与现有的标记方法相比,本发明无需提前将循环细胞外囊泡从外周血中分离出来,可有效避免超速离心等方法对循环细胞外囊泡结构的破坏,最大限度地保持循环细胞外囊泡自身的理化性能,该方法具有普适性,适用于标记和快速分离多种动物、细胞来源的循环细胞外囊泡。(The invention discloses an in-situ labeling and rapid separation method of circulating extracellular vesicles, which comprises the steps of injecting phospholipid polyethylene glycol or a substance modified by derivatives thereof into a blood circulation system of a subject, and automatically assembling the substance modified by the phospholipid polyethylene glycol or the derivatives thereof on a cell membrane phospholipid bilayer of the circulating extracellular vesicles in a free state. The labeling method can be applied to aspects of monitoring the metabolism dynamics of the circulating extracellular vesicles and rapidly separating the circulating extracellular vesicles from living bodies, and is favorable for basic research and clinical application of the circulating extracellular vesicles. Compared with the existing labeling method, the invention does not need to separate the circulating extracellular vesicles from peripheral blood in advance, can effectively avoid the damage of ultracentrifugation and other methods to the circulating extracellular vesicle structure, and furthest keeps the physical and chemical properties of the circulating extracellular vesicles.)

1. A method for non-invasive in situ labeling of circulating extracellular vesicles for non-medical purposes, comprising: injecting phospholipid polyethylene glycol or a substance modified by the derivative thereof into the blood circulation system of a subject, wherein the phospholipid polyethylene glycol or the substance modified by the derivative thereof is self-assembled on a cell membrane phospholipid bilayer of the circulating extracellular vesicle in a free state.

2. Use of a phospholipid polyethylene glycol or a derivative thereof modified substance for the preparation of a product for in situ labelling of circulating extracellular vesicles in a subject, characterized in that: the product is used for making phospholipid polyethylene glycol or substance modified by derivative thereof enter the blood circulation system of a subject.

3. The method for the non-invasive in situ labelling of circulating extracellular vesicles of non-medical purpose according to claim 1 or the use according to claim 2, characterized in that: the subject is a human or non-human mammal.

4. The method for the non-invasive in situ labelling of circulating extracellular vesicles of non-medical purpose according to claim 1 or the use according to claim 2, characterized in that: the phospholipid polyethylene glycol or the substance modified by the derivative of the phospholipid polyethylene glycol is at least one of phospholipid-polyethylene glycol-biotin, phospholipid-polyethylene glycol-folic acid, phospholipid-polyethylene glycol-biotin-fluorescent agent, phospholipid-polyethylene glycol-nano particles, phospholipid-polyethylene glycol-medicine and phospholipid-polyethylene glycol-polypeptide.

5. The method for the non-invasive in situ labelling of circulating extracellular vesicles of non-medical purpose according to claim 1 or the use according to claim 2, characterized in that: the amount of the phospholipid polyethylene glycol or the substance modified by the phospholipid polyethylene glycol derivative entering a blood circulation system is 5-300 mg/kg.

6. The method for the non-invasive in situ labelling of circulating extracellular vesicles of non-medical purpose according to claim 1 or the use according to claim 2, characterized in that: the phospholipid polyethylene glycol or the substance modified by the phospholipid polyethylene glycol derivative is injected into the bodies of the non-human mammals or the human bodies in an intravenous injection or intraperitoneal injection mode.

7. The method for the non-invasive in situ labelling of circulating extracellular vesicles of non-medical purpose according to claim 1 or the use according to claim 2, characterized in that: and dissolving the substance modified by the phospholipid polyethylene glycol or the derivative thereof by using dimethyl sulfoxide, and then diluting by using sterile saline to obtain the injection of the substance modified by the phospholipid polyethylene glycol or the derivative thereof.

8. A method for non-invasive tracking of circulating extracellular vesicles for non-medical purposes, comprising the steps of:

(1) injecting phospholipid-polyethylene glycol-biotin into a subject;

(2) at different time points, arterial blood was taken from the subject and circulating extracellular vesicles were isolated from the blood;

(3) labeling circulating extracellular vesicles by using streptavidin-coupled fluorescent dye and flow antibodies of different differentiation antigens;

(4) detecting the labeled product of step (3) by using a flow cytometer.

9. The method for the non-invasive tracing of circulating extracellular vesicles of non-medical purpose according to claim 8, wherein: the step of separating the circulating extracellular vesicles from the blood comprises the following steps: centrifuging the blood at 4 deg.C and 1550g, collecting upper layer plasma, and repeating at least twice to remove cells and cell debris; centrifuging the supernatant at 4 deg.C for 20,000g to obtain precipitate as circulating extracellular vesicle.

10. A method for the non-invasive rapid separation of circulating extracellular vesicles for non-medical purposes, comprising the steps of:

injecting phospholipid-polyethylene glycol-biotin into a subject;

taking blood from an artery of a subject; centrifuging blood, removing cells and cell debris in the blood, adding streptavidin coupled iron oxide nanoparticles into supernatant, mixing uniformly, and incubating at 37 ℃;

and separating out the circulating extracellular vesicles with the membrane surface modified by the iron oxide nanoparticles by utilizing the magnet adsorption.

Technical Field

The invention relates to the field of molecular biology and biotechnology, in particular to a method for labeling circulating extracellular vesicles (C-EVs) by a living body, and also relates to application of the method in aspects of monitoring the metabolic kinetics of the circulating extracellular vesicles and rapidly separating the circulating extracellular vesicles from a living body, and the like, and the method is favorable for basic research and clinical application of the circulating extracellular vesicles.

Background

Extracellular vesicles are vesicular bodies having a bilayer membrane structure, which are shed from cell membranes or secreted from cells, have diameters of 40 to 1000nm, and are widely present in cell culture supernatants and various body fluids (blood, lymph, saliva, urine, semen, milk). Extracellular vesicles in peripheral blood, also called circulating microvesicles (C-EVs), are a mixture of extracellular vesicles secreted by a variety of cells such as platelets, erythrocytes, lymphocytes, and endothelial cells. The extracellular vesicles inherit abundant biological information molecules of the mother cells, such as proteins, lipids, DNA, mRNA, miRNA and the like, serve as intercellular information transfer carriers in various pathophysiological behaviors, and participate in processes such as intercellular communication, cell migration, angiogenesis, immunoregulation and the like. In addition, based on their natural delivery properties and good biological safety, extracellular vesicles are considered as promising gene or drug delivery vehicles for the treatment of a variety of diseases.

Despite the promising results, the current knowledge about the basic properties of circulating extracellular vesicles is still seriously insufficient, especially the in vivo metabolic kinetics, the tissue distribution and clearance rules, and the comparison of different subtypes of extracellular vesicles, and the like, which remain to be further studied. At present, the research on the biological behavior in the circulating extracellular vesicles needs to firstly separate and purify the blood circulating extracellular vesicles by ultracentrifugation and other methods, and then the blood circulating extracellular vesicles are labeled in vitro and then are infused back into a human body for subsequent research. Although this strategy plays an important role in studying the biological behavior of circulating extracellular vesicles in vivo, it has a number of drawbacks: 1. the strong centrifugal force of the in vitro ultracentrifugation can cause the structural damage of the circulating extracellular vesicle; 2. in-vitro labeling strategies such as fluorescent dye labeling and quantum dot labeling, and in-vitro vector construction such as electrochemical transfection can change the physicochemical properties of the circulating extracellular vesicles, thereby influencing the objective removal efficiency of the circulating microvesicles; 3. the stability of an in vitro labeling strategy is poor, fluorescent dyes such as DiI, DiO, PKH and the like have strong light bleaching performance and weak light intensity, and quantum dots are easy to cause leakage and the like, so that signal loss or diffusion and other adverse effects are caused; 4. in vitro labeling only enables characterization of total circulating extracellular vesicles in the blood. Therefore, in order to research the metabolism dynamics and the in vivo distribution rule of the circulating extracellular vesicles, a method which is biologically friendly, simple, convenient and quick and can realize nondestructive marking of the circulating extracellular vesicles is urgently needed to be developed.

Disclosure of Invention

In order to solve the problems, the invention provides an in-situ labeling and rapid separation method for circulating extracellular vesicles, which does not need in-vitro lengthy centrifugal separation and static incubation, can directly inject phospholipid-polyethylene glycol-Biotin (DSPE-PEG-Biotin) into a blood circulation system to complete in-situ labeling of the circulating extracellular vesicles, has high labeling efficiency, and is less interfered by other components in blood; more importantly, the in situ labeling method does not affect the physical properties (size, morphology) and the characteristic molecular expression of the circulating extracellular vesicles; the in-situ labeling method is not based on an immunoaffinity mode, so that extracellular vesicles derived from different cells or different species can react with DSPE-PEG-Biotin, the application range is wide, and the method has universality. In addition, the DSPE-PEG-Biotin has better biological friendliness and has no obvious toxic or side effect on main metabolic organs of an organism such as liver, kidney and the like.

The technical scheme provided by the invention is as follows:

in a first aspect, the present invention provides a non-invasive method for the in vivo labeling of circulating extracellular vesicles for non-medical purposes, comprising injecting into the blood circulation system of a subject a substance modified with phospholipid polyethylene glycol or a derivative thereof, which self-assembles in a free state onto the membrane phospholipid bilayer of the circulating extracellular vesicles.

In a second aspect, the present invention provides the use of a phospholipid polyethylene glycol or a derivative thereof modified substance for the manufacture of a product for in vivo labeling of circulating extracellular vesicles in a subject, the product being for use in bringing a phospholipid polyethylene glycol or a derivative thereof modified substance into the blood circulation system.

Preferably, the subject is a human or non-human mammal.

Preferably, the phospholipid polyethylene glycol or the derivative thereof is at least one of phospholipid-polyethylene glycol-biotin, phospholipid-polyethylene glycol-folic acid, phospholipid-polyethylene glycol-biotin-fluorescent agent, phospholipid-polyethylene glycol-nanoparticles, phospholipid-polyethylene glycol-drug, and phospholipid-polyethylene glycol-polypeptide.

Preferably, the amount of phospholipid polyethylene glycol or phospholipid polyethylene glycol derivative modified substance entering the blood circulation system is 5-300 mg/kg.

Preferably, the phospholipid polyethylene glycol or the modified substance thereof is injected into the non-human mammal or human body by intravenous injection or intraperitoneal injection.

Preferably, the phospholipid polyethylene glycol or the derivative thereof modified substance is dissolved in dimethyl sulfoxide and then diluted with sterile saline to obtain the injection of the phospholipid polyethylene glycol or the derivative thereof modified substance.

In a third aspect, the present invention provides a method for the in vivo tracking of non-invasive circulating extracellular vesicles for non-medical purposes, comprising the steps of:

(1) injecting phospholipid-polyethylene glycol-biotin into a subject;

(2) at different time points, arterial blood is taken from the subject, and circulating extracellular vesicles are separated from the blood;

(3) labeling circulating extracellular vesicles by using streptavidin-coupled fluorescent dye and flow antibodies of different differentiation antigens;

(4) detecting the labeled product of step (3) by using a flow cytometer.

Preferably, the step of separating the circulating extracellular vesicles from the blood comprises: centrifuging the blood at 4 deg.C and 1550g, collecting upper layer plasma, and repeating at least twice to remove cells and cell debris; centrifuging the supernatant at 4 deg.C for 20,000g to obtain precipitate as circulating extracellular vesicle.

In a fourth aspect, the present invention provides a non-invasive method for rapidly isolating circulating extracellular vesicles from a living body for non-medical purposes, comprising the steps of:

injecting phospholipid-polyethylene glycol-biotin into a subject;

taking blood from an artery of a subject, centrifuging, removing cells and cell debris in the blood, adding streptavidin coupled iron oxide nanoparticles into supernatant, mixing uniformly, and incubating at 37 ℃;

and separating out the circulating extracellular vesicles with the membrane surface modified by the iron oxide nanoparticles by utilizing the magnet adsorption.

Compared with the prior art, the invention has the following advantages:

1. the membrane phospholipid replacement strategy is used for directly marking the circulating extracellular vesicles in the blood circulation system in vivo, so that the tedious and tedious method of in vitro centrifugation, fluorescent dye marking and then re-infusion into the body is avoided, the advantages of simplicity, convenience, rapidness and high efficiency are achieved, the adverse effects of structural damage of the circulating extracellular vesicles, easiness in light bleaching of fluorescent signals and the like are effectively avoided, and the in vivo circulation rule of the circulating extracellular vesicles is conveniently and accurately researched.

2. The in-situ living body marking method provided by the invention has strong biological friendliness, stably introduces the marking agent on the surface of the circulating extracellular vesicle, does not influence the physicochemical property of the circulating extracellular vesicle, has no obvious toxicity to main metabolic organs of a human body such as liver, kidney and the like, and is convenient for developing human body research and clinical outcome transformation.

3. The living body marking method provided by the invention is suitable for research and analysis of different objects, has strong universality and is suitable for extracellular vesicles of other species or other body fluid sources.

Drawings

FIG. 1 shows the chemical structure of DSPE-PEG-Biotin, wherein O represents oxygen atom, HO represents hydroxyl, N represents nitrogen atom, NH represents imino, NH₂Represents amino, NH₄ ⁺Represents an ammonium group, C represents a carbon atom, P represents a phosphorus atom, S represents a sulfur atom, and n represents-OCH₂CH₂-the number of the cells.

FIG. 2 shows the mice after injection of DSPE-PEG-Biotin, FIG. 2(a) shows the body weight change curve, FIG. 2(b) shows the liver weight change, and FIG. 2(c) shows the kidney weight change.

FIG. 3 shows the biochemical index change of blood (PLT, platelets; RBC, red blood cells; WBC, white blood cells; HGB, hemoglobin; Lymph, lymphocytes; MCH, mean corpuscular hemoglobin content, MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PDW, platelet distribution breadth; Gran, granulocytes) after injection of DSPE-PEG-Biotin in mice, and the results show no significant difference.

FIG. 4 shows the relationship between the detection of circulating extracellular vesicle biotinylation level by flow cytometry and the dosage of DSPE-PEG-Biotin.

Fig. 5 shows the effect of DSPE-PEG-Biotin on the appearance and particle size distribution of circulating extracellular vesicles, fig. 5(a) and 5(b) are transmission electron micrographs of circulating extracellular vesicles before and after labeling with DSPE-PEG-Biotin in sequence, fig. 5(c) is a particle size distribution diagram for NTA analysis, and fig. 5(d) is the average particle size of circulating extracellular vesicles before and after labeling with DSPE-PEG-Biotin.

FIG. 6 shows the effect of DSPE-PEG-Biotin labeling on circulating extracellular vesicle distribution expression; FIG. 6(a) is the result of flow cytometry, and FIG. 6(b) is the result of immunoblotting.

FIG. 7 shows the dynamic change of Biotin levels on circulating extracellular vesicles after DSPE-PEG-Biotin injection into the blood circulation.

FIG. 8 shows circulating extracellular vesicles isolated using streptavidin-coupled iron oxide nanoparticles after DSPE-PEG-Biotin injection into the blood circulation.

Detailed Description

The invention is further explained below with reference to examples and figures. The examples and drawings are merely illustrative and are not intended to limit the scope of the invention. It will be understood by those skilled in the art that certain well-known structures and descriptions thereof may be omitted from the embodiments and drawings.

The previous research shows that DSPE-PEG-Biotin is Biotin-functionalized phosphatidylethanolamine, can be assembled on a cell membrane phospholipid bilayer in a free state, and does not influence the biological behavior of the circulating extracellular vesicles. Therefore, a certain dose of DSPE-PEG-Biotin is injected into an animal body through a tail vein or an abdominal cavity, so that the efficient and lossless Biotin labeling of the circulating extracellular vesicles is realized in a living body, and the in-vivo metabolism time and the in-vivo metabolism rule of the circulating extracellular vesicles are firstly researched on the basis.

On the other hand, the invention solves the bottleneck that the circulating extracellular vesicles are difficult to separate. The most common method currently used to isolate circulating extracellular vesicles is ultracentrifugation. The ultracentrifugation method is time-consuming and labor-consuming, large ultracentrifugation equipment is needed without a standardized operation process, and the method is not favorable for clinical popularization and application; more importantly, the efficiency of ultracentrifugation for separating extracellular vesicles is less than 30%, resulting in a low yield of circulating extracellular vesicles. In addition, a great deal of research has proved that the circulating extracellular vesicles separated by ultracentrifugation are low in purity, and a great amount of protein polymers in peripheral blood are separated along with the extracellular vesicles during ultracentrifugation, so that the purity of the circulating extracellular vesicles is greatly interfered. The invention realizes the in-situ biotin labeling of the circulating extracellular vesicles by using a method for labeling the circulating extracellular vesicles with living bodies, and on the basis, the rapid magnetic separation of the circulating extracellular vesicles can be realized by specifically combining biotin with magnetic nanoparticles. Thereby avoiding a plurality of defects of the current ultracentrifugation and providing a simple and rapid method for separating the circulating extracellular vesicles.

DSPE-PEG-Biotin used in the present invention is available from Avanti Polar Lipids, Inc., CAS number: 385437-57-0, formula: c₁₄₂H₂₈₀N₅O₅₆PS, molecular weight: 3016.815. the phospholipid-polyethylene glycol-folic acid, the phospholipid-polyethylene glycol-fluorescent agent, the phospholipid-polyethylene glycol-nano particles, the phospholipid-polyethylene glycol-medicine and the phospholipid-polyethylene glycol-polypeptide are sequentially shown in figure 1The biotin is replaced by folic acid, fluorescent agent, nanoparticles, medicine, and polypeptide.

The term "non-medical purpose" of the present invention refers to "non-therapeutic purpose and non-diagnostic purpose".

The technical scheme of the invention is explained in detail by the following specific examples:

example 1

This example provides a method for in vivo labeling of circulating extracellular vesicles:

(1) preparing DSPE-PEG-Biotin injection: heating in 55 deg.C water bath to dissolve DSPE-PEG-Biotin (structural formula shown in figure 1) in dimethyl sulfoxide (DMSO), diluting with sterile 0.9% physiological saline by vortex, and storing at 4 deg.C.

(2) The C57BL/6 mice were divided, weighed, anesthetized and fixed, and then DSPE-PEG-Biotin solution was injected into C57BL/6 mice by tail vein injection according to dosage standards of 25mg/kg, 75mg/kg and 150mg/kg, the total volume of the injection solution per mouse was not more than 200. mu.L, and the control group was injected with 200. mu.L of sterile 0.9% physiological saline.

(3) The weight of the mice is weighed at different time points, 0.5-1mL of blood is taken from the inner canthus artery after the mice are anesthetized, and the main tissue organs are harvested after the mice are sacrificed.

(4) And (4) taking part of the blood obtained in the step (3) to carry out conventional blood analysis, embedding the tissues and organs, then slicing, dyeing the slices by a conventional hematoxylin-eosin (HE) method, and observing the slices by a common optical microscope. The results show that the in situ Biotin labeling method has no obvious influence on indexes such as the body weight (shown in figure 2(a)), the liver and kidney functions (shown in figure 2(b), figure 2(c)), the blood routine (shown in figure 3), the tissue morphology and the like of the mouse, and prove that the DSPE-PEG-Biotin has good biological friendliness.

(5) Centrifuging the blood obtained in the step (3) at 4 ℃ and 1550g for 20min to collect upper plasma, and repeating twice to remove cells and cell debris; centrifuging the supernatant at 4 deg.C for 2 hr under 20,000g condition, retaining the supernatant for use, precipitating to obtain circulating extracellular vesicles, resuspending with appropriate amount of sterile PBS, and storing at-80 deg.C.

(6) And (3) diluting the supernatant obtained in the step (5) by PBS, adding the diluted supernatant into a streptavidin (SA-FITC) solution (2.5 mu g/mL), incubating at room temperature for 10min, and measuring the fluorescence intensity at 511-515nm by using a fluorescence spectrophotometer, wherein the result shows that the fluorescence intensity is in positive correlation with the injection dose of the DSPE-PEG-Biotin, and the DSPE-PEG-Biotin can be effectively combined within 12 h.

(7) And (3) taking samples of main tissues and organs such as liver, kidney, heart and the like obtained in the step (3), labeling the samples with Cy3 labeled streptavidin (SA-Cy3), observing the samples under a fluorescence microscope, and calculating the relative fluorescence intensity by image J software. The results show that the free DSPE-PEG-Biotin is mainly distributed in the liver, the kidney, the heart and the like, and suggest that the DSPE-PEG-Biotin is mainly metabolized by the liver and the kidney.

(8) And (3) performing morphology and particle size characterization and membrane surface biomolecule and biotinylation efficiency detection on the circulating extracellular vesicles obtained in the step (5) by using a transmission electron microscope, a fluorescence microscope, a Nanoparticle Tracking Analysis (NTA) technology, an immunoblot and a flow cytometer, and displaying that the circulating extracellular vesicle surface biotinylation efficiency is in positive correlation with the injected DSPE-PEG-Biotin dose (figure 4). Furthermore, in situ biotinylation labels did not affect the morphology of circulating extracellular vesicles (see fig. 5(a), 5(b)), particle size (see fig. 5(c), 5(d)) and expression of characteristic molecules (fig. 6).

Example 2

The present example provides the application of a method for in vivo labeling of circulating extracellular vesicles in the study of biological behavior in circulating extracellular vesicles:

(1) preparing DSPE-PEG-Biotin injection: heating in water bath at 55 deg.C to dissolve DSPE-PEG-Biotin in DMSO, diluting with sterile salt solution by vortex, and storing at 4 deg.C.

(2) The mice were divided, weighed, anesthetized and fixed, and then the DSPE-PEG-Biotin solution was injected into C57BL/6 mice by tail vein injection at a dose of 150mg/kg, and the total volume of the solution per mouse was not more than 200. mu.L.

(3) Anesthetizing the mice at different time points, taking 0.5-1mL of blood through the inner canthus artery, centrifuging the obtained blood at 4 ℃ for 20min to collect upper plasma, and repeating twice to remove cells and cell debris; centrifuging the supernatant at 4 deg.C for 2h at 20,000g to obtain precipitate as circulating extracellular vesicle, resuspending with appropriate amount of sterile PBS, and storing at-80 deg.C.

(4) And (3) after the circulating extracellular vesicles obtained in the step (3) are marked by SA-FITC and flow antibodies aiming at different surface marker molecules, detecting the circulating life of the total circulating extracellular vesicles and the proportion and the circulating life of the circulating extracellular vesicles of each cell subset by using a flow cytometer. The results showed that the total circulating extracellular vesicle metabolism time was about 3 days (fig. 7); circulating extracellular vesicles of erythrocyte origin have a longer cycle life than extracellular vesicles of other cell origin.

Example 3

This example provides a method for rapidly isolating circulating extracellular vesicles from a living body:

(1) preparing DSPE-PEG-Biotin injection: heating the DSPE-PEG-Biotin in a metal bath at 55 ℃ to dissolve the DSPE-PEG-Biotin in DMSO, then diluting the DSPE-PEG-Biotin by using a sterile salt solution in a vortex mode to prepare a DSPE-PEG-Biotin salt solution, and storing the DSPE-PEG-Biotin salt solution at 4 ℃.

(2) The mice were grouped, weighed, anesthetized and fixed, and then the DSPE-PEG-Biotin solution was injected into C57BL/6 mice by tail vein injection according to dosage standards of 25mg/kg, 75mg/kg and 150mg/kg, and the total volume of the solution injected into each mouse was not more than 200. mu.L.

(3) The mouse living body is subjected to blood sampling of 0.5-1mL through an inner canthus artery, centrifugation is carried out for 20min twice at 4 ℃ and 1550g, cells and cell fragments are removed, supernatant is taken and added with a proper amount of streptavidin coupled iron oxide nanoparticle (SA-IONPs) solution (10 mug/muL), the mixed solution is gently mixed by a vortex instrument, then the mixed solution is incubated in an incubator at 37 ℃ for 28-32min, then a magnet (100 x 50 x 20mm, 0.6T) is used for separating circulating extracellular vesicles successfully labeled by SA-IONPs in the mixed solution, the circulating extracellular vesicles are resuspended and eluted for a plurality of times by PBS, finally the precipitate is resuspended by PBS and frozen at-80 ℃, and circulating extracellular vesicles with membrane surfaces modified by iron oxide nanoparticles are obtained (shown in figure 8).

The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.