CN113583954B - In-situ labeling and rapid separation method for circulating extracellular vesicles - Google Patents
In-situ labeling and rapid separation method for circulating extracellular vesicles Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
The invention discloses an in-situ labeling and rapid separation method for circulating extracellular vesicles, which comprises the steps of injecting a substance modified by phospholipid polyethylene glycol or a derivative thereof into a blood circulation system of a subject, and automatically assembling the substance modified by the phospholipid polyethylene glycol or the derivative thereof onto a cell membrane phospholipid bilayer of the circulating extracellular vesicles in a free state. The marking method can be applied to the aspects of monitoring the metabolism dynamics of the circulating extracellular vesicles, rapidly separating the circulating extracellular vesicles from living bodies and the like, and is beneficial to the basic research and clinical application of the circulating extracellular vesicles. Compared with the existing marking method, the method does not need to separate the circulating extracellular vesicles from peripheral blood in advance, can effectively avoid the damage of methods such as ultracentrifugation and the like to the circulating extracellular vesicles, and can keep the physical and chemical properties of the circulating extracellular vesicles to the maximum extent.
Description
Technical Field
The invention relates to the field of molecular biology and biotechnology, in particular to a method for labeling circulating extracellular vesicles (circulating extracellular vesicles, C-EVs) by a living body, and also relates to application of the method in aspects of monitoring the metabolism dynamics of the circulating extracellular vesicles, rapidly separating the circulating extracellular vesicles from the living body and the like, which is beneficial to basic research and clinical application of the circulating extracellular vesicles.
Background
Extracellular vesicles are vesicle-like bodies with a bilayer membrane structure, which are shed from the cell membrane or secreted by the cell, and have diameters between 40-1000nm, which are widely present in cell culture supernatants and in various body fluids (blood, lymph, saliva, urine, semen, milk). Extracellular vesicles in peripheral blood, also called circulating microvesicles (C-EVs), are extracellular vesicle mixtures secreted by various cells such as platelets, erythrocytes, lymphocytes, endothelial cells, etc. Extracellular vesicles inherit the biological information molecules rich in blast cells, such as proteins, lipids, DNA, mRNA, miRNA, and the like, and serve as intercellular information transfer carriers in various pathophysiological behaviors, and participate in the processes of intercellular communication, cell migration, angiogenesis, immune regulation, and the like. In addition, based on its natural cargo properties and good biosafety, extracellular vesicles are considered to be very promising gene or drug delivery vehicles for the treatment of a variety of diseases.
Despite the good prospects, the current knowledge about the basic properties of circulating extracellular vesicles is still severely deficient, especially in terms of their in vivo metabolism kinetics, tissue distribution and clearance, and comparison of different subtypes of extracellular vesicles, etc. are still under further investigation. At present, the research of biological behaviors in the circulating extracellular vesicles requires that the circulating extracellular vesicles are separated and purified by methods such as ultracentrifugation and the like, marked in vitro and then are input back into human bodies for subsequent research. Although this strategy plays an important role in studying the in vivo biological behavior of circulating extracellular vesicles, it has a number of drawbacks: 1. the strong centrifugal force of the in vitro ultracentrifugation separation can cause the structural damage of the circulating extracellular vesicles; 2. in vitro labeling strategies such as fluorescent dye labeling and quantum dot labeling, in vitro vector construction such as electrochemical transfection and the like can change the physical and chemical properties of the circulating extracellular vesicles, thereby influencing the objective removal efficiency of the circulating microvesicles; 3. the stability of the in vitro labeling strategy is poor, the optical bleaching property of fluorescent dyes such as DiI, diO, PKH and the like is strong, the light intensity is weak, the quantum dots are easy to leak, and the like, so that adverse effects such as signal loss or diffusion are caused; 4. in vitro labeling only allows characterization of the total circulating extracellular vesicles of the blood. Therefore, in order to study the metabolism dynamics and in-vivo distribution rule of the circulating extracellular vesicles, development of a method which is biologically friendly, simple, convenient and rapid and can realize nondestructive marking of the circulating extracellular vesicles is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides an in-situ labeling and rapid separation method of circulating extracellular vesicles, which can directly inject phospholipid-polyethylene glycol-Biotin (DSPE-PEG-Biotin) into a blood circulation system without lengthy centrifugal separation and static incubation in vitro, 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 characteristic molecular expression of the circulating extracellular vesicles; the in-situ labeling method is not based on an immunoaffinity mode, so that extracellular vesicles from different cells or different species can react with DSPE-PEG-Biotin, the application range is wide, and the method has universality. In addition, DSPE-PEG-Biotin has better biological friendliness and has no obvious toxic or side effect on main metabolic organs of organisms 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 non-medical in vivo labelling of circulating extracellular vesicles, comprising injecting a phospholipid polyethylene glycol or a derivative thereof modified substance into the blood circulation system of a subject, the phospholipid polyethylene glycol or derivative thereof modified substance self-assembling 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 derivative modified substance thereof for the preparation of a product for in vivo labelling of a circulating extracellular vesicle of a subject, the product for bringing the phospholipid polyethylene glycol or derivative modified substance into the blood circulation system.
Preferably, the subject is a human or non-human mammal.
As a preferable mode of the above technical scheme, the substance modified by phospholipid polyethylene glycol or its derivative is at least one of phospholipid-polyethylene glycol-biotin, phospholipid-polyethylene glycol-folic acid, phospholipid-polyethylene glycol-biotin-fluorescent agent, phospholipid-polyethylene glycol-nanoparticle, phospholipid-polyethylene glycol-drug, phospholipid-polyethylene glycol-polypeptide.
As a preferable mode of the above technical scheme, the amount of the phospholipid polyethylene glycol or the derivative modified substance entering the blood circulation system is 5-300 mg/kg.
Preferably, the phospholipid polyethylene glycol or the derivative modified substance thereof is injected into the non-human mammal or human body by intravenous injection or intraperitoneal injection.
Preferably, the above technical scheme is that the phospholipid polyethylene glycol or the derivative modified substance is dissolved in dimethyl sulfoxide and diluted with sterile saline to obtain the injection of the phospholipid polyethylene glycol or the derivative modified substance.
In a third aspect, the present invention provides a method for non-invasively tracking circulating extracellular vesicles in vivo for non-medical purposes, comprising the steps of:
(1) Injecting phospholipid-polyethylene glycol-biotin into a subject;
(2) At various time points, arterial blood is taken from the subject, and circulating extracellular vesicles are separated from the blood;
(3) Labeling circulating extracellular vesicles with streptavidin-conjugated fluorescent dye and flow-type antibodies to different differentiation antigens;
(4) Detecting the marked product of step (3) by using a flow cytometer.
Preferably, the step of separating the circulating extracellular vesicles from the blood comprises: centrifuging blood at 4deg.C and 1550g, collecting upper plasma, repeating at least twice to remove cells and cell debris; the supernatant was centrifuged at 20,000g at 4℃and the pellet obtained was the circulating extracellular vesicles.
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 fragments in the blood, adding streptavidin-coupled ferric oxide nano particles into the supernatant, uniformly mixing, and incubating at 37 ℃;
and (3) separating the circulating extracellular vesicles with the membrane surface modified by the ferric oxide nano particles by using magnet adsorption.
Compared with the prior art, the invention has the following advantages:
1. the method directly marks the circulating extracellular vesicles in the blood circulation system in the living body based on the membrane phospholipid replacement strategy, avoids the tedious and tedious method of in-vitro centrifugation and re-inputting into the body after fluorescent dye marking, has the advantages of simplicity, convenience, rapidness and high efficiency, effectively avoids the adverse effects of structural damage, fluorescent signals Yi Guangpiao and the like of the circulating extracellular vesicles, and is convenient for accurately researching the internal circulation rule of the circulating extracellular vesicles.
2. The in-situ labeling method for living bodies provided by the invention has strong biological friendliness, does not influence the physicochemical properties of the circulating extracellular vesicles while stably introducing the labeling agent on the surfaces of the circulating extracellular vesicles, has no obvious toxicity to main metabolic organs of human bodies such as livers, kidneys and the like, and is convenient for developing human body researches and clinical result 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 from other species or other body fluid sources.
Drawings
FIG. 1 shows the chemical structural formula of DSPE-PEG-Biotin, wherein O represents an oxygen atom, HO represents a hydroxyl group, N represents a nitrogen atom, NH represents an imino group, NH 2 Represents amino, NH 4 + Represents an ammonium radical, C represents a carbon atom, P represents a phosphorus atom, S represents a sulfur atom, and n represents-OCH 2 CH 2 -number of.
Fig. 2 shows the mice after injection of DSPE-PEG-Biotin, with fig. 2 (a) showing the weight change curve, fig. 2 (b) showing the liver weight change, and fig. 2 (c) showing the kidney weight change.
FIG. 3 shows the change in biochemical indicators of blood (PLT, platelets, RBC, red blood cells, WBC, white blood cells, HGB, hemoglobin, lymph, lymphocytes, MCH, mean red blood cell hemoglobin concentration, MCHC, mean red blood cell hemoglobin concentration, MCV, mean red blood cell volume, PDW, platelet distribution width, gran, granulocytes) of mice after injection of DSPE-PEG-Biotin, and shows no significant difference in results.
FIG. 4 shows the relationship between circulating extracellular vesicle biotinylation levels detected by flow cytometry and DSPE-PEG-Biotin doses.
FIG. 5 shows the effect of DSPE-PEG-Biotin on the appearance and particle size distribution of circulating extracellular vesicles, wherein FIG. 5 (a) and FIG. 5 (b) are transmission electron microscope images of circulating extracellular vesicles before and after DSPE-PEG-Biotin labeling, FIG. 5 (c) is a particle size distribution diagram of NTA analysis, and FIG. 5 (d) is the average particle size of circulating extracellular vesicles before and after DSPE-PEG-Biotin labeling.
FIG. 6 shows the effect of DSPE-PEG-Biotin markers on circulating extracellular vesicle distribution expression; FIG. 6 (a) shows the results of flow cytometry, and FIG. 6 (b) shows the results of immunoblotting.
FIG. 7 shows the dynamic changes in Biotin levels on circulating extracellular vesicles after injection of DSPE-PEG-Biotin into the blood circulation.
FIG. 8 shows the isolation of the resulting circulating extracellular vesicles using streptavidin-coupled iron oxide nanoparticles after injection of DSPE-PEG-Biotin into the blood circulation.
Detailed Description
The invention is further explained below with reference to examples and figures. Wherein the examples and drawings are for illustrative purposes only and are not intended to limit the scope of the present invention. It will be appreciated by those skilled in the art that certain well-known structures in the examples and figures and descriptions thereof may be omitted.
Our earlier studies found that DSPE-PEG-Biotin is a Biotin-functionalized phosphatidylethanolamine which can self-assemble onto the membrane phospholipid bilayer in the free state without affecting the biological behavior of circulating extracellular vesicles. Therefore, by injecting a certain dose of DSPE-PEG-Biotin into an animal body through a tail vein or an abdominal cavity, the high-efficiency and nondestructive Biotin marking of the circulating extracellular vesicles is realized in the living body, and the metabolism time and the rule in the circulating extracellular vesicles are studied for the first time on the basis.
On the other hand, the invention solves the bottleneck that the isolation of the circulating extracellular vesicles is difficult. The most common method of isolating circulating extracellular vesicles is currently ultracentrifugation. The ultracentrifugation method is time-consuming and labor-consuming, does not need large-scale ultracentrifugation equipment in a standardized operation flow, and is not beneficial to clinical popularization and application; more importantly, the ultracentrifugation method has an efficiency of separating extracellular vesicles of less than 30%, resulting in lower yields of circulating extracellular vesicles. In addition, a great deal of research has demonstrated that the purity of the circulating extracellular vesicles isolated by ultracentrifugation is low, and a great deal of protein polymers in peripheral blood are separated along with the extracellular vesicles during ultracentrifugation, thus greatly interfering with the purity of the circulating extracellular vesicles. The invention realizes the in-situ biotin marking of the circulating extracellular vesicles by using a method for marking the circulating extracellular vesicles by living bodies, and on the basis, the rapid magnetic separation of the circulating extracellular vesicles can be realized by specifically combining the biotin with the magnetic nano particles. Thus avoiding a plurality of defects of the current ultracentrifugation and providing a simple and rapid method for separating the circulating extracellular vesicles.
The DSPE-PEG-Biotin used in the invention is purchased from Avanti Polar Lipids company, CAS number: 385437-57-0, molecular formula: c (C) 142 H 280 N 5 O 56 PS, molecular weight: 3016.815. the phospholipid-polyethylene glycol-folic acid, phospholipid-polyethylene glycol-fluorescent agent, phospholipid-polyethylene glycol-nanoparticle, phospholipid-polyethylene glycol-drug and phospholipid-polyethylene glycol-polypeptide of the invention sequentially refer to substances in which biotin is replaced by folic acid, fluorescent agent, nanoparticle, drug and polypeptide in figure 1.
The term "non-medical purpose" of the present invention refers to "non-therapeutic purpose and non-diagnostic purpose".
The following describes the technical scheme of the present invention in detail through specific embodiments:
example 1
The present embodiment provides a method for in vivo labeling of circulating extracellular vesicles:
(1) Preparing DSPE-PEG-Biotin injection: the DSPE-PEG-Biotin (structural formula shown in figure 1) is dissolved in dimethyl sulfoxide (DMSO) by heating in water bath at 55deg.C, diluted with sterile 0.9% physiological saline by vortex, and stored at 4deg.C.
(2) C57BL/6 mice are grouped, weighed, anesthetized and fixed, then DSPE-PEG-Biotin solution is injected into the C57BL/6 mice according to the dosage standard of 25mg/kg, 75mg/kg and 150mg/kg in a tail vein injection mode, the total volume of the injected solution of each mouse is not more than 200 mu L, and 200 mu L of sterile 0.9% physiological saline is injected into a control group.
(3) Weighing the mice at different time points, taking 0.5-1mL of blood from the inner canthus artery after anesthetizing the mice, and harvesting main tissues and organs after sacrifice.
(4) Taking part of blood obtained in the step (3), performing conventional blood analysis, embedding tissues and organs, slicing, staining the slices by using a hematoxylin-eosin staining method (HE), and observing the stained slices by using a common optical microscope. The results show that the in-situ Biotin labeling method has no obvious influence on the indexes of body weight (see fig. 2 (a)), liver and kidney functions (see fig. 2 (b), fig. 2 (c)), blood routine (fig. 3), tissue morphology and the like of the mice, and proves 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 the steps twice to remove cells and cell fragments; centrifuging the supernatant at 4deg.C for 2 hr at 20,000g, retaining the supernatant for use, and standing to obtain precipitate which is the circulating extracellular vesicle, re-suspending with appropriate amount of sterile PBS, and storing at-80deg.C.
(6) The supernatant obtained in the step (5) is diluted by PBS and added into a fluorescein isothiocyanate labeled streptavidin (SA-FITC) solution (2.5 mug/mL), and incubated for 10min at room temperature, and the fluorescence intensity is measured at 511-515nm by a fluorescence spectrophotometer, so that the result shows that the fluorescence intensity is positively correlated with the injection dose of DSPE-PEG-Biotin, and the DSPE-PEG-Biotin can be effectively combined within 12 h.
(7) Taking samples of major tissues and organs obtained in the step (3), such as liver, kidney, heart and the like, labeling the samples with Cy 3-labeled streptavidin (SA-Cy 3), observing the samples under a fluorescence microscope, and calculating the relative fluorescence intensity by image J software. The results showed that free DSPE-PEG-Biotin was mainly distributed in liver, kidney, heart, etc., suggesting that DSPE-PEG-Biotin is mainly metabolized by liver, kidney, etc.
(8) Characterization of morphology and particle size, detection of membrane surface biomolecules and biotinylation efficiency of the circulating extracellular vesicles obtained in step (5) was performed by using transmission electron microscopy, fluorescence microscopy, nanoparticle Tracking Analysis (NTA) technology, immunoblotting and flow cytometry, and the results showed that the biotinylation efficiency of the circulating extracellular vesicles was positively correlated with the injected DSPE-PEG-Biotin dose (fig. 4). Furthermore, the in situ biotinylated markers 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 the characteristic molecules (fig. 6).
Example 2
The present embodiment provides an application of a method for in vivo labeling of circulating extracellular vesicles in research of biological behaviors in circulating extracellular vesicles:
(1) Preparing DSPE-PEG-Biotin injection: the DSPE-PEG-Biotin is dissolved in DMSO by heating in a water bath at 55 ℃, and the DSPE-PEG-Biotin is diluted by vortexing with sterile saline solution and stored at 4 ℃.
(2) The mice were grouped, weighed, anesthetized and fixed, and then DSPE-PEG-Biotin solution was injected into C57BL/6 mice by tail vein injection at a dose level of 150mg/kg, with the total volume of solution injected per mouse not exceeding 200 μl.
(3) Collecting upper plasma by centrifuging at 4deg.C and 1550g for 20min after anesthetizing mice at different time points, and repeating twice to remove cells and cell fragments; centrifuging the supernatant at 4deg.C for 2 hr at 20,000g, precipitating to obtain circulating extracellular vesicles, re-suspending with sterile PBS, and storing at-80deg.C.
(4) 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, the proportion of the circulating extracellular vesicles of each cell subset and the circulating life by a flow cytometer. The results show that total circulating extracellular vesicle metabolism time was about 3 days (fig. 7); the circulating extracellular vesicles of erythrocyte origin have a longer circulating life than the extracellular vesicles of other cell origin.
Example 3
The present example provides a method for rapid isolation of circulating extracellular vesicles from a living body:
(1) Preparing DSPE-PEG-Biotin injection: dissolving DSPE-PEG-Biotin in DMSO by heating in 55deg.C metal bath, diluting DSPE-PEG-Biotin by vortex with sterile saline solution, preparing into DSPE-PEG-Biotin saline solution, and preserving at 4deg.C.
(2) The mice were grouped, weighed, anesthetized and fixed, and then DSPE-PEG-Biotin solution was injected into C57BL/6 mice by tail vein injection at a dose level of 25mg/kg, 75mg/kg, 150mg/kg, with the total volume of injected solution per mouse not exceeding 200. Mu.L.
(3) The living body of the mice is subjected to blood collection through inner canthus artery for 0.5-1mL, centrifugation is carried out at 4 ℃ for 20min for two times to remove cells and cell fragments, a proper amount of solution (10 mug/mug) of streptavidin-coupled ferric oxide nano particles (SA-IONPs) is added into the supernatant, the mixture is gently mixed by a vortex instrument, then the mixture is incubated for 28-32min in a 37 ℃ incubator, then a magnet (100X 50X 20mm, 0.6T) is used for separating the circulating extracellular vesicles marked successfully by SA-IONPs in the mixture, the mixture is resuspended and eluted for a plurality of times by PBS, finally the sediment is resuspended by PBS and frozen at-80 ℃ to obtain the circulating extracellular vesicles with the membrane surface modified by the ferric oxide nano particles (shown in figure 8).
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the 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.
Claims (1)
1. A method for rapid isolation of circulating extracellular vesicles, comprising the steps of:
injecting phospholipid-polyethylene glycol-biotin into a subject; the amount of the phospholipid-polyethylene glycol-biotin entering the blood circulation system is 5-300 mg/kg; the subject is a non-human mammal;
taking blood from the artery of the subject within 12 h-3 days after injection; centrifuging blood, removing cells and cell fragments in the blood, adding streptavidin-coupled ferric oxide nano particles into the supernatant, uniformly mixing, and incubating at 37 ℃;
and (3) separating the circulating extracellular vesicles with the membrane surface modified by the ferric oxide nano particles by using magnet adsorption.
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