Preparation method of reusable crown ether modified magnetic beads and application of reusable crown ether modified magnetic beads in exosome extraction

Abstract

The invention discloses a preparation method of reusable crown ether modified magnetic beads and application of the reusable crown ether modified magnetic beads in exosome extraction.The preparation method comprises the steps that EDC is added into carboxylated magnetic graphene oxide, NHS is added after stirring, and a mixed solution is obtained after stirring; and adding an aqueous solution of crown ether into the mixed solution, carrying out amidation reaction, and after the reaction is finished, carrying out magnetic suction washing and drying to obtain the reusable crown ether modified magnetic beads. According to the method, MGO is used as a substrate and has high magnetism, and crown ether modified magnetic beads have broad-spectrum capturing capacity, are not limited by specific membrane protein and can be combined with general phospholipid bilayers of various exosomes. The magnetic beads recovered after the exosome is captured and separated can be repeatedly used, so that the cost is reduced. The exosome capturing magnetic bead is convenient to extract and short in time consumption, the purity of the extracted exosome is high, and the biological activity of the exosome is not damaged.

Description

Technical Field

[0001] The present invention belongs to the field of nano-biomaterials and technologies, and particularly relates to a method for preparing reusable crown ether-modified magnetic beads and an application thereof in exosome extraction. Background Art

[0002] Exosomes are nanoscale vesicles (30-150 nm in diameter) released by the fusion of intracellular multivesicular bodies (MVBs) with the cell membrane. They possess a typical phospholipid bilayer structure. Their surfaces are rich in markers such as tetraspanins (CD9 / CD63 / CD81), heat shock proteins (HSP70 / HSP90), and integrins, while their interiors carry functional proteins, mRNA, miRNA, and DNA fragments. According to the International Society for Extracellular Vesicles (ISEV) guidelines (MISEV2018), exosomes must be triple-validated by transmission electron microscopy (TEM) for morphological observation, nanoparticle tracking analysis (NTA) for particle size measurement, and Western blot for marker protein detection. Exosomes, as key mediators of intercellular communication, participate in the following physiological and pathological processes: 1) Disease diagnosis: Tumor-derived exosomes carry biomarkers such as PD-L1 and EGFRvIII, making them valuable in liquid biopsies; 2) Drug delivery: The natural targeting properties of exosomes make them ideal vehicles for siRNA / mRNA delivery; and 3) Tissue repair: Mesenchymal stem cell (MSC) exosomes promote angiogenesis by delivering miR-21, VEGF, and other proteins. Therefore, the isolation and purification of exosomes is a challenging task.

[0003] Existing technologies for isolating exosomes:

[0004] (1) Traditional ultracentrifugation: As the gold standard method for exosome isolation, differential centrifugation (300g→200g→10,000g→100,000g) is used to gradually remove cell debris and finally ultracentrifuge the exosomes. However, this method is highly equipment-dependent (ultracentrifuge is required, costing more than $100,000 USD); the recovery rate is low (30-50%), and high-speed centrifugation leads to exosome aggregation; there are many co-precipitated pollutants (such as lipoproteins and protein aggregates); and the integrity of exosomes is easily destroyed.

[0005] (2) Commercial polymer precipitation reagents (such as PEG 8000): PEG dehydration is used to precipitate exosomes from the solution, but the purity is extremely poor (impurity protein accounts for >60%); subsequent purification steps (such as density gradient centrifugation) are required, and the operation is cumbersome; PEG residues interfere with downstream analysis (such as a decrease in the signal-to-noise ratio of mass spectrometry detection).

[0006] (3) Size exclusion chromatography (SEC): separation is achieved based on the particle size of exosomes through a porous gel column; however, the sample is severely diluted (the recovery volume is increased by 3-5 times); the column efficiency is easily clogged (viscous samples such as plasma require pre-filtration); and the equipment throughput is low (single processing volume <1 mL).

[0007] (4) Immunoaffinity capture method: by coupling antibodies to magnetic beads, exosomes are captured by antigen-antibody binding specificity; or by modifying the surface with aptamers, exosomes are captured by phosphatidylserine binding specificity. However, antibodies are expensive and require large amounts of antibody. After antibody coupling, activity is easily lost (activity retention is usually <60%); reusability is poor (efficiency drops by >50% after ≤3 times); and elution conditions are harsh (low pH or high salt can easily destroy the integrity of the exosome membrane).

[0008] (5) Microfluidics: Exosomes are separated on a chip through acoustic waves, electric fields, or nanostructures; however, the equipment is highly customized and difficult to scale up; the processing volume is limited (μL-level samples) and the reproducibility is poor (CV>15%).

[0009] (6) Patents CN 118258991 A and CN 117095895 A have problems in actual application, such as insufficient process reproducibility and low raw material utilization. These technical bottlenecks have caused the pilot-scale scale-up costs of the patented technology to exceed the expected value by 38%, seriously restricting its industrial application prospects. Summary of the Invention

[0010] To overcome the high cost problem caused by the non-reusability of kits for isolating exosomes in the prior art, the present invention aims to construct a method for preparing reusable crown ether-modified magnetic beads and their application in exosome extraction. In the absence of specialized equipment, exosomes can be harvested from cell culture supernatant at low cost and with reduced contamination by impurity proteins. The magnetic beads can be reused, effectively reducing costs.

[0011] To achieve the above-mentioned purpose, the technical solution provided by the present invention is:

[0012] A method for preparing reusable crown ether-modified magnetic beads comprises the following steps:

[0013] Adding EDC to the carboxylated magnetic graphene oxide, stirring, then adding NHS, and stirring to obtain a mixed solution;

[0014] An aqueous solution of crown ether is added to the mixture to carry out an amidation reaction. After the reaction is completed, the beads are washed by magnetic absorption and dried to obtain reusable crown ether-modified magnetic beads.

[0015] Furthermore, the mass ratio of carboxylated magnetic graphene oxide to EDC is 1:5-20; the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:5-20.

[0016] Furthermore, the stirring time after adding EDC to the carboxylated magnetic graphene oxide is at least 30 minutes, and the stirring time after adding NHS is at least 30 minutes.

[0017] Furthermore, the molar ratio of the crown ether to EDC is 1:1-50; the molar ratio of the crown ether to NHS is 1:1-50; and the molar ratio of the crown ether to carboxylated magnetic graphene oxide is 20:1-10.

[0018] Furthermore, carboxylated magnetic graphene oxide is prepared by the following process: dispersing magnetic graphene oxide in pure water by ultrasonication, then adding sodium hydroxide and chloroacetic acid, performing bath ultrasonication, and washing by magnetic adsorption to obtain carboxylated magnetic graphene oxide.

[0019] Furthermore, the mass ratio of magnetic graphene oxide to sodium hydroxide is 1:20-50, the mass ratio of magnetic graphene oxide to chloroacetic acid is 1:20-30, and the bath ultrasonic reaction time is at least 2 hours.

[0020] A reusable crown ether-modified magnetic bead.

[0021] Application of reusable crown ether-modified magnetic beads in the extraction of exosomes.

[0022] An exosome extraction kit comprises reusable crown ether-modified magnetic beads and an eluent.

[0023] A method for extracting exosomes, comprising the following steps:

[0024] Reusable crown ether-modified magnetic beads were added to the exosome sample and incubated in an ice bath to obtain a magnetic bead-exosome complex;

[0025] After the supernatant in the magnetic bead-exosome complex was removed by magnetic attraction, the magnetic bead-exosome complex was redispersed in pure water, the exosomes were eluted by ultrasound, and then magnetic separation was performed to obtain exosomes.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] In the present invention, the oxygen atom on the crown ether ring has a high electronegativity, forming a dipole. +) carries a positive charge. This interaction between positive and negative charges, known as the ion-dipole interaction, is the primary force driving the binding of crown ethers to phospholipids. Experimental data indicate that the binding energy of this interaction is approximately 15-25 kJ / mol, indicating a considerable binding strength. Through this ion-dipole interaction, crown ethers can stably bind to the phospholipid headgroups on the surface of the exosome membrane, potentially influencing the structure and function of the exosome. The ring cavity size of the crown ether matches the effective diameter of the phosphocholine headgroup, allowing guest molecules to fit well within the crown ether ring cavity, thereby minimizing steric hindrance and increasing the stability of the system. The presence of methylene (-CH2-) units in the crown ether molecule may generate hydrophobic interactions with the alkyl chains of the phospholipid molecules. Although this interaction may be relatively weak, it still acts as a secondary factor to enhance the binding between the crown ether and the exosome membrane. Using the method of the present invention to extract exosomes, exosomes measuring 30-150 nm can be obtained. Furthermore, the method utilizes magnetic nanoparticles, which are low-cost, fast to prepare, and possess strong magnetic properties, enabling complete separation from the solution within 30 seconds. The present invention utilizes MGO as a substrate, which has strong magnetic properties. The reusable crown ether-modified magnetic beads have a broad-spectrum capture capability, are not limited to specific membrane proteins, and can bind to the universal phospholipid bilayer of various exosomes. The magnetic beads recovered after capturing and separating exosomes can be reused, reducing costs. At the same time, it avoids the need for expensive equipment during the extraction process and has good application prospects in the field of exosome extraction. The crown ether-phospholipid complex dissociation constant KD≈10 -4 M, only mild salt solution (such as 0.5 mol / L NaCl solution) is needed to release exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the SEM image of MGO-crown ether;

[0029] Figure 2 TEM images of exosomes;

[0030] Figure 3 Zeta potential diagram of exosomes. DETAILED DESCRIPTION

[0031] To facilitate understanding of the present invention, the present invention will be described more fully below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present invention. However, the present invention may be implemented in a variety of different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present invention.

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and biological materials described are commercially available unless otherwise specified.

[0033] 1. Preparation of crown ether modified magnetic beads

[0034] 1. Preparation of magnetic graphene oxide by hydrothermal method

[0035] A single layer of graphene oxide is dispersed in ethylene glycol to obtain a graphene oxide solution, to which ferric chloride, sodium acetate and polyethylene glycol are added. After mixing and stirring, a hydrothermal reaction (high temperature and high pressure reaction) is carried out. After the reaction is completed, the product is collected by centrifugation and magnetically washed with ethanol and pure water in sequence to obtain magnetic graphene oxide.

[0036] 2. Preparation of carboxylated magnetic graphene oxide

[0037] The magnetic graphene oxide obtained in step (1) is ultrasonically dispersed in pure water, and then sodium hydroxide and chloroacetic acid are added to react in a bath ultrasonic manner. After the reaction is completed, ethanol and pure water are used for magnetic washing in sequence to obtain carboxylated magnetic graphene oxide.

[0038] 3. Crown ether coupling

[0039] EDC is added to the carboxylated magnetic graphene oxide obtained in step (2) to activate the carboxyl groups, and then NHS is added and reacted in an ice bath to obtain a mixed solution. Crown ether is dissolved in pure water and added to the mixed solution to undergo amidation reaction. After the reaction, the crown ether is washed with pure water by magnetic suction and vacuum dried to obtain crown ether modified magnetic beads, i.e., MGO-crown ether magnetic beads.

[0040] Furthermore, in step (1), the mass ratio of the monolayer graphene oxide to ferric chloride is 1:2-10; the mass ratio of the monolayer graphene oxide to sodium acetate is 1:20-80; the mass ratio of the monolayer graphene oxide to polyethylene glycol is 1:1-10; the mixture is stirred for at least 1 hour; after stirring, the reaction vessel (Teflon high-temperature and high-pressure reactor) is placed in a forced air drying oven for reaction at a reaction temperature of 180-200°C for at least 6 hours.

[0041] Furthermore, in step (2), the mass ratio of the magnetic graphene oxide to sodium hydroxide is 1:20-50, the mass ratio of the magnetic graphene oxide to chloroacetic acid is 1:20-30, and the bath ultrasonic reaction is carried out for at least 2 hours.

[0042] Furthermore, in step (3), the mass ratio of the carboxylated magnetic graphene oxide to EDC is 1:5-20; the mass ratio of the carboxylated magnetic graphene oxide to NHS is 1:5-20; the reaction is carried out for at least 30 minutes after the addition of EDC, and the reaction is carried out for at least 30 minutes after the addition of NHS; and the ratio of the amount of crown ether to the carboxylated magnetic graphene oxide obtained in step (2) is 20:1-10.

[0043] 2. Exosome Extraction Method

[0044] The crown ether-modified magnetic beads prepared by the above method can capture exosomes.

[0045] 1. Initial isolation of exosomes

[0046] Collect cell culture supernatant, blood, urine, or other biological fluids and centrifuge at 3000g for 10 minutes. Discard the pellet (to remove nuclei, heavy mitochondria, and large cell debris) and collect the supernatant. Centrifuge the collected supernatant again at 10,000g for 10-40 minutes. Discard the pellet (to remove nuclei, heavy mitochondria, and large cell debris) and collect the supernatant. Aspirate the supernatant and filter it through a 0.22μm syringe filter to exclude molecules larger than 0.22μm. This will yield the primary exosome sample and store it at 4°C until exosome isolation.

[0047] 2. Magnetic adsorption of exosomes using MGO-crown ether

[0048] Take 200-800 μL (1-4 mg) of crown ether modified magnetic beads and 1-5 mL of the exosome sample initially isolated in step (1) and incubate them in an ice bath for 10-30 min.

[0049] 3. Exosome elution

[0050] After magnetically removing the supernatant, redisperse the magnetic beads in the tube in 1-5 mL of pH 7.5 wash buffer and sonicate for 10-30 minutes to elute the exosomes. Remove the beads using a magnet and recover the exosomes from the supernatant. Recovered exosomes are filtered through a 0.22 μm filter, placed in cryovials, and stored at -80°C.

[0051] This method achieves efficient separation and recovery of exosomes through specific capture by magnetic beads combined with gentle elution.

[0052] 4. Magnetic bead recycling

[0053] The magnetic beads were washed with ethanol and pure water in sequence, stored in pure water, and the efficiency remained >80% after 10 cycles.

[0054] 3. An exosome extraction kit

[0055] The kit includes reusable crown ether modified magnetic beads and water with a pH of 7.5.

[0056] The magnetic beads of the present invention exhibit excellent dispersion stability (hydrated diameter 150±20nm, PDI<0.15) in aqueous media (including deionized water, PBS buffer and cell culture medium) through an optimized crown ether co-modified interface, while maintaining high magnetic responsiveness (saturation magnetization intensity ≥45emu / g), and can achieve rapid separation under the action of a conventional magnetic frame (recovery rate >98% within 30 seconds). The crown ether cavity selectively binds to the choline group of the exosome membrane phospholipids through host-guest interaction (binding constant Ka=10 3 -10 4 M-1), while the steric hindrance of the ring structure effectively inhibits nonspecific adsorption of serum proteins (reduced by 62±5% compared to traditional magnetic beads). Gentle exosome release is achieved by treatment with 0.1 mol / L KCl solution for 3 minutes. After 10 regeneration cycles, the magnetic beads maintain over 92% of their initial capture efficiency, and exosome integrity is >95% (verified by nanoflow cytometry).

[0057] The exosome capture function of crown ether modified magnetic beads is that crown ether, due to its cavity structure, can selectively bind to the choline groups on the phospholipids of exosomes through the host-guest recognition mechanism. The affinity of this binding process is high, and the binding constant (Ka) ranges from 10 3 -10 4 M-1. The presence of methylene (-CH2-) units in the crown ether molecule generates hydrophobic interactions, enhancing binding between the crown ether and the exosome membrane. Furthermore, the steric hindrance created by the crown ether's cyclic structure significantly reduces the nonspecific adsorption of serum proteins, reducing nonspecific adsorption by 62±5% compared to traditional magnetic beads, demonstrating the method's enhanced specificity in complex biological matrices.

[0058] The exosome separation capability of crown ether-modified magnetic beads lies in the ability to induce gentle dissociation or release of exosomes from the magnetic bead surface under mild dissociation conditions, using 0.1 mol / L potassium chloride solution or pH 7.5 buffer for less than 15 minutes. This method provides a simple and non-destructive means for enriching or isolating cellular exosomes.

[0059] The reusable crown ether-modified magnetic bead system's reusability lies in its ability to combine the superparamagnetic properties of Fe₃O₄ with the beads' reusability and structural stability. The magnetic bead-based enrichment strategy for targeted exosomes has been demonstrated to possess excellent reusability. After 10 consecutive regeneration cycles, the capture efficiency of the magnetic beads remained above 92% of the initial value, demonstrating the method's excellent stability and durability. Furthermore, analysis using nanoflow cytometry revealed that the integrity of the exosomes remained above 95%, indicating that the regeneration process had minimal impact on the structure and function of the enriched exosomes, demonstrating their excellent stability and durability. These results confirm the feasibility and reliability of this material for practical applications.

[0060] Principle of the present invention:

[0061] The present invention prepares magnetic beads based on a graphene oxide substrate, resulting in a magnetic bead system with a high surface area and multiple binding sites. Crown ethers are introduced through a chemical reaction to construct a novel magnetic bead system that can rapidly and efficiently isolate exosomes from cell culture supernatants, reduce impurity adsorption, and release the exosomes in a manner that does not compromise the exosome's intrinsic activity, enabling their use in subsequent related applications. Furthermore, the resulting magnetic bead system exhibits stability and reproducibility. The principle of selective binding is a key mechanism for the application of crown ethers in exosome targeting. This principle is based on the unique molecular structure of crown ethers and their high affinity for specific phospholipid molecules. Specifically, the cyclic structure of crown ethers forms a cavity of a specific size that matches the diameter of the phospholipid headgroups in the exosome membrane. For example, the headgroup diameter of phosphatidylcholine (PC) is approximately 0.45 nm, which exhibits good steric complementarity with certain crown ether cavities of specific sizes. This steric complementarity enables crown ethers to form stable complexes with phospholipid headgroups through host-guest interactions. Host-guest chemistry refers to the process in which a host molecule (such as a crown ether) binds to a guest molecule (such as a phospholipid head group) through non-covalent bonds. + ) is the primary driver of binding. This interaction involves electrostatic attraction between the negatively charged ring formed by the crown ether's oxygen atoms and the positively charged quaternary ammonium groups. Experimental data indicate that the binding energy of this ion-dipole interaction is typically between 15-25 kJ / mol, sufficient to ensure the formation of a stable complex between the crown ether and specific phospholipid molecules, thereby achieving selective targeting to exosomes. Furthermore, other non-covalent interactions, such as van der Waals forces, may also influence the binding of crown ethers to phospholipids, contributing to the stable formation of the complex. By rationally designing the structure and size of crown ethers, their affinity for specific phospholipids on the exosome membrane can be further optimized, improving the specificity and efficiency of targeting.

[0062] The following are specific examples.

[0063] Example 1: A method for preparing reusable crown ether-modified magnetic beads, comprising the following steps:

[0064] (1) Preparation of magnetic graphene oxide. A single layer of graphene oxide was dissolved in ethylene glycol solvent and added to a Teflon high temperature and high pressure reactor. Ultrasonic dispersion was performed for 8 minutes. Then, ferric chloride, sodium acetate, and polyethylene glycol 8000 were added. After mixing and stirring for 3 hours, the reactor was transferred to a forced air drying oven and reacted at 200°C for 10 hours. After the reaction, the product was magnetically washed three times with ethanol and pure water respectively, and vacuum dried at 60°C to obtain magnetic graphene oxide.

[0065] The mass ratio of the monolayer graphene oxide to ferric chloride is 1:2, the mass ratio of the monolayer graphene oxide to sodium acetate is 1:25, and the mass ratio of the monolayer graphene oxide to polyethylene glycol 8000 is 1:1.5.

[0066] (2) Preparation of carboxylated magnetic graphene oxide: The magnetic graphene oxide obtained in step (1) was ultrasonically dispersed in pure water for 8 minutes, and then sodium hydroxide and chloroacetic acid were added and subjected to ultrasonic bath reaction for 2 hours. After the reaction, the mixture was washed with ethanol and pure water in sequence to obtain carboxylated magnetic graphene oxide.

[0067] The mass ratio of the magnetic graphene oxide to the sodium hydroxide is 1:35, and the mass ratio of the magnetic graphene oxide to the chloroacetic acid is 1:25.

[0068] (3) Crown ether coupling: EDC was added to the carboxylated magnetic graphene oxide obtained in step (2) and the reaction was continued for 30 min. NHS was then added and the reaction was continued in an ice bath for 1 h. After the reaction, the solution was washed with pure water by magnetic suction to obtain a magnetic bead solution. The crown ether was dissolved in pure water and then added to the magnetic bead solution. The solution was placed on a shaker and amidation reaction was carried out for 24 h. After the reaction, the solution was washed with pure water by magnetic suction and vacuum dried to obtain crown ether-modified magnetic beads.

[0069] Among them, the mass ratio of carboxylated magnetic graphene oxide to EDC is 1:15, the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:10, and the mass ratio of crown ether to carboxylated magnetic graphene oxide is 20:2.

[0070] The SEM characterization of the prepared magnetic beads can be found in Figure 1 It can be seen that the graphene-grafted material crown ether is rich in amino groups, and the EDS results show that the MGO-crown ether surface has a uniform N distribution, which proves that the crown ether is successfully bound to MGO-COOH through amidation reaction, and the binding of the crown ether is relatively uniform.

[0071] Example 2: A method for extracting extracellular vesicles, comprising the following steps:

[0072] (1) Initial isolation of exosomes: Breast cancer cell culture supernatant was collected and centrifuged at 3000 g for 10 min. The precipitate (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The collected supernatant was centrifuged at 10000 g for 30 min. The precipitate (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The supernatant was aspirated and filtered with a 0.22 μm syringe filter to exclude molecules larger than 0.22 μm and stored in a refrigerator at 4 °C before exosome isolation.

[0073] (2) Extracting exosomes using crown ether-modified magnetic beads: 500 μL (3 mg) of crown ether-modified magnetic beads and 1 mL of the exosome sample initially isolated in step (1) were incubated in an ice bath for 20 min to obtain a magnetic bead-exosome complex.

[0074] (3) Exosome elution: After removing the supernatant by magnetic aspiration, the magnetic bead-exosome complex in the test tube was redispersed in 1 mL of pH 7.5 pure water and the exosomes were eluted by sonication for 20 min. The magnetic beads were removed using a magnet and the exosomes were recovered from the supernatant. The recovered exosomes were filtered through a 0.22 μm filter membrane and stored in cryovials at -80°C.

[0075] (4) Magnetic bead recycling: Use ethanol and pure water to magnetically wash the magnetic beads in turn, store them in pure water, and the efficiency remains >80% after recycling 10 times.

[0076] Identification of exosomes:

[0077] (1) Exosomes obtained by electron microscopy:

[0078] 10 μL of the exosome extract obtained in Example 2 was added dropwise to a copper mesh, adsorbed at room temperature for 10 minutes, and excess liquid was removed with filter paper. 10 μL of a 2% phosphotungstic acid solution was then added dropwise to the copper mesh, and the mesh was stained at room temperature for 2 minutes. Excess liquid was removed with filter paper, and the mesh was air-dried at room temperature. The observation voltage was set to 120 kV, and transmission electron microscopy was performed. It was observed that the obtained extracellular vesicles had a uniform particle size and a double membrane conformation, which was consistent with the morphology of exosomes. TEM characterization of the exosomes obtained and separated can be found in Figure 2 , it can be seen that the diameter of the separated exosomes is about 102 nm, the biological morphology is complete, the exosomes present a typical cup-shaped or biconcave disc-shaped structure, the membrane structure is complete and the boundaries are clear.

[0079] (2) Identification and extraction of exosomes using Zeta potential analyzer:

[0080] The exosome proteins extracted in Example 2 were identified. The extracted exosomes were diluted to 10 8 -10 9Particles / mL (protein concentration determined by BCA assay is approximately 0.1-1 μg / μL). Use a dedicated folded capillary electrophoresis cell (DTS1070) or a transparent disposable electrode cup to inject the sample until the electrode is submerged (approximately 1 mL). Avoid air bubbles (which can be eliminated by gently tapping the cuvette). Let the sample stand for 2 minutes to stabilize the temperature. The zeta potential is measured using a zeta potential meter. The zeta potential of the exosomes obtained and separated is characterized as follows: Figure 3 , it can be seen that the Zeta potential of the exosomes after separation is -23.3 mV. The Zeta potential of exosomes in the literature is distributed between -10 and -25 mV, so the Zeta potential of the product is consistent with the results in the literature.

[0081] It can be seen that the exosome capture magnetic beads prepared by the present invention can effectively extract exosomes with high purity, and the extraction and separation methods adopted will not cause damage to the exosomes. The extracted exosomes are intact in morphology and can be used for downstream applications.

[0082] Example 3: A method for preparing crown ether modified magnetic beads, comprising the following steps:

[0083] (1) Preparation of magnetic graphene oxide: Monolayer graphene oxide was dissolved in ethylene glycol solvent, added to a Teflon high-temperature and high-pressure reactor, and ultrasonically dispersed for 8 minutes. Then, ferric chloride, sodium acetate and polyethylene glycol 8000 were added and mixed and stirred for 2 hours. The reactor was transferred to a forced air drying oven and reacted at 180°C for 12 hours. After the reaction was completed, it was magnetically washed three times with ethanol and pure water respectively, and vacuum dried at 60°C to obtain magnetic graphene oxide.

[0084] The mass ratio of the single-layer graphene oxide to ferric chloride is 1:10, the mass ratio of the single-layer graphene oxide to sodium acetate is 1:80, and the mass ratio of the single-layer graphene oxide to polyethylene glycol 8000 is 1:1.

[0085] (2) Preparation of carboxylated magnetic graphene oxide: The magnetic graphene oxide obtained in step (1) was ultrasonically dispersed in pure water for 8 minutes, and then sodium hydroxide and chloroacetic acid were added and subjected to ultrasonic bath reaction for 2 hours. After the reaction, the mixture was washed with ethanol and pure water in sequence to obtain carboxylated magnetic graphene oxide.

[0086] The mass ratio of the magnetic graphene oxide to the sodium hydroxide is 1:20, and the mass ratio of the magnetic graphene oxide to the chloroacetic acid is 1:30.

[0087] (3) Crown ether coupling: EDC was added to the carboxylated magnetic graphene oxide obtained in step (2) and the reaction was continued for 30 min. NHS was then added and the reaction was continued in an ice bath for 1 h. After the reaction, the solution was washed with pure water by magnetic suction to obtain a magnetic bead solution. The crown ether was dissolved in pure water and then added to the magnetic bead solution. The solution was placed on a shaker and amidation reaction was carried out for 24 h. After the reaction, the solution was washed with pure water by magnetic suction and vacuum dried to obtain crown ether-modified magnetic beads.

[0088] The mass ratio of carboxylated magnetic graphene oxide to EDC is 1:15, the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:10, and the mass ratio of crown ether to carboxylated magnetic graphene oxide is 20:1.

[0089] Example 4: A method for preparing crown ether modified magnetic beads, comprising the following steps:

[0090] (1) Preparation of magnetic graphene oxide. A single layer of graphene oxide was dissolved in ethylene glycol solvent and added to a Teflon high temperature and high pressure reactor. Ultrasonic dispersion was performed for 8 minutes. Then, ferric chloride, sodium acetate, and polyethylene glycol 8000 were added. After mixing and stirring for 1 hour, the reactor was transferred to a forced air drying oven and reacted at 200°C for 6 hours. After the reaction, the product was magnetically washed three times with ethanol and pure water respectively, and vacuum dried at 60°C to obtain magnetic graphene oxide.

[0091] The mass ratio of the single-layer graphene oxide to ferric chloride is 1:5, the mass ratio of the single-layer graphene oxide to sodium acetate is 1:50, and the mass ratio of the single-layer graphene oxide to polyethylene glycol 8000 is 1:10.

[0092] (2) Preparation of carboxylated magnetic graphene oxide: The magnetic graphene oxide obtained in step (1) was ultrasonically dispersed in pure water for 8 minutes, and then sodium hydroxide and chloroacetic acid were added and subjected to bath ultrasonic reaction for 3 hours. After the reaction, the mixture was washed with ethanol and pure water in sequence to obtain carboxylated magnetic graphene oxide.

[0093] The mass ratio of the magnetic graphene oxide to the sodium hydroxide is 1:30, and the mass ratio of the magnetic graphene oxide to the chloroacetic acid is 1:23.

[0094] (3) Crown ether coupling: EDC was added to the carboxylated magnetic graphene oxide obtained in step (2) and the reaction was continued for 30 min. NHS was then added and the reaction was continued in an ice bath for 1 h. After the reaction, the solution was washed with pure water by magnetic suction to obtain a magnetic bead solution. The crown ether was dissolved in pure water and then added to the magnetic bead solution. The solution was placed on a shaker and amidation reaction was carried out for 24 h. After the reaction, the solution was washed with pure water by magnetic suction and vacuum dried to obtain crown ether-modified magnetic beads.

[0095] The mass ratio of carboxylated magnetic graphene oxide to EDC is 1:15, the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:10, and the mass ratio of crown ether to carboxylated magnetic graphene oxide is 20:10.

[0096] Example 5: A method for preparing crown ether modified magnetic beads, comprising the following steps:

[0097] (1) Preparation of magnetic graphene oxide. A single layer of graphene oxide was dissolved in ethylene glycol solvent and added to a Teflon high temperature and high pressure reactor. Ultrasonic dispersion was performed for 8 minutes. Then, ferric chloride, sodium acetate, and polyethylene glycol 8000 were added. After mixing and stirring for 4 hours, the reactor was transferred to a forced air drying oven and reacted at 190°C for 8 hours. After the reaction, the product was magnetically washed three times with ethanol and pure water respectively, and vacuum dried at 60°C to obtain magnetic graphene oxide.

[0098] The mass ratio of the single-layer graphene oxide to ferric chloride is 1:8, the mass ratio of the single-layer graphene oxide to sodium acetate is 1:35, and the mass ratio of the single-layer graphene oxide to polyethylene glycol 8000 is 1:7.

[0099] (2) Preparation of carboxylated magnetic graphene oxide: The magnetic graphene oxide obtained in step (1) was ultrasonically dispersed in pure water for 8 minutes, and then sodium hydroxide and chloroacetic acid were added and subjected to bath ultrasonic reaction for 4 hours. After the reaction, the mixture was washed with ethanol and pure water in sequence to obtain carboxylated magnetic graphene oxide.

[0100] The mass ratio of the magnetic graphene oxide to the sodium hydroxide is 1:50, and the mass ratio of the magnetic graphene oxide to the chloroacetic acid is 1:20.

[0101] (3) Crown ether coupling: EDC was added to the carboxylated magnetic graphene oxide obtained in step (2) and the reaction was continued for 30 min. NHS was then added and the reaction was continued in an ice bath for 1 h. After the reaction, the solution was washed with pure water by magnetic suction to obtain a magnetic bead solution. The crown ether was dissolved in pure water and then added to the magnetic bead solution. The solution was placed on a shaker and amidation reaction was carried out for 24 h. After the reaction, the solution was washed with pure water by magnetic suction and vacuum dried to obtain crown ether-modified magnetic beads.

[0102] The mass ratio of carboxylated magnetic graphene oxide to EDC is 1:15, the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:10, and the mass ratio of crown ether to carboxylated magnetic graphene oxide is 20:5.

[0103] Example 6: A method for extracting extracellular vesicles, comprising the following steps:

[0104] (1) Initial isolation of exosomes: Blood was collected and centrifuged at 3000 g for 10 min. The pellet (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The collected supernatant was centrifuged at 10000 g for 30 min. The pellet (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The supernatant was aspirated and filtered with a 0.22 μm syringe filter to exclude molecules larger than 0.22 μm and stored in a refrigerator at 4 °C before exosome isolation.

[0105] (2) Extracting exosomes using crown ether-modified magnetic beads: 600 μL of crown ether-modified magnetic beads and 3 mL of the exosome sample initially separated in step (1) were incubated in an ice bath for 20 min to obtain a magnetic bead-exosome complex.

[0106] (3) Exosome elution: After removing the supernatant by magnetic aspiration, the magnetic bead-exosome complex in the test tube was redispersed in 1 mL of pH 7.5 pure water and the exosomes were eluted by sonication for 10 min. The magnetic beads were removed using a magnet and the exosomes were recovered from the supernatant. The recovered exosomes were filtered through a 0.22 μm filter membrane and stored in cryovials at -80°C.

[0107] Example 7: A method for extracting extracellular vesicles, comprising the following steps:

[0108] (1) Initial isolation of exosomes: Urine was collected and centrifuged at 3000 g for 10 min. The precipitate (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The collected supernatant was centrifuged at 10000 g for 30 min. The precipitate (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The supernatant was aspirated and filtered with a 0.22 μm syringe filter to exclude molecules larger than 0.22 μm and stored in a refrigerator at 4 °C before exosome isolation.

[0109] (2) Extracting exosomes using crown ether-modified magnetic beads: 200 μL of crown ether-modified magnetic beads and 1 mL of the exosome sample initially separated in step (1) were incubated in an ice bath for 20 min to obtain a magnetic bead-exosome complex.

[0110] (3) Exosome elution: After removing the supernatant by magnetic aspiration, the magnetic bead-exosome complex in the test tube was redispersed in 1 mL of pH 7.5 pure water and the exosomes were eluted by sonication for 20 min. The magnetic beads were removed using a magnet and the exosomes were recovered from the supernatant. The recovered exosomes were filtered through a 0.22 μm filter membrane and stored in cryovials at -80°C.

[0111] Example 8: A method for extracting extracellular vesicles, comprising the following steps:

[0112] (1) Initial isolation of exosomes: Saliva was collected and centrifuged at 3000 g for 10 min. The pellet (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The collected supernatant was centrifuged at 10000 g for 30 min. The pellet (nuclei, heavy mitochondria, and large cell debris) was discarded and the supernatant was collected. The supernatant was aspirated and filtered with a 0.22 μm syringe filter to exclude molecules larger than 0.22 μm and stored in a refrigerator at 4 °C before exosome isolation.

[0113] (2) Extracting exosomes using crown ether-modified magnetic beads: 800 μL of crown ether-modified magnetic beads and 5 mL of the exosome sample initially separated in step (1) were incubated in an ice bath for 20 min to obtain a magnetic bead-exosome complex.

[0114] (3) Exosome elution: After removing the supernatant by magnetic aspiration, the magnetic bead-exosome complex in the test tube was redispersed in 1 mL of pH 7.5 pure water and the exosomes were eluted by sonication for 30 min. The magnetic beads were removed using a magnet and the exosomes were recovered in the supernatant. The recovered exosomes were filtered through a 0.22 μm filter membrane and stored in cryovials at -80°C.

[0115] The exosome capture magnetic beads of the present invention are convenient to extract, take a short time, and the extracted exosomes are of high purity without damaging the biological activity of the exosomes.

[0116] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with this technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present invention, and these modifications or replacements should all be included in the scope of protection of the present invention.

Claims

1. A method for preparing reusable crown ether modified magnetic beads, characterized in that: The following steps are involved: Adding EDC to the carboxylated magnetic graphene oxide, stirring, then adding NHS, and stirring to obtain a mixed solution; An aqueous solution of crown ether is added to the mixture to carry out an amidation reaction. After the reaction is completed, the beads are washed by magnetic absorption and dried to obtain reusable crown ether-modified magnetic beads.

2. The method for preparing reusable crown ether modified magnetic beads according to claim 1, wherein The mass ratio of carboxylated magnetic graphene oxide to EDC is 1:5-20; the mass ratio of carboxylated magnetic graphene oxide to NHS is 1:5-20.

3. The method for preparing reusable crown ether modified magnetic beads according to claim 1, wherein The stirring time after adding EDC to the carboxylated magnetic graphene oxide is at least 30 minutes, and the stirring time after adding NHS is at least 30 minutes.

4. The method for preparing reusable crown ether modified magnetic beads according to claim 1, wherein The molar ratio of the crown ether to EDC is 1:1-50; the molar ratio of the crown ether to NHS is 1:1-50; and the molar ratio of the crown ether to carboxylated magnetic graphene oxide is 20:1-10.

5. The method for preparing reusable crown ether modified magnetic beads according to claim 1, wherein The carboxylated magnetic graphene oxide is prepared by the following process: dispersing the magnetic graphene oxide in pure water by ultrasonication, then adding sodium hydroxide and chloroacetic acid, performing bath ultrasonication, and washing by magnetic adsorption to obtain the carboxylated magnetic graphene oxide.

6. The method for preparing reusable crown ether modified magnetic beads according to claim 5, wherein The mass ratio of magnetic graphene oxide to sodium hydroxide is 1:20-50, the mass ratio of magnetic graphene oxide to chloroacetic acid is 1:20-30, and the bath ultrasonic reaction time is at least 2 hours.

7. A reusable crown ether modified magnetic beads prepared according to the method according to any one of claims 1 to 16.

8. Use of reusable crown ether-modified magnetic beads prepared according to the method according to any one of claims 1 to 16 in extracting exosomes.

9. An exosome extraction kit, characterized in that: Comprising reusable crown ether modified magnetic beads and an eluent prepared by the method described in any one of claims 1 to 16.

10. A method for extracting exosomes, characterized in that: The following steps are involved: Adding the reusable crown ether-modified magnetic beads prepared by the method according to any one of claims 16 to the exosome sample, incubating the sample in an ice bath to obtain a magnetic bead-exosome complex; After the supernatant in the magnetic bead-exosome complex was removed by magnetic attraction, the magnetic bead-exosome complex was redispersed in pure water, the exosomes were eluted by ultrasound, and then magnetic separation was performed to obtain exosomes.

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