Preparation of temperature response composite magnetic bead and application of temperature response composite magnetic bead in exosome separation
By preparing temperature-responsive composite magnetic beads, the problems of low exosome separation efficiency and incomplete release in the prior art are solved, efficient capture and gentle release are achieved, and high yield and high purity exosomes are obtained, which are suitable for a variety of sample sources.
Patent Information
- Application Number
- CN202510735302.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-04
- Publication Date
- 2025-08-29
AI Technical Summary
The existing magnetic bead separation method is difficult to take into account the high yield, high purity and biological activity of exosomes, and the release process is incomplete, which affects its downstream application.
Temperature-responsive composite magnetic beads are prepared to achieve efficient capture and gentle release of exosomes by modifying polar functional groups on the surface of magnetic particles, covering the shell of phase change material, and coupling aptamers.
It significantly improves the capture and release efficiency of exosomes, obtains high yield and high purity exosomes, has a complete morphology, is suitable for a variety of sample sources, and maintains biological activity.
Smart Images
Figure CN120555419A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biotechnology, and particularly relates to the preparation of temperature-responsive composite magnetic beads and their application in exosome separation. Background Art
[0002] Exosomes, extracellular vesicles with a diameter of approximately 30-200 nm secreted by living cells, carry functional molecules such as nucleic acids, proteins, and lipids, making them highly promising new biomarker carriers. In the field of exosome separation technology, efficiently obtaining high-yield, high-purity, morphologically intact exosomes that maintain biological activity is crucial for clinical disease detection, the construction of drug delivery systems, and basic medical research. However, separation methods based on density, size, charge, and affinity, such as ultracentrifugation, magnetic bead separation, and polymer precipitation, struggle to balance yield, purity, morphology, and biological activity, thus limiting the expansion of downstream applications.
[0003] Magnetic bead separation has attracted considerable attention due to its advantages, including high specificity, high purity, intact morphology, and ease of use. However, it faces the dual challenges of insufficient capture affinity and difficulty in efficient and gentle release, resulting in limited exosome yield, impaired bioactivity, and morphological disruption. Combining enhanced capture efficiency with an efficient and gentle release mechanism could make magnetic bead separation an effective approach for isolating exosomes with high yield, purity, high bioactivity, and morphological integrity. Therefore, the development of novel composite magnetic bead materials and separation methods with optimized performance is urgently needed to overcome the bottlenecks of existing technologies.
[0004] The main methods for capturing exosomes based on magnetic bead separation include antibody-antigen recognition, ligand-receptor recognition, phospholipid bilayer recognition, and aptamer-antigen recognition. Among them, aptamers are products of in vitro chemical synthesis with specificity and affinity comparable to antibodies. They are easy to screen, modify, and couple, and can be mass-produced. In addition, the synergistic affinity of single / multiple aptamers can improve the efficiency of exosome capture. In the process of capturing exosomes using magnetic beads, the magnetic bead-exosome complex formed may cause specific active sites on the exosome surface to be shielded or the local spatial structure to be destroyed due to the physical adsorption of the magnetic beads, thereby functionally limiting its downstream applications. Therefore, research on the efficient release of exosomes from magnetic bead carriers is of key significance for preserving the natural structural integrity of exosomes, restoring the activity of their surface functional sites, and expanding practical application scenarios. Existing methods for exosome release based on magnetic bead separation primarily include elution, enzyme-catalyzed treatment, direct chemical reaction, and competitive substitution. These methods typically disconnect the exosomes from the composite magnetic beads, resulting in incomplete release and compromised bioactivity, making efficient and gentle release difficult. New release methods that can improve release efficiency, thereby increasing exosome yield while maintaining bioactivity are rarely reported. Summary of the Invention
[0005] The present invention aims to provide a preparation method of temperature-responsive composite magnetic beads and their application in exosome separation. The temperature-responsive composite magnetic beads can improve the capture efficiency and release efficiency of exosomes, increase the yield and purity of exosomes, and maintain the intact morphology and biological activity of exosomes.
[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0007] A method for preparing temperature-responsive composite magnetic beads comprises the following steps:
[0008] S1. preparing magnetic microparticles with surface modified with polar functional groups;
[0009] S2, coating the surface of the magnetic microparticles obtained in S1 with a phase change material shell, resuspending the particles in an amphiphilic molecule solution, and subjecting the particles to a rotation reaction to obtain temperature-responsive composite magnetic beads having polar functional groups on the surface;
[0010] S3. The surface of the temperature-responsive composite magnetic beads having polar functional groups obtained in S2 is coupled with an aptamer to obtain aptamer-coupled temperature-responsive composite magnetic beads.
[0011] Preferably, in S1, the method for preparing magnetic particles whose surfaces are modified with polar functional groups comprises the following steps:
[0012] S11, mixing an inorganic iron salt and a polyol, heating and stirring until the mixture is clear, adding a surfactant and an alkaline solvent to dissolve the mixture, and then reacting the mixture by a solvothermal method to obtain magnetic beads;
[0013] S12. Evenly mix the magnetic beads prepared in S11 with anhydrous ethanol and ultrapure water, and sequentially add 25-28% by mass of ammonia water and a silicon source precursor, and react by stirring to obtain magnetic particles with polar functional groups modified on the surface.
[0014] Preferably, the inorganic iron salt is selected from one of ferric chloride hexahydrate and cobalt chloride hexahydrate.
[0015] Preferably, the polyol is selected from ethylene glycol or propylene glycol.
[0016] Preferably, the surfactant is selected from one of trisodium citrate and vinyl pyrrolidone.
[0017] Preferably, the alkaline solvent is selected from anhydrous sodium acetate or sodium hydroxide.
[0018] Preferably, in S11, the solvent thermal reaction conditions are: reaction at 180-260° C. for 6-48 hours, and the reaction product is washed with ultrapure water and anhydrous ethanol in sequence and then dried.
[0019] In S12, the silicon source precursor is selected from one of methyl orthosilicate and ethyl orthosilicate.
[0020] Preferably, in S2, the phase change material shell is coated by the following method:
[0021] S21, mixing and melting fatty acid compounds in a certain mass ratio to obtain a multi-component fatty acid blend;
[0022] S22. Dissolve the multi-component fatty acid blend obtained in S21 in an organic solvent, mix it with the magnetic particles obtained in S1, and react it with ultrasound and rotation to obtain magnetic particles with a phase change material shell layer coated on the surface.
[0023] Preferably, in S21, the fatty acid compound is selected from two or more of lauric acid, palmitic acid, stearic acid, butyric acid, and propionic acid.
[0024] Preferably, in S21, the mass ratio of the fatty acid compounds is 1-100:1-100.
[0025] Preferably, in S2, the amphiphilic molecule is one of a polyethylene glycol derivative, a polystyrene-polyethylene glycol block copolymer, a polyacrylic acid-polystyrene block copolymer, and a lipid-polyethylene glycol conjugate.
[0026] Preferably, in S3, the coupling buffer is selected from one of PBS, Tris-HCl, PB (pH 8.0) or MES; the aptamer is selected from Apt CD63 、Apt EpCAM 、Apt CD9 、Apt CD81 、Apt EGFR 、Apt PD-L1 、Apt CD109 、Apt MUC1 、Apt CA125 One or more of .
[0027] The present invention also provides temperature-responsive composite magnetic beads prepared by the preparation method.
[0028] The present invention also provides the use of the temperature-responsive composite magnetic beads in exosome separation.
[0029] The present invention also provides a method for isolating exosomes, which uses the temperature-responsive composite magnetic beads for separation, comprising the following steps:
[0030] T1, incubating the temperature-responsive composite magnetic beads with a biological sample, and enriching the sample by magnetic separation to obtain an exosome-magnetic bead complex;
[0031] T2, heating the exosome-magnetic bead complex obtained in T1 to release the exosomes, recovering the magnetic beads, collecting the supernatant, centrifuging, discarding the precipitate, and obtaining purified exosomes.
[0032] Preferably, in T1, the biological sample is selected from one of cell culture supernatant, blood, urine, saliva, cerebrospinal fluid, milk, ascites and pleural effusion.
[0033] Preferably, in T1, the co-incubation time is 0.5-3 h, and the co-incubation times are 1-3 times.
[0034] Preferably, in T2, the heating method includes one of metal bath heating, water bath heating, air bath heating, magnetic heating, light heating or microwave heating, the heating temperature is 20-40°C, and the heating time is 5-60 min.
[0035] The present invention also provides a use of the exosomes isolated by the method in the preparation of in vitro diagnostic reagents, drug delivery vectors, regenerative medicine materials, immunotherapy compositions, cosmetic raw materials or basic research tools.
[0036] The present invention also provides evaluation indicators of exosomes isolated by the method, including capture efficiency, release efficiency, purity, morphological integrity, protein expression, particle size, and particle concentration.
[0037] Compared with the prior art, the present invention has the following advantages and technical effects:
[0038] The present invention discloses the preparation of temperature-responsive composite magnetic beads and their application in exosome separation. The application of the temperature-responsive composite magnetic beads in exosome separation can significantly improve the efficiency of exosome capture and release, and is gentle and non-destructive, simple to operate, and low in cost. The obtained exosomes have high yield and purity, intact morphology, and do not affect downstream analysis. The beads are suitable for separating exosomes from various sample sources and can effectively solve the problem that existing technologies have difficulty in balancing the yield and purity of exosomes while maintaining biological activity.
[0039] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 This is a process flow chart for isolating exosomes according to the present invention;
[0041] Figure 2 TEM image of the magnetic bead-exosome complex obtained in Example 2;
[0042] Figure 3 This is a characterization diagram of the exosome particle concentration and particle size measured by NanoFCM after exosome separation using temperature-responsive composite magnetic beads in Example 2;
[0043] Figure 4 This is a TEM image of the exosome morphology after the temperature-responsive composite magnetic beads were used to separate the exosomes in Example 2;
[0044] Figure 5 This is a protein expression graph of exosomes verified by Western blot after exosomes were separated using temperature-responsive composite magnetic beads in Example 2;
[0045] Figure 6 This is a graph characterizing the purity of exosomes measured by nanoflow cytometry after exosomes were separated using temperature-responsive composite magnetic beads in Example 2. DETAILED DESCRIPTION
[0046] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.
[0047] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.
[0048] Source of test materials:
[0049] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in this field and can be purchased through commercial channels.
[0050] Example 2 Reference Figure 1 process flow chart.
[0051] Example 1
[0052] A temperature-responsive composite magnetic bead preparation method comprises the following steps:
[0053] S1. Preparing magnetic particles whose surfaces are modified with polar functional groups, the preparation method comprises the following steps:
[0054] S11. 30.0 g of ferric chloride hexahydrate and 300 mL of ethylene glycol were added to a 1 L beaker in sequence, and the beaker was placed on a 60 ° C constant temperature magnetic stirrer. Stir vigorously at 1000 rpm until clear. 12.0 g of trisodium citrate and 6.0 g of anhydrous sodium acetate were added, and vigorous stirring was continued until the crystalline particle reagent was completely dissolved. The mixture was transferred to a polytetrafluoroethylene liner while hot, and the liner and reactor were assembled as required. The mixture was placed in a 200 ° C oven for reaction for 18 h. After the reactor was cooled to room temperature, the polytetrafluoroethylene liner in the reactor was vertically removed, the supernatant in the liner was discarded, and the precipitate was washed with ultrapure water and anhydrous ethanol in sequence, and dried at 70 ° C to obtain magnetic beads.
[0055] S12. Place 1000mg of the magnetic beads prepared in S11 in a 1L three-necked flask. Add 400mL of anhydrous ethanol and 100mL of ultrapure water in sequence. Fix the three-necked flask on a cantilevered stirring paddle and stir. Add 5mL of 25% ammonia water from one side of the three-necked flask and continue stirring for 5min. Pipette 300μL of tetraethyl orthosilicate and 10mL of anhydrous ethanol into a 50mL centrifuge tube and vortex mix thoroughly. Add the mixture dropwise to the reaction system and increase the speed to 800rpm to continue the reaction. From the time of adding tetraethyl orthosilicate, the timer is 12h. After the reaction is completed, collect the magnetic beads and wash them with anhydrous ethanol magnetic separation 5 times to obtain silica magnetic particles rich in hydroxyl groups on the surface. Store at 4℃ for later use.
[0056] S2. The surface of the magnetic particles obtained in S1 is coated with a phase change material shell, wherein the phase change material shell is coated by the following method:
[0057] S21, placing 3.25 g of lauric acid and 1.75 g of palmitic acid in a 50 mL beaker on a heated magnetic stirrer at 60° C., stirring at 500 rpm to melt, and after complete melting, continuing the reaction for 30 min to obtain a multi-component fatty acid blend. After the reaction is completed, the mixture is placed on a table to cool and solidify. The obtained multi-component fatty acid blend solid is freeze-dried and stored at 4° C.
[0058] S22. Dissolve the multi-component fatty acid blend solid obtained in S21 with anhydrous ethanol to 0.1 mg / mL, take 0.25 mg of the above solution and mix it with 1 mg of the magnetic particles prepared in S1, heat and ultrasonicate at 40°C for 5 minutes, place it on a rotary mixer, and fix it at 4°C for 3 hours. After the reaction is completed, wash it with ultrapure water for three times, and finally disperse it in ultrapure water for later use.
[0059] Distearoylphosphatidylethanolamine-polyethylene glycol-carboxyl is dissolved in 4% ethanol solution by ultrasonication to 0.1 mg / mL, 0.1 mg of the above solution is taken to resuspend the above 1 mg of the surface-coated phase change material shell magnetic particles, and after slight ultrasonication to ensure that they are well dispersed, they are placed on a rotary mixer to react at 4°C for 3 hours. After the reaction is completed, the supernatant is discarded by magnetic separation, and the beads are washed three times with ultrapure water and dispersed in ultrapure water to obtain temperature-responsive composite magnetic beads containing carboxyl groups on the surface;
[0060] S3. The temperature-responsive composite magnetic beads containing carboxyl groups on the surface obtained by S2 at 1 mg / mL were magnetically washed three times with 0.02M MES buffer, and then dispersed in an equal volume of 0.02M MES buffer. 20μL, 25mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 20μL, 25mg / mL N-hydroxysuccinimide activation working solution were added. After vortex mixing, the beads were placed on a rotary mixer for activation at room temperature for 30 minutes. After the activation was completed, the supernatant was discarded by magnetic separation. The composite magnetic beads were magnetically washed three times with PB8.0 buffer and then resuspended. 1mg of the resuspended magnetic beads were mixed with 2μL of Apt with a final concentration of 100μM. CD63 / Apt Epcam The dual aptamer solution was vortexed and placed on a rotary mixer for coupling at 4°C for 2 hours. After the reaction, the supernatant was discarded after magnetic separation, and the pellet was resuspended in 0.03g / mL BSA blocking buffer, vortexed, and placed on a rotary mixer for blocking at 4°C for 2 hours. After the reaction, the composite magnetic beads were washed with PBS and resuspended to obtain temperature-responsive composite magnetic beads.
[0061] Example 2
[0062] A method for isolating exosomes, using the temperature-responsive composite magnetic beads for separation, comprises the following steps:
[0063] T1. Culture A549 cells in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. When the cell density reaches 70%, wash the cell surface three times with sterile PBS and starve the cells in serum-free RPMI 1640 medium for 48 h. Collect the cell supernatant and centrifuge at 3000 g for 20 min at 4°C. Filter through a 0.22 μM filter to remove impurities such as dead cells, cell debris, membrane particles, and apoptotic bodies.
[0064] The temperature-responsive composite magnetic beads prepared in Example 1 were mixed with the pretreated and concentrated cell supernatant, and the mixture was slowly incubated at 4°C for 1 hour on a rotary mixer. After the incubation, the supernatant was magnetically separated and discarded to obtain the exosome-magnetic bead complex, which was resuspended in PBS for storage;
[0065] T2. Set the metal bath temperature to 40°C. After the temperature stabilizes, place the exosome-magnetic bead complex suspension in the metal bath and heat-release for 15 minutes. Immediately perform magnetic separation on the sample after the end, recover the magnetic beads. The supernatant after magnetic separation is the released exosome solution. The solution is further centrifuged at 4°C and 12,000g for 5 minutes, and the supernatant is retained to obtain purified exosomes.
[0066] Example 3
[0067] The exosome isolation method is the same as in Example 2, except that the biological sample is the supernatant of cells cultured from multiple sources of starvation.
[0068] The effect of Example 2 was verified by the following experiments.
[0069] TEM, NanoFCM and Western blot were used to characterize the morphology, particle size, particle concentration, purity and protein expression of exosome particles. Figure 2-Figure 6 shown.
[0070] Depend on Figure 2 It can be seen that during the co-incubation stage of the temperature-responsive composite magnetic beads and the A549 cell supernatant, obvious magnetic bead-exosome complexes appeared, indicating that the temperature-responsive composite magnetic beads prepared by this method have the ability to specifically capture exosomes.
[0071] Depend on Figure 3 It can be seen that the average particle size of exosomes in the supernatant of A549 cells separated by temperature-responsive composite magnetic beads is 62.7±22.5nm, and the particle concentration is 1.08×10 11 / mL.
[0072] Depend on Figure 4 It can be seen that the particle size of exosomes separated by temperature-responsive composite magnetic beads is 30-150nm under electron microscopy, with complete morphology and few impurities.
[0073] Depend on Figure 5 It can be seen that the exosomes separated by temperature-responsive composite magnetic beads expressed TSG101, CD63, and CD9 proteins, but did not express Calnexin protein, which is consistent with the "three positive and one negative" characteristics.
[0074] Depend on Figure 6 It can be seen that the exosomes separated by temperature-responsive composite magnetic beads were characterized by nanoflow cytometry to determine the proportion of membrane-bearing particles, and the calculated purity was 83.4%.
[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. A method for preparing temperature-responsive composite magnetic beads, characterized in that: The following steps are involved: S1. preparing magnetic microparticles with surface modified with polar functional groups; S2, coating the surface of the magnetic microparticles obtained in S1 with a phase change material shell, resuspending the particles in an amphiphilic molecule solution, and subjecting the particles to a rotation reaction to obtain temperature-responsive composite magnetic beads having polar functional groups on the surface; S3. The surface of the temperature-responsive composite magnetic beads having polar functional groups obtained in S2 is coupled with an aptamer to obtain aptamer-coupled temperature-responsive composite magnetic beads.
2. The preparation method according to claim 1, characterized in that In S1, the method for preparing magnetic particles whose surfaces are modified with polar functional groups comprises the following steps: S11, mixing an inorganic iron salt and a polyol, heating and stirring until the mixture is clear, adding a surfactant and an alkaline solvent to dissolve the mixture, and then reacting the mixture by a solvothermal method to obtain magnetic beads; S12. Evenly mix the magnetic beads prepared in S11 with anhydrous ethanol and ultrapure water, add 25-28% ammonia water and a silicon source precursor in sequence, stir and react, and obtain magnetic particles with polar functional groups modified on the surface.
3. The preparation method according to claim 2, characterized in that: In S11, the solvothermal reaction conditions are: reaction at 180-260° C. for 6-48 hours, and the reaction product is washed with ultrapure water and anhydrous ethanol in sequence and then dried.
4. The preparation method according to claim 1, characterized in that In S2, the phase change material shell is coated by the following method: S21, mixing and melting the fatty acid compounds according to a mass ratio to obtain a multi-component fatty acid blend; S22. Dissolve the multi-component fatty acid blend obtained in S21 in an organic solvent, mix it with the magnetic particles obtained in S1, and react it with ultrasound and rotation to obtain magnetic particles with a phase change material shell layer coated on the surface.
5. The preparation method according to claim 1, characterized in that: In S2, the amphiphilic molecule is one of a polyethylene glycol derivative, a polystyrene-polyethylene glycol block copolymer, a polyacrylic acid-polystyrene block copolymer, and a lipid-polyethylene glycol conjugate.
6. The temperature-responsive composite magnetic beads prepared by the preparation method according to any one of claims 1 to 5.
7. Use of the temperature-responsive composite magnetic beads as claimed in claim 6 in exosome separation.
8. A method for isolating exosomes, characterized in that: The separation is performed using the temperature-responsive composite magnetic beads described in claim 6, comprising the following steps: T1. Co-incubating the temperature-responsive composite magnetic beads according to claim 6 with a biological sample, and enriching the sample by magnetic separation to obtain an exosome-magnetic bead complex; T2, heating the exosome-magnetic bead complex obtained in T1 to release the exosomes, recovering the magnetic beads, collecting the supernatant, centrifuging, discarding the precipitate, and obtaining purified exosomes.
9. The separation method according to claim 8, characterized in that In T2, the heating method includes one of metal bath heating, water bath heating, air bath heating, magnetic heating, light heating or microwave heating, the heating temperature is 20-40° C., and the heating time is 5-60 min.
10. Use of the exosomes isolated by the method according to any one of claims 8 to 9 in the preparation of in vitro diagnostic reagents, drug delivery vectors, regenerative medicine materials, immunotherapy compositions, cosmetic raw materials or basic research tools.