Exosome separation material taking CLIKKPF as ligand and preparation method of exosome separation material

Abstract

The invention relates to the technical field of bioengineering, in particular to an exosome separation material with CLIKKPF as a ligand and a preparation method of the exosome separation material. A novel exosome affinity medium is developed by utilizing the Ca < 2 + > dependency recognition characteristic between CLIKKPF and PS, specific adsorption of the exosome is realized by virtue of a water-soluble polymer and a brush-like structure thereof in combination with polypeptide CLIKKPF, and meanwhile, rapid separation of the target exosome is realized by utilizing nano magnetic beads as supporting particles.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to an exosome separation material using CLIKKPF as a ligand and a preparation method thereof. Background Art

[0002] Exosomes are bilayer vesicles secreted by living cells and bacteria, richly loaded with proteins and nucleic acids. As crucial mediators of long-distance cellular communication, exosomes are closely implicated in the development and progression of numerous diseases, including tumors and inflammation. The isolation and enrichment of exosomes in complex biological systems is crucial for disease diagnosis, prognosis, detection, and molecular mechanism studies. However, the small size of exosome particles, approximately 30 to 150 nm in diameter, and their widespread presence in complex biological samples, including blood, tissue fluid, and urine, present significant challenges in their isolation and enrichment.

[0003] Currently, the main exosome isolation and enrichment technologies used in complex biological systems include ultracentrifugation, size exclusion chromatography, ultrafiltration, and polymer precipitation. However, these methods mostly rely on physical properties of exosomes, such as density, size, or solubility, making it difficult to effectively distinguish interfering particles such as lipoprotein aggregates in living systems.

[0004] To improve the selectivity of exosome separation and enrichment, current research focuses on molecular markers on the exosome surface, using affinity recognition ligands as targeting units to develop affinity separation strategies based on molecular recognition. CLIKKPF is a polypeptide sequence with phospholipid-specific recognition capabilities. For example, Chinese patent CN117731790A, "Encapsulated complexes for spontaneous in situ directional encapsulation of cell membranes, their preparations, and applications," utilizes the specific affinity between the inner membrane molecule PS and a PS-targeting peptide-modified nanodrug to spontaneously self-assemble within the inner leaflet of the cell membrane. This approach eliminates the need for additional co-extrusion methods to produce biomimetic nanodrugs based on PS's universal spontaneous in situ directional encapsulation of the cell membrane. However, whether CLIKKPF can be used to achieve efficient exosome separation remains unexplored. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the main exosome separation and enrichment technologies applied to complex living systems include ultracentrifugation, size exclusion chromatography, ultrafiltration and polymer precipitation, but most of these methods rely on the physical properties of exosomes such as density, size or solubility, and it is difficult to effectively distinguish interfering particles such as lipoprotein aggregates in living systems.

[0006] In order to solve the above problems, the present invention utilizes Ca between CLIKKPF and PS 2+The novel exosome affinity medium was developed based on its dependent recognition properties. The specific adsorption of exosomes was achieved by combining water-soluble polymers and their brush-like structures with the peptide CLIKKPF. Meanwhile, nanomagnetic beads were used as support particles to achieve the rapid separation of target (marine-derived) exosomes.

[0007] To achieve the above-mentioned purpose, the technical solution of the present invention is: a method for preparing an exosome separation material using CLIKKPF as a ligand, comprising the following steps: (1) Preparation of magnetic nanoparticles (MNPs) by hydrothermal method; (2) Preparation of magnetic nanoparticles MNP@Si coated with silicon nanoparticles; (3) Preparation of amino-functionalized magnetic nanoparticles MNP@NH2; (4) Preparation of bromine-functionalized magnetic nanoparticles MNP@Br; (5) Preparation of magnetic nanoparticles MNP@poly(NIPAm- co -AGE); (6) Preparation of azide-functionalized magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@N3; (7) Preparation of CLIKKPF-modified magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@CLIKKPF.

[0008] In the presence of calcium ions, the affinity recognition relationship between the peptide CLIKKPF and exosomes is mainly based on the targeted binding mechanism of phosphatidylserine (PS) on the membrane surface. This biopolymer medium can exhibit good affinity adsorption behavior. Figure 1 As shown, bare nanomagnetic beads were prepared via hydrothermal synthesis. Their surfaces were then modified with tetraethyl orthosilicate to introduce reactive amino groups. 2-Bromoisobutyryl bromide was then used to successfully construct a SI-ATRP initiator system. Using water-soluble NIPAm and AGE as monomers, a surface-initiated atom transfer radical polymerization (SI-ATRP) reaction was performed in aqueous solution, resulting in the in situ growth of a brush-like polymer outer layer. The water-soluble polymer and its brush-like structure, combined with the peptide CLIKKPF, enabled specific adsorption of exosomes. The nanomagnetic beads served as support particles for rapid isolation of target exosomes.

[0009] Furthermore, in step (1), the preparation method of magnetic nanoparticles is as follows: ferric chloride and sodium acetate are dispersed in 50 mL of ethylene glycol, stirred at room temperature until the solid is completely dissolved, and when the solution turns brown, the solution is poured into a high-pressure reactor and reacted at 220°C; after the reaction is completed, the reactor is taken out and cooled at room temperature, and the solution is poured into a beaker, and the nanomagnetic beads are collected through the beaker with a strong magnet, the supernatant is discarded, and the sample is repeatedly rinsed with ultrapure water and anhydrous ethanol, respectively, and the sample is stored in anhydrous ethanol to obtain magnetic nanoparticles MNP.

[0010] Furthermore, in step (2), the preparation method of magnetic nanoparticles wrapped by silicon nanoparticles is as follows: MNPs are dispersed in an ethanol aqueous solution and ultrasonicated; after the ultrasonication, ammonia water is added to the system and stirred; during the stirring process, ethyl orthosilicate is added, and then stirring is continued at room temperature; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and are washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol to obtain magnetic nanoparticles MNP@Si wrapped by silicon nanoparticles.

[0011] Furthermore, in step (3), the preparation method of amino-functionalized magnetic nanoparticles is as follows: ultrasonically disperse MNP@Si in APTES-ethanol solution; pour the mixed solution into a round-bottom flask, add glass beads, fix the round-bottom flask on a rotary evaporator, and stir at room temperature; after the reaction is completed, use a strong magnet to adsorb the nanomagnetic beads through the flask wall, wash them with anhydrous ethanol and ultrapure water respectively, and then store the nanomagnetic beads in anhydrous ethanol to obtain amino-functionalized magnetic nanoparticles MNP@NH2.

[0012] Furthermore, in step (4), the preparation method of bromine-functionalized magnetic nanoparticles is as follows: MNP@NH2 is dispersed in tetrahydrofuran, sealed with a sealing film, and ultrasonically dispersed; then triethylamine is added, and the sample is quickly placed in an ice-water mixture for cooling, and then 2-bromoisobutyryl bromide is added under stirring, and the system is quickly sealed with a sealing film and placed on a shaker at room temperature overnight; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol to obtain bromine-functionalized magnetic nanoparticles MNP@Br.

[0013] Furthermore, in step (5), the preparation method of magnetic nanoparticles modified with temperature-responsive polymer brushes is as follows: MNP@Br, N-isopropylacrylamide, allyl glycidyl ether, copper bromide, and cuprous bromide are added to isopropanol, and glass beads are placed; after ultrasonicating the system, nitrogen is blown, and then tris[2-(dimethylamino)ethyl]amine is added and nitrogen is blown again; the sample is then sealed and placed on a shaker, and after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and washed with ethylenediaminetetraacetic acid aqueous solution, anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol to obtain magnetic nanoparticles MNP@poly(NIPAm- co -AGE).

[0014] Furthermore, in step (6), the preparation method of the azide-functionalized magnetic nanoparticles is as follows: MNP@poly(NIPAm- co -AGE), ammonium chloride, and sodium azide were added to N, N-dimethylformamide, and the system was sealed and placed on a shaker. After the reaction was completed, the nanoparticles were adsorbed through the wall of the flask with a strong magnet, and the nanoparticles were washed with anhydrous ethanol and ultrapure water respectively. The nanoparticles were then stored in anhydrous ethanol and recorded as azide-functionalized magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@N3.

[0015] Furthermore, in step (7), the preparation method of the magnetic nanoparticles modified with CLIKKPF is as follows: saturated copper sulfate solution and sodium ascorbate are added to a methanol aqueous solution; after the system is fully mixed, MNP@poly(NIPAm- co -AGE)@N3 and alkynylated CLIKKPF; the system was placed on a shaker and reacted in the dark, at room temperature, and at 100 rpm; after the reaction was completed, the nanomagnetic beads were adsorbed through the wall of the flask with a strong magnet, washed with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol, which was recorded as magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@CLIKKPF.

[0016] A magnetic nanoparticle MNP@poly(NIPAm- co -AGE)@CLIKKPF.

[0017] The magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@CLIKKPF is applied in the isolation of marine-derived exosomes.

[0018] The beneficial effects of the present invention are: (1) High separation selectivity and strong specificity. Based on the affinity mechanism of molecular recognition, the present invention can effectively distinguish exosomes from interfering particles such as lipoprotein aggregates in biological samples, solving the problem that traditional separation methods (such as ultracentrifugation and ultrafiltration) that rely on physical properties (such as density and size) are difficult to eliminate impurities.

[0019] (2) High separation efficiency and strong enrichment capacity. The magnetic nanoparticles of the present invention serve as a substrate, providing a large specific surface area and capable of loading more ligands. The surface-modified temperature-responsive polymer brush (poly (NIPAm-co-AGE)) forms a brush-like structure, increasing the contact sites with exosomes and enhancing affinity adsorption capacity. The specific reaction between the azide group and the alkyne-modified CLIKKPF ensures stable fixation of the ligand, further improving the binding efficiency.

[0020] (3) The separation process is fast, convenient, and easy to operate. The magnetic properties of the material of the present invention (based on a magnetic nanoparticle substrate) enable the separation process to be completed through the steps of "adsorption-magnetic separation-elution", without relying on large equipment such as ultracentrifugation, greatly shortening the separation time. The operation steps are simple and easy to standardize. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Flow chart of the isolation of exosomes according to the present invention.

[0023] Figure 2 The TEM characterization results of magnetic nanoparticles at different synthesis stages are shown in Figure 1, where A is MNP, B is MNP@Si, C is MNP@NH2, D is MNP@Br, and E is MNP@poly(NIPAm- co -AGE), F is MNP@poly(NIPAm- co -AGE)@N3, G is MNP@poly(NIPAm- co -AGE)@CLIKKPF.

[0024] Figure 3 These are the FT-IR characterization results of magnetic nanoparticles at different synthesis stages.

[0025] Figure 4 This is the thermogravimetric analysis diagram of magnetic nanoparticles at different synthesis stages.

[0026] Figure 5 The DLS analysis of magnetic nanoparticles at different synthesis stages, where A is MNP, B is MNP@Si, C is MNP@NH2, D is MNP@Br, and E is MNP@poly(NIPAm- co -AGE), F is MNP@poly(NIPAm- co -AGE)@N3, G is MNP@poly(NIPAm- co -AGE)@CLIKKPF.

[0027] Figure 6 The Zeta potential characterization diagram of magnetic nanoparticles at different synthesis stages, where A is MNP, B is MNP@Si, C is MNP@NH2, D is MNP@Br, and E is MNP@poly(NIPAm- co -AGE), F is MNP@poly(NIPAm- co -AGE)@N3, G is MNP@poly(NIPAm- co -AGE)@CLIKKPF.

[0028] Figure 7 It is MNP@poly(NIPAm- co -AGE)@CLIKKPF TEM characterization results of exosomes purified from kelp extract.

[0029] Figure 8 It is MNP@poly(NIPAm- co -AGE)@CLIKKPF (A) and MNP@poly(NIPAm- co -AGE)@N3(B) on the adsorption effect of exosomes from large yellow croaker muscle stem cells.

[0030] Figure 9 Is the pH effect on MNP@poly(NIPAm- co -AGE)@CLIKKPF adsorbs the effects of large yellow croaker muscle stem cell exosomes.

[0031] Figure 10 The effect of temperature on MNP@poly(NIPAm- co -AGE)@CLIKKPF adsorbs the effects of large yellow croaker muscle stem cell exosomes.

[0032] Figure 11 The effect of medium dosage on MNP@poly(NIPAm- co -AGE)@CLIKKPF adsorbs the effects of large yellow croaker muscle stem cell exosomes.

[0033] Figure 12Is the binding time to MNP@poly(NIPAm- co -AGE)@CLIKKPF adsorbs the effects of large yellow croaker muscle stem cell exosomes.

[0034] Figure 13 The effect of eluent concentration on MNP@poly(NIPAm- co -AGE)@CLIKKPF adsorbs the effects of large yellow croaker muscle stem cell exosomes.

[0035] Figure 14 Ultracentrifugation (A) and MNP@poly(NIPAm- co -AGE)@CLIKKPF (B) Particle size distribution of exosomes from large yellow croaker muscle stem cells extracted by affinity adsorption method.

[0036] Figure 15 Ultracentrifugation (A) and MNP@poly(NIPAm- co -AGE)@CLIKKPF (B) Zeta potential of large yellow croaker muscle stem cell exosomes extracted by affinity adsorption method.

[0037] Figure 16 It is MNP@poly(NIPAm- co -AGE)@CLIKKPF TEM characterization results of exosomes purified from microalgae supernatant, where A is Spirulina and B is Chlorella.

[0038] Figure 17 It is MNP@poly(NIPAm- co -AGE)@CLIKKPF TEM characterization results of exosomes purified from large yellow croaker cell culture medium.

[0039] Figure 18 It is MNP@poly(NIPAm- co -AGE)@CLIKKPF TEM characterization results of exosomes purified from scallop homogenate. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available or can be prepared by existing methods unless otherwise specified.

[0046] Example 1: A method for preparing an exosome separation material using CLIKKPF as a ligand comprises the following steps: (1) Preparation of magnetic nanoparticles (MNPs) by hydrothermal method: 11 g of ferric chloride and 10-35 g of sodium acetate were dispersed in 50-500 mL of ethylene glycol. The mixture was stirred at room temperature until the solid was completely dissolved and the solution turned brown. The solution was then poured into a high-pressure reactor and reacted at 150-250°C for 10-15 h. After the reaction, the reactor was removed and cooled to room temperature. The solution was then poured into a beaker and the nanoparticles were collected through the beaker with a strong magnet. The supernatant was discarded and the sample was rinsed repeatedly with ultrapure water and anhydrous ethanol for 2-6 times. The sample was then stored in anhydrous ethanol to obtain magnetic nanoparticles, which were recorded as MNPs.

[0047] (2) Preparation of magnetic nanoparticles (MNP@Si) coated with silicon nanoparticles: 0.1-1 g of MNPs were dispersed in 200-800 mL of ethanol-water solution (85%, v:v) and ultrasonicated for 0.5-1.5 h to allow the MNPs to be fully dispersed in the system. After the ultrasonication, 5-35 mL of ammonia water (25%, v:v) was added to the system and stirred at 100-280 rpm for 10-30 min. During the stirring process, 5-50 mL of ethyl orthosilicate was added and the stirring was continued at room temperature for 1-2 h. After the reaction was completed, the nanoparticles were adsorbed through the wall of the flask with a strong magnet and washed 2-6 times with anhydrous ethanol and ultrapure water respectively. The nanoparticles were then stored in anhydrous ethanol and recorded as magnetic nanoparticles coated with silicon nanoparticles (MNP@Si).

[0048] (3) Preparation of amino-functionalized magnetic nanoparticles (MNP@NH2): 0.1-1 g of MNP@Si was ultrasonically dispersed in 50-500 mL of APTES-ethanol solution (4%, v:v). The mixture was poured into a 500 mL round-bottom flask and 20 glass beads (1-3 mm in diameter) were added. The round-bottom flask was fixed on a rotary evaporator and stirred at 100-280 rpm for 1-4 h at room temperature. After the reaction, the nanoparticles were adsorbed through the wall of the flask using a strong magnet. The nanoparticles were washed 2-4 times with anhydrous ethanol and ultrapure water, respectively, and then stored in anhydrous ethanol. These nanoparticles were designated as amino-functionalized magnetic nanoparticles (MNP@NH2).

[0049] (4) Preparation of bromine-functionalized magnetic nanoparticles MNP@Br: Disperse 0.1-1 g of MNP@NH2 in 5-100 mL of tetrahydrofuran, seal with parafilm, and ultrasonically disperse for 10-40 min. Then, add 5-30 mL of triethylamine and quickly place the sample in an ice-water mixture to cool it down. Then, add 2.5-8 mL of 2-bromoisobutyryl bromide while stirring at 100-280 rpm. Quickly seal with parafilm and place the system on a shaker at 100-280 rpm at room temperature overnight. After the reaction is completed, use a strong magnet to adsorb the nanoparticles through the wall of the flask. Wash with anhydrous ethanol and ultrapure water 2-4 times respectively, and then store the nanoparticles in anhydrous ethanol. The nanoparticles are recorded as bromine-functionalized magnetic nanoparticles (MNP@Br).

[0050] (5) Preparation of magnetic nanoparticles MNP@poly(NIPAm- co-AGE): Add 0.1-1 g of MNP@Br, 1-8 g of N-isopropylacrylamide (NIPAm), 1.5-7.5 mL of allyl glycidyl ether (AGE), 0.01-0.1 g of copper bromide, and 0.01-0.1 g of cuprous bromide to 50-150 mL of isopropanol and 20 glass beads. Ultrasonicate the system for 5-60 min, then purge with nitrogen for 5-40 min. Then, add 0.01-0.2 g of tris[2-(dimethylamino)ethyl]amine and continue purging with nitrogen for 5-40 min. Then, seal the sample and place it on a shaker for 12-30 h at 25-40°C and 100-280 rpm. After the reaction is completed, the nanoparticles are adsorbed through the wall of the flask with a strong magnet, and washed 2-6 times with 2-8% ethylenediaminetetraacetic acid aqueous solution, anhydrous ethanol and ultrapure water respectively. The nanoparticles are then stored in anhydrous ethanol and recorded as magnetic nanoparticles modified with temperature-responsive polymer brushes (MNP@poly(NIPAm- co -AGE)).

[0051] (6) Preparation of azide-functionalized magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@N3: 0.1-1 g of MNP@poly(NIPAm- co -AGE), 1-8 g of ammonium chloride, 0.1-2 g of sodium azide or potassium azide are added to 20-150 mL of pure water and 20 glass beads are placed. After ultrasonicating the system for 5-60 minutes, the sample is sealed and placed on a shaker. The reaction is carried out at 15-40 ° C and 100-280 rpm for 10-30 hours. After the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, washed three times with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol. The nanomagnetic beads are recorded as azide-functionalized magnetic nanoparticles (MNP@poly(NIPAm- co -AGE)@N3).

[0052] (7) Preparation of CLIKKPF-modified magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@CLIKKPF: Add 0.05-0.2 mL of saturated copper sulfate solution and 0.5-4 g of sodium ascorbate to 100-400 mL of methanol aqueous solution (35-60%). After the system is thoroughly mixed, add 0.1-1 g of MNP@poly(NIPAm- co-AGE)@N3 and 0.05-0.3 g alkynylated CLIKKPF. The system was placed on a shaker and allowed to react for 8-24 hours at room temperature and 100-280 rpm in the dark. After the reaction, the nanoparticles were adsorbed through the flask wall using a strong magnet. The beads were then washed 2-6 times with anhydrous ethanol and then ultrapure water. The nanoparticles were then stored in anhydrous ethanol and labeled as CLIKKPF-modified magnetic nanoparticles (MNP@poly(NIPAm- co -AGE)@CLIKKPF).

[0053] Example 2: A method for preparing an exosome separation material using CLIKKPF as a ligand comprises the following steps: (1) Preparation of magnetic nanoparticles (MNPs) by hydrothermal method: 11 g of ferric chloride and 24 g of sodium acetate were dispersed in 100 mL of ethylene glycol and stirred at room temperature until the solid was completely dissolved. When the solution turned brown, the solution was poured into a high-pressure reactor and reacted at 220°C for 13 h. After the reaction, the reactor was removed and cooled to room temperature. The solution was poured into a beaker and the nanomagnetic beads were collected through the beaker with a strong magnet. The supernatant was discarded and the sample was rinsed repeatedly with ultrapure water and anhydrous ethanol for 6 times. The sample was then stored in anhydrous ethanol to obtain magnetic nanoparticles, which were recorded as MNPs.

[0054] (2) Preparation of silicon nanoparticle-coated magnetic nanoparticles MNP@Si: 0.3 g of MNP was dispersed in 500 mL of ethanol-water solution (85%, v:v) and ultrasonicated for 1.5 h to allow the MNP to be fully dispersed in the system. After the ultrasonication, 15 mL of ammonia water (25%, v:v) was added to the system and stirred at 180 rpm for 15 min. During the stirring process, 15 mL of ethyl orthosilicate was added and the stirring was continued at room temperature for 1.5 h. After the reaction was completed, the nanomagnetic beads were adsorbed through the wall of the flask with a strong magnet and washed with anhydrous ethanol and ultrapure water six times respectively. The nanomagnetic beads were then stored in anhydrous ethanol and recorded as silicon nanoparticle-coated magnetic nanoparticles (MNP@Si).

[0055] (3) Preparation of amino-functionalized magnetic nanoparticles (MNP@NH2): 400 mg of MNP@Si was ultrasonically dispersed in 200 mL of APTES-ethanol solution (4%, v:v). The mixture was poured into a 500 mL round-bottom flask and 20 glass beads (1 mm in diameter) were added. The round-bottom flask was fixed on a rotary evaporator and stirred at 120 rpm for 3 h at room temperature. After the reaction, the nanoparticles were adsorbed on the wall of the flask using a strong magnet. The nanoparticles were washed six times with anhydrous ethanol and ultrapure water, respectively, and then stored in anhydrous ethanol. These nanoparticles were designated as amino-functionalized magnetic nanoparticles (MNP@NH2).

[0056] (4) Preparation of bromine-functionalized magnetic nanoparticles MNP@Br: 300 mg of MNP@NH2 was dispersed in 50 mL of tetrahydrofuran, sealed with a sealing film, and ultrasonically dispersed for 15 min. Then, 15 mL of triethylamine was added, and the sample was quickly placed in an ice-water mixture to cool down. Then, 3.5 mL of 2-bromoisobutyryl bromide was added while stirring at 100 rpm. The mixture was quickly sealed with a sealing film and placed on a shaker at 150 rpm at room temperature overnight. After the reaction was completed, the nanoparticles were adsorbed through the wall of the flask with a strong magnet, washed 6 times with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol. The nanoparticles were recorded as bromine-functionalized magnetic nanoparticles (MNP@Br).

[0057] (5) Preparation of magnetic nanoparticles MNP@poly(NIPAm- co -AGE): 0.5 g of MNP@Br, 3 g of N-isopropylacrylamide (NIPAm), 3.5 mL of allyl glycidyl ether (AGE), 0.02 g of copper bromide, and 0.04 g of cuprous bromide were added to 50 mL of isopropanol and 20 glass beads were placed. After ultrasonicating the system for 25 min, nitrogen was blown for 15 min, and then 0.02 g of tris[2-(dimethylamino)ethyl]amine was added and nitrogen was blown for another 15 min. The sample was then sealed and placed on a shaker for 24 h at 37°C and 100 rpm. After the reaction was completed, the nanomagnetic beads were adsorbed through the wall of the flask with a strong magnet and washed three times with 5% ethylenediaminetetraacetic acid aqueous solution, anhydrous ethanol, and ultrapure water. The nanomagnetic beads were then stored in anhydrous ethanol and recorded as magnetic nanoparticles modified with temperature-responsive polymer brushes (MNP@poly(NIPAm- co -AGE)).

[0058] (6) Preparation of azide-functionalized magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@N3: 0.5g of MNP@poly(NIPAm- co -AGE), 3g of ammonium chloride, 0.2g of sodium azide or potassium azide were added to 50 mL of pure water and 20 glass beads were placed. After the system was ultrasonicated for 30 min, the sample was sealed and placed on a shaker and reacted at room temperature and 120 rpm for 10 h. After the reaction was completed, the nanomagnetic beads were adsorbed through the wall of the flask with a strong magnet, washed three times with anhydrous ethanol and ultrapure water respectively, and then stored in anhydrous ethanol. They were recorded as azide-functionalized magnetic nanoparticles (MNP@poly(NIPAm- co -AGE)@N3).

[0059] (7) Preparation of CLIKKPF-modified magnetic nanoparticles MNP@poly(NIPAm- co -AGE)@CLIKKPF: 0.1 mL of saturated copper sulfate solution and 2 g of sodium ascorbate were added to 300 mL of methanol aqueous solution (57%). After the system was thoroughly mixed, 0.2 g of MNP@poly(NIPAm- co -AGE)@N3 and 0.1 g alkynylated CLIKKPF. The system was placed on a shaker and allowed to react for 18 h at 100 rpm in the dark at room temperature. After the reaction was completed, the nanoparticles were adsorbed through the flask wall using a strong magnet. The beads were washed six times with anhydrous ethanol and then ultrapure water, respectively. The nanoparticles were then stored in anhydrous ethanol and labeled as CLIKKPF-modified magnetic nanoparticles (MNP@poly(NIPAm- co -AGE)@CLIKKPF).

[0060] Material characterization: TEM results of nanoparticles at different preparation stages are shown in Figure 2. Figure 2 As shown, compared with the bare ball ( Figure 2 -A), magnetic nanoparticles after being wrapped with silicon nanoparticles ( Figure 2 -B), changes the surface activity of magnetic nanoparticles, making them more cohesive and causing the magnetic beads to aggregate. Figure 2 -C), bromine ( Figure 2 -D), polymer brush ( Figure 2 -E), azido ( Figure 2 -F) and CLIKKPF ( Figure 2 -G), whose appearance has not changed significantly.

[0061] FT-IR spectroscopy was used to characterize the nanoparticles at different preparation stages. Figure 3 As shown, the MNP nanoparticles have a peak at 585 cm -1 The Fe-O vibration absorption peak appears at 1096.4 cm -1 The Si-O-Si stretching vibration absorption peak was observed at 952.7 cm -1 、798.4 cm -1 The absorption signal at 468.7 cm comes from the bending vibration of -Si-OH and the stretching vibration of Si-O, indicating that the silicon-based reagent has successfully wrapped on the surface of the MNP nanoparticle core to form a silica gel layer. -1 The Si-O absorption peak at 1096.4 cm -1The Si-O-Si absorption peaks were significantly weakened. Due to the low amino density on the surface of the nanoparticles, FT-IR did not produce any significant signal changes. In the infrared spectrum of MNP@Br, a peak at 2356.6 cm -1 The absorption peak at 1635.2 cm indicates that the bromination reaction may produce cyanide compounds due to the presence of nitrogen-containing substances in the previous step. -1 The absorption signal at cams from the amide bond, indicating that the SI-ATRP initiator was successfully modified on the surface of the nanoparticles through the amide reaction. co -AGE) in the infrared spectrum, 2356.6 cm -1 The absorption peak at 1096.4 cm -1 , 585 cm -1 、468.7 cm -1 The absorption peaks at 1096.4 cm -1 The stretching vibration and methylene shear bands originating from the ester carbonyl group indicate that the brush polymer was successfully synthesized on the surface of the nanoparticles. -1 The weak signal at 2356.6 cm is derived from the symmetrical vibration of the AGE epoxy group. -1 The obvious stretching vibration at 2356.6 cm is derived from the higher vibration frequency of the nitrogen-nitrogen triple bond, indicating the formation of high-density azide groups on the surface of the nanoparticles. Through the specific reaction of azide and carbonyl groups, CLIKKPF was successfully modified onto the polymer surface. -1 、1635.2 cm -1 The absorption peak at 1096.4 cm -1 A strong absorption peak can be clearly observed at the center, and this spectral change feature is consistent with the expected structure, indicating that the CLIKKPF modification process was successfully completed and the target material was successfully prepared.

[0062] Thermogravimetric analysis was used to characterize the MNP, MNP@Si, MNP@NH2, MNP@Br, MNP@poly(NIPAm- co -AGE)、MNP@poly(NIPAm- co -AGE)@N3, MNP@poly(NIPAm- co -AGE)@CLIKKPF and its organic components and some thermally stable substances were characterized. Figure 4As shown, when the temperature gradually increased to 200°C, the TGA images of the seven tested nanomagnetic beads all showed a significant downward trend with almost the same slope, and their weight loss rate was about 0.5%. This is due to the evaporation of water absorbed in the sample, crystal water, and some organic reagents. The TGA curve of MNP is clearly different from that of other tested magnetic beads. Over the entire temperature range, the weight loss rate of unmodified MNP reached 0.47%, which is attributed to the rich oxygen-containing functional groups on the surface of the nanomagnetic beads and their high-temperature resistance. During the nitrogen flow protection process, Fe3O4 magnetic beads generally remain stable below 600°C, while FeO is thermodynamically stable above 570°C. Therefore, when the temperature is between 600-800°C, a phase transition process from Fe3O4 to FeO will occur, causing certain fluctuations in the TGA curve. For MNP@Si, as the temperature rises, its thermal weight loss rate gradually slows down. This is mainly due to the dehydration of organic reagents adsorbed on the sample surface and some hydroxyl groups in the SiO2 layer at high temperature. However, the overall weight loss rate is slightly higher than that of other magnetic beads to be tested because less organic polymers are introduced than other magnetic beads. It can be further observed from the figure that the weight loss rate of MNP@Br is the highest among all magnetic beads. This may be because the chemical bond energy between the bromine atoms and the surface of the magnetic beads is relatively low, which makes it easier to break during heating, causing the related groups to detach from the surface of the magnetic beads. It may also be because its thermal decomposition temperature is low, and it decomposes earlier in the heating process to produce volatility. Similarly, the weight loss rate of MNP@NH2 is second only to MNP@Br at 9.40%, which is attributed to its own instability, while the nanomagnetic beads MNP@poly(NIPAm- co -AGE)、MNP@poly(NIPAm- co -AGE)@N3 and MNP@poly(NIPAm- co -AGE)@CLIKKPF weight loss rates were 8.05%, 6.78% and 8.28% respectively. The high overall weight loss rate was mainly due to the introduction of more organic matter on its surface, which also proved that the brush polymer was successfully grafted onto the surface of the nanomagnetic particles. co -AGE)@N3 exhibits a relatively low mass loss rate, which may be due to the steric hindrance effect of the epoxy groups introduced by the AGE monomer, which effectively reduces the interaction between polymer chain segments and thus improves the thermal stability.

[0063] Dynamic light scattering (DLS) technology was used to systematically characterize the microstructure and particle size distribution of nanomagnetic beads at different preparation stages. Figure 5 shown. MNP, MNP@Si, MNP@NH2, MNP@Br, MNP@poly(NIPAm- co -AGE)、MNP@poly(NIPAm- co-AGE)@N3, MNP@poly(NIPAm- co -AGE)@CLIKKPF are all spherical or quasi-spherical, with relatively uniform particle size distribution, but some samples have a certain degree of aggregation. The surface of the MNP magnetic nanoparticles is smooth, and its hydrodynamic particle size is 899.3±69.83 nm (PDI = 0.330±0.011) ( Figure 5 -A), the hydrodynamic particle size is larger, which is attributed to the fact that the magnetic nanoparticles are insoluble in water, resulting in poor dispersion in water and easy aggregation. At the same time, its large sedimentation coefficient also makes it more likely to aggregate during the measurement process, but its polydispersity index (PDI) is small, indicating that the particle size distribution is relatively narrow and the particle size is relatively uniform. The hydrodynamic size of MNP@Si increased to 3208.67±1879.68 nm (PDI= 0.638±0.327). The increased particle size is partly due to the silica gel layer modified on the surface of the nanoparticles ( Figure 5 -B), and on the other hand, it is due to the hydration of hydrophilic silica gel on the surface of nanoparticles. In addition, the modification of the silica gel layer leads to a certain degree of agglomeration, thereby reducing its dispersibility. After the MNP@Si nanoparticles were modified with amino, azido, ATRP initiator and polymer brush, dynamic light scattering test showed that its hydrodynamic particle size increased compared with MNP ( Figure 5 -C to 5-F), this change is attributed to the surface modified functional groups and the grafted polymer layer, while the higher PDI value reflects the uneven distribution of polymer on the nanoparticle surface. Figure 5 As shown in Figure 5-G, the introduction of CLIKKPF further increases its PDI, indicating that CLIKKPF is not evenly distributed on the surface of magnetic nanoparticles, resulting in uneven size of functionalized magnetic nanoparticles.

[0064] The zeta potential of the prepared nanoparticles was measured using a Malvern particle size analyzer electrophoresis method. Zeta potential is the key to understanding their distribution and aggregation process, and can be used to understand the stability of the dispersion and its surface charge characteristics and behavior. Figure 6 It can be seen that the Zeta potential of MNP is positive and the absolute value distribution is relatively concentrated ( Figure 6-A). This phenomenon shows that the surface of the MNP magnetic beads carries more positive charges and the charge density is relatively high. Since like charges repel each other, the higher positive charge density generates a stronger electrostatic repulsion between the particles. This repulsion effectively prevents the aggregation of the magnetic beads in the solution, thereby improving their dispersion stability. This result is highly consistent with the TEM image and DLS analysis results. Theoretically, the smaller the particle size, the greater the ratio of its surface area to volume. A larger surface area means that more surface charges can be carried per unit volume, resulting in an increase in the Zeta potential, which further explains the good dispersion stability exhibited by the MNP magnetic beads. The Zeta potential of MNP@Si is -31.90±0.82 mV ( Figure 6 -B), is negative and has a large absolute value, indicating that its surface is negatively charged and has a high density. Although it can maintain dispersion to a certain extent, it may have a strong electrostatic attraction with positively charged substances. Its surface is relatively rough due to the modification of the silica gel layer, which may have an impact on the surface charge distribution. The functional group coating may increase the surface charge, thereby increasing the Zeta potential. The Zeta potential of MNP@NH2 is close to 0 ( Figure 6 -C), at this time, the surface charge of the magnetic beads is small, the electrostatic repulsion is very weak, the magnetic beads are prone to aggregation, and the stability is poor. The introduction of bromine atoms changes the surface charge distribution of the magnetic beads ( Figure 6 -D), so that there is a certain electrostatic repulsion between MNP@Br particles to maintain dispersion. MNP@poly(NIPAm- co -AGE)) has a zeta potential as high as 37.63±0.93 mV ( Figure 6 -E), is the highest positive value among all magnetic beads, indicating that its surface positive charge density is large. The modification of the polymer layer may significantly increase the surface positive charge, making the electrostatic repulsion between particles very strong, which is extremely beneficial to its dispersion stability in solution. co -AGE)@N3( Figure 6 -F)、MNP@poly(NIPAm- co -AGE)@CLIKKPF( Figure 6 -G) is due to the influence of the azide group on the peptide chain, which produces a negative potential.

[0065] Application Example 1: Sample preparation: Take the large yellow croaker cell culture medium, pass it through a 70-mesh sieve, a 200-mesh sieve, a 0.8 μm filter membrane, and a 0.22 μm filter membrane in sequence, and collect the filtrate in a sterile bottle for later use.

[0066] Sample preparation: Accurately weigh 0.1 g of MNP@poly(NIPAm- co-AGE)@CLIKKPF magnetic nanoparticles were placed in a 50 mL centrifuge tube. MNP@poly(NIPAm- co -AGE)@CLIKKPF. After washing, use a strong magnet to adsorb the nanomagnetic beads through the flask wall and pour out the supernatant. Repeat this three times and wait for the sample to be loaded. Add 35 mL of treated large yellow croaker cell culture medium to a centrifuge tube, and then add 10 mL of adsorption buffer. Then shake at 4°C for 40 minutes. After the adsorption is completed, use a strong magnet to isolate the centrifuge tube and place the MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed and the solution in the tube was discarded. Then 5 mL of PBS balance solution was added and shaken for 1 min. The MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed, and the PBS balance solution was discarded. This was repeated twice to remove non-specifically bound exosomes. Finally, 1 mL of elution solution was added and shaken for 5 minutes. The MNP@poly(NIPAm- co -AGE)@CLIKKPF fixed, the eluate was collected and stored at 4°C. The eluate was sent to TEM for examination to determine whether it contained exosome particles. The results were as follows Figure 7 As shown, the eluate from the culture medium of large yellow croaker cells contains abundant exosome particles (black circles). This demonstrates that the CLIKKPF-functionalized magnetic nanoparticles can effectively separate and purify exosome particles in actual animal cell culture fluid samples.

[0067] To verify the MNP@poly(NIPAm- co -AGE)@CLIKKPF on the adsorption effect of large yellow croaker muscle stem cell exosomes, with MNP@poly(NIPAm- co -AGE)@N3 was used as a control to conduct parallel experiments, and NTA was used to quantify the particle size distribution characteristics and concentration of exosomes in the sample, and its adsorption efficiency was further calculated. Figure 8 MNP@poly(NIPAm- co -AGE)@CLIKKPF( Figure 8 -A) has a much higher adsorption capacity for large yellow croaker muscle stem cell EVs than MNP@poly(NIPAm- co -AGE)@N3( Figure 8 -B). It is worth noting that there is no significant difference in the particle size distribution of exosomes extracted from the two materials, proving that MNP@poly(NIPAm- co -AGE)@CLIKKPF has a specific adsorption effect on large yellow croaker muscle stem cell exosomes.

[0068] PBS buffer with pH 5.5-9.5 was used as the sample buffer to verify the exosomes in the culture medium concentrate of large yellow croaker in the presence of MNP@poly(NIPAm- co -AGE)@CLIKKPF nanoparticle surface binding effect will be affected by pH. Figure 9 As shown in the figure, as the pH gradually increases, the particle size of the extracted large yellow croaker muscle stem cell exosome particles shows an overall downward trend. This change may be because the pH affects the charge properties and hydration layer of the nanoparticle surface. Under acidic conditions, the surface of the nanoparticles may be highly protonated, the electrostatic repulsion between charges is relatively weak, or close to the isoelectric point of the exosomes, and the particles are prone to aggregation, and the particle size is larger; as the pH increases, the charge properties of the particle surface change, the electrostatic repulsion is enhanced, and the hydration layer thickens, which increases the distance between the particles, reduces the degree of aggregation, and reduces the particle size. However, they are all within the particle size range of extracellular vesicles, and the adsorption capacity is also on a downward trend. Combined with MNP@poly(NIPAm- co -AGE)@CLIKKPF has a negative Zeta potential. In a suitable acidic environment, the configuration and charge distribution of the surface active sites of the nanoparticles are more conducive to binding with exosomes. Therefore, in general, when the pH is 5.5, MNP@poly(NIPAm- co -AGE)@CLIKKPF has a better adsorption effect on exosomes of large yellow croaker muscle stem cells.

[0069] Effect of temperature on MNP@poly(NIPAm- co -AGE)@CLIKKPF adsorption of large yellow croaker muscle stem cell exosomes Figure 10 As shown. Exosomes from large yellow croaker muscle stem cells extracted at different temperatures exhibited some variation, with particle sizes ranging from 100 to 120 nm. This fluctuation may be due to temperature affecting exosome activity and intermolecular interactions. At low temperatures, molecular motion is slow, and exosomes may aggregate through weak interactions, resulting in larger particle sizes. As temperatures rise, molecular motion intensifies, partially destroying the aggregated structure and reducing the particle size. However, excessively high temperatures may trigger surface structural changes, further promoting particle aggregation and increasing particle size. Regarding binding capacity, as the temperature increases from 4°C to 37°C, the binding capacity decreases, with a slight increase at 45°C. This is because temperature changes affect the conformation of active sites on the nanoparticle surface and the intermolecular interactions associated with exosome binding. Overall, adsorption capacity is better at 18°C.

[0070] With the development of MNP@poly(NIPAm- co-AGE)@CLIKKPF nanomagnetic bead medium dosage increased from 5 mg to 100 mg, and the adsorbed and extracted large yellow croaker muscle stem cell exosomes showed a trend of first increasing and then decreasing, all located at 140-170 nm. At lower dosages, the nanoparticles are relatively dispersed. As the dosage increases, the distance between the particles decreases, and the distance between the adsorbed exosomes is further reduced, so that even after desorption, they may aggregate through some weak interactions, resulting in an increase in particle size. However, when the dosage is too high, factors such as steric hindrance may inhibit excessive aggregation, causing the particle size to decrease. The binding ability decreases with the increase in the amount of medium, perhaps because when the amount of medium is low, the active sites on the surface of the nanoparticles can more fully contact and bind to the target (large yellow croaker muscle stem cell exosomes), and the binding ability is stronger. As the amount of medium increases, the number of nanoparticles increases, and the active sites of some particles may be blocked, or the diffusion of the target between the particles may be hindered, resulting in a decrease in effective binding with the target and a decrease in binding ability. From Figure 11 From the average trend, a lower medium dosage (5 mg) may be more conducive to maintaining a higher binding capacity.

[0071] Time is MNP@poly(NIPAm- co -AGE)@CLIKKPF nanomagnetic beads are one of the key factors for the binding of large yellow croaker muscle stem cell exosomes. Large yellow croaker muscle stem cell exosomes and nanomagnetic beads MNP@poly(NIPAm -co -AGE)@CLIKKPF nanomagnetic beads with different reaction time results Figure 12 As shown in the figure, as the reaction time is extended from 15 min to 120 min, the particle size of the large yellow croaker muscle stem cell exosomes shows a trend of first increasing and then decreasing. As the reaction time increases, the nanomagnetic beads gradually combine with the exosomes, the particle density increases, the intermolecular distance decreases, and the interaction force increases, resulting in a slight increase in the degree of aggregation, which shows an increase in the measured particle size. Subsequently, as some binding sites are saturated and the reaction becomes stable, the bound particles disaggregate, and the particle size decreases. As time continues to increase, the particle size changes further, which may form a new structure between the exosomes. And MNP@poly(NIPAm- co The overall binding capacity of the exosomes (-AGE)@CLIKKPF) increased over time, then decreased slightly. Longer reaction times allow more time for the nanoparticles to interact with the exosomes, allowing more active sites to participate in binding, thereby enhancing binding capacity. However, a slight decrease in binding capacity at 120 minutes may be due to the shedding of some bound exosomes caused by the prolonged reaction, or to changes in active sites on the nanoparticle surface, which impaired further binding. The trends in the figure suggest that a reaction time of approximately 90 minutes may be optimal, as this provides strong binding capacity and a relatively suitable particle size.

[0072] MNP@poly(NIPAm- co -AGE)@CLIKKPF nanomagnetic beads bind to large yellow croaker muscle stem cell exosomes in a pH-mediated adsorption / desorption process. The effect of eluent pH on the desorption efficiency of large yellow croaker muscle stem cell exosomes was further verified. The pH value of the eluent was adjusted using different concentrations of ammonia water, such as Figure 13 As shown, as the ammonia concentration increases, the exosome particle size reaches its maximum at 9% ammonia, while the exosome concentration after elution is highest at 6% ammonia. Experiments verifying the effect of pH on adsorption revealed that under a suitable acidic environment, the configuration and charge distribution of the nanoparticle surface active sites are more conducive to binding with exosomes. The pH of the desorption solution at 6% ammonia is greater than 11, facilitating debinding. 6% ammonia concentration results in the highest exosome concentration after elution while maintaining exosome integrity, making it the optimal desorption concentration.

[0073] Using dynamic light scattering (DLS) technology, it was found that ultracentrifugation ( Figure 14 -A) with MNP@poly(NIPAm- co -AGE)@CLIKKPF affinity adsorption method ( Figure 14 -B) The hydrated particle sizes of the extracted large yellow croaker muscle stem cell exosomes were 127.50±1.76 nm and 59.33±0.55 nm ( Figure 14 -B), among which the large yellow croaker muscle stem cell exosomes extracted by ultracentrifugation had a relatively low polydispersity index (PDI, 0.263±0.009), indicating that they had higher size uniformity. In contrast, the PDI value of the exosomes extracted by affinity adsorption was 0.495±0.005, which was relatively high, indicating that their particle size distribution was more dispersed, but mainly located between 50-150 nm, which was consistent with the basic size of exosomes.

[0074] Zeta potential is often used to characterize the surface charge of particles in colloidal dispersion systems, thereby reflecting the interaction between particles and can be used to evaluate the stability of nanoparticles. Overall, ultracentrifugation ( Figure 15 -A) The extracted large yellow croaker muscle stem cell exosomes were more abundant than MNP@poly(NIPAm- co -AGE)@CLIKKPF affinity adsorption method ( Figure 15 -B) The absolute value of the zeta potential of the large yellow croaker muscle stem cell exosomes extracted is larger, and the greater the electrostatic repulsion between particles, the more stable the system. However, the data dispersion of the ultracentrifugation group is greater, indicating that there are large individual differences in the exosome samples extracted by ultracentrifugation, and the surface charge properties of different particles vary greatly; while MNP@poly(NIPAm- co-AGE)@CLIKKPF The zeta potential of the large yellow croaker muscle stem cell exosomes extracted by affinity adsorption method is relatively concentrated, indicating that the surface charge properties of the exosomes extracted by affinity adsorption method are relatively uniform.

[0075] Application Example 2: Sample preparation: Take the culture medium of Spirulina and Chlorella, centrifuge it at 10,000 rpm for 15 min, filter it through 0.8 μm filter membrane and 0.22 μm filter membrane, and collect the filtrate in a sterile bottle for later use.

[0076] Sample preparation: Accurately weigh 0.1 g of MNP@poly(NIPAm- co -AGE)@CLIKKPF magnetic nanoparticles were placed in a 50 mL centrifuge tube. MNP@poly(NIPAm- co -AGE)@CLIKKPF. After washing, use a strong magnet to adsorb the nanomagnetic beads through the flask wall and pour off the supernatant. Repeat this three times before loading. Add 35 mL of treated microalgae culture medium to a centrifuge tube and add 10 mL of adsorption buffer. Then shake at 4°C for 40 minutes. After adsorption, use a strong magnet to isolate the centrifuge tube and place the MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed and the solution in the tube was discarded. Then 5 mL of PBS balance solution was added and shaken for 1 min. The MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed, and the PBS balance solution was discarded. This was repeated twice to remove non-specifically bound exosomes. Finally, 1 mL of elution solution was added and shaken for 5 minutes. The MNP@poly(NIPAm- co -AGE)@CLIKKPF fixed, the eluate was collected and stored at 4°C. The eluate was sent to TEM for examination to determine whether it contained exosome particles. The results were as follows Figure 16 As shown, Spirulina ( Figure 16 -A) with Chlorella ( Figure 16 -B) The eluate contains abundant exosome particles (black circles), demonstrating that the CLIKKPF-functionalized magnetic nanoparticles can effectively separate and purify exosome particles from various microalgae samples.

[0077] Application Example 3: Sample preparation: The kelp processing solution is prepared by crushing the dried kelp, dissolving it in physiological saline, and centrifuging it at 1000 r / min at 6°C for 20 min to remove the free kelp cells in the sample; taking the supernatant, and then centrifuging it at 10000 r / min at 6°C for 30 min to remove the broken kelp cell fragments in the sample; finally, taking the supernatant and filtering it with a 0.22 μm filter into a new container for later use.

[0078] Sample preparation: Accurately weigh 0.1 g of MNP@poly(NIPAm- co -AGE)@CLIKKPF magnetic nanoparticles were placed in a 50 mL centrifuge tube. MNP@poly(NIPAm- co -AGE)@CLIKKPF. After washing, use a strong magnet to adsorb the nanomagnetic beads through the flask wall and pour off the supernatant. Repeat this three times before loading. Add 35 mL of kelp extract filtered through a 0.22 μm filter to a centrifuge tube, followed by 10 mL of adsorption buffer (Tris-HCl 10 mM, pH 7.4; calcium chloride 0.5 mM; sodium chloride 120 mM). Then shake at 4°C for 40 minutes. After adsorption, use a strong magnet to isolate the core tube and place the MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed and the solution in the tube was discarded. Then 5 mL of PBS balance solution was added and shaken for 1 min. The MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed, and the PBS balance solution was discarded. This was repeated twice to remove non-specifically bound exosomes. Finally, 1 mL of elution solution (3% ammonia solution) was added and shaken for 5 minutes. The MNP@poly(NIPAm- co -AGE)@CLIKKPF fixed, the eluate (the eluate is rich in exosome particles) was taken and stored at 4 ° C. The eluate was sent to TEM to determine whether it contained exosome particles. The results are as follows Figure 17 As shown in the figure, the eluate contains abundant exosome particles (black circles), which indicates that the CLIKKPF-functionalized magnetic nanoparticles can effectively separate and purify exosome particles in actual dried plant samples.

[0079] Application Example 4: Sample Preparation: 5 g of scallop adductor muscle was minced and added to 50 mL of normal saline, followed by homogenization. The homogenization was then centrifuged at 10,000 rpm for 40 min, filtered through a 0.8 μm filter, and then filtered through a 0.22 μm filter. The filtrate was collected in a sterile bottle for later use.

[0080] Sample preparation: Accurately weigh 0.1 g of MNP@poly(NIPAm- co -AGE)@CLIKKPF magnetic nanoparticles were placed in a 50 mL centrifuge tube. MNP@poly(NIPAm- co -AGE)@CLIKKPF. After washing, use a strong magnet to adsorb the nanomagnetic beads through the flask wall and pour off the supernatant. Repeat this three times and wait for sample loading. Add 35 mL of treated scallop tissue fluid to a centrifuge tube and then add 10 mL of adsorption buffer. Then shake at 4°C for 40 minutes. After adsorption, use a strong magnet to isolate the centrifuge tube and place the MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed and the solution in the tube was discarded. Then 5 mL of PBS balance solution was added and shaken for 1 min. The MNP@poly(NIPAm- co -AGE)@CLIKKPF was fixed, and the PBS balance solution was discarded. This was repeated twice to remove non-specifically bound exosomes. Finally, 1 mL of elution solution was added and shaken for 5 minutes. The MNP@poly(NIPAm- co -AGE)@CLIKKPF fixed, the eluate was collected and stored at 4°C. The eluate was sent to TEM for examination to determine whether it contained exosome particles. The results were as follows Figure 18 As shown, scallop muscle tissue fluid contains abundant exosome particles (white circles). This demonstrates that the CLIKKPF-functionalized magnetic nanoparticles can effectively separate and purify exosome particles in actual animal tissue samples.

[0081] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing an exosome separation material using CLIKKPF as a ligand, characterized in that The following steps are involved: (1) Preparation of magnetic nanoparticles (MNPs) by hydrothermal method; (2) Preparation of magnetic nanoparticles MNP@Si coated with silicon nanoparticles; (3) Preparation of amino-functionalized magnetic nanoparticles MNP@NH2; (4) Preparation of bromine-functionalized magnetic nanoparticles MNP@Br; (5) Preparation of magnetic nanoparticles MNP@poly(NIPAm-co-AGE) modified with temperature-responsive polymer brushes; (6) Preparation of azide-functionalized magnetic nanoparticles MNP@poly(NIPAm-co-AGE)@N3; (7) Preparation of CLIKKPF-modified magnetic nanoparticles MNP@poly(NIPAm-co-AGE)@CLIKKPF.

2. The preparation method according to claim 1, wherein: In step (1), the preparation method of magnetic nanoparticles is as follows: ferric chloride and sodium acetate are dispersed in 50 mL of ethylene glycol, stirred at room temperature until the solid is completely dissolved, and when the solution turns brown, the solution is poured into a high-pressure reactor and reacted at 220°C; after the reaction is completed, the reactor is taken out and cooled at room temperature, and the solution is poured into a beaker, and the nanomagnetic beads are collected through the beaker with a strong magnet, and the supernatant is discarded. The sample is repeatedly rinsed with ultrapure water and anhydrous ethanol, and the sample is stored in anhydrous ethanol to obtain magnetic nanoparticles MNP.

3. The preparation method according to claim 1, wherein: In step (2), the preparation method of magnetic nanoparticles wrapped by silicon nanoparticles is as follows: MNPs are dispersed in an ethanol aqueous solution and ultrasonicated; after the ultrasonication, ammonia water is added to the system and stirred; during the stirring process, ethyl orthosilicate is added, and then stirring is continued at room temperature; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and are washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol to obtain magnetic nanoparticles MNP@Si wrapped by silicon nanoparticles.

4. The preparation method according to claim 1, wherein: In step (3), the preparation method of amino-functionalized magnetic nanoparticles is as follows: MNP@Si is ultrasonically dispersed in an APTES-ethanol solution; the mixed solution is poured into a round-bottom flask, and glass beads are added, the round-bottom flask is fixed on a rotary evaporator, and stirred at room temperature; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and the nanomagnetic beads are washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol to obtain amino-functionalized magnetic nanoparticles MNP@NH2.

5. The preparation method according to claim 1, wherein: In step (4), the preparation method of bromine-functionalized magnetic nanoparticles is as follows: MNP@NH2 is dispersed in tetrahydrofuran, sealed with a sealing film, and ultrasonically dispersed; then triethylamine is added, and the sample is quickly placed in an ice-water mixture for cooling, and then 2-bromoisobutyryl bromide is added under stirring, and the system is quickly sealed with a sealing film and placed on a shaker at room temperature overnight; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and are washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol to obtain bromine-functionalized magnetic nanoparticles MNP@Br.

6. The preparation method according to claim 1, wherein: In step (5), the preparation method of magnetic nanoparticles modified with temperature-responsive polymer brushes is as follows: MNP@Br, N-isopropylacrylamide, allyl glycidyl ether, copper bromide, and cuprous bromide are added to isopropanol, and glass beads are placed; after ultrasonicating the system, nitrogen is blown, and then tri[2-(dimethylamino)ethyl]amine is added and nitrogen is blown again; the sample is then sealed and placed on a shaker. After the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and the nanomagnetic beads are washed with ethylenediaminetetraacetic acid aqueous solution, anhydrous ethanol, and ultrapure water, respectively. The nanomagnetic beads are then stored in anhydrous ethanol to obtain magnetic nanoparticles MNP@poly(NIPAm-co-AGE) modified with temperature-responsive polymer brushes.

7. The preparation method according to claim 1, wherein: In step (6), the preparation method of azide-functionalized magnetic nanoparticles is as follows: MNP@poly(NIPAm-co-AGE), ammonium chloride, and sodium azide are added to N, N-dimethylformamide or pure water, the system is sealed and placed on a shaker; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, and the nanomagnetic beads are washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol, which is recorded as modified azide-functionalized magnetic nanoparticles MNP@poly(NIPAm-co-AGE)@N3.

8. The preparation method according to claim 1, wherein: In step (7), the preparation method of magnetic nanoparticles modified with CLIKKPF is as follows: saturated copper sulfate solution and sodium ascorbate are added to a methanol aqueous solution; after the system is thoroughly mixed, MNP@poly(NIPAm-co-AGE)@N3 and alkynylated CLIKKPF are added; the system is placed on a shaker and reacted at 100 rpm in the dark at room temperature; after the reaction is completed, the nanomagnetic beads are adsorbed through the wall of the flask with a strong magnet, washed with anhydrous ethanol and ultrapure water respectively, and then the nanomagnetic beads are stored in anhydrous ethanol, and recorded as magnetic nanoparticles modified with CLIKKPF MNP@poly(NIPAm-co-AGE)@CLIKKPF.

9. Magnetic nanoparticles MNP@poly(NIPAm-co-AGE)@CLIKKPF prepared by the method of any one of claims 1 to 8.

10. Use of the magnetic nanoparticles MNP@poly(NIPAm-co-AGE)@CLIKKPF according to claim 9 in the isolation of marine-derived exosomes.

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