Anti-adhesion cationic golf ball-shaped microspheres and methods for separating and purifying small extracellular vesicles

Abstract

The present invention discloses anti-adhesive cationic golf-shaped microspheres and a method for separating and purifying small extracellular vesicles using the same. The anti-adhesive cationic golf-shaped microspheres are prepared by heterogeneously agglomerating soft silicone oil droplets and amphoteric polystyrene beads with a particle size of 100 to 200 nm, followed by adhesion of a phase-transition bovine serum albumin nanofilm and rapid etching with tetrahydrofuran. The anti-adhesive cationic golf-shaped microspheres of the present invention can quickly and easily separate small extracellular vesicles from a sample through size and electrostatic effects, and are of great significance in the field of small extracellular vesicle separation. Compared to the current separation technology based on a single characteristic commonly used for the separation of small extracellular vesicles, the present invention overcomes the shortcomings of low purity of small extracellular vesicle separation and poor product integrity, providing a guarantee for the separation and enrichment of small extracellular vesicles in biological samples such as blood, cell culture fluid, and urine.

Description

Technical Field

[0001] The present invention belongs to the technical field of extracellular vesicle separation, and relates to a method for separating small extracellular vesicles based on size and charge and resistance to nonspecific adsorption. Background Art

[0002] Extracellular vesicles (EVs), which carry proteins, nucleic acids, and lipids, have emerged as important mediators of intercellular communication, particularly in many pathophysiological conditions, such as cancer progression and metastasis. Small extracellular vesicles (sEVs), in particular, are less than 200 nm in size and are released by virtually all cells and detected in all body fluids. These nanosized EVs can modulate anti-tumor immune responses and monitor tumor progression and metastasis. Growing evidence suggests that sEVs can serve as messengers of health and disease, making them ideal specimens for liquid biopsies.

[0003] sEVs are being considered as non-invasive diagnostic biomarkers for many diseases. Therefore, rapid isolation of sEVs for downstream analysis is highly desirable. Currently, commercially available sEV phenotyping techniques, including Western blotting, enzyme-linked immunosorbent assay (ELISA), and flow cytometry, have limited multiplexing capabilities and sensitivity. These techniques often require time-consuming and expensive sEV isolation procedures, which limits their use in clinical applications. Therefore, simple and efficient sEV isolation procedures are highly desirable for highly selective and sensitive phenotyping of cancer-derived sEVs.

[0004] The main technologies for sEV separation include ultracentrifugation, ultrafiltration, polymer precipitation, size exclusion chromatography, and immunoaffinity-based separation technology. Ultracentrifugation is currently the "gold standard" method for sEV separation. It can separate large volumes of body fluid samples with high yields, but the equipment is expensive, the operation is complex and time-consuming, and the structure and function of sEVs are easily damaged. Ultrafiltration is simple to operate and has high enrichment efficiency, but because proteins and other molecules smaller than the membrane pore size can pass through the filter membrane, the purity of the separated sEVs will be insufficient, and membrane pore blockage will also affect the separation efficiency of extracellular vesicles. Polymer precipitation uses the property of polymers to "hijack" water molecules to reduce the solubility of sEVs, combined with low-speed centrifugation or other auxiliary means to achieve sEV enrichment. It can handle large doses of samples, but the purity of the sEVs obtained is not high. Immunoaffinity methods offer strong selectivity and high separation purity, enabling the isolation of specific sEV subtypes. However, due to the heterogeneity of sEVs and their significant influence on the microenvironment, immunoaffinity methods cannot fully isolate specific sEVs. Furthermore, immunoaffinity methods have drawbacks such as high separation costs and demanding reagent and storage requirements. This method is not the best choice for researchers who do not require high purity or specific sEV subtypes.

[0005] Therefore, seeking a separation technology that can efficiently and simply separate sEVs and ensure the purity of sEVs without affecting the morphological structure and physiological function of sEVs is an urgent problem that needs to be solved in this technical field. Summary of the Invention

[0006] The purpose of the present invention is to solve the problem of low separation purity of sEVs separation methods in the existing technology, which affects the functional analysis and engineering application of sEVs. It provides anti-adhesion cationic golf-shaped microspheres and a method for separating and purifying sEVs using the microspheres. This method can achieve high-purity separation of sEVs without affecting the structural morphology and physiological function of sEVs, and the operation is simple and fast.

[0007] To achieve the above objectives, the anti-adhesion cationic golf-shaped microspheres provided by the present invention are prepared using the following technical solutions:

[0008] Step 1: Under alkaline conditions, the silanol groups in 3-(methacryloyloxy)propyltrimethoxysilane are dehydrated and condensed to prepare soft silicone oil droplets;

[0009] Step 2: using azobis(N-hydroxyisobutylamidine) hydrate to initiate polymerization of styrene to prepare amphoteric polystyrene beads with a particle size of 100 to 200 nm;

[0010] Step 3: Under acidic conditions, amphoteric polystyrene beads with a particle size of 100-200 nm are added to soft silicone oil droplets for heterogeneous coagulation, and then potassium persulfate is added to initiate polymerization to form raspberry-shaped particles;

[0011] Step 4: The raspberry-like particles are uniformly dispersed in a bovine serum albumin (BSA) aqueous solution, followed by adding a tris-(2-carboxyethyl)phosphine aqueous solution to reduce the BSA, incubating and centrifuging to obtain raspberry-like particles adhered to a phase-transition BSA nanofilm;

[0012] Step 5: The raspberry-like particles attached to the phase-transition BSA nanofilm were uniformly dispersed in tetrahydrofuran, and the amphoteric polystyrene beads with a particle size of 100 to 200 nm were etched away, and the beads were washed by centrifugation with deionized water to obtain anti-adhesive golf ball-shaped microspheres;

[0013] Step 6: Disperse the anti-adhesive golf ball-shaped microspheres in water and add polylysine. After incubation at room temperature, centrifuge to obtain the precipitate and wash to obtain the anti-adhesive cationic golf ball-shaped microspheres.

[0014] In the above step 2, polyvinyl pyrrolidone is preferably added to water, stirred evenly, and then styrene is added. The mixture is stirred at 30-40°C and 300-500 rpm for 15-30 min, then the temperature is raised to 80-90°C and stirred at a constant temperature for 15-30 min while bubbling with nitrogen. Then, azobis(N-hydroxyisobutylamidine) hydrate is added, the mixture is stirred for 5-6 hours, and then cooled to obtain amphoteric polystyrene beads with a particle size of 100-200 nm. The addition ratio of polyvinyl pyrrolidone to water, styrene, and azobis(N-hydroxyisobutylamidine) hydrate is 1.0 g: 30-50 mL: 3.0-5.0 mL: 0.10-0.20 g.

[0015] In the above step 3, the soft silicone oil droplets are preferably uniformly dispersed in water, the pH is adjusted to 2-6 with hydrochloric acid, and then amphoteric polystyrene beads with a particle size of 100-200 nm are added, and stirred at room temperature for 10-30 minutes for electrostatic heterogeneous coagulation, followed by nitrogen gas for 30-50 minutes and heating to 60-80°C, followed by addition of potassium persulfate and polymerization reaction for 10-12 hours to obtain raspberry-shaped particles; wherein the addition ratio of the soft silicone oil droplets to water, amphoteric polystyrene beads with a particle size of 100-200 nm, and potassium persulfate is 1.0 mL: 40-60 mL: 3.0-5.0 mL: 0.03-0.05 g.

[0016] In the above step 4, the addition ratio of the raspberry-shaped particles to the BSA aqueous solution and the tris-(2-carboxyethyl)phosphine aqueous solution is preferably 10 mg: 2.0-6.0 mL: 2.0-6.0 mL, the concentration of the BSA aqueous solution is 2.0-5.0 mg / mL, the concentration of the tris-(2-carboxyethyl)phosphine aqueous solution is 20-60 mmol / L, and the pH is 5.0; the incubation is carried out at room temperature for 1-2 h.

[0017] In the above step 5, the ratio of the raspberry-shaped particles adhered to the phase-transition BSA nanofilm to tetrahydrofuran is preferably 1.0 mg: 0.30-1.0 mL.

[0018] In the above step 6, the anti-adhesion golf-shaped microspheres, water, and polylysine are preferably added in a ratio of 1 mg: 1.0-2.0 mL: 2.0-4.0 mg; and the incubation is performed at room temperature for 24-48 hours.

[0019] The above-mentioned anti-adhesion cationic golf-shaped microspheres have a particle size of 1 to 5 μm, and have an imprint with a pore size of 100 to 200 nm on the surface. The imprinted part is positively charged, and there is a BSA film on the outside that resists nonspecific adsorption. The polylysine in the imprinted part and the phase-change BSA nanofilm on the outside are both adsorbed through physical action.

[0020] The method for separating and purifying sEVs using the anti-adhesion cationic golf-shaped microspheres of the present invention is as follows: the anti-adhesion cationic golf-shaped microspheres are uniformly dispersed in the cell culture supernatant, incubated with shaking at 35-38°C for 20-30 minutes, and then centrifuged and washed in 10 mmol / L PBS buffer at pH 7.4. The obtained precipitate is mixed with ammonia water with a mass concentration of 4% to 8%, and after shaking elution at 4°C, the supernatant is collected to obtain the separated and purified small extracellular vesicles.

[0021] Alkaline solvents (pH 10-12) are typically used to disrupt electrostatic interactions. In alkaline solutions, the surface of the anti-adhesion cationic golf-shaped microspheres is negatively charged, sharing the same charge as the phosphate groups on the surface of sEVs, resulting in charge repulsion. sEVs fall off the surface of the anti-adhesion cationic golf-shaped microspheres. However, when the solution is too alkaline, components such as channel proteins, transport proteins, and signaling pathway proteins on the membrane lose their normal function, causing the sEV membrane to lose its selective permeability and irreversible damage. Therefore, the present invention uses 4% to 8% ammonia water to elute sEVs adsorbed on the surface of the anti-adhesion cationic golf-shaped microspheres.

[0022] In the above-mentioned method for separating and purifying sEVs using anti-adhesive cationic golf-shaped microspheres, the dosage ratio of the anti-adhesive cationic golf-shaped microspheres to cell culture supernatant and ammonia water with a mass concentration of 4% to 8% is preferably 1.0 mg: 0.30 to 1.0 mL: 0.01 to 0.05 mL.

[0023] In the above isolation and purification method, the cell culture supernatant is incubated directly with the anti-adhesion cationic golf-ball-shaped microspheres without any treatment. Both the incubation and elution processes are performed with gentle agitation on a shaker. The resulting sEVs are cryopreserved at 80°C for long-term storage or at 4°C for storage of only one week.

[0024] The present invention uses 100-200 nm amphoteric polystyrene beads as imprinting templates. Under acidic conditions, the zeta potential of the 100-200 nm amphoteric polystyrene beads is positive, while the zeta potential of the soft silicone oil droplets is negative. The two form raspberry-like particles through electrostatic heterogeneous coagulation. The raspberry-like particles are adhered to a phase-transition BSA nanofilm, and the 100-200 nm amphoteric polystyrene beads are then rapidly etched to produce anti-adhesive golf-shaped microspheres. The anti-adhesive golf-shaped microspheres are then modified with polylysine to produce anti-adhesive cationic golf-shaped microspheres, which can be used for the separation and purification of sEVs. Compared with the prior art, the present invention has the following advantages:

[0025] This invention describes a method for separating sEVs based on size and electrostatic interactions. The prepared anti-adhesive cationic golf-ball-shaped microspheres have 100-200 nm imprints on their surfaces, partially modified with polylysine and externally coated with a phase-shift BSA nanofilm. The 100-200 nm imprints are size-selective, restricting the entry of bioparticles larger than 200 nm. The polylysine polymerized within the pores eliminates the adsorption of positively charged bioparticles. The phase-shift BSA nanofilm on the exterior of the imprints reduces the nonspecific adsorption of interfering components in the sample.

[0026] 2. Compared with the current separation technology based on a single characteristic of sEVs, the anti-adhesion cationic golf-shaped microspheres of the present invention are based on the synergistic effect of multiple physical properties of sEVs. The time for separating sEVs is only 30 minutes, the capture rate of sEVs can reach more than 90%, and the recovery rate can reach more than 80%. The morphological structure and biological function of sEVs are not changed, overcoming the shortcomings of low sEV separation purity and poor product integrity, and providing a guarantee for the separation and enrichment of sEVs in biological samples such as blood, cell culture fluid and urine.

[0027] 3. The anti-adhesion cationic golf-shaped microspheres of the present invention can bind to sEVs through size and electrostatic effects. Anti-adhesion cationic golf-shaped microspheres sEVs can be quickly and easily separated by low-speed centrifugation, and almost all sEVs in body fluid samples can be separated. At the same time, the surface imprinting of the anti-adhesion cationic golf-shaped microspheres and the external phase-transition BSA nanofilm can resist nonspecific adsorption and improve the purity of the enriched sEVs. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 3 is a scanning electron microscope image of the anti-adhesion cationic golf ball-shaped microspheres prepared in Example 1.

[0029] Figure 2 This is a scanning electron micrograph of the anti-adhesive cationic golf-shaped microspheres adsorbing sEVs in Example 2.

[0030] Figure 3 Scanning electron microscopy (A) and nanoparticle tracking (B) characterization images of adsorbed sEVs eluted with 8% ammonia water.

[0031] Figure 4 is the concentration of sEVs in all operating solutions during the capture of sEVs by anti-adhesive cationic golf ball-shaped microspheres. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the present invention more clear, the present invention will be clearly and completely described below in conjunction with the embodiments and drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0033] Example 1

[0034] Preparation of Anti-adhesive Cationic Golf-shaped Microspheres

[0035] Step 1: Bubble 50 mL of deionized water with nitrogen for 15 minutes, then add 0.50 mL of ammonia water and mix evenly. Add 2.0 mL of 3-(methacryloyloxy)propyltrimethoxysilane at 35°C, magnetically stir at 35°C and 500 rpm for 20 minutes, and then let it stand at 35°C for 24 hours to obtain soft silicone oil droplets.

[0036] Step 2: First, thoroughly wash 40 mL of styrene with 10 mL of a 10 wt.% aqueous NaOH solution and then deionized water to remove the stabilizer. The washed styrene is stored in the dark until further use. 1.25 g of polyvinylpyrrolidone is added to a 150 mL three-necked round-bottom flask containing 35 mL of water. After stirring at room temperature, 4.0 mL of the washed styrene is added. The mixture is stirred at 35°C and 350 rpm for 20 minutes. The temperature is then raised to 90°C and magnetically stirred for 20 minutes while bubbling with nitrogen. Then, 0.15 g of azobis(N-hydroxyisobutylamidine)hydrate is quickly added to initiate the polymerization of the styrene. The mixture is stirred at this temperature for 6 hours and then cooled to obtain monodisperse amphoteric polystyrene spheres with a particle size of 100 to 200 nm.

[0037] Step 3: Add 1.0 mL of soft silicone oil droplets to 50 mL of water, disperse evenly, and adjust the pH of the soft silicone oil droplet aqueous solution to 3.0 with hydrochloric acid. Add 4.0 mL of amphoteric polystyrene beads and stir at 35°C for 15 minutes for electrostatic heterogeneous coagulation. Then, pass nitrogen for 30 minutes and heat to 70°C. When the temperature stabilizes, add 0.04 g of potassium persulfate and react for 12 hours to obtain raspberry-shaped particles.

[0038] Step 4: Using water as the solvent, prepare 2.0 mg / mL BSA aqueous solution and 50 mmol / L tri-(2-carboxyethyl)phosphine aqueous solution respectively, and adjust the pH of the tri-(2-carboxyethyl)phosphine aqueous solution to 5.0 with 1.0 mol / L NaOH aqueous solution. Evenly disperse 10 mg of the raspberry-like particles prepared above in 5.0 mL of 2.0 mg / mL BSA aqueous solution, then add 5.0 mL of 50 mmol / L tri-(2-carboxyethyl)phosphine aqueous solution (pH 5.0), mix well, incubate at room temperature for 1 hour, and centrifuge to obtain raspberry-like particles adhered to the phase-transition BSA nanofilm.

[0039] Step 5: 9.0 mg of raspberry-like particles attached to the phase-change BSA nanofilm were dispersed in 3.0 mL of tetrahydrofuran and shaken for 20 min. The amphoteric polystyrene beads with a particle size of 100-200 nm were quickly etched and washed by centrifugation with deionized water to obtain anti-adhesive golf-shaped microspheres.

[0040] Step 6: Take 5.0 mg of anti-adhesive golf-shaped microspheres and evenly disperse them in 5.0 mL of water. Add 15 mg of polylysine and incubate at room temperature and 100 rpm for 24 hours. Wash them by centrifugation with deionized water and dry them at room temperature to obtain anti-adhesive cationic golf-shaped microspheres.

[0041] The morphology of anti-adhesion cationic golf ball-shaped microspheres was characterized by scanning electron microscopy. Figure 1 As shown, it can be seen that there are imprints with a pore size of 100 to 200 nm on the surface of the material, and the outside of the imprint is rough. At the same time, the laser particle size Zeta potential instrument measured that after the anti-adhesion golf-shaped microspheres were modified with polylysine, the Zeta potential changed from -35.39 mV to 14.18 mV, proving the successful modification of polylysine and indicating the successful preparation of anti-adhesion cationic golf-shaped microspheres.

[0042] Example 2

[0043] Isolation and purification of sEVs using anti-adhesive cationic golf-ball-shaped microspheres

[0044] 1. MCF-7 cell culture

[0045] Step a: First, preheat the MCF-7 cell culture medium in a 37°C CO2 constant temperature incubator. Then, quickly take out the MCF-7 cells frozen in a -80°C refrigerator and thaw them in a 37°C water bath. Then, in a clean operating table, thaw about 1.5×10 6Add 10 MCF-7 cells to a centrifuge tube containing 4.0 mL of MCF-7 cell culture medium. Centrifuge at 1000 rpm for 4 minutes. Discard the supernatant and add 4.0 mL of preheated MCF-7 cell culture medium. Mix thoroughly by pipetting, then transfer to a culture dish. Observe under an inverted microscope and incubate in a 37°C, 5% CO2 incubator. Observe cell attachment and morphology daily. Replace with fresh MCF-7 cell culture medium when the initially reddish cell culture medium turns yellow.

[0046] Step b: When the cells reach 90%, the old culture medium is collected and washed twice with sterile PBS buffer (pH 7.4 10mM) to remove dead cells and cell debris. 1.0mL of 0.25% trypsin-EDTA solution is added to enzymatically dissociate the cells. After 5 minutes, the culture dish is gently shaken and placed under an inverted microscope for observation to see if the adherent cells are free and dispersed. After the cell enzymatic dissociation is completed, 2.0mL of MCF-7 cell culture medium is added to terminate the digestion. The enzymatically dissociated cells are transferred to a centrifuge tube and centrifuged at 1500rpm for 4 minutes. After removing the supernatant, 8.0mL of MCF-7 cell culture medium is added to the centrifuge tube, pipetting and mixing, and then transferred to a culture dish for continued culture. When the cell confluence reaches more than 90%, the MCF-7 cell culture supernatant is continued to be collected. The obtained MCF-7 cell culture supernatant can be used for MCF-7 sEVs isolation.

[0047] 2. Isolation and purification of sEVs

[0048] 10 mg of anti-adhesive cationic golf-shaped microspheres were evenly dispersed in 3.0 mL of the MCF-7 cell culture supernatant obtained above and incubated on a shaker at 100 rpm at 37°C for 20 min to allow the anti-adhesive cationic golf-shaped microspheres to adsorb sEVs. The microspheres were then washed two to three times with 10 mmol / L PBS buffer (pH 7.4) to remove nonspecifically adsorbed impurities and ensure the purity of the resulting sEVs. The washed sEV-adsorbed anti-adhesive cationic golf-shaped microspheres were mixed with 0.30 mL of 8% ammonia water and shaken at 500 rpm at 4°C for 10 min. The anti-adhesive cationic golf-shaped microspheres precipitated due to gravity, while the sEVs dissolved in the 8% ammonia water. The sEVs and anti-adhesive cationic golf-shaped microspheres were successfully separated by solid-liquid separation, and the liquid fraction was the sEV extract.

[0049] The anti-adhesive cationic golf-shaped microspheres adsorbing sEVs were diluted with water and dropped onto a clean silicon wafer. The microspheres were fixed with 4% paraformaldehyde for 30 min in the dark, then dehydrated with a series of alcohol concentrations (10%, 30%, 50%, 70%, 90%, and 100%) for 10 min each, and dried at 37°C overnight. The anti-adhesive cationic golf-shaped microspheres after adsorbing sEVs were observed using a scanning electron microscope. Figure 2 Scanning electron microscopy results showed that after the anti-adhesion cationic golf-shaped microspheres were co-incubated with the MCF-7 cell culture supernatant, sEVs were clearly present in the imprints on the surface of the anti-adhesion cationic golf-shaped microspheres. The size of the captured sEVs was within the size range of sEVs reported in previous studies, confirming that the anti-adhesion cationic golf-shaped microspheres of the present invention can selectively capture sEVs.

[0050] The morphology and particle size of sEVs after elution with 8% ammonia were characterized by field emission scanning electron microscopy and nanoparticle tracer analysis. Figure 3 It can be seen that the separated sEVs are round with an average diameter of 104±29 nm, which is consistent with the literature reports, proving that the sEVs adsorbed by the anti-adhesion cationic golf ball-shaped microspheres can be successfully eluted by 8% ammonia water without destroying their morphology and structure.

[0051] In order to verify the capture ability of anti-adhesive cationic golf-shaped microspheres, the nanoparticle tracing technology was used to characterize the number of sEVs in all operating solutions during the capture of standard sEVs by anti-adhesive cationic golf-shaped microspheres, including standard solution, adsorption supernatant, two washing solutions and eluted recovered sEVs samples. The specific method is: 10 mg of anti-adhesive cationic golf-shaped microspheres are evenly dispersed in 3.0 mL of standard sEVs solution, incubated on a shaker at 37°C and 100 rpm for 20 min, centrifuged, and then washed twice with 10 mmol / L PBS buffer at pH 7.4. The washed anti-adhesive cationic golf-shaped microspheres adsorbing sEVs are mixed with 0.30 mL of 8% ammonia water and eluted at 500 rpm at 4°C for 10 min. Figure 4 As shown, the concentration of the sEVs standard solution used for adsorption was 2.60×10 10 After the adsorption operation was completed using anti-adhesive cationic golf ball-shaped microspheres, the particle concentration of the supernatant obtained by centrifugation was reduced to 1.96×10 9 The two PBS buffer washes also showed a low number of sEVs. When desorbed with 8% ammonia, the concentration of the released sEV solution was 1.98×10 10This study demonstrated that sEVs adsorbed by anti-adhesive cationic golf ball-shaped microspheres could be recovered by elution with 8% ammonia. Therefore, anti-adhesive cationic golf ball-shaped microspheres can be used for sEV separation, with capture rates exceeding 90% and recovery rates exceeding 80%.

[0052] To verify the lipoprotein contamination of the recovered sEVs, an ApoE enzyme-linked immunosorbent assay (ELISA) was used to measure the lipoprotein content of sEVs isolated by ultracentrifugation (the gold standard) and this method. The results showed that the lipoprotein content of sEVs obtained by ultracentrifugation was 6360 pg / mL, while the lipoprotein content of sEVs obtained by this method was 4568 pg / mL. These results demonstrate that anti-adhesive cationic golf ball-shaped microspheres have a relatively high purity for isolating sEVs from cell culture supernatants.

Claims

1. An anti-adhesion cationic golf ball-shaped microsphere, characterized in that The microspheres are prepared by the following steps: Step 1: Under alkaline conditions, the silanol groups in 3-(methacryloyloxy)propyltrimethoxysilane are dehydrated and condensed to prepare soft silicone oil droplets; Step 2: using azobis(N-hydroxyisobutylamidine) hydrate to initiate polymerization of styrene to prepare amphoteric polystyrene beads with a particle size of 100 to 200 nm; Step 3: Under acidic conditions, amphoteric polystyrene beads with a particle size of 100-200 nm are added to soft silicone oil droplets for heterogeneous coagulation, and then potassium persulfate is added to initiate polymerization to form raspberry-shaped particles; Step 4: uniformly dispersing the raspberry-like particles in a bovine serum albumin aqueous solution, then adding a tri-(2-carboxyethyl)phosphine aqueous solution to reduce the bovine serum albumin, incubating, and centrifuging to obtain raspberry-like particles adhered to a phase-transition bovine serum albumin nanofilm; Step 5: The raspberry-shaped particles adhered to the phase-transition bovine serum albumin nanofilm are uniformly dispersed in tetrahydrofuran, and the amphoteric polystyrene beads with a particle size of 100 to 200 nm are etched away, and the beads are centrifuged and washed with deionized water to obtain anti-adhesive golf ball-shaped microspheres; Step 6: Disperse the anti-adhesive golf ball-shaped microspheres in water and add polylysine. After incubation at room temperature, centrifuge to obtain the precipitate and wash to obtain the anti-adhesive cationic golf ball-shaped microspheres.

2. The anti-adhesion cationic golf-shaped microspheres according to claim 1, characterized in that: In step 2, polyvinyl pyrrolidone is added to water and stirred evenly, and then styrene is added. The mixture is stirred at 30-40° C. and a rotation speed of 300-500 rpm for 15-30 minutes, and then the temperature is raised to 80-90° C. and stirred at a constant temperature for 15-30 minutes while bubbling nitrogen. Then, azobis(N-hydroxyisobutylamidine)hydrate is added, and the mixture is stirred for 5-6 hours and then cooled to obtain amphoteric polystyrene beads with a particle size of 100-200 nm. The addition ratio of the polyvinyl pyrrolidone to water, styrene, and azobis(N-hydroxyisobutylamidine)hydrate is 1.0 g:30-50 mL:3.0-5.0 mL:0.10-0.20 g.

3. The anti-adhesion cationic golf-shaped microspheres according to claim 1, wherein: In step 3, the soft silicone oil droplets are evenly dispersed in water, the pH is adjusted to 2.0-6.0 with hydrochloric acid, and then amphoteric polystyrene beads with a particle size of 100-200 nm are added, and the mixture is stirred at room temperature for 10-30 minutes for electrostatic heterogeneous coagulation. Subsequently, nitrogen is passed through for 30-50 minutes and heated to 60-80°C, and potassium persulfate is added. The polymerization reaction is carried out for 10-12 hours to obtain raspberry-shaped particles; wherein the addition ratio of the soft silicone oil droplets to water, amphoteric polystyrene beads with a particle size of 100-200 nm, and potassium persulfate is 1.0 mL: 40-60 mL: 3.0-5.0 mL: 0.03-0.05 g.

4. The anti-adhesion cationic golf-shaped microspheres according to claim 1, wherein: In step 4, the addition ratio of the raspberry-shaped particles to the bovine serum albumin aqueous solution and the tris-(2-carboxyethyl)phosphine aqueous solution is 10 mg:2.0-6.0 mL:2.0-6.0 mL, the concentration of the bovine serum albumin aqueous solution is 2.0-5.0 mg / mL, the concentration of the tris-(2-carboxyethyl)phosphine aqueous solution is 20-60 mmol / L, and the pH is 5.0; the incubation is carried out at room temperature for 1-2 h.

5. The anti-adhesion cationic golf-shaped microspheres according to claim 1, characterized in that: In step 5, the ratio of the raspberry-shaped particles adhered to the phase-transition bovine serum albumin nanofilm and tetrahydrofuran is 1.0 mg: 0.30-1.0 mL.

6. The anti-adhesion cationic golf-shaped microspheres according to claim 1, wherein: In step 6, the anti-adhesion golf-shaped microspheres, water, and polylysine are added in a ratio of 1.0 mg: 1.0-2.0 mL: 2.0-4.0 mg; and the incubation is performed at room temperature for 24-48 hours.

7. The anti-adhesion cationic golf-shaped microspheres according to claim 1, wherein: The particle size of the anti-adhesion cationic golf ball-shaped microspheres is 1 to 5 μm.

8. A method for separating and purifying small extracellular vesicles using the anti-adhesive cationic golf ball-shaped microspheres according to any one of claims 1 to 7, characterized in that: The anti-adhesion cationic golf-shaped microspheres were evenly dispersed in the cell culture supernatant, incubated with shaking at 35-38°C for 20-30 minutes, and then centrifuged and washed in 10 mmol / L PBS buffer at pH 7.

4. The resulting precipitate was mixed with 4%-8% ammonia water and eluted with shaking at 4°C. The supernatant was collected to obtain the isolated and purified small extracellular vesicles.

9. The method for separating and purifying small extracellular vesicles using anti-adhesive cationic golf-ball-shaped microspheres according to claim 8, characterized in that: The dosage ratio of the anti-adhesion cationic golf ball-shaped microspheres, cell culture supernatant, and ammonia water with a mass concentration of 4% to 8% is 1.0 mg: 0.30 to 1.0 mL: 0.01 to 0.05 mL.

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