Exosome enrichment method based on boric acid modified capture material

Abstract

The invention provides an exosome enrichment method based on a boric acid modified capture material. The method comprises the following steps: adding a boric acid modified capture material into a solution to be enriched, adjusting the pH value to 7.8-8.5 with Tris-HCl, carrying out mild oscillation incubation at 2-8 DEG C for 5-60 min, centrifuging, and collecting the precipitate; and gently washing the precipitate by using a PBS (Phosphate Buffer Solution) containing fructose or glucose, adding an eluent, eluting at 2-8 DEG C for 5-30 minutes, and centrifuging to obtain a supernatant rich in exosome. The exosome enrichment method disclosed by the invention is simple, efficient, rapid and low in cost; the obtained exosome is intact in form and high in purity, the biological activity of the exosome is not affected, and the exosome has important practical value and popularization prospects.

Description

Technical Field

[0001] The present invention belongs to the field of separation analysis, and in particular relates to an exosome enrichment method based on boric acid-modified capture materials. Background Art

[0002] Exosomes are biomarkers for dynamic monitoring of disease progression and are also important tools for intercellular communication under normal physiological and pathological conditions. Exosomes are stably present in a variety of body fluids, such as blood, urine, saliva, and cerebrospinal fluid. Almost all types of cells can produce exosomes, and their formation process is complex, involving continuous invagination of the plasma membrane to form endosomes and intraluminal vesicles; subsequently, some mature multivesicular bodies containing intraluminal vesicles fuse with the plasma membrane to release cup-shaped extracellular vesicles with a diameter of 30-150 nm, which are exosomes. During the exosome biogenesis process, the contents of donor cells and some extracellular components are loaded into exosomes, including proteins, lipids, nucleic acids, carbohydrates, and metabolites, which transmit substances and information to recipient cells through receptor-ligand interactions, thereby mediating many physiological and pathological processes. The information carried by exosomes can reflect the functional status of parent cells. As a biomarker, exosomes can provide rich, stable, sensitive and specific biological information. By analyzing the proteins in exosomes that are related to specific diseases, non-invasive detection can be achieved, which is helpful for early detection and diagnosis of diseases.

[0003] In order to achieve more accurate and reliable diagnosis and treatment based on exosomes, it is crucial to separate exosomes with high efficiency and purity. Exosomes can be separated according to their size, density, surface charge or membrane protein type. Exosome separation strategies include ultracentrifugation, ultrafiltration, immunoaffinity capture, polymer-based precipitation, size exclusion and microfluidics. Ultracentrifugation is the most widely used method and is regarded as the "gold standard" for exosome separation. Its disadvantages are that it is easy to produce protein aggregation and lipoprotein contamination problems. The exosomes obtained are usually of low purity, low separation efficiency, and require expensive equipment and are time-consuming. Size exclusion can retain the biological activity of exosomes, but the separated exosomes contain impurities of similar size and are difficult to scale up. Ultrafiltration-based exosome separation is simple to operate and does not require special equipment, but it may cause clogging of the separation membrane, affecting the separation rate, and the exosomes may be damaged and broken. Polymer-induced precipitation does not require complex equipment and is easy to scale up, suitable for large sample processing, but it may co-precipitate non-exosome contaminants, has low purity, and residual polymer materials will also affect downstream analysis. The immunoaffinity capture method can efficiently enrich exosomes, but it relies on specific markers and is costly. The dissociation of captured exosomes is still a big challenge. For example, Chinese patent CN 113652391A discloses an integrated method for exosome capture and analysis based on boric acid-directed immunoaffinity coupling, which labels antibodies corresponding to different exosome membrane proteins such as CD63, EpCAM and HER2 onto Wulff-type graphite phase carbon nitride to capture exosomes for in situ analysis. This method is not only costly, but also very difficult to release the captured exosomes intact, which limits its application scope. Microfluidics technology has the characteristics of being fast, portable, and easy to automate, but the amount of samples that can be processed is small and there is a lack of standardized separation methods. The ideal exosome separation technology should be fast, simple, and high-yield, but due to the nanoscale size and high heterogeneity of exosomes, it is still challenging to efficiently obtain exosomes from complex biological fluids. Summary of the invention

[0004] The purpose of the present invention is to provide an exosome enrichment method based on boric acid modified capture materials. The exosome enrichment method is simple, efficient, rapid, and low-cost, and the obtained exosomes have intact morphology and high purity, and do not affect their biological activity, which has important practical value and promotion prospects.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A method for enriching exosomes based on boronic acid-modified capture materials, comprising the following steps: adding the boronic acid-modified capture material to a solution to be enriched, adjusting the pH to 7.8-8.5 with Tris-HCl, incubating with gentle shaking at 2-8°C for 5-60 minutes, centrifuging, and collecting the precipitate; gently washing the precipitate with a PBS buffer containing fructose or glucose, adding an eluent to elute at 2-8°C for 5-30 minutes, and centrifuging to obtain a supernatant rich in exosomes.

[0006] Furthermore, the boronic acid modified capture material is a boronic acid functionalized material formed by introducing boronic acid groups into the surface or structure of amino metal organic framework materials, amino functionalized graphene oxide or amino modified silica microspheres.

[0007] Furthermore, in the solution to be enriched, the concentration of the boronic acid modified capture material is 1-6 mg / mL.

[0008] Furthermore, the solution to be enriched is plasma, cell culture supernatant or urine.

[0009] Furthermore, the pH of the Tris-HCl is 8.5-9.0, and the concentration is 0.1-1 M; the pH of the PBS buffer is 6.6-7.2, and the concentration is 0.01 M; wherein the concentration of fructose or glucose in the PBS buffer is 20-100 mM.

[0010] Furthermore, the precipitate was gently washed three times with PBS buffer containing fructose or glucose.

[0011] Furthermore, the boronic acid modified capture material can be prepared by the following method: Carboxyphenylboronic acid was ultrasonically dissolved in dimethyl sulfoxide, and then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added, and the mixture was stirred at room temperature for 40 min to obtain an activated carboxyphenylboronic acid solution; the amino-functionalized material was activated at 120 ℃ and ultrasonically dispersed in dimethyl sulfoxide to obtain an activated amino-functionalized material; the activated amino-functionalized material was dropped into the activated carboxyphenylboronic acid solution, and the reaction was stirred at room temperature for 24 h; the precipitate was collected by centrifugation, washed with dimethyl sulfoxide and ethanol in turn, and dried in vacuum at 60 ℃ to obtain the boronic acid modified capture material.

[0012] Furthermore, the carboxyphenylboric acid is 4-carboxyphenylboric acid or 3-carboxyphenylboric acid.

[0013] Furthermore, the amino-functionalized material is an amino-metal organic framework material, amino-functionalized graphene oxide or amino-modified silica microspheres.

[0014] Furthermore, the mass ratio of the amino-functionalized material to carboxyphenylboronic acid is 1:2-8.

[0015] Furthermore, the concentration of the carboxyphenylboronic acid is 2-5 mg / mL; the molar ratio of the carboxyphenylboronic acid to EDC·HCl is 1:1-10; the molar ratio of the EDC·HCl to NHS is 1:2-5; and the concentration of the amino-functionalized material is 0.25-2.5 mg / mL.

[0016] Furthermore, the activation time of the amino-functionalized material is ≥6 h.

[0017] The beneficial effects of the present invention are: The exosome enrichment method based on boric acid modified capture material provided by the present invention has the advantages of high purity, high efficiency (short enrichment time) and low breakage rate. It has less nonspecific adsorption, mild conditions throughout the process, little damage to exosomes, and retains the complete morphology and biological activity of exosomes. Compared with ultracentrifugation, it can improve the purity of exosome enrichment, shorten the enrichment time and have low cost, solve the problems of high cost, low exosome purity, long time and high breakage rate commonly found in traditional methods, and can more conveniently obtain high-quality exosomes, which is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a transmission electron microscopy characterization image of the N2aSW cell exosomes captured in Example 1.

[0019] Figure 2 This is the particle size distribution diagram of the captured N2aSW cell exosomes characterized by nanoparticle tracking analysis in Example 1.

[0020] Figure 3 This is the result of immunoblotting analysis of the captured exosome characteristic proteins HSC70, TSG101, CD81 and CD9 in N2aSW cells in Example 2.

[0021] Figure 4 This is a comparison of the purity of exosomes separated by boric acid modification capture method and ultracentrifugation method.

[0022] Figure 5 is the effect of capture time on exosome capture efficiency.

[0023] Figure 6 These are the experimental results showing that the addition of exosomes promoted the healing of N2aSW cell scratches 24 h and 36 h later. DETAILED DESCRIPTION

[0024] Example 1 223 mg (1.0 mmol) zirconium chloride and 181 mg (1.0 mmol) 2-aminoterephthalic acid were weighed and added to 30 mL N,N-dimethylformamide (DMF), and ultrasonicated for 20 min. Then 4.3 mL acetic acid and 0.3 mL water were added to the mixed solution, and ultrasonicated for 10 min. Then, the mixture was poured into a high-pressure reactor with a 100 mL polytetrafluoroethylene liner and heated in a 120 ℃ oven for 24 h. After the reaction was completed, the mixture was naturally cooled to room temperature, and the precipitate was collected by centrifugation at 12,000 rpm for 10 min, washed with DMF and methanol three times, and dried in vacuum at 60 ℃ to obtain an amino metal organic framework material.

[0025] 40 mg (0.2 mmol) of 4-carboxyphenylboronic acid was weighed and dissolved in 10 mL of dimethyl sulfoxide, and ultrasonicated for 20 min. 77 mg (0.4 mmol) of EDC·HCl and 115 mg (1.0 mmol) of NHS were added to the above solution, and the carboxyl groups were activated by stirring at room temperature for 40 min. 10 mg of the prepared amino metal organic framework material was activated at 120 °C for 6 h, added to 10 mL of dimethyl sulfoxide, ultrasonicated for 30 min until completely dispersed, and then added dropwise to the activated carboxyphenylboronic acid solution, and stirred at 150 rpm for 24 h at room temperature. Centrifuged at 12,000 rpm for 10 min, washed with dimethyl sulfoxide and ethanol three times in sequence, and dried in vacuum at 60 °C to obtain the metal organic framework material modified with boronic acid.

[0026] Collect 4 mL of N2aSW cell culture medium, centrifuge at 2,000 g and 10,000 g for 10 min, filter through a 0.22 μm filter, add 16 mg of boric acid-modified capture material, and adjust the pH to 7.8 with 0.1 M Tris-HCl at pH = 8.8. Incubate at 4 °C with gentle shaking for 20 min, collect the boric acid-modified capture material with captured exosomes by centrifugation, and gently wash three times with 100 μL 1× PBS washing solution. Add 100 μL of PBS eluent (pH = 6.8) containing 60 mM fructose, elute at 4 °C for 10 min, and centrifuge to obtain the supernatant rich in exosomes.

[0027] The obtained exosomes were observed under transmission electron microscopy. Figure 1 As shown in Figure 2, the phospholipid bilayer membrane of the exosomes is clearly visible, showing a typical disc-like structure with a complete morphology. The particle size and distribution of the exosomes were determined by nanoparticle tracking analysis, as shown in Figure 2. Figure 2 As shown in the figure, the size distribution of most particles is between 30-150 nm and the particle size distribution range is narrow, which is consistent with the size of exosomes.

[0028] Example 2 Weigh 0.2 g of graphene oxide and add it to 40 mL of DMF. Ultrasonic dispersion is performed for 3 h to obtain a uniform suspension. 9.0 g of ethylenediamine is slowly added under vigorous stirring and reacted at room temperature for 24 h. After the reaction is completed, 100 mL of anhydrous ethanol is added and allowed to stand overnight. The supernatant is removed, vacuum filtered, repeatedly washed with ethanol and deionized water, and vacuum dried at 60 °C for 24 h to obtain amino-functionalized graphene oxide.

[0029] 2.5 mg of amino-functionalized graphene oxide was activated at 120 °C for 6 h, added to 10 mL of dimethyl sulfoxide solution, and ultrasonically dispersed for 1 h. 20 mg (0.1 mmol) of 4-carboxyphenylboronic acid was weighed and dissolved in 10 mL of dimethyl sulfoxide, 192 mg (1.0 mmol) of EDC·HCl and 230 mg (2.0 mmol) of NHS were added, activated for 40 min, and the amino-functionalized graphene oxide was added dropwise under stirring, and the stirring reaction was continued for 24 h. The precipitate was collected by centrifugation, washed with dimethyl sulfoxide and ethanol three times in sequence, and dried under vacuum at 60 °C to obtain boric acid-modified graphene oxide.

[0030] 4 mL of N2aSW cell culture medium was collected and centrifuged at 2,000 g for 10 min to remove cells and dead cells. The supernatant was centrifuged at 10,000 g for 10 min to remove cell debris, and then filtered through a 0.22 μm filter. 8 mg of boric acid-modified graphene oxide was added, gently mixed, and the pH was adjusted to 8.5 with Tris-HCl at pH = 9.0. Incubate at 4 °C with gentle shaking for 30 min, centrifuge at 8,000 rpm for 5 min, discard the supernatant, and wash three times with 100 μL 1× PBS. Add 100 μL of PBS eluent containing 30 mM glucose, elute at 4 °C for 30 min, and centrifuge to obtain the supernatant rich in exosomes.

[0031] Add RIPA lysis buffer containing 1% PMSF and 2% SDS to the extracted exosome samples, lyse the exosomes on ice, and measure the exosome protein concentration using the BCA kit. Add 4×Loading and boil at 100°C for 10 min to fully denature the protein, take 15 μL and load it into the 12% SDS-PAGE gel loading well for electrophoresis analysis. Transfer the electrophoretically separated proteins from the gel to the PVDF membrane and block it with 5% skim milk powder for 1.5 h. Dilute TSG101, HSC70, CD81, and CD9 primary antibodies with TBST at 1:1000, incubate the blocked PVDF membrane on a shaker at 4°C overnight, wash with TBST, add the corresponding secondary antibody and incubate at room temperature for 1.5 h, and wash away the excess secondary antibody. Develop the membrane with ECL luminescent solution and image it under a luminometer. The results are as follows Figure 3As shown, TSG101, HSC70, CD81 and CD9 exosome marker proteins can be effectively detected in the exosome eluate of N2aSW cell culture supernatant.

[0032] Example 3 Weigh 0.1 g of spherical silica and activate it at 110 °C for 4 h, resuspend it in 20 mL of anhydrous toluene, disperse it evenly by ultrasonication, then add 0.15 mL of 3-aminopropyltriethoxysilane, condense and reflux at 80 °C, and stir to react for 24 h. After the reaction is completed, cool to room temperature, centrifuge at 6,000 rpm for 5 min, wash the precipitate with anhydrous ethanol 5 times, and dry it at 60 °C to constant weight to obtain amino-modified silica microspheres.

[0033] 50 mg (0.3 mmol) of 4-carboxyphenylboronic acid was dissolved in 10 mL of dimethyl sulfoxide by ultrasonication, and 345 mg (1.8 mmol) of EDC·HCl and 978 mg (8.5 mmol) of NHS were added and stirred for activation for 40 min. 25 mg of amino-modified silica microspheres were activated at 120 °C for 9 h and added to 10 mL of dimethyl sulfoxide. After ultrasonic dispersion, they were added dropwise to the activated carboxyphenylboronic acid solution and stirred at 120 rpm for 24 h at room temperature. The precipitate was collected by centrifugation, washed with dimethyl sulfoxide and ethanol three times in sequence, and dried under vacuum at 25 °C to obtain boric acid-modified silica microspheres.

[0034] Peripheral blood from patients with Alzheimer's disease was drawn into EDTA K2 anticoagulant tubes and centrifuged at 3000 g for 15 min at 4 °C to collect plasma. 300 μL of plasma was diluted to 3 mL with 1×PBS, centrifuged at 2,000 g for 10 min, the supernatant was collected, filtered through a 0.22 μm filter, boric acid-modified silica microspheres were added, shaken, and pH = 8.2 was adjusted with Tris-HCl at pH = 8.5. Incubated at 4 °C for 20 min, centrifuged at 6,000 rpm for 5 min, the supernatant was discarded, and gently washed three times with PBS at pH = 7.8. 30 μL of PBS eluent containing 100 mM fructose was added to release exosomes at 4 °C, and the exosome-rich supernatant was collected by centrifugation, which can be used for the early diagnosis of downstream Alzheimer's disease.

[0035] Example 4 The exosomes in the cell culture supernatant were enriched by boric acid modification capture method and ultracentrifugation method, respectively. The number of enriched exosome particles and total protein concentration were detected by nanoparticle tracking analysis technology and BCA method. The relative purity was expressed as the ratio of the number of exosome particles to the amount of protein. Figure 4As shown, the relative purity of exosomes obtained by the boric acid modification capture method is 3.0 times higher than that by the ultracentrifugation method, indicating that the exosomes enriched by the enrichment method of the present invention are of high purity.

[0036] Example 5 The exosomes separated by ultracentrifugation were used as exosome standards, 16 mg of boric acid-modified metal organic framework material was added, the pH was adjusted to 7.8 with 0.1 M Tris-HCl at pH = 8.8, and the supernatant was incubated for 1 min, 5 min, 10 min, 20 min, 30 min, and 60 min at 4 °C, and the exosome protein concentration in the supernatant was measured. The capture efficiency was expressed as the ratio of the enriched exosome protein concentration to the exosome standard protein concentration, as shown in Figure 5 As shown in the figure, the capture efficiency can reach 80.3% in the 5th minute, and the capture equilibrium is reached in the 20th minute, with a capture efficiency of 87.6%, indicating that the enrichment method of the present invention has high capture efficiency.

[0037] Example 6 Scratch the N2aSW cells and add 10 10 The exosomes enriched by the boric acid-modified capture material were cultured and the closure of the scratch was observed. Figure 6 As shown in the figure, 24 h and 36 h after the addition of exosomes, the degree of wound closure of the cells gradually increased. Compared with the control group without exosomes, the degree of wound closure was higher and the scratch size was significantly reduced, indicating that the exosomes obtained by the boric acid modification capture method have better structural integrity, less exosome damage, and better retention of the biological activity of the exosomes.

[0038] The protection scope of the present invention is not limited to the above-mentioned specific embodiments. Any equivalent substitution or modification made by any technician familiar with the art after understanding the technical solution of the present invention shall be deemed to be within the protection scope of the present invention as long as it complies with the claims.

Claims

1. A method for enriching exosomes based on boric acid-modified capture materials, characterized in that: The following steps are involved: Add the boric acid-modified capture material to the solution to be enriched, adjust the pH to 7.8-8.5 with Tris-HCl, incubate with gentle shaking at 2-8°C for 5-60 min, centrifuge, and collect the precipitate; gently wash the precipitate with PBS buffer containing fructose or glucose, add the eluent and elute at 2-8°C for 5-30 min, centrifuge, and obtain the supernatant rich in exosomes.

2. The exosome enrichment method according to claim 1, characterized in that: The boric acid modified capture material is a boric acid functional modified material formed by introducing boric acid groups into the surface or structure of amino metal organic framework materials, amino functionalized graphene oxide or amino modified silica microspheres.

3. The exosome enrichment method according to claim 1, characterized in that: In the solution to be enriched, the concentration of the boronic acid modified capture material is 1-6 mg / mL.

4. The exosome enrichment method according to claim 1, characterized in that The solution to be enriched is plasma, cell culture supernatant or urine.

5. The exosome enrichment method according to claim 1, characterized in that: The pH of the Tris-HCl is 8.5-9.0, and the concentration is 0.1-1 M; the pH of the PBS buffer is 6.6-7.2, and the concentration is 0.01 M; wherein the concentration of fructose or glucose in the PBS buffer is 20-100 mM.

6. The exosome enrichment method according to claim 1, characterized in that: The boronic acid modified capture material is prepared by the following method: The carboxyphenylboronic acid is ultrasonically dissolved in dimethyl sulfoxide, and then EDC·HCl and NHS are added, and the reaction is stirred at room temperature to obtain an activated carboxyphenylboronic acid solution; the amino-functionalized material is activated at high temperature and then ultrasonically dispersed in dimethyl sulfoxide to obtain an activated amino-functionalized material; the activated amino-functionalized material is dropped into the activated carboxyphenylboronic acid solution, and the reaction is stirred at room temperature, and the precipitate is collected by centrifugation, washed with dimethyl sulfoxide and ethanol in turn, and then vacuum dried to obtain a boronic acid modified capture material.

7. The exosome enrichment method according to claim 6, characterized in that: The carboxyphenylboronic acid is 4-carboxyphenylboronic acid or 3-carboxyphenylboronic acid.

8. The method for enriching exosomes according to claim 6, characterized in that: The amino-functionalized material is an amino-metal organic framework material, an amino-functionalized graphene oxide or an amino-modified silica microsphere.

9. The method for enriching exosomes according to claim 6, characterized in that: The mass ratio of the amino functional material to carboxyphenylboronic acid is 1:2-8.

10. The method for enriching exosomes according to claim 6, characterized in that: The concentration of the carboxyphenylboronic acid is 2-5 mg / mL; the molar ratio of the carboxyphenylboronic acid to EDC·HCl is 1:1-10; the molar ratio of the EDC·HCl to NHS is 1:2-5; and the concentration of the amino-functionalized material is 0.25-2.5 mg / mL.

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