Nanomaterials for separating exosomes and preparation methods and applications thereof

Abstract

The present invention proposes a nanomaterial for separating exosomes and a preparation method and application thereof, which belongs to the technical field of exosome separation. The preparation method of the present invention comprises the following steps: obtaining a cellulose nanomaterial; immobilizing streptavidin on the cellulose nanomaterial by covalent bond coupling; adding an antibody for capturing exosomes to the cellulose nanomaterial immobilized with streptavidin, and immobilizing the antibody on the nanomaterial after reaction to obtain a nanomaterial for separating exosomes. The present invention utilizes the advantages of cellulose nanomaterials having high specific surface area and multiple modification sites, immobilizes exosome-specific capture antibodies on cellulose nanomaterials, and can significantly increase the chance of contact between exosomes and capture antibodies, while ensuring specificity and having high capture efficiency, taking into account specificity and yield, and having a simple preparation process, a high separation efficiency for exosomes, no need for large-scale separation equipment, easy operation, and low cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial synthesis and exosome separation, and specifically relates to a nanomaterial for separating exosomes, a preparation method of the nanomaterial for separating exosomes, and an application of the nanomaterial. Background Art

[0002] Exosomes are vesicles with a size of 30nm-150nm, which can be secreted by almost all cells, including animals and plants, eukaryotes and prokaryotes. It has a double-layer membrane structure, and its shape is a saucer-shaped or oval with a depression in the middle under a transmission electron microscope. Exosomes were originally considered to be garbage secreted by cell metabolism and had no specific physiological functions. However, with the continuous research of scientists, it has been found that it has important physiological functions and is an important tool for cell-to-cell communication.

[0003] However, due to its small size and high heterogeneity, its separation and purification is very challenging. Currently, the commonly used separation methods are: (1) ultracentrifugation and density gradient centrifugation. Both methods require large centrifugation equipment and generally take more than 10 hours. The yield of exosomes is 5%-25%, which is relatively low. The purity of exosomes separated by ultracentrifugation is moderate, and the purity of exosomes obtained by density gradient centrifugation is very high, but it takes longer and the yield is much lower. (2) Size exclusion method. The advantage of this method is that it does not require large equipment and takes much less time. Generally speaking, the yield and purity of exosomes separated by it are higher than those of ultracentrifugation. However, this method cannot separate particles of similar size to exosomes, and its specificity is not high. (3) Polymer precipitation method. This method uses the hydrophilic nature of polymers to separate exosomes by sedimentation. The advantage is that the yield is higher, but the purity is poor because there will be polymer impurities, and the specificity is not high. (4) Microfluidic method. This method requires complex equipment design and its low throughput is not conducive to large sample separation.

[0004] One of the existing technologies, Chinese patent application CN108865971A, proposes a method for separating exosomes and a separation device thereof, which uses an anodized aluminum film corresponding to the size of the exosomes for filtration. This method has two shortcomings. First, this method cannot separate miscellaneous proteins of the same size as exosomes, such as low-density lipoproteins or some chylomicron particles, and the specificity is not high. Second, the filter membrane in the device is at risk of clogging, and needs to be cleaned regularly, which is not convenient enough.

[0005] One of the existing technologies, Chinese patent application CN109576210A, proposes a method for rapid separation of exosomes, which uses polyethylene glycol as a spacer to synthesize superparamagnetic nanoparticles and ligands to form a complex, and then captures the exosomes. However, the synthesis of this particle complex is relatively complicated, and the capture efficiency is not very ideal.

[0006] One of the existing technologies, Chinese patent application CN110231207A, proposes a method for isolating exosomes. This method is similar to the PEG polymer exosome separation kit currently on the market. It will also obtain a lot of non-exosome impurities, and there is also the problem of polymer contamination of samples, and the specificity is low.

[0007] In summary, due to the size of exosomes and their high heterogeneity, current separation methods have disadvantages such as low purity, low specificity, or low yield. To address this, the present invention proposes a novel and convenient nanomaterial for separating exosomes, and a preparation method and application thereof. Summary of the invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art and provide a nanomaterial for separating exosomes and a preparation method and application thereof.

[0009] In one aspect of the present invention, a method for preparing a nanomaterial for separating exosomes is provided, the method comprising the following steps:

[0010] Obtaining cellulose nanomaterials;

[0011] immobilizing streptavidin on the cellulose nanomaterial by covalent bonding;

[0012] An antibody for capturing exosomes is added to the cellulose nanomaterial immobilized with streptavidin, and the antibody is immobilized on the cellulose nanomaterial after reaction to obtain a nanomaterial for separating exosomes.

[0013] Optionally, obtaining the cellulose nanomaterial comprises:

[0014] The cellulose nanomaterial is obtained by oxidizing lignin with 2,2,6,6-tetramethylpiperidine-1-oxide.

[0015] Optionally, the concentration range of the 2,2,6,6-tetramethylpiperidine-1-oxide is 1.0% to 2.0%, and the carboxyl content of the cellulose nanomaterial is 1.2 mmol / g to 2.3 mmol / g.

[0016] Optionally, the step of immobilizing streptavidin on the cellulose nanomaterial by covalent bonding comprises:

[0017] Under the coupling reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, the carboxyl group of the cellulose nanomaterial forms a covalent bond with the amino group of streptavidin, so that the streptavidin is immobilized on the cellulose nanomaterial.

[0018] Optionally, the concentration of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is in the range of 0.13 mol / L to 1.3 mol / L; and / or,

[0019] The concentration range of the N-hydroxysuccinimide is 0.10 mol / L to 1.15 mol / L; and / or,

[0020] The concentration range of the streptavidin is 100 μg / mL to 800 μg / mL; and / or,

[0021] The temperature range of the coupling reaction is 4° C. to 25° C., and the time range of the coupling reaction is 4 h to 48 h.

[0022] Optionally, the antibody is any one of CD63, CD9, CD81, EpCAM, EGFR; and / or,

[0023] The antibody concentration range is 10 μg / mL to 40 μg / mL; and / or,

[0024] The reaction time range of streptavidin and antibody is 4h~6h, and the temperature range is 2℃~10℃.

[0025] In another aspect of the present invention, a nanomaterial for isolating exosomes is provided, which is prepared according to the method described above.

[0026] Another aspect of the present invention provides an application of a nanomaterial for separating exosomes, wherein the nanomaterial described above is applied to separate exosomes.

[0027] Optionally, the method for isolating exosomes comprises the following steps:

[0028] The nanomaterial immobilized with the antibody is added to the exosome solution to be separated, and the exosomes to be separated are captured by centrifugation after capture, and the exosomes to be separated are captured on the nanomaterial.

[0029] Optionally, the concentration range of the nanomaterial is 0.11 g / mL to 0.52 g / mL; and / or,

[0030] The capture time ranges from 0.5h to 4h.

[0031] The present invention proposes a nanomaterial for separating exosomes, and a preparation method and application thereof. The present invention utilizes the advantages of cellulose nanomaterials having a high specific surface area and multiple modification sites, and immobilizes exosome-specific capture antibodies on the cellulose nanomaterials, which can significantly increase the chance of contact between exosomes and capture antibodies, has high capture efficiency while ensuring specificity, takes both specificity and yield into account, has a simple preparation process, has a high separation efficiency for exosomes, has a high yield, does not require large-scale separation equipment, is easy to operate, and has a low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A flowchart of a method for preparing a nanomaterial according to an embodiment of the present invention;

[0033] Figure 2 This is a flow chart of the coupling reaction between streptavidin and cellulose nanofiber material according to another embodiment of the present invention;

[0034] Figure 3 This is an infrared characterization diagram of the coupling product of another embodiment of the present invention;

[0035] Figure 4 This is a transmission electron microscopy image of a model exosome according to another embodiment of the present invention;

[0036] Figure 5 This is a nanoparticle tracking analysis diagram of a model exosome according to another embodiment of the present invention. DETAILED DESCRIPTION

[0037] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the technical terms or scientific terms used in the present invention shall have the usual meanings understood by persons with ordinary skills in the field to which the present invention belongs. The terms "including" or "comprising" used in the present invention neither limit the shapes, numbers, steps, actions, operations, components, originals and / or their groups mentioned, nor exclude the presence or addition of one or more other different shapes, numbers, steps, actions, operations, components, originals and / or their groups. In addition, the terms "first" and "second" are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number and order of the indicated technical features.

[0039] In some descriptions of the invention, unless otherwise expressly specified and limited, the terms "install", "connect", "connected" or "fixed" and the like are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect through an intermediate medium, internal connections between two elements or mutual interaction between two elements. Also, the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating positions or positional relationships based on the positions or positional relationships shown in the drawings, are only used to indicate relative positional relationships, and when the absolute position of the described object changes, the relative positional relationship may also change accordingly.

[0040] like Figure 1 As shown, in one aspect of the present invention, a method S100 for preparing a nanomaterial for separating exosomes is provided, comprising the following steps S110 to S130:

[0041] S110, obtaining cellulose nanomaterials, specifically comprising: oxidizing lignin using 2,2,6,6-tetramethylpiperidine-1-oxide (TEMPO) to obtain cellulose nanomaterials.

[0042] The concentration of 2,2,6,6-tetramethylpiperidine-1-oxide in this embodiment ranges from 1.0% to 2.0%, and the carboxyl content in the cellulose nanomaterial ranges from 1.2 mmol / g to 2.3 mmol / g.

[0043] In some preferred embodiments, the concentration range of 2,2,6,6-tetramethylpiperidine-1-oxide may be further preferably 1.0%-1.2%, and the content of carboxyl groups in the cellulose nanomaterial may be further preferably 1.3 mmol / g to 1.5 mmol / g.

[0044] The cellulose nanofiber material used in this embodiment has a very high specific surface area and many functional groups that can be modified, such as carboxyl and hydroxyl groups. The above-mentioned modifiable functional groups react with streptavidin to facilitate the immobilization of exosome-specific antibodies on the cellulose nanomaterial.

[0045] S120, immobilizing streptavidin on the cellulose nanomaterial by covalent bonding, specifically comprising:

[0046] In the coupling reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), the carboxyl group of the cellulose nanomaterial forms a covalent bond with the amino group of streptavidin, so that streptavidin is immobilized on the cellulose nanomaterial. The specific reaction flow chart is as follows: Figure 2 As shown, and the infrared results of the material before and after coupling are as follows Figure 3 As shown, Figure 2 and Figure 3 The TOCNF in the figure is a TEMPO-oxidized cellulose nanomaterial, Streptavidin (SA) is streptavidin, and TOCNF-SA is a novel nanomaterial immobilized with antibodies obtained in this example.

[0047] In this embodiment, the concentration range of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.13 mol / L to 1.3 mol / L, the concentration range of N-hydroxysuccinimide is 0.10 mol / L to 1.15 mol / L, the concentration range of streptavidin is 100 ug / mL to 800 ug / mL, the temperature range of the coupling reaction is 4°C to 25°C, and the time range of the coupling reaction is 4h to 48h.

[0048] In some preferred embodiments, the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide can be further preferably 0.26 mol / L to 0.52 mol / L, the concentration of N-hydroxysuccinimide can be further preferably 0.20 mol / L to 0.69 mol / L, the concentration of streptavidin can be further preferably 200 ug / mL to 450 ug / mL, the temperature of the coupling reaction can be further preferably 4°C to 10°C, and the time of the coupling reaction can be further preferably 12h to 24h.

[0049] In this embodiment, the carboxyl functional groups on the cellulose nanomaterial are activated under the EDC / NHS reaction conditions, and then covalently bonded with the amino groups on the streptavidin to form amide bonds, so that the streptavidin is immobilized on the cellulose nanomaterial.

[0050] It should be noted that the inventors have found through long-term research that the conditions of the coupling reaction are crucial to the yield of isolated exosomes. If the reaction conditions are not well controlled, streptavidin may not bind or the yield may be extremely low. Therefore, this embodiment has determined the optimal reaction conditions through a series of condition screening and optimization.

[0051] S130, adding an antibody for capturing exosomes to the cellulose nanomaterial immobilized with streptavidin, and immobilizing the antibody on the nanomaterial after the reaction to obtain a nanomaterial for separating exosomes, that is, obtaining a fiber nanomaterial modified with a specific capture antibody.

[0052] The antibody of this embodiment uses any one of CD63, CD9, CD81, EpCAM, and EGFR, the antibody concentration range is 10ug / mL to 40ug / mL, the reaction time range of streptavidin and antibody is 4h to 6h, and the temperature is 2°C to 10°C.

[0053] In some preferred embodiments, the antibody concentration range may further preferably be 20 ug / mL to 30 ug / mL.

[0054] In this embodiment, biotinylated antibodies are immobilized on cellulose nanomaterials to capture exosomes, thereby achieving separation of exosomes.

[0055] The nanomaterial formed by the present invention has the advantages of high specific surface area and multiple modification sites. Immobilizing the exosome-specific capture antibody on the nanomaterial can significantly increase the chance of contact between the exosome and the capture antibody, while ensuring specificity and having a high capture efficiency, taking both specificity and yield into account, and can overcome the defects of existing exosome separation technology such as the need for large equipment and low separation efficiency, and has low separation cost, is simple and effective.

[0056] In another aspect of the present invention, a nanomaterial for separating exosomes is provided, which is prepared by the method described above. For the specific preparation process, please refer to the above description and will not be repeated here.

[0057] The nanomaterial obtained by the present invention can efficiently separate exosomes and has the characteristics of good specificity and high capture efficiency without the need for large-scale equipment.

[0058] Another aspect of the present invention provides an application of a nanomaterial for separating exosomes, wherein the nanomaterial described above is applied to the separation of exosomes.

[0059] Specifically, the method for separating exosomes using the nanomaterial of the present invention comprises the following steps:

[0060] The nanomaterials loaded with antibodies are added to the exosome solution to be separated, so as to separate the exosomes (the electron microscopy image of the exosomes is as follows Figure 4 As shown in the figure, the exosomes to be separated are captured by centrifugation after incubation, and the exosomes to be separated are captured on the nanomaterials. The fiber nanomaterials that capture the exosomes will gather at the bottom of the test tube. At the same time, this embodiment can use the change in the concentration of exosome particles before and after capture to evaluate the optimization of capture conditions. The nanoparticle tracking results of exosomes are as follows Figure 5 shown.

[0061] In this embodiment, the concentration range of the nanomaterial used to capture the exosomes to be separated is 0.11 g / mL to 0.52 g / mL, and the capture time range is 0.5-4 h.

[0062] In some preferred embodiments, the concentration of the nanomaterial used to capture the exosomes to be separated may be further preferably 0.12 g / mL to 0.24 g / mL, and the capture time may be further preferably 1 h to 2.5 h.

[0063] It should be noted that the exosomes of this embodiment can be formed by the following steps:

[0064] First, cells were cultured with full culture medium (DMEM medium). When the proliferation density reached about 60%-70%, they were washed three times with 1X phosphate buffered saline (PBS), replaced with exosome-free serum and cultured for another 48 hours, and then the culture medium was collected.

[0065] Second, the dead cells and cell debris were removed at a low speed, and then filtered with a 0.2um filter membrane for later use as the pretreated cell culture supernatant, which contained large vesicles and small vesicles;

[0066] Third, a 100KDa ultrafiltration tube was used for concentration until the sample volume was 1 mL. Then, the SEC separation column was used to separate the model exosomes (the transmission electron microscopy results of the model exosomes are shown in Figure 4 ), used for validation of the platform method.

[0067] It should be noted that the cell line used in this embodiment is a cancer cell line, which can be a breast cancer cell line (such as MDA-MB-231, MCF-7) or a lung cancer cell line (such as H1299, A549).

[0068] The present invention uses the modified nanomaterials to capture exosomes in cell culture supernatant, which can achieve efficient separation of exosomes and has the characteristics of good specificity and high capture efficiency.

[0069] The following will further illustrate the preparation method and specific application of nanomaterials in conjunction with several specific examples:

[0070] Example 1

[0071] The preparation method of cellulose nanomaterials in this example includes the following steps:

[0072] First, the streptavidin was immobilized on the cellulose nanomaterial through coupling reaction: 2.82 g of TEMPO-oxidized cellulose nanofibers with a solid content of 1.07% were weighed and placed in a 15 mL centrifuge tube, and then 2 mL of 0.26 M EDC solution and 2.26 mL of 0.23 M NHS solution were added. The reaction was first allowed to react at room temperature for 30 min, and then 400 ug / mL of streptavidin was added. The reaction solution was placed at 4 ° C for 48 h. After the reaction was completed, the unreacted reagents were removed by centrifugation 3 times with PBS. After the removal was completed, a portion was freeze-dried for infrared characterization (see the reaction process). Figure 2 ).

[0073] Second, the antibody was immobilized on the cellulose nanomaterial: 0.24 g of the cellulose nanomaterial obtained above was obtained, 500 μL of 20 μg / mL biotinylated CD63 antibody was added, and after reacting at 4°C for 2 hours, the unreacted antibody was removed by centrifugation to obtain the nanomaterial for separating exosomes.

[0074] Example 2

[0075] This embodiment proposes an application of nanomaterials, and the specific process is as follows:

[0076] The antibody-loaded nanomaterial obtained in Example 1 was added to 2 mL of exosome solution (the exosome solution was diluted with PBS) and reacted at 4°C for 1 h. After the reaction, the nanomaterial that captured the exosomes will sink to the bottom by centrifugation. At this time, the capture rate was calculated by the particle concentration of exosomes in the supernatant (tested by NTA) and the particle concentration before and after the reaction to be 83.3%.

[0077] Example 3

[0078] This embodiment proposes an application of nanomaterials, and the specific process is as follows:

[0079] The antibody-loaded nanomaterial obtained in Example 1 was added to 2 mL of exosome solution (diluted with PBS) and reacted at 4°C for 2.5 h. After the reaction, the nanomaterial that captured the exosomes will sink to the bottom by centrifugation. At this time, the capture rate was calculated by the particle concentration of exosomes in the supernatant (tested by NTA) and the particle concentration before and after the reaction to be 86.5%.

[0080] Example 4

[0081] This embodiment proposes an application of nanomaterials, and the specific process is as follows:

[0082] The antibody-loaded nanomaterial obtained in Example 1 was added to 1 mL of the exosome solution (diluted with PBS) and reacted at 4°C for 1 h. After the reaction, the nanomaterial that captured the exosomes will sink to the bottom by centrifugation. At this time, the capture rate was calculated by the particle concentration of the exosomes in the supernatant (tested by NTA) and the particle concentration before and after the reaction to be 82.7%.

[0083] Example 5

[0084] This embodiment proposes an application of nanomaterials, and the specific process is as follows:

[0085] The antibody-loaded nanomaterial obtained in Example 1 was added to 1 mL of exosome solution (diluted with PBS) and reacted at 4°C for 2 h. After the reaction, the nanomaterial that captured the exosomes will sink to the bottom by centrifugation. At this time, the capture rate was calculated by the particle concentration of exosomes in the supernatant (tested by NTA) and the particle concentration before and after the reaction to be 84.7%.

[0086] The present invention provides a nanomaterial for separating exosomes and a preparation method and application thereof, which have the following beneficial effects:

[0087] First, by utilizing the advantages of nanomaterials with high specific surface area and multiple modification sites, the exosome-specific capture antibodies are immobilized on the nanomaterials, which can significantly increase the chance of contact between the exosomes and the capture antibodies, while ensuring specificity and having high capture efficiency, taking into account both specificity and yield.

[0088] Second, the separation method of the present invention does not require large-scale separation equipment. Compared with expensive exosome separation kits, it has low cost and is simple and convenient to operate.

[0089] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a nanomaterial for separating exosomes, characterized in that: The preparation method comprises the following steps: Access to cellulose nanomaterials, including: oxidizing lignin by using 2,2,6,6-tetramethylpiperidine-1-oxide to obtain the cellulose nanomaterial, wherein the cellulose nanomaterial has a carboxyl group; The streptavidin is immobilized on the cellulose nanomaterial by covalent bond coupling, comprising: in a coupling reaction of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide, the carboxyl group of the cellulose nanomaterial and the amino group of the streptavidin form an amide bond covalent bond, so that the streptavidin is immobilized on the cellulose nanomaterial; the temperature range of the coupling reaction is 4° C. to 25° C., and the time range of the coupling reaction is 4 h to 48 h; An antibody for capturing exosomes is added to a cellulose nanomaterial immobilized with streptavidin, and the antibody is immobilized on the cellulose nanomaterial after reaction to obtain a nanomaterial for separating exosomes; the nanomaterial has a high specific surface area and multiple modification sites.

2. The method according to claim 1, characterized in that The concentration range of the 2,2,6,6-tetramethylpiperidine-1-oxide is 1.0% to 2.0%, and the content of carboxyl groups in the cellulose nanomaterial is 1.2 mmol / g to 2.3 mmol / g.

3. The method according to claim 1, characterized in that The concentration range of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 0.13 mol / L to 1.3 mol / L; and / or, The concentration range of the N-hydroxysuccinimide is 0.10 mol / L to 1.15 mol / L; and / or, The concentration range of the streptavidin is 100 μg / mL to 800 μg / mL.

4. The method according to claim 1, characterized in that: The antibody is any one of CD63, CD9, CD81, EpCAM, and EGFR; and / or, The antibody concentration range is 10 μg / mL to 40 μg / mL; and / or, The reaction time range of streptavidin and antibody is 4h~6h, and the temperature range is 2℃~10℃.

5. A nanomaterial for separating exosomes, characterized in that: Prepared according to the method according to any one of claims 1 to 4.

6. An application of a nanomaterial for separating exosomes, characterized in that: The nanomaterial according to claim 5 is used in exosome separation.

7. The use according to claim 6, characterized in that: The method for isolating exosomes comprises the following steps: The nanomaterial loaded with antibodies is added to the exosome solution to be separated, and the exosomes to be separated are captured by centrifugation after capture, and the exosomes to be separated are captured on the nanomaterial.

8. The use according to claim 7, characterized in that: The concentration range of the nanomaterial is 0.11 g / mL to 0.52 g / mL; and / or, The capture time ranges from 0.5h to 4h.

Images