CN113667638B - Functionalized red blood cell based on surface modification, preparation method thereof and application thereof in exosome separation - Google Patents

Abstract

The invention discloses a functionalized erythrocyte based on surface modification, a preparation method thereof and application thereof in exosome separation. The preparation method comprises the following steps: mixing anti-RBC with red blood cells, reacting at 40-50 ℃ for 1-2 h, activating, and adding an aptamer with a nucleotide sequence shown as SEQ ID NO.1 for modification after activation. The invention constructs an exosome separation system and a method which can be used without ultracentrifugation, exosome antibodies and complex extraction reagents on the basis of natural biomaterial red blood cells.

Description

A functionalized erythrocyte based on surface modification and its preparation method and application in isolating exosomes

technical field

The invention belongs to the technical field of cell engineering, and in particular relates to a method for preparing functionalized red blood cells based on surface modification and its application in separating exosomes.

Background technique

Extracellular vesicles (EVs) are nanoscale lipid bilayer membrane vesicles actively secreted by various cells in the body. According to their synthesis, size and biological characteristics, extracellular vesicles can be further classified as exocrine body (exosomes) and microvesicles (microvesicles). Among them, exosomes with a diameter of about 40-150 nm widely exist and are distributed in blood and various body fluids; through the signal molecules carried, exosomes constitute a new intercellular information communication system in the body, which not only affects the normal function of cells Physiological functions, and participate in the occurrence and development of many diseases. In particular, exosomes derived from tumor cells contain abundant and stable biological substances (such as proteins, mRNA, miRNA and DNA, etc.). Exosomes have unique biological properties that provide non-invasive molecular information for liquid biopsy of tumors. However, most of the existing exosome isolation techniques require specific equipment (ultracentrifuge), low separation purity (affinity adsorption), and rely on antibodies with complex preparation processes (exosome separation magnetic beads). These methods are difficult to meet The clinical demand for tumor-derived exosomes greatly limits its clinical application. Therefore, the development of simple, fast and efficient exosome centrifugal separation and enrichment technology has become the primary problem to be solved in liquid biopsy to realize exosome detection.

Red blood cells are a natural and easy-to-obtain biological material. Due to the lipids in the membrane structure and the rich negative charges on the surface, the red blood cells in the circulation can be independent of each other and not aggregated together, showing good suspension stability. Erythrocytes have good plasticity and can deform under the action of external forces such as centrifugal force to reduce the damage to their structure by mechanical force. They can act as a "sponge pad" to protect the integrity of the exosome structure during the process of separating exosomes. Mature erythrocytes do not have nuclei, so they can be lysed together with exosomes to extract nucleic acids without elution, so as to preserve the number of captured exosomes to the greatest extent, and then use them for downstream analysis of nucleic acid molecules such as DNA and miRNA derived from exosomes. Moreover, there are abundant glycoproteins, simple proteins, and membrane contractile proteins on the red blood cell membrane. Some of these proteins protrude on the cell surface, like branches sticking out from the ground, such as ABH antigen; some are embedded in the cell membrane, such as Rh antigen. The erythrocyte membrane protein has a large number of modification sites, which is convenient for the functional transformation of erythrocytes by using biomolecules.

On the other hand, traditional exosome separation magnetic beads use antibodies to capture exosomes, but proteins as probe molecules are susceptible to denaturation and expensive synthesis due to environmental factors such as pH and temperature, and aptamers are composed of DNA or RNA ( It is mainly DNA), which is smaller than protein. After screening and enrichment, it can have a sensitivity comparable to the antigen-antibody reaction, and at the same time, the synthesis is easier and the stability is better. Nucleic acid aptamers can bind to a variety of target substances with high specificity and high selectivity, and the use of functionalized nucleic acids instead of antibodies to specifically recognize target substances has also brought new impetus to research in this field. Some studies have used the aptamer (aptamer) with a specific recognition function for the surface cluster differentiation antigen CD63 of exosomes to successfully recognize tumor exosomes.

Contents of the invention

Aiming at the above-mentioned deficiencies in the prior art, the present invention provides a functionalized red blood cell based on surface modification and its preparation method and its application in the isolation of exosomes. Exosome isolation systems and methods for ultracentrifugation, exosome antibodies, and complex extraction reagents.

In order to achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:

A method for preparing functionalized erythrocytes based on surface modification, mixing anti-RBC with erythrocytes, reacting at 40-50°C for 1-2 hours, and then activating, adding exosome-specific aptamers after activation grooming.

Further, the specific aptamer is Apt-CD63, and the specific sequence of the aptamer is as follows: CACCCCACCTCGCTCCCGTGACACTAATGCTATTTTTTTTTT (SEQ ID NO.1).

Further, the aptamer can also be other aptamers capable of specifically binding to exosomes.

Further, the following steps are included:

(1) Prepare MES buffer

(2) separate and wash red blood cells;

(3) Mix red blood cells and anti-RBC at a volume ratio of 1:5-50, react at 40-50°C for 1-2 hours, and invert the reaction vessel 5-6 times during the reaction;

(4) Mix the erythrocyte/anti-RBC complex, EDC, NHS, and MES obtained in step (3) evenly, leave it at room temperature for 15-20 minutes to activate, then add the aptamer, and place it at 4°C for 10-12 hours. Mix once every 15 minutes;

(5) Centrifuge the solution obtained in step (4) at 4° C. at 4000-5000 rpm, and then collect the functionalized red blood cells in the lower layer.

Further, the volume ratio of red blood cell/anti-RBC complex, EDC, NHS and aptamer in step (4) is 15-20:1-2:1-2:1.

Further, the concentration of EDC is 5 mM; the concentration of NHS is 12.5 mM; the concentration of aptamer is 100 μM; the concentration of red blood cell/anti-RBC complex is 5×10 ¹² /L.

The functionalized red blood cells prepared by the above method.

The method for isolating exosomes by using the above-mentioned functionalized red blood cells comprises the following steps:

(1) Dilute and mix the plasma with an equal volume of PBS;

(2) After mixing the functionalized red blood cells and plasma, centrifuge at 3000-4000rpm at 4°C for 1-5min and collect the solid phase product;

(3) Mix the solid-phase product with PBS, then add nuclease, centrifuge at 3000-4000 rpm for 5-10 min at 4°C, and collect the centrifuged liquid phase to obtain exosomes.

A kit for isolating exosomes, comprising the above-mentioned functionalized red blood cells.

Design principle of the present invention is as follows:

Combining the advantages of natural biomaterial red blood cells and nucleic acid aptamers, aptamers are used to functionalize red blood cells to achieve efficient enrichment and isolation of tumor cell-derived exosomes. First, the anti-erythrocyte antibody specifically binds to erythrocytes, and the polypeptide of the antibody provides enough carboxyl groups on the surface of erythrocytes as aptamer binding sites for modifying amino groups; The exosomes are immobilized on the surface of red blood cells to form an exosome capture platform for red blood cells/antibodies/aptamers. It is only necessary to mix the functionalized red blood cells with the body fluid samples taken from the patient, and the free exosomes can be bound by the aptamer, and then the exosomes attached to the surface of the red blood cells can be separated together by simple centrifugation to achieve the concentration of exosomes. Efficient separation and enrichment. The nucleic acid extraction of exosomes can directly use the lysate to decompose the erythrocyte-exosome conjugate without worrying about the interference from the erythrocytes; if complete exosomes are to be obtained, nucleases can be used to hydrolyze the aptamer chain to release the captured exosomes. Exosomes, at this time, red blood cells are deposited at the bottom of the tube with centrifugation, and exosomes exist in the supernatant due to their low density.

Beneficial effects of the present invention:

1. The novel "aptamer-functionalized red blood cells" constructed by the present invention can isolate and enrich exosomes simply and quickly.

2. The present invention does not require complex steps such as elution and ultracentrifugation, and the isolated exosomes can be directly used for subsequent analysis of biomolecules such as DNA, mRNA, and miRNA, making the research on exosomes easier and faster.

3. For research that needs to keep the structure of exosomes independent and complete, the present invention can also maintain the integrity and high purity of the obtained exosomes to the greatest extent.

4. Compared with other methods such as immunomagnetic beads, the biocompatibility is better, and the raw materials are easy to obtain, which has the advantages of low cost and good separation effect.

Description of drawings

Fig. 1 is a schematic diagram of the preparation of functionalized red blood cells according to the method of the present invention;

Figure 2 is the characterization of aptamer functionalized red blood cells;

Figure 3 is the exosomes captured by aptamer functionalized red blood cells;

Figure 4 is the exosomes captured by red fluorescent dye-labeled aptamer functionalized red blood cells;

Figure 5 is the separation of captured exosomes from functionalized erythrocytes.

Detailed ways

The specific embodiments of the present invention are described below so that those skilled in the art can understand the present invention, but it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes Within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions and creations using the concept of the present invention are included in the protection list.

Example 1

The principle of constructing functionalized red blood cells that can be used for exosome isolation is shown in Figure 1, and the specific process is as follows:

1. Prepare MES buffer

(1) Add 0.5325g of MES powder (molecular weight 195.28) into 20mL of H ₂ O;

(2) adjust the pH of the above solution to 6.0 with NaOH solution;

(3) Add 0.225g NaCl solution;

(4) Adjust the total volume to 50 mL with H ₂ O.

2. Separation and washing of RBC (red blood cells)

(1) Centrifuge anticoagulated whole blood at a low speed of 3500rpm for 5min (sodium citrate), absorb the upper layer of plasma and leave red blood cells;

(2) Add an equal volume of PBS (0.01M) to the remaining erythrocytes, invert and mix well, centrifuge again at the speed in step (1) and discard the supernatant, leaving the erythrocytes. Repeat this operation three times;

(3) The remaining red blood cells were resuspended in an equal volume of PBS. (The RBC concentration at this time is about 5×10 ¹² /L).

3. The reaction between anti-RBC (anti-erythrocyte antibody) and RBC

RBC and anti-RBC were mixed, wherein, the volume of RBC was 20 μL, and the volume of anti-RBC was 1 μL; reacted at 40° C. for 1 h, during which time the mixing container was inverted 5 times.

4. Modification of Apt-CD63 on RBC

(1) Raw materials

The amount of RBC/anti-RBC obtained in step 3 depends on the need;

EDC (2μL, 1mg+1mLH ₂ O to make 5mM)

NHS (2μL, 1mg+1mLH ₂ O to make 12.5mM)

MES buffer (450 μL or 400 μL)

DNA (Apt-CD63) 1 μL, 100 μM.

(2) Modification process

a. Mix RBC/anti-RBC complex, EDC, NHS, and MES evenly, and place it at room temperature for 15 minutes to activate "-COOH"; Mix once every 15 minutes;

b. Centrifuge the solution at 4000rpm for 10min at 4°C, leave the layer of functionalized RBC, and wash once with 2 times the volume of PBS;

c. Store the remaining F-RBC (functionalized red blood cells) in a refrigerator at 4°C, and add 200 μL of PBS for storage.

The prepared functionalized erythrocytes were detected, and the erythrocytes obtained in the preparation process without adding anti-RBC antibody were used as a control, and the characterization results are shown in FIG. 2 .

As shown in Figure 2, in Figure A in Figure 2, you can see a green fluorescent circular structure with a diameter of about 7 μm (the size of red blood cells) (the attached picture is black and white, so the fluorescence is not shown in the attached drawing, the same below). This is because the erythrocytes are first bound to the anti-RBC antibody, and the "-COOH" on the antibody is used as the anchor point, and then the aptamer modified with "-NH ₂ " at the 3' end is covalently linked. Since the 5' end of the aptamer is also labeled with the FAM fluorescent group, the functionalized erythrocytes that fluoresce green can be seen under a fluorescence microscope.

In Figure B, there is basically no fluorescence. This is because no anti-RBC antibody was added during the preparation of functionalized red blood cells. Although the aptamer modified with “-NH ₂ ” was added later, due to the lack of antibodies as bridges, the surface of red blood cells failed. Modify the aptamer. This shows that the strategy of using anti-RBC antibody binding to red blood cells as the anchor point and then linking the aptamer is successful.

Example 2

Using the functionalized red blood cells constructed in Example 1 to separate exosomes, the specific process is as follows:

(1) Dilute the plasma (500 μL) with an equal volume of PBS, add 200 μL of the prepared F-RBC into it and shake and mix for 30 minutes;

(2) Let stand for 5 minutes, centrifuge at 3000rpm at 4°C for 1 minute, and absorb the supernatant to retain the precipitate. At this time, the exosomes exist in the precipitate separated at the bottom of the tube;

(3) Pipette and mix the sediment at the bottom of the tube with 200 μL PBS, add 1 μL, 0.1 U/μL DNase I, mix well and let stand for ten minutes;

(4) Centrifuge at 3,000 rpm for five minutes at 4°C, and suck up the supernatant with a pipette. At this time, exosomes are present in the supernatant.

Example 3

1. Characterization of functionalized erythrocytes capturing exosomes using staining techniques and fluorescence microscopy

Using PKH67 green fluorescent dye, the successfully separated exosomes were first stained and marked, and the fluorescently labeled exosomes were separated by ultracentrifugation. Then the functionalized erythrocytes were added to the exosome suspension, mixed and centrifuged at 3500rpm for 5min, and the precipitate was imaged under a fluorescence microscope. Figure 3A shows that the exosomes were successfully labeled with the green fluorescent lipophilic dye PKH67, which appeared as green spots under the fluorescence microscope. In Figure 3B, the functionalized erythrocytes captured green fluorescently labeled exosomes, and these exosomes attached to the surface of the erythrocytes formed a "circular aperture". On the contrary, the erythrocytes in Figure 3C could not capture without the modified aptamer Exosomes therefore do not show any fluorescence.

In order to further prove that exosomes were captured by functionalized erythrocytes, functionalized erythrocytes and unfunctionalized erythrocytes were stained with red fluorescent dye PKH26, and green fluorescently labeled exosomes were captured and imaged again. Figure 4A clearly shows that red fluorescent functionalized erythrocytes capture green fluorescent-labeled exosomes, while the red blood cells in Figure 4B cannot capture exosomes without modified aptamers, so only red fluorescent-labeled erythrocytes can be seen .

2. In order to obtain purer and more complete exosomes, the aptamer chain was hydrolyzed with nuclease DNase I, and the supernatant was taken for fluorescence imaging. Figure 5A clearly shows that red fluorescent functionalized red blood cells capture green fluorescent-labeled exosomes, while Figure 5B shows functionalized red blood cells treated with nucleases, almost no exosomes remain on the surface of red blood cells, proving that exosomes Secretosomes have been eluted successfully. Figure 5C is an imaging of the supernatant, and green fluorescently labeled exosomes scattered like star spots can be seen, which proves that complete exosomes have been obtained.

sequence listing

<110> Affiliated Hospital of North Sichuan Medical College

Chongqing Hospital of Traditional Chinese Medicine

<120> A functionalized erythrocyte based on surface modification and its preparation method and application in exosome isolation

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

caccccacct cgctcccgtg acactaatgc tatttttttt tt 42

Claims (4)

1. A preparation method of functionalized erythrocytes based on surface modification is characterized in that anti-RBC is mixed with erythrocytes, the mixture is activated after reaction is carried out for 1 to 2h at the temperature of 40 to 50 ℃, and a specific aptamer of an exosome is added for modification after the activation is finished; the specific aptamer is Apt-CD63, and the nucleotide sequence of the specific aptamer is shown in SEQ ID NO. 1; the preparation process comprises the following steps:

(1) Preparing MES buffer solution

(2) Separating and washing red blood cells;

(3) Mixing the red blood cells and the anti-RBC according to the volume ratio of 1 to 5 to 50, reacting at 40 to 50 ℃ for 1 to 2h, and turning over the reaction vessel for 5 to 6 times during the reaction;

(4) Uniformly mixing the erythrocyte/anti-RBC compound obtained in the step (3), EDC, NHS and MES, standing and activating at room temperature for 15-20min, then adding a specific aptamer, standing at 4 ℃ for 10-12h, and uniformly mixing every 15 min; wherein the volume ratio of the erythrocyte/anti-RBC complex to the EDC to the NHS to the aptamer is 15 to 20;

the concentration of EDC is 5mM; the concentration of NHS was 12.5mM; the concentration of the aptamer is 100 mu M; the concentration of the red blood cell/anti-RBC complex is 5X 10 ¹² /L

(5) And (4) centrifuging the solution obtained in the step (4) at 4 ℃ to 4000 to 5000rpm, and collecting the functionalized erythrocytes in the lower layer.

2. Functionalized red blood cells produced by the method of claim 1.

3. A method for the isolation of exosomes using the functionalized red blood cells of claim 2, comprising the steps of:

(1) Diluting and mixing plasma with PBS (phosphate buffer solution) with the same volume;

(2) Uniformly mixing the functionalized erythrocytes with the blood plasma, centrifuging at 4 ℃ and 3000 to 4000rpm for 1 to 5min, and collecting a solid-phase product;

(3) And (3) blowing and uniformly mixing the solid-phase product with PBS, adding nuclease, centrifuging at 4 ℃ and 3000-4000 rpm for 5-10min, and collecting a centrifugal liquid phase to obtain the exosome.

4. A kit for exosome isolation comprising the functionalized red blood cells of claim 2.

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