Immunomagnetic substrate for targeted destruction of tumor exosome and preparation method and application thereof

Abstract

The invention relates to an immunomagnetic substrate for targeted destruction of tumor exosomes and a preparation method and application thereof. The immunomagnetic substrate for targeted destruction of the tumor exosome comprises a Fe3O4 magnetic bead, and the Fe3O4 magnetic bead is sequentially modified with a dendrimer PAMAM, a trimethylamine nitrogen oxide TMAO, an EpCAM aptamer and a curvature sensing peptide AHD. Compared with the prior art, the preparation method has the advantages that the EpCAM aptamer and the curvature sensing peptide AHD are modified on the substrate at the same time, so that the tumor exosome can be specifically recognized and captured, and the tumor exosome can be broken to prevent the tumor exosome from playing an immunosuppressive role. Compared with an antibody and an aptamer, the size of the aptamer is smaller, the EpCAM aptamer is modified on the substrate, it is guaranteed that the substrate has a large number of exosome affinity sites, and the targeting performance on tumor exosomes is improved.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to an immunomagnetic substrate for specifically targeting and disrupting tumor exosomes, a preparation method thereof, and an application thereof. Background Art

[0002] Exosomes are lipid bilayer vesicles with a particle size of 30 - 150 nm secreted by cells for communication with other nearby or distant cells. Exosomes contain a large number of nucleic acids, proteins, lipids, and metabolic molecules, especially some components from the parental cells. More notably, tumor exosomes in cancer patients can carry a large number of carcinogenic molecules related to tumorigenesis and progression to normal cells, forming an immunosuppressive tumor microenvironment, and subsequently promoting tumor metastasis. Moreover, tumor exosomes carrying immunosuppressive molecules can interact with immune cells, causing immune cell dysfunction and promoting the immune escape of tumor cells. Therefore, reducing the level of tumor exosomes in cancer patients helps to improve the immunosuppressive tumor microenvironment and inhibit exosome-induced tumor metastasis.

[0003] Currently, the strategies for reducing the level of tumor exosomes in patients mainly directly inhibit exosome biogenesis. However, since the biogenesis processes of exosomes in normal cells and tumor cells are very similar, this strategy will interfere with normal exosome-mediated intercellular communication and affect vital activities. Although the curvature-sensing peptide AHD has been shown to disrupt exosomes in vivo, it lacks targeting to tumor exosomes and inevitably also disrupts exosomes derived from non-tumor cells. In order to reduce the level of tumor exosomes without affecting the intercellular communication mediated by other types of exosomes, a technology that can specifically target and disrupt tumor exosomes, inhibit their ability to promote tumor cell migration, and at the same time not disrupt exosomes derived from non-tumor cells is now needed. Summary of the Invention

[0004] Based on the lack of an effective technical solution in the prior art for specifically targeting and disrupting tumor exosomes, inhibiting their ability to promote tumor cell migration, and at the same time not disrupting exosomes derived from non-tumor cells, the object of the present invention is to overcome the defects of the existing technology and provide an immunomagnetic substrate for specifically targeting and disrupting tumor exosomes, a preparation method thereof, and an application thereof.

[0005] The immunomagnetic substrate for specifically targeting and disrupting tumor exosomes provided by the present invention can specifically target, recognize, and bind to tumor exosomes, and then rupture them, thereby inhibiting their ability to promote tumor cell migration.

[0006] The solution of the present invention solves the problem that the existing methods for inhibiting tumor exosomes lack targeting. Based on the characteristics that the EpCAM aptamer recognizes the corresponding antigen on the surface of tumor exosomes and the curvature-sensing peptide AHD disrupts the membrane structure of exosomes, the present invention designs and prepares an immunomagnetic substrate, which can target and recognize tumor exosomes and then destroy them, effectively reducing the ability of tumor exosomes to induce the migration of tumor cells.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] The present invention provides an immunomagnetic substrate for targeting and destroying tumor exosomes, including Fe3O4 magnetic beads, on which dendrimer PAMAM, trimethylamine N-oxide (TMAO), EpCAM aptamer, and curvature-sensing peptide AHD are sequentially modified.

[0009] The immunomagnetic substrate for targeting and destroying tumor exosomes provided by the present invention is also denoted as Fe3O4@PAMAM@TMAO@Aptamer-AHD, abbreviated as FPTA-AHD.

[0010] In one embodiment of the present invention, the surface of the Fe3O4 magnetic beads is modified with carboxyl groups.

[0011] In one embodiment of the present invention, the dendrimer PAMAM is selected from commercially available 5th-generation amino-terminated polyamidoamine.

[0012] In one embodiment of the present invention, the trimethylamine N-oxide (TMAO) is obtained by oxidizing dimethylaminopropyl acrylamide with hydrogen peroxide, and has strong hydrophilicity, which can effectively avoid interfering with the non-specific adsorption of proteins to the substrate.

[0013] In one embodiment of the present invention, the 5'-end of the EpCAM aptamer is modified with carboxyl groups, and its nucleotide sequence is:

[0014] COOH-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG, which can specifically target the relevant antigen on the surface of tumor exosomes.

[0015] In one embodiment of the present invention, the constituent amino acids of the curvature-sensing peptide AHD are all D-amino acids, and the sequence is SGSWLRDVWDWICTVLTDFKTWLQSKL-NH2, which can disrupt the membrane structure within the size range of exosomes.

[0016] In one embodiment of the present invention, the crosslinking type between PAMAM and Fe3O4 is the crosslinking between carboxyl and amino groups, the crosslinking type between TMAO and PAMAM is the crosslinking between amino and carbon-carbon double bond groups, the crosslinking type between EpCAM aptamer and PAMAM is the crosslinking between carboxyl and amino groups, and the crosslinking type between AHD and PAMAM is the crosslinking between amino groups through glutaraldehyde.

[0017] In the immunomagnetic substrate for targeted disruption of tumor exosomes provided by the present invention, PAMAM provides a large number of binding sites, increases the amount of aptamer modification, and improves the affinity of the substrate for the target. TMAO has strong hydrophilicity, effectively avoiding non-specific adsorption of other proteins and exosomes. The EpCAM aptamer is used to recognize and capture tumor exosomes, and the curvature-sensing peptide AHD contacts the captured tumor exosomes and causes them to rupture.

[0018] The present invention also provides a preparation method of the immunomagnetic substrate for targeted disruption of tumor exosomes, comprising the following steps:

[0019] PAMAM is modified onto Fe3O4 magnetic beads through crosslinking between carboxyl and amino groups, and then TMAO, EpCAM aptamer, and AHD are respectively modified onto PAMAM through crosslinking between carbon-carbon double bond and amino groups, crosslinking between carboxyl and amino groups, and crosslinking between amino groups through glutaraldehyde, to obtain the immunomagnetic substrate for targeted disruption of tumor exosomes.

[0020] In one embodiment of the present invention, the preparation method of the immunomagnetic substrate for targeted disruption of tumor exosomes comprises the following steps:

[0021] (1) Preparation of Fe3O4@PAMAM: Disperse Fe3O4 solid powder in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, add N-hydroxysuccinimide ester (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to activate carboxyl groups, then wash the activated product with phosphate (PBS) buffer solution, add PAMAM, react at room temperature, and wash the reaction product with PBS to obtain Fe3O4@PAMAM;

[0022] (2) Preparation of Fe3O4@PAMAM@TMAO@Aptamer (FPTA): Add TMAO solution to the Fe3O4@PAMAM dispersion, react at room temperature, and wash with PBS to obtain

[0023] Fe3O4@PAMAM@TMAO; Add N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) to the EpCAM aptamer solution to activate the carboxyl group, and then add Fe3O4@PAMAM@TMAO to the EpCAM aptamer solution for reaction. Wash the product with PBS to obtain Fe3O4@PAMAM@TMAO@Aptamer (FPTA).

[0024] (3) Preparation of Fe3O4@PAMAM@TMAO@Aptamer-AHD (FPTA-AHD): Disperse the prepared Fe3O4@PAMAM@TMAO@Aptamer in glutaraldehyde solution to activate the amino group at room temperature. Wash with PBS, and then add AHD solution for reaction. Wash the product to obtain

[0025] Fe3O4@PAMAM@TMAO@Aptamer-AHD (FPTA-AHD).

[0026] In one embodiment of the present invention, the dosage relationship of Fe3O4 solid powder, PAMAM, TMAO, EpCAM aptamer, and AHD solution is 10 mg: 20 μL: 100 μL: 60 μL: 400 μL AHD.

[0027] In one embodiment of the present invention, in step (1), the concentration of N-hydroxysuccinimide is 0.15 M, the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 0.1 M, and PAMAM is dissolved in methanol to form a solution with a mass fraction of 5%. In step (2), TMAO is dissolved in a 0.138 M sodium chloride solution to form a solution with a mass fraction of 10%, the concentration of EpCAM aptamer is 10 μM, the concentration of N-hydroxysuccinimide is 0.1 M, and the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide is 0.4 M. In step (3), the concentration of the curvature-sensing peptide AHD is 100 μM.

[0028] In one embodiment of the present invention, in the above steps, for all washing operations, the solid is separated from the supernatant using a magnet, and clean washing buffer (PBS) is re-added to wash away excess unreacted substances.

[0029] The present invention further provides the application of the immunomagnetic substrate for targeted destruction of tumor exosomes, and the application of the immunomagnetic substrate for targeted destruction of tumor exosomes in the preparation of drugs for treating tumors.

[0030] In one embodiment of the present invention, the application of the immunomagnetic substrate for targeted destruction of tumor exosomes in the preparation of drugs for inhibiting tumor cell migration.

[0031] In one embodiment of the present invention, the application of the immunomagnetic substrate for targeted destruction of tumor exosomes in the preparation of a medicament for treating breast cancer.

[0032] The present invention further verifies the effect of the immunomagnetic substrate (FPTA-AHD) for targeted destruction of tumor exosomes on the specific recognition and destruction of tumor exosomes. The specific method is described as follows:

[0033] 1 mg of FPTA and 1 mg of FPTA-AHD are respectively added to the exosome solution, incubated at room temperature for 30 min, and then the supernatant is separated from the solid by a magnet. The morphology of the exosomes after being captured or destroyed is observed by transmission electron microscopy. The exosomes in the supernatant are quantified using a nanoparticle tracking analyzer, and the proportion of the remaining exosomes to the initial amount of exosomes is calculated.

[0034] In one embodiment of the present invention, the amount of exosomes is 200 μL, 1×10 9 particles / mL.

[0035] In one embodiment of the present invention, the quantification of the exosomes in the supernatant using a nanoparticle tracking analyzer includes the following steps: diluting the exosomes to a suitable concentration, injecting 3 mL of the solution into the instrument with a syringe, and the instrument detects the exosome concentration, and calculates the proportion of the remaining exosomes to the initial amount of exosomes.

[0036] In one embodiment of the present invention, the morphology of the exosomes after being captured or destroyed is observed by transmission electron microscopy, including the following steps: separating the solid after the reaction from the supernatant by a magnet, then redispersing the solid in physiological saline, taking 5 μL and dropping it on a copper mesh, standing for 5 min, sucking off the excess liquid with a blotting paper, taking 5 μL of 2% uranyl acetate solution, dropping it on the above copper mesh, standing for 5 min, sucking off the excess liquid with a blotting paper, and after air drying, observing with a transmission electron microscope.

[0037] In one embodiment of the present invention, the tumor exosomes are derived from the breast cancer cell line MCF-7 and the plasma of breast cancer patients.

[0038] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0039] (1) Strong anti-protein adsorption ability: The zwitterionic TMAO has strong hydrophilicity and can effectively avoid the interference caused by non-specific adsorption. Therefore, the present invention designs to modify TMAO on the surface of the substrate, avoiding the non-specific adsorption of other proteins and exosomes to the substrate. Compared with other hydrophilic compounds such as PEG, etc., TMAO has a smaller molecular weight and a smaller steric effect, facilitating the modification of more aptamers.

[0040] (2) High targeting to tumor exosomes: Compared with antibodies, aptamers are smaller in size. In the present invention, EpCAM aptamers are modified onto the substrate, ensuring that the substrate has a large number of exosome affinity sites and improving the targeting to tumor exosomes.

[0041] (3) Targeted destruction of tumor exosomes: In the present invention, EpCAM aptamers and the curvature-sensing peptide AHD are simultaneously modified onto the substrate, which can specifically recognize and capture tumor exosomes while breaking them, preventing tumor exosomes from exerting immunosuppressive effects. Brief Description of the Drawings

[0042] Figure 1 It is a schematic diagram of the preparation and application principle of the immunomagnetic substrate for targeted destruction of tumor exosomes in the present invention.

[0043] Figure 2 It is the ultraviolet spectrum diagram of the curvature-sensing peptide AHD in the solution before and after reacting with FPTA in Example 1.

[0044] Figure 3 It is the transmission electron microscope image of tumor exosomes captured by FPTA and destroyed by FPTA-AHD in Example 1. Figure 3 a is the transmission electron microscope image of tumor exosomes captured by FPTA, Figure 3 b is the transmission electron microscope image of tumor exosomes destroyed by FPTA-AHD.

[0045] Figure 4 It is the destruction efficiency of tumor exosomes with different addition amounts of FPTA-AHD in Example 1.

[0046] Figure 5 It is the transmission electron microscope image of the supernatant and solid after non-tumor exosomes are incubated with FPTA-AHD in Example 2. Figure 5 a is the transmission electron microscope image of the supernatant after non-tumor exosomes are incubated with FPTA-AHD, Figure 5 b is the transmission electron microscope image of the solid after non-tumor exosomes are incubated with FPTA-AHD.

[0047] Figure 6 It is the result diagram of the scratch experiment of MCF-7 cells in Example 3.

[0048] Figure 7 It is the result diagram of the scratch experiment of MDA-MB-231 cells in Example 3. Detailed Embodiments

[0049] The present invention will be described in detail below with reference to the drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and the detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0050] In the following examples, unless otherwise specified, raw materials or processing technologies are conventional commercially available raw material products or conventional processing technologies in the art.

[0051] Example 1:

[0052] An immunomagnetic substrate for targeted destruction of tumor exosomes, the preparation and application principle of which refer to Figure 1 , and the specific preparation steps are as follows:

[0053] (1) Preparation of Fe3O4@PAMAM:

[0054] Disperse 10 mg of Fe3O4 solid powder in 1 mL of 2-morpholinoethanesulfonic acid (MES) buffer solution (pH = 5.5), add N-hydroxysuccinimide ester (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), activate the carboxyl group at 37 °C for 30 min, then wash the activated product 3 times with phosphate (PBS) buffer solution, disperse it in 200 μL of phosphate buffer solution, PAMAM is purchased from Rosebio Ruoshe Biology (P17078 - 200 mg), and is made into a 5% methanol solution. Take 20 μL of PAMAM solution and add it to the above dispersion, react at room temperature for 16 h, wash the reaction product 3 times with PBS to obtain Fe3O4@PAMAM.

[0055] (2) Preparation of Fe3O4@PAMAM@TMAO@Aptamer (FPTA):

[0056] TMAO is first synthesized through the following steps: Add diethylenetriaminepentaacetic acid (DTPA) (800 mg) to 30 mL of ultrapure water and stir. Then, slowly add 4.31 mL of 30% H2O2 to the above solution and heat to 60 °C. Dissolve 14.4 g of dimethylaminopropylacrylamide (DMAPAA) in 10 mL of ultrapure water and drop it into the above mixture while stirring, and react for 6 h. After the reaction is completed and cooled to room temperature, add acetone to precipitate the product. The bottom product is a colorless viscous liquid, and the supernatant is discarded to obtain TMAO.

[0057] Dissolve 100 mg of TMAO in 1 mL of 0.138 M sodium chloride solution. Take 100 μL of the TMAO solution and add it to the Fe3O4@PAMAM dispersion. React at room temperature for 48 h, and wash twice with PBS to obtain Fe3O4@PAMAM@TMAO. Add N-hydroxysuccinimide ester (NHS) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) to 60 μL of the EpCAM aptamer solution, activate the carboxyl group at 37 °C for 30 min, and then add 2.4 mg of Fe3O4@PAMAM@TMAO to the above solution and react for 16 h. Wash the product twice with PBS to obtain Fe3O4@PAMAM@TMAO@Aptamer (FPTA).

[0058] (3) Preparation of Fe3O4@PAMAM@TMAO@Aptamer-AHD (FPTA-AHD):

[0059] Disperse the above FPTA in 1 mL of 5% glutaraldehyde solution, activate the amino group at room temperature for 2 h, wash three times with PBS, then add 400 μL of AHD solution, react for 4 h, and wash the product three times to obtain Fe3O4@PAMAM@TMAO@Aptamer-AHD (FPTA-AHD).

[0060] (4) Detection of AHD modification on FPTA by ultraviolet spectrophotometer:

[0061] Prepare two tubes of AHD solutions with the same concentration and volume. Add one tube to PBS as the control group, and add the other tube to the FPTA suspension with the same volume. After incubation, collect the solutions of the two systems and measure the absorbance with an ultraviolet spectrophotometer. The results are as Figure 2 shown. Compared with the control group, after reacting with FPTA, the absorbance of AHD decreased significantly, indicating that AHD was successfully modified onto FPTA.

[0062] (5) Extract tumor exosomes from breast cancer cell culture medium:

[0063] Culture breast cancer cells MCF-7 in a humidified atmosphere containing 5% CO2 at 37 °C. Use high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Passage the cells every three days. When the cell density reaches about 80%, discard the original medium, wash the cells twice with PBS, then add medium without fetal bovine serum, and after culturing for 24 h, collect the cell culture supernatant for exosome extraction.

[0064] First, the cell culture supernatant was centrifuged at 3500 g for 20 min to remove cell debris and large particles, then filtered using a 0.22-μm filter membrane, ultrafiltered and concentrated using an ultrafiltration tube with a molecular weight cut-off of 100 kDa, centrifuged at 3000 g for 20 min, and finally replaced with PBS twice to obtain the concentrated solution.

[0065] Tumor exosomes were isolated and purified from the concentrated solution using high-performance size-exclusion chromatography. The chromatographic column KW-804 was connected to the chromatographic system Shimadzu LC-2010A, the mobile phase was PBS, the flow rate was 1 ml / min, the wavelength of the ultraviolet detector was set at 280 nm, the column temperature was 25 °C, and the fractions at 5 - 6.5 min were collected. The collected fractions were concentrated in a 100-kDa ultrafiltration tube to obtain pure tumor exosomes.

[0066] (6) Specific recognition and destruction of tumor exosomes by FPTA-AHD:

[0067] 1 mg of FPTA and 1 mg of FPTA-AHD were respectively added to the exosome solution, incubated at room temperature for 30 min, then the supernatant was separated from the solid using a magnet, and the morphology of the exosomes after being captured or destroyed was observed using a transmission electron microscope.

[0068] (7) Verification of the destruction of tumor exosomes using a transmission electron microscope:

[0069] The solid after the reaction was separated from the supernatant using a magnet, then the solid was redispersed in physiological saline, 5 μL was taken and adsorbed on a copper mesh for sample preparation, and observed using a 120-kV transmission electron microscope. As Figure 3 shown in a, the exosomes captured on the surface of FPTA presented a complete cup-shaped vesicle structure (indicated by the black arrow). As Figure 3 shown in b, the surface of FPTA-AHD adhered to the membrane of the destroyed exosomes, and the exosomes were no longer in a complete vesicle structure.

[0070] (8) Optimization of the addition amounts of FPTA-AHD and tumor exosomes:

[0071] 0, 10, 30, 60, and 120 μg of FPTA-AHD were respectively added to the exosome solution, incubated for 30 min, the supernatant was separated from the solid using a magnet, and the remaining exosome concentration in the supernatant was measured using a nanoparticle tracking analyzer, and the proportion of the remaining exosomes was calculated. As Figure 4 shown, when the addition amount of FPTA-AHD was 60 μg, the destruction efficiency reached the highest, and when the amount of FPTA-AHD was further increased, the remaining exosome concentration basically did not decrease.

[0072] Example 2:

[0073] Verify that the immunomagnetic substrate provided by the present invention for targeting and destroying tumor exosomes has no obvious effect on non-tumor exosomes. The specific steps are as follows:

[0074] (1) Purification of urinary exosomes (non-tumor exosomes):

[0075] Collect morning urine and dispense it into 50 mL centrifuge tubes. First, centrifuge at 3500 g for 20 min to remove cell debris and large particles, then filter using a 0.22 μm filter membrane, ultrafiltrate and concentrate the filtrate using an ultrafiltration tube with a molecular weight cut-off of 100 kDa, centrifuge at 3000 g for 20 min, and finally replace with PBS twice to obtain a concentrated urine solution.

[0076] Separate and purify exosomes from the concentrated urine solution using high-performance size exclusion chromatography. Connect the chromatographic column KW-804 to the chromatographic system Shimadzu LC-2010A, with the mobile phase being PBS, the flow rate being 1 mL / min, the wavelength of the ultraviolet detector being set at 280 nm, the column temperature being 25 °C, and collect the fractions at 5 - 6.5 min. Concentrate the collected fractions in a 100 kDa ultrafiltration tube to obtain pure urinary exosomes (non-tumor exosomes).

[0077] (2) Incubation of non-tumor exosomes with FPTA-AHD:

[0078] Add 1 mg of FPTA-AHD to the non-tumor exosome solution, incubate at room temperature for 30 min, then separate the supernatant from the solid using a magnet, and use a transmission electron microscope to observe whether the non-tumor exosomes are adsorbed and destroyed.

[0079] (3) Transmission electron microscopy characterization:

[0080] Separate the solid from the supernatant after the reaction using a magnet, then redisperse the solid in physiological saline. Take 5 μL of the supernatant and the solid dispersion solution respectively and adsorb them on copper grids for sample preparation. Use a 120 kV transmission electron microscope to observe the morphology of non-tumor exosomes in the supernatant and whether FPTA-AHD can adsorb and destroy non-tumor exosomes. The results are as Figure 5 shown. After incubation of non-tumor exosomes with FPTA-AHD, they still maintain a complete cup-shaped vesicle structure, and no vesicle structure or damaged membrane fragments are observed on the surface of the solid.

[0081] Example 3:

[0082] Verify that the method of the present invention can destroy tumor exosomes in the plasma of breast cancer patients, thereby inhibiting the role of tumor exosomes in promoting the migration of breast cancer cells. The operating method is described as follows:

[0083] (1) Extract the total exosome crude fraction from the plasma of breast cancer patients:

[0084] The plasma was centrifuged at 12,000 g for 10 min to remove cell debris, and then the supernatant was filtered through a 0.22-μm filter membrane. 200 μL of the filtered plasma was loaded onto a mini-SEC chromatography column packed with agarose 4B beads, with PBS as the mobile phase. Every 500 μL of liquid was taken as a fraction, and fractions 7 - 11 containing exosomes were collected and concentrated using an ultrafiltration tube with a molecular weight cut-off of 100 kDa to obtain the total crude exosome fraction.

[0085] (2) Scratch assay:

[0086] Well-growing low-metastatic breast cancer cells MCF-7 and high-metastatic breast cancer cells MDA-MB-231 were selected. After trypsin digestion, the digestion reaction was terminated by adding medium. Then the cells were gently pipetted and mixed evenly, and the cells were seeded evenly in a six-well plate. The cells were incubated in an incubator at 37 °C with 5% CO2. When the cell confluence reached about 90%, a 200-μL pipette tip was used to draw a straight line at the bottom of the culture dish, keeping the pipette tip perpendicular to the culture dish. After scratching, the cells were gently washed 3 times with PBS to remove the scratched cells, and 2 mL of complete medium was added. Plasma exosomes from breast cancer patients (tumor exosome group) or the supernatant after incubation of plasma exosomes from breast cancer patients with FPTA-AHD (supernatant group) were added to the medium respectively. At the same time, PBS (PBS group) was added to another dish of cells for control. The cells at the scratched area were observed under a microscope at different times to see if they were covered with cells, and photos were taken. As Figure 6 and 7 , both MCF-7 cells and MDA-MB-231 cells co-incubated with tumor exosomes showed more obvious migration. The migration of the control group was comparable to that of the supernatant group. This indicates that tumor exosomes have the ability to promote the migration of breast cancer cells, and when tumor exosomes are destroyed by FPTA-AHD, the migratory-promoting ability is basically lost.

[0087] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. Obviously, those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. An immunomagnetic substrate for targeted disruption of tumor exosomes, characterized in that, It includes Fe3O4 magnetic beads, on which dendrimer PAMAM, trimethylamine N-oxide (TMAO), EpCAM aptamer and curvature-sensing peptide AHD are successively modified.

2. The immunomagnetic substrate for targeting and destroying tumor exosomes according to claim 1, wherein The surface of the Fe3O4 magnetic beads is modified with carboxyl groups.

3. An immunomagnetic substrate for targeted destruction of tumor exosomes according to claim 1, characterized in that, The dendrimer PAMAM is selected from commercially available 5th-generation amino-terminated polyamidoamine.

4. The immunomagnetic substrate for targeted destruction of tumor exosomes according to claim 1, characterized in that The trimethylamine N-oxide (TMAO) is obtained by oxidizing dimethylaminopropylacrylamide with hydrogen peroxide.

5. An immunomagnetic substrate for targeted destruction of tumor exosomes according to claim 1, characterized in that, The nucleotide sequence of the EpCAM aptamer is: COOH-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG.

6. The immunomagnetic substrate for targeted destruction of tumor exosomes according to claim 1, characterized in that, The sequence of the curvature-sensing peptide AHD is SGSWLRDVWDWICTVLTDFKTWLQSKL-NH2.

7. The preparation method of the immunomagnetic substrate for targeting and destroying tumor exosomes according to any one of claims 1-6, characterized in that, It includes the following steps: PAMAM is modified onto the Fe3O4 magnetic beads through crosslinking between carboxyl and amino groups, and then TMAO, EpCAM aptamer and AHD are respectively modified onto PAMAM through crosslinking of carbon-carbon double bond with amino group, crosslinking of carboxyl with amino group, and crosslinking of amino groups through glutaraldehyde to obtain the immunomagnetic substrate for targeted destruction of tumor exosomes.

8. The preparation method of the immunomagnetic substrate for targeting and destroying tumor exosomes according to claim 7, characterized in that, It includes the following steps: (1) Preparation of Fe3O4@PAMAM: Disperse Fe3O4 solid powder in 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, add N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to activate carboxyl groups, then wash the activated product with phosphate buffer solution, add PAMAM, react at room temperature, and wash the reaction product with PBS to obtain Fe3O4@PAMAM; (2) Preparation of Fe3O4@PAMAM@TMAO@Aptamer: Add TMAO solution to the Fe3O4@PAMAM dispersion, react at room temperature, and wash with PBS to obtain Fe3O4@PAMAM@TMAO; Add N-hydroxysuccinimide ester and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to the EpCAM aptamer solution to activate carboxyl groups, then add Fe3O4@PAMAM@TMAO to the EpCAM aptamer solution for reaction, and wash the product with PBS to obtain Fe3O4@PAMAM@TMAO@Aptamer; (3) Preparation of Fe3O4@PAMAM@TMAO@Aptamer-AHD: Disperse the prepared Fe3O4@PAMAM@TMAO@Aptamer in glutaraldehyde solution, activate amino groups at room temperature, wash with PBS, then add AHD solution, react, and wash the product to obtain Fe3O4@PAMAM@TMAO@Aptamer-AHD, which is the immunomagnetic substrate for targeted destruction of tumor exosomes.

9. Use of the immunomagnetic substrate for targeted destruction of tumor exosomes according to any one of claims 1-6 in the preparation of a drug for treating tumors.

10. The application according to claim 9, characterized in that, Use of the immunomagnetic substrate for targeted destruction of tumor exosomes in the preparation of a drug for treating breast cancer.

Images