A method for programming extracellular vesicle-cell interactions based on DNA-encoded receptor ligands

Abstract

The present invention provides a method for programming the interaction between extracellular vesicles and cells based on DNA-encoded receptor ligands, the method comprising: inserting a cholesterol-modified DNA single strand as a ligand into the phospholipid bilayer of the extracellular vesicle by oscillating with the extracellular vesicle; mixing three cholesterol-modified DNA single strands and a DNA containing a complementary pairing sequence with the ligand, annealing, and forming a receptor with a DNA tetrahedral structure; the receptor is inserted into the cell membrane through the cholesterol at three vertices, and recognizes the extracellular vesicles functionalized with the DNA ligand, thereby achieving efficient and specific uptake of extracellular vesicles from different sources by the cell. According to the present invention, a programmable, universal, efficient, and specific method for manipulating the interaction between extracellular vesicles and cells is provided, providing a new tool for applications such as live cell delivery and cell communication regulation.

Description

Technical Field

[0001] The present invention relates to the field of cell engineering, and more specifically to a method for programming the interaction between extracellular vesicles and cells based on DNA-encoded receptor ligands. Background Art

[0002] Extracellular vesicles are a class of highly heterogeneous natural biological vesicles with a phospholipid bilayer structure secreted by cells. Extracellular vesicles contain bioactive substances such as proteins, lipids, and nucleic acids, which can be secreted by donor cells and delivered to recipient cells to change the phenotype and function of recipient cells. The "extracellular vesicle-cell" interaction is involved in various normal physiological reactions and pathological processes. Extracellular vesicles have unique advantages such as a wide range of sources, high biocompatibility, and rich contents. They have received extensive attention in the fields of drug delivery, regenerative medicine, tissue repair, and synthetic biology. Therefore, reprogramming the interaction between extracellular vesicles and cells is not only crucial to elucidating related biological processes, but also helps to promote the construction of extracellular vesicles as information transfer units in synthetic biology. In recent years, great progress has been made in the study of artificial intervention in the interaction between extracellular vesicles and cells. For example, the expression of VSV-G protein (vesicularstomatitis virus G protein) on the surface of extracellular vesicles by genetic engineering has been used to promote its uptake in target cells; the modification of c(RGDyK) peptide on the surface of extracellular vesicles by copper-free click chemistry can promote its aggregation in brain vascular endothelial cells; and the functionalization of macrophage-derived extracellular vesicles by AS1411 nucleic acid aptamers can improve their ability to target glioma cells. However, the specificity, programmability and versatility of these methods are still limited. Summary of the invention

[0003] The purpose of the present invention is to provide a method for programming the interaction between extracellular vesicles and cells based on DNA-encoded receptor ligands, thereby solving the problems of limited specificity, programmability and versatility of existing technologies for reprogramming the interaction between extracellular vesicles and cells.

[0004] In order to solve the above problems, the present invention adopts the following technical solutions:

[0005] Provided is a method for programming the interaction between extracellular vesicles and cells based on DNA-encoded receptor ligands, the method comprising: inserting a cholesterol-modified DNA single strand as a ligand into the phospholipid bilayer of the extracellular vesicle by oscillating with the extracellular vesicle; mixing three cholesterol-modified DNA single strands and a DNA containing a complementary pairing sequence to the ligand, annealing, and forming a receptor with a DNA tetrahedral structure; the receptor is inserted into the cell membrane through the cholesterol at three vertices, and recognizes the extracellular vesicles functionalized with the DNA ligand, thereby achieving efficient and specific uptake of extracellular vesicles from different sources by the cell.

[0006] According to a preferred embodiment of the present invention, the method comprises the following steps: S1: preparing DNA-encoded artificial receptors and ligands respectively; S2: modifying cells with the DNA-encoded artificial receptors prepared in step S1; S3: isolating and extracting extracellular vesicles, and modifying the extracellular vesicles with the DNA-encoded artificial ligands prepared in step S1; S4: adding the extracellular vesicles modified with the DNA-encoded artificial ligands to the cells modified with the DNA-encoded artificial receptors, incubating at 37°C for 2h to 3h, and observing the DNA receptor ligand-mediated extracellular vesicle uptake with a laser confocal microscope or a flow cytometer.

[0007] According to a preferred solution of the present invention, step S1 includes the following sub-steps:

[0008] (1) dissolving magnesium chloride hexahydrate in a phosphate buffer solution so that the final concentration of magnesium ions is 1-5 mM;

[0009] (2) Dissolve four DNA tetrahedral short chains in the solution prepared in step (1) respectively, and mix them in a PCR tube. The final concentration of each short chain ranges from 0.5 to 2 μM.

[0010] (3) placing the PCR tube in step (2) in a PCR instrument, heating at 95° C. for 10 minutes, rapidly cooling to 4° C. to obtain a DNA-encoded artificial receptor, and storing at 4° C. for later use;

[0011] (4) Dissolve the ligand DNA-chol short chain in the solution prepared in step (1) to a concentration of 80-120 μM to prepare a DNA-encoded artificial ligand, which is stored at 4°C for future use.

[0012] According to a preferred embodiment of the present invention, step S2 includes the following sub-steps:

[0013] (5) The cells were seeded in a glass-bottomed cell culture dish, the culture medium was removed, and PBS was added for washing;

[0014] (6) adding the DNA tetrahedron solution obtained in step (2) to serum-free culture medium;

[0015] (7) adding the solution in step (6) to the cells in step (5), and incubating at room temperature for 8 to 12 minutes to obtain cells modified with the DNA-encoded artificial receptor;

[0016] (8) Wash the cells in (7) three times with PBS and add serum-free culture medium.

[0017] 5. The method according to claim 4, characterized in that step S3 comprises the following sub-steps:

[0018] (9) The cells from which extracellular vesicles are to be extracted are inoculated and cultured in several T75 cell culture flasks. When the cell growth area reaches about 80%, they are washed three times with 10 mL of PBS, and finally 10 mL of serum-free medium is added to each flask. After culturing for 24 hours, about 100 mL of cell culture supernatant is obtained; the dead cells are removed by centrifugation at 300 g for 5 to 10 minutes, and the supernatant is taken; the cell debris is removed by centrifugation at 3000 g for 10 to 20 minutes, and the supernatant is taken; the cell organelles and large cell vesicles are removed by centrifugation at 10000 g for 20 to 30 minutes, and the supernatant is taken; the lower layer of solution is filtered through a 0.22 μm filter; the upper layer of liquid is gently removed by centrifugation at 100000 g for 80 to 90 minutes, and the bottom is resuspended with 500 μL of PBS, which is the extracellular vesicles, and stored at -80°C for later use.

[0019] According to a preferred solution of the present invention, step S3 further includes the following sub-steps:

[0020] (10) Take 100 μL of extracellular vesicles for rapid dissolution, add 1 μL of Deep-red extracellular vesicle staining reagent, and mix well; incubate at 37°C in the dark for 30 to 40 minutes; transfer all the solution to a filter tube and centrifuge at 3000 g for 3 to 5 minutes at room temperature; add 100 μL of PBS to the filter tube and centrifuge at 3000 g for 3 to 5 minutes, repeat once; centrifuge at 3000 g for 3 to 5 minutes at room temperature to remove the remaining solution; aspirate 50 μL of PBS into the tube and gently blow to resuspend the extracellular vesicles;

[0021] (11) Take 50 μL of the solution in step (10) and add 5.6 μL of ligand DNA-chol short chain with a concentration of 80-120 μM;

[0022] (12) placing the solution in step (11) on a vortex shaker at 600 rpm / min for 3 to 5 minutes;

[0023] (13) Add the solution in step (12) to the sleeve of the ultrafiltration tube, add 350 μL of PBS solution, centrifuge at 5000 g for 3 to 5 minutes, and remove the bottom solution;

[0024] (14) Add another 400 μL of PBS solution to the ultrafiltration tube sleeve, centrifuge at 5000 g for 5 min, and repeat the washing twice;

[0025] (15) Pipette about 20 μL of the residual solution in the ultrafiltration tube sleeve in step (14), add it to a 500 μL centrifuge tube, and place it at 4°C for later use.

[0026] According to a preferred embodiment of the present invention, step S4 includes the following sub-steps:

[0027] (16) taking 20 μL of the DNA-chol short-chain modified extracellular vesicles prepared in step (15) and adding it to the DNA tetrahedron modified cell serum-free culture medium prepared in step (8);

[0028] (17) Place the cells in cell culture medium and incubate at 37°C for 2–3 h.

[0029] (18) Remove the cell culture medium in step (17), add 1 mL of culture medium containing 5 μL PlasMem Bright Green cell membrane dye and 10 μL Hoechst 33342 (100×) cell nuclear dye, and incubate at 37°C for 20 minutes;

[0030] (19) removing the culture medium containing the dye in step (18), washing twice with PBS, and adding cell culture medium again;

[0031] (20) DNA receptor ligand-mediated extracellular vesicle uptake was detected using confocal laser scanning microscopy or flow cytometry.

[0032] Preferably, the extracellular vesicles from different sources include: extracellular vesicles from STO cells, watermelon, and Escherichia coli.

[0033] Preferably, the cells include human cells, mouse cells, etc., and can be selected from: MDA-MB-231 cells, HeLa cells, etc.

[0034] According to a preferred embodiment of the present invention, the cell's ability to uptake extracellular vesicles can be adjusted by changing the length of the complementary DNA pairing chain, and the extracellular vesicle uptake capacity increases with the increase in the number of complementary DNA pairing bases.

[0035] The present invention relates to a new method for manipulating the interaction between extracellular vesicles and cells, specifically, a single-stranded DNA is inserted into the surface of the extracellular vesicle through cholesterol as a ligand, a complementary strand is extended from the vertex of the DNA tetrahedron as a receptor, and the remaining three vertices are inserted into the surface of the cell membrane through cholesterol, so that the cell can efficiently and specifically take up the extracellular vesicle, and manipulate the interaction between the extracellular vesicle and the cell. The schematic diagram of such a DNA-encoded receptor ligand programming "extracellular vesicle-cell" interaction provided by the present invention is as follows Figure 1 shown.

[0036] It should be known that the prior art has never disclosed the technology of the present invention for the interaction between extracellular vesicles encoding receptor ligands and cells. The present invention solves the problem of controllable interaction between extracellular vesicles from different species and cell reprogramming by adopting such a technical solution.

[0037] Compared with the prior art, the present invention has the following significant advantages:

[0038] 1) The artificially synthesized receptors and ligands in the present invention are all encoded by DNA. Thanks to the DNA-specific base complementary pairing and rich programmable sequences, multiple groups of specific recognition units can be constructed by changing the base sequences of the DNA tetrahedron-extended DNA single strands on the cells and the DNA single strands on the extracellular vesicles, so that the "extracellular vesicle-cell" interaction programmed by the DNA-encoded receptor ligand has a highly specific and predictable orthogonal reaction effect;

[0039] 2) Because extracellular vesicles from different sources and different types of cells all have a phospholipid bilayer as their basic membrane structure, and DNA-encoded receptor ligands are inserted into the phospholipid bilayer of the biological membrane by utilizing the hydrophobic effect of cholesterol, this operation does not require complex covalent modification and genetic engineering and is easy to operate. Therefore, this "extracellular vesicle-cell" interaction programmed by DNA-encoded receptor ligands has wide versatility.

[0040] 3) The tetrahedral structure of DNA provides a stable basis for the DNA receptors on cells and will not be rapidly internalized by cells.

[0041] 4) The present invention provides a universal platform for cell engineering to customize the exchange of substances and signals between cells across species.

[0042] In summary, according to the present invention, a programmable, universal, efficient and specific method for controlling the interaction between extracellular vesicles and cells is provided, which provides a new tool for applications such as living cell delivery and cell communication regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1Schematic diagram of the "extracellular vesicle-cell" interaction programmed by the DNA-encoded receptor ligand designed for the present invention;

[0044] Figure 2 TEM image (a) and particle size analysis (b), dynamic light scattering results (c), and Chol-DNA ligand image (d) of the extracellular vesicles after extraction in Example 1;

[0045] Figure 3 The polyacrylamide gel electrophoresis diagram of the DNA tetrahedral receptor after synthesis in Example 1 (a), the laser confocal microscopy result of its modification on the MDA-MB-231 cell membrane (b) and the statistical diagram of fluorescence change over time (c);

[0046] Figure 4 The confocal laser scanning microscopy results of DNA-encoded extracellular vesicle uptake in Example 2 (a) and the statistical graph of fluorescence changes over time (b);

[0047] Figure 5 Flow cytometric fluorescence images of extracellular vesicle uptake by cells with paired and unpaired DNA in Example 2;

[0048] Figure 6 This is a laser confocal fluorescence analysis of the localization of extracellular vesicles and endocytosis in Example 2;

[0049] Figure 7 This is a flow cytometry fluorescence graph of extracellular vesicle uptake of DNA encoding different base lengths in Example 2;

[0050] Figure 8 This is a graph showing the uptake analysis of extracellular vesicles from mouse STO cells, watermelon, and E. coli encoded by the DNA in Example 3 in MDA-MB-231 cells;

[0051] Fig. 9 This is a graph showing the selective uptake analysis of DNA-encoded HeLa cells and watermelon-derived extracellular vesicles in HeLa cells in Example 4. DETAILED DESCRIPTION

[0052] The present invention is further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0053] Example 1 Preparation of DNA-encoded artificial receptors and ligands

[0054] In this Example 1, the preparation of DNA-encoded artificial receptors and ligands comprises the following steps:

[0055] (1) Dissolve magnesium chloride hexahydrate (MgCl2·6H2O, 10.16 mg) in phosphate buffered saline (PBS, 10 mL).

[0056] (2) Four DNA tetrahedral short chains (40 μM, 2.5 μL) were dissolved in the solution (1) (90 μL) and mixed in a PCR tube. The sequences are shown in Table 1.

[0057] (3) Place the PCR tube in (2) in a PCR instrument, heat at 95°C for 10 minutes, and quickly cool to 4°C to allow the four DNA single strands to assemble into a DNA tetrahedron structure. Store at 4°C before use.

[0058] (4) Ligand cholesterol-modified DNA short chain A'-chol (L1) (100 μM) was stored at 4°C before use.

[0059] (5) 6% polyacrylamide gel was used in 1×TAE-Mg 2+ DNA tetrahedron was run in a buffer solution (40mM Tris, 2mM EDTA-NH2, 12.5mM MgAc·4H2O, and 20mM HAc, pH 8.0) with a running voltage of 100V and a running time of 1h. The gel was stained with Gel red. The synthesis effect of DNA tetrahedron was imaged and analyzed by gel imaging.

[0060] In this embodiment, the method of modifying cells using the artificial receptor encoded by the above DNA comprises the following steps:

[0061] (1) MDA-MB-231 cells were seeded in a glass-bottomed cell culture dish, the culture medium was removed, and 2 mL of PBS was added to wash once.

[0062] (2) Add 100 μL of DNA tetrahedron solution (1 μM) to 300 μL of serum-free culture medium.

[0063] (3) Add the solution in (2) to the cells in (1) and incubate at room temperature for 10 minutes.

[0064] (4) Wash the cells in (3) three times with 2 mL PBS and finally add serum-free culture medium. Figure 1 As shown, cholesterol-modified DNA (Chol-DNA) is inserted into the phospholipid bilayer of extracellular vesicles as a ligand by oscillating with the extracellular vesicles; three cholesterol-modified DNA single strands and a DNA containing a complementary pairing sequence with the ligand are mixed and annealed to form a receptor with a DNA tetrahedral structure. The DNA receptor is inserted into the cell membrane through the cholesterol at the three vertices and recognizes the extracellular vesicles functionalized with the DNA ligand.

[0065] The DNA sequences used in this Example 1 are shown in Table 1 below.

[0066] Table 1. DNA sequences

[0067]

[0068] like Figure 2 As shown, extracellular vesicles derived from MDA-MB-231 cells were obtained by ultracentrifugation, and the extracellular vesicles were negatively stained with uranyl acetate. TEM results (a) showed that the extracellular vesicles extracted from MDA-MB-231 cells showed a typical spherical cup-shaped structure; nanoparticle tracking analysis (b) showed that the diameter of the extracellular vesicles was mainly 50-150nm; dynamic light scattering results (c) showed that the hydrated particle size of the extracellular vesicles increased after DNA modification; the ligand Chol-DNA was modified with the FAM fluorescent group, and the microplate reader detected (d) that the extracellular vesicles had an obvious increase in fluorescence signal. After DNaseⅠ digestion, the fluorescence signal disappeared, proving that the ligand Chol-DNA was successfully inserted into the extracellular vesicle membrane.

[0069] like Figure 3 As shown, polyacrylamide gel electrophoresis (a) showed that the DNA tetrahedral structure receptor was successfully assembled and could bind complementarily to the ligand DNA; laser confocal microscopy results (b, c) showed that the DNA tetrahedral receptor with cholesterol modified at three vertices could be successfully inserted into the cell membrane, and 55% of the fluorescence signal was still on the cell membrane after 2 hours.

[0070] Example 2 DNA-encoded receptor ligands program MDA-MB-231 cell "extracellular vesicle-cell" interactions

[0071] In this Example 2, the method of programming the "extracellular vesicle-cell" interaction of MDA-MB-231 cells with a DNA-encoded receptor ligand comprises the following steps:

[0072] (1) MDA-MB-231 cells were cultured in 10 T75 cell culture flasks. When the cells grew to 80%-90% of the area of ​​the culture dish, they were washed three times with PBS and cultured in serum-free medium for 24 hours. The supernatant was collected, and the extracellular vesicles were separated from the culture medium by ultracentrifugation and resuspended in 500 μL PBS.

[0073] (2) Extracellular vesicle staining: Take 100 μL of extracellular vesicles in step (1), add 1 uL of Deep-red extracellular vesicle staining reagent, and mix well. Incubate at 37°C in the dark for 30 minutes. Transfer all the solution to a filter tube and centrifuge at 3000g for 5 minutes at room temperature. Add 100 μL of PBS to the filter tube and centrifuge at 3000g for 5 minutes. Repeat once. Centrifuge at 3000g for 5 minutes at room temperature to remove the remaining solution. Pipette 60 μL of PBS into the tube and gently pipette to resuspend the extracellular vesicles.

[0074] (3) DNA modification of extracellular vesicles: Take 60 μL of the solution in step (2) and divide it into two equal parts, each of which is 30 μL. In one group (DNA modification group), 10 μL of ligand DNA-chol short chain (40 μM) is added to 30 μL, placed on a vortex oscillator, and shaken at 600 rpm / min for 5 minutes. After the reaction, the solution is added to the ultrafiltration tube sleeve (100k MWCO), and 350 μL of PBS solution is added. Centrifuge at 5000g for 5 minutes. Remove the bottom solution and repeat the washing twice. Repeat the above operation for the other group (control group). Finally, each group obtains about 20 μL of the same residual solution, which is placed at 4°C for use. In this way, DNA ligand-modified extracellular vesicles are obtained.

[0075] (4) Cell modification with DNA tetrahedron: MDA-MB-231 cells were seeded in two glass-bottomed cell culture dishes, the culture medium was removed, and 2 mL of PBS was added to wash once. For one group of cells, 100 μL of DNA tetrahedron solution (1 μM) was added to 300 μL of serum-free culture medium and incubated at room temperature for 10 minutes. The cells were washed three times with 2 mL of PBS, and finally serum-free culture medium was added to obtain cells modified with DNA tetrahedron receptors.

[0076] (5) DNA receptor ligand-encoding extracellular vesicles interact with cells: The two groups of extracellular vesicles in step (3) were added to the two groups of cells in (4), respectively, and incubated at 37°C for 2 h in serum-free medium. The medium was removed, and 1 mL of medium containing 5 μL PlasMem Bright Green cell membrane dye and 10 μL Hoechst 33342 (100×) cell nuclear dye was added, and incubated at 37°C for 20 min. The medium containing the dye was removed, and the cells were washed twice with PBS. The cell culture medium was added again.

[0077] (6) Observe DNA receptor ligand-mediated extracellular vesicle uptake using laser confocal microscopy or flow cytometry.

[0078] The DNA sequences used in this Example 2 are shown in Table 2 below.

[0079] Table 2. DNA sequences

[0080]

[0081]

[0082] Table 3. DNA sequences with different pair lengths

[0083]

[0084]

[0085] like Figure 4 As shown, the laser confocal microscopy results (a, b) showed that after 2 hours of co-incubation, the extracellular vesicle uptake in the DNA encoding group was significantly increased compared with the extracellular vesicle uptake in the natural state, and the fluorescence intensity increased by about 6.5 times.

[0086] like Figure 5 As shown, the results of fluorescence intensity measurement by flow cytometry showed that cells can efficiently take up extracellular vesicles only when the DNA receptor ligand bases are complementary paired. When the receptor ligand bases are not paired, efficient extracellular vesicle uptake cannot be achieved.

[0087] like Figure 6 As shown, the cells used ECGreen dye to trace the cell endocytosis process, and laser confocal microscopy imaging showed that after 2 hours of co-incubation, under the natural state (control group), extracellular vesicles were mainly co-localized with endocytic vesicles, and extracellular vesicles encoded by DNA receptor ligands were distributed on the cell membrane, cytoplasm and endocytic vesicles.

[0088] like Figure 7 As shown, by changing the length of the complementary DNA pairing chain (see Table 3 for DNA sequences of different lengths), the cell's ability to uptake extracellular vesicles can be adjusted, and the extracellular vesicle uptake ability increases with the increase in the number of complementary DNA pairing bases.

[0089] Example 3 DNA-encoded receptor ligands program extracellular vesicles from different sources to interact with cells

[0090] In this Example 3, the DNA-encoded receptor ligand programming of extracellular vesicles from different sources to interact with cells includes the following steps:

[0091] (1) Extraction of extracellular vesicles from mouse STO cells: As shown in the MDA-MB-231 cell method in Example 2, extracellular vesicles from mouse STO cells were obtained.

[0092] (2) Extraction of extracellular vesicles from watermelon: A piece of watermelon weighing about 500 g was squeezed by physical squeezing, and 50 mL of watermelon juice was taken. It was diluted with PBS at a ratio of 1:2, and the extracellular vesicles were separated by differential ultracentrifugation. The specific steps are as follows: the solution was first centrifuged at 1000 g for 10 minutes, the supernatant was taken, and then centrifuged at 3000 g for 20 minutes, repeated twice, and the supernatant was taken. The supernatant was centrifuged at 10000 g for 30 minutes, the supernatant was taken, repeated twice, and filtered with a 0.22 μm filter. Using a Beckman ultracentrifuge (Optima x-90) and a 50.2Ti rotor, centrifuged at 100000 g for 90 minutes at 4°C, the supernatant was carefully discarded, and the extracellular vesicles were resuspended in PBS to collect.

[0093] (3) Extraction of extracellular vesicles from E. coli: 50 mL of E. coli LB (Luria-Bertani) liquid culture medium was centrifuged at 10,000 g for 30 minutes, and the supernatant was taken. Repeat twice. Filter twice through a 0.22 μm filter to remove cells and cell debris in the solution. Extracellular vesicles from E. coli were collected by ultracentrifugation at 100,000 g for 120 minutes at 4°C. Finally, the vesicles were resuspended in 500 μL PBS.

[0094] (4) The extracellular vesicles from different sources separated in steps (1-3) were labeled and stained as described in Example 2, and modified with DNA ligands (A'-chol, L1). They were co-incubated with MDA-MB-231 cells modified with DNA receptors (R1). The DNA sequences are detailed in Table 4 below.

[0095] (5) Use laser confocal microscopy or flow cytometry to observe the uptake of extracellular vesicles from different sources mediated by DNA receptor ligands.

[0096] like Figure 8 As shown in the figure, in addition to extracellular vesicles from MDA-MB-231, we also extracted extracellular vesicles from mouse STO cells, watermelon, and E. coli to study the versatility of DNA-encoded receptor ligands. TEM images show that extracellular vesicles from mouse STO cells, watermelon, and E. coli all present cup-shaped and round structures. MDA-MB-231 cells and extracellular vesicles from different sources were modified with DNA receptor ligands and co-incubated for 2 hours. The results of laser confocal fluorescence images and flow cytometric analysis showed that DNA-encoded receptor ligands can significantly promote the uptake of extracellular vesicles from different sources by MDA-MB-231 cells.

[0097] Example 4 DNA-encoded receptor ligands program the selective uptake of HeLa cells and watermelon-derived extracellular vesicles in HeLa cells

[0098] In this Example 4, the selective uptake of HeLa cells and watermelon-derived extracellular vesicles by programming the receptor ligand encoded by DNA in HeLa cells includes the following steps:

[0099] (1) Extracellular vesicles from HeLa cells and watermelon were isolated and obtained according to the method in Example 3, and labeled and stained. Different sequence DNAs were modified to serve as different ligands A'-chol (L1) or B'-chol (L2). The specific DNA sequences are shown in Table 4.

[0100] (2) According to the method in Example 2, HeLa cells are simultaneously modified with tetrahedrons carrying different DNA receptors R1 and R2.

[0101] (3) Based on Fig. 9 The experiment was designed to co-incubate the HeLa cells modified or not with the DNA ligand in step (1) and the extracellular vesicles derived from watermelon with the HeLa cells modified with the DNA receptor in step (2) in serum-free medium.

[0102] (4) Laser confocal microscopy was used to photograph the selective uptake of DNA-encoded receptor ligands programmed by HeLa cells and watermelon-derived extracellular vesicles in HeLa cells.

[0103] Table 4. DNA sequences

[0104]

[0105] Thanks to DNA-specific base pairing and rich programmable sequences, we can construct multiple sets of specific recognition units by changing the base sequences of DNA tetrahedron-extended DNA strands on cells and DNA strands on extracellular vesicles. We extracted extracellular vesicles from HeLa cells and watermelon, and used them to study the selective uptake of extracellular vesicles from different sources by HeLa cells by changing the DNA recognition sequences of receptor ligands.

[0106] like Fig. 9As shown, HeLa cells and watermelon-derived extracellular vesicles with different fluorescent labels were modified with different DNA ligands L1 or L2, and HeLa cells were modified with the corresponding receptors R1 and R2. After 2 hours of co-incubation, the laser confocal fluorescence images showed that in the mixed system, cells could efficiently take up extracellular vesicles only when the base sequences of the receptor ligands encoded by DNA met the complementary pairing. In the control group, HeLa cells and watermelon-derived extracellular vesicles were not modified, and the HeLa cells took up very little. When HeLa cell-derived extracellular vesicles were modified with DNA ligand L1, HeLa cells could efficiently take up HeLa vesicles. When watermelon-derived extracellular vesicles were modified with DNA ligand L2, HeLa cells could efficiently take up watermelon-derived vesicles. When HeLa cells and watermelon-derived extracellular vesicles were modified with DNA ligands L1 and L2, respectively, HeLa cells could efficiently take up HeLa and watermelon-derived vesicles in the system.

[0107] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for programming the interaction between extracellular vesicles and cells based on DNA-encoded receptor ligands, not for the diagnosis and treatment of diseases, characterized in that: The method comprises: inserting a cholesterol-modified DNA single strand as a ligand into the phospholipid bilayer of the extracellular vesicle by oscillating with the extracellular vesicle to obtain an extracellular vesicle modified with a DNA-encoded artificial ligand; mixing three cholesterol-modified DNA single strands and a DNA single strand containing a complementary pairing sequence with the ligand, annealing, and forming a receptor with a DNA tetrahedral structure; using the receptor-modified cells to obtain cells modified with a DNA-encoded artificial receptor; adding the extracellular vesicle modified with the DNA-encoded artificial ligand into the cells modified with the DNA-encoded artificial receptor, wherein the receptor is inserted into the cell membrane through the cholesterol at three vertices to recognize the extracellular vesicle functionalized with the DNA ligand, thereby realizing efficient and specific uptake of extracellular vesicles from different sources by the cell.

2. The method according to claim 1, characterized in that The method comprises the following steps: S1: Prepare DNA-encoded artificial receptors and ligands respectively; S2: cells modified with the artificial receptor encoded by the DNA prepared in step S1; S3: isolating and extracting extracellular vesicles, and modifying the extracellular vesicles with the artificial ligand encoded by the DNA prepared in step S1; S4: Add extracellular vesicles modified with DNA-encoded artificial ligands to cells modified with DNA-encoded artificial receptors, incubate at 37°C for 2h~3h, and observe DNA receptor ligand-mediated extracellular vesicle uptake using laser confocal microscopy or flow cytometry.

3. The method according to claim 2, characterized in that Step S1 includes the following sub-steps: (1) Dissolve magnesium chloride hexahydrate in phosphate buffer solution to make the final concentration of magnesium ions 1-5 mM; (2) Dissolve three cholesterol-modified DNA single strands and one DNA single strand containing a sequence complementary to the ligand in the solution prepared in step (1), and mix them in a PCR tube. The final concentration of each of the four DNA single strands is in the range of 0.5-2 μM. (3) Place the PCR tube in step (2) in a PCR instrument, heat at 95°C for 10 minutes, and quickly cool to 4°C to obtain a DNA tetrahedral structure, i.e., the DNA-encoded artificial receptor, which is stored at 4°C for future use; (4) Dissolve a cholesterol-modified DNA single strand in the solution prepared in step (1) to a concentration of 80-120 μM to prepare a DNA-encoded artificial ligand, which is then stored at 4°C for future use.

4. The method according to claim 2 or 3, characterized in that: Step S2 includes the following sub-steps: (5) Seed the cells in a glass-bottomed cell culture dish, remove the culture medium, and add PBS to wash; (6) adding the DNA-encoded artificial receptor to serum-free culture medium; (7) adding the solution containing the DNA-encoded artificial receptor in step (6) to the cells in step (5), and incubating at room temperature for 8 to 12 minutes to obtain cells modified with the DNA-encoded artificial receptor; (8) Wash the cells in step (7) with PBS and add serum-free culture medium.

5. The method according to claim 2, characterized in that: Step S3 includes the following sub-steps: (9) Inoculate the cells from which extracellular vesicles are to be extracted into several T75 cell culture flasks. When the cell growth area reaches 80%, wash them three times with 10 mL PBS. Finally, add 10 mL of serum-free medium to each flask. After culturing for 24 h, obtain 100 mL of cell culture supernatant; centrifuge at 300 g for 5-10 minutes to remove dead cells and take the supernatant; centrifuge at 3000 g for 10-20 minutes to remove cell debris and take the supernatant; centrifuge at 10000 g for 20-30 minutes to remove organelles and large cell vesicles and take the supernatant; filter with a 0.22 μm filter and take the lower layer of solution; centrifuge at 100000 g for 80-90 minutes, gently remove the upper layer of liquid, resuspend the bottom with 500 μL PBS, which is the extracellular vesicles, and store at -80 °C for later use.

6. The method according to claim 5, characterized in that Step S3 also includes the following sub-steps: (10) Take 100 μL of extracellular vesicles for rapid dissolution, add 1 μL of Deep-red extracellular vesicle staining reagent, and mix well; incubate at 37 °C in the dark for 30 to 40 minutes; transfer all the solution to a filter tube and centrifuge at 3000 g for 3 to 5 minutes at room temperature; add 100 μL of PBS to the filter tube and centrifuge at 3000 g for 3 to 5 minutes, repeat once; centrifuge at 3000 g for 3 to 5 minutes at room temperature to remove the remaining solution; aspirate 50 μL of PBS into the tube and gently blow to resuspend the extracellular vesicles; (11) Take 50 μL of the extracellular vesicle solution in step (10) and add 5.6 μL of cholesterol-modified DNA single-stranded ligand with a concentration of 80-120 μM; (12) Place the solution in step (11) on a vortex shaker and shake at 600 rpm / min for 3 to 5 minutes; (13) Add the solution in step (12) to the ultrafiltration tube sleeve, add 350 μL of PBS solution, centrifuge at 5000 g for 3-5 minutes, and remove the bottom solution; (14) Add another 400 μL of PBS solution to the ultrafiltration tube sleeve, centrifuge at 5000 g for 5 min, and repeat the wash; (15) Pipette 20 μL of the residual solution in the ultrafiltration tube sleeve in step (14) and add it to a 500 μL centrifuge tube to obtain DNA-encoded artificial ligand-modified extracellular vesicles, which are then placed at 4°C for later use.

7. The method according to claim 2, characterized in that Step S4 includes the following sub-steps: (16) Take 20 μL of extracellular vesicles modified with DNA-encoded artificial ligands and add them to the serum-free culture medium of cells modified with DNA-encoded artificial receptors; (17) Place the cells in cell culture medium and incubate at 37 °C for 2–3 h. (18) Remove the cell culture medium in step (17), add 1 mL of culture medium containing 5 μL of PlasMem Bright Green cell membrane dye and 10 μL of 100× Hoechst 33342 cell nuclear dye, and incubate at 37 °C for 20 min; (19) Remove the culture medium containing the dye in step (18), wash twice with PBS, and add cell culture medium again; (20) Detect DNA receptor ligand-mediated extracellular vesicle uptake using confocal laser scanning microscopy or flow cytometry.

8. The method according to claim 1, characterized in that The extracellular vesicles from different sources are selected from: extracellular vesicles from STO cells, watermelon, and Escherichia coli.

9. The method according to claim 1, characterized in that: The cells are selected from human and mouse cells.

10. The method according to claim 9, characterized in that The cells are selected from: MDA-MB-231 cells and HeLa cells.

11. The method according to claim 1, characterized in that: Changing the length of the complementary DNA pairing chain regulates the cell's ability to uptake extracellular vesicles. The extracellular vesicle uptake ability increases with the increase in the number of complementary DNA pairing bases.

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