Preparation method for engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells, and use of same

Abstract

A preparation method for an engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells, and the use of same, belonging to the technical field of biomedicines. The present invention provides an engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells, which is obtained by collecting a supernatant of cells that stably overexpress a protein containing ofCSbps and separating same, the sequence of the ofCSbps being one of or a combined polypeptide sequence of more than one of SEQ ID NO. 1-7, or a derived sequence taking one polypeptide sequence of SEQ ID NO. 1-7 as a backbone, or a derived sequence taking a combined polypeptide sequence of more than one of SEQ ID NO. 1-7 as a backbone. The engineered extracellular vesicle of the present invention has the characteristic of targeting ofCS-positive cells. Since extracellular vesicles have nanoscale liposome encapsulation structures, the present invention is suitable for basic research and clinical diagnosis and treatment application on various diseases, such as placenta-derived physiological / pathological and tumor / cancer diseases, in humans and animals.

Description

Preparation method and application of engineered extracellular vesicles targeting oncoembryonic chondroitin sulfate-positive cells Technical Field

[0001] The present invention belongs to the field of biomedical technology and specifically relates to an engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells, and a preparation method and application thereof. Background Art

[0002] Extracellular vesicles (EVs) are a general term for various membrane-bound vesicles released by cells. Due to their varying diameters and modes of formation, they are categorized as exosomes (30–150 nm in diameter), microvesicles (100–1000 nm in diameter), apoptotic bodies (100–5000 nm in diameter), and oncosomes (1–10 μm in diameter). EVs possess a nanoscale lipid-encapsulated structure, encapsulating a variety of proteins, nucleic acids, and metabolites, regulating the biological functions of target cells and enabling intercellular communication.

[0003] Extracellular vesicles (EVs) possess numerous properties, including barrier penetration, excellent biocompatibility, and low immunogenicity, making them ideal drug delivery vehicles. Engineered EVs secreted by engineered cells offer the added advantage of tissue and organ targeting.

[0004] Oncofetal chondroitin sulfate (ofCS) was reported in Cancer Cell in 2015 by Ali Salanti's team at the University of Copenhagen, Denmark. They discovered that a common class of glycosaminoglycan molecules exists in most human cancer cells and placental trophoblasts. Furthermore, studies on malaria have revealed that the Plasmodium-derived protein VAR2CSA binds to ofCS, and that VAR2CSA contains several shortest ofCS-binding peptides (ofCS-BPs). Recombinant VAR2CSA protein or synthetic ofCS-BPs can be used as probes for the diagnosis and treatment of placental diseases and cancers.

[0005] The invention patents with Chinese authorization numbers CN109589416B, CN109568289B and CN109589413B disclose the preparation method and application of nanoparticle carriers targeting ofCS based on ofCS-BPs. However, the assembly steps of artificially synthesized ofCS-BPs and nanoparticles are cumbersome and costly, and the obtained nanoparticle carriers themselves have certain toxicity. To overcome and optimize these defects, we constructed a method for preparing ofCS-targeted engineered extracellular vesicles, overexpressed ofCSbps-Lamp2b-eGFP fusion protein in HEK293F cells through genetic engineering methods and collected the extracellular vesicles secreted therefrom. The engineered extracellular vesicles were successfully targeted and enriched in placental trophoblast cells.

[0006] Summary of the Invention

[0007] In view of the defects in the above-mentioned prior art, the purpose of the present invention is to design and provide a method for preparing engineered extracellular vesicles targeting ofCS-positive cells. The present invention utilizes a lentiviral vector system to stably overexpress ofCSbps-Lamp2b-eGFP in HEK293F cells, collects the cell supernatant, and obtains extracellular vesicles by ultracentrifugation. The engineered extracellular vesicles have the characteristics of targeting ofCS-positive cells (such as placental trophoblast cells, tumor / cancer cells, etc.). Because the extracellular vesicles have a nanoscale lipid inclusion structure, diagnostic and therapeutic related molecules that have been clinically applied or are being studied in preclinical studies can be delivered to the target site. The present invention is suitable for basic research and clinical diagnosis and treatment applications of various diseases such as placental physiology / pathology and tumors / cancer in humans and animals.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] In one aspect, the present invention provides an engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells, wherein the engineered extracellular vesicle is obtained by collecting and isolating the supernatant of cells stably overexpressing a fusion protein plasmid vector containing ofCSbps;

[0010] The sequence of the ofCSbps is selected from one or more of the combined polypeptide sequences of SEQ ID NOs. 1-7, or a derivative sequence using one or more of the combined polypeptide sequences of SEQ ID NOs. 1-7 as a backbone. Sequences SEQ ID NOs. 1-7 are all shortest binding polypeptides that contain several ofCS segments in VAR2CSA.

[0011] The engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells, wherein the fusion protein plasmid vector containing ofCSbps contains a protein for expression and localization on the surface of the extracellular vesicles;

[0012] Preferably, the protein used for extracellular vesicle surface expression and localization is selected from one of Lamp2b, CD9, CD63, CD47, CD81, APMAP, TSPAN14, TSG101, Alix, Flotillin-1, Syntenin-1, and HSP70.

[0013] The engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells, the fusion protein plasmid vector containing ofCSbps contains proteins for cell transfection efficiency detection, screening, purification and tracing;

[0014] Preferably, the protein used for cell transfection efficiency detection, screening, purification and tracing is selected from one of eGFP, GFP, eYFP, mRFP1, mCherry, Luciferase, 6*His, Flag, GST and c-Myc.

[0015] The engineered extracellular vesicles targeting oncofetal chondroitin sulfate-positive cells are selected from the group consisting of cell lines, primary cells, organoid cells, single cells, and living animal and plant cells;

[0016] Preferably, the cell line is HEK293F cells.

[0017] The engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells are transfected by a lentiviral infection method, a liposome transfection method, a calcium phosphate co-precipitation method or an electroporation method.

[0018] The engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells have a size of 30-200 nm, a shape of a saucer or a hemispherical shape with one side concave, and the surface of the extracellular vesicles contains ofCSbps or a peptidomimetic of ofCSbps.

[0019] In a second aspect, the present invention provides a method for preparing engineered extracellular vesicles targeting oncofetal chondroitin sulfate-positive cells as described in any one of the preceding claims, comprising the following steps:

[0020] S1. Construction of a fusion protein plasmid vector containing ofCSbps;

[0021] S2. Infecting the plasmid vector obtained in step S1 into cells via lentivirus, and screening to obtain protein-positive cell monoclonal cell lines for cell transfection efficiency detection, screening, purification, and tracing;

[0022] S3. Collect the cell supernatant obtained in step S2 and obtain engineered extracellular vesicles by separation.

[0023] The preparation method is characterized in that the screening method is one of flow cytometry sorting or limiting dilution method; the separation method is one of ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, tangential flow filtration or immunoaffinity capture.

[0024] Use of any one of the engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells in the preparation of drugs or preparations for researching, diagnosing, treating or preventing tumors, placental-derived physiological or pathological diseases, or uterine-derived physiological or pathological diseases.

[0025] The application described above, wherein the engineered extracellular vesicles are used as delivery vehicles to load components for studying, detecting, preventing or treating tumors, placental-derived physiological or pathological diseases, or uterine-derived physiological or pathological diseases;

[0026] Preferably, the component is selected from one of polypeptides, proteins, lipids, carbohydrates, RNA, DNA or small molecule compounds;

[0027] Preferably, the placental physiological or pathological disease is one of early miscarriage, recurrent miscarriage, ectopic pregnancy, placental abruption, intrauterine growth restriction, gestational hypertension, preeclampsia, premature birth, gestational diabetes, placenta accreta disease, and gestational trophoblastic disease;

[0028] Preferably, the uterine-derived physiological or pathological disease is one of endometriosis, endometrial adenomyosis, intrauterine adhesions, uterine fibroids, and uterine fibroids;

[0029] Preferably, the loading method is one of genetic engineering, chemical modification, electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion, and surfactant.

[0030] The specific construction process of the fusion protein plasmid vector containing ofCSbps in step S1 is as follows:

[0031] (1) Primer design and PCR cloning of target fragments;

[0032] (2) Gel excision and recovery of target fragments;

[0033] (3) GoldenGate reaction to construct intermediate vector and sequencing identification;

[0034] (4) LR reaction, transformation and identification of final vector-positive clones.

[0035] When ultracentrifugation is used to separate and obtain extracellular vesicles in step S3, the specific operation process is as follows:

[0036] (1) Collect the cell supernatant and centrifuge at 300 g for 10 min at 4°C to remove the cells;

[0037] (2) Centrifugation at 2000 g for 20 min at 4°C to remove dead cells;

[0038] (3) Centrifugation at 10,000 g for 30 min at 4°C to remove cell debris;

[0039] (4) Filter with a 0.22 μm filter to remove impurities and bacteria;

[0040] (5) Centrifuge at 120,000 g for 120 min, discard the supernatant, and wash once with PBS;

[0041] (6) Centrifuge at 120,000 g for 60 min, discard the supernatant, and precipitate the extracellular vesicles. Resuspend in PBS and quantify with BCA. Aliquot at 50 μg / tube and store in a -80°C refrigerator.

[0042] Compared with the prior art, the method of the present invention has the following beneficial effects:

[0043] 1. The steps of the present invention are simple and the preparation cost is relatively low.

[0044] 2. Compared with drug-targeted nanoparticle carriers obtained by chemical synthesis and assembly, the extracellular vesicles prepared by the scheme provided by the present invention have the advantages of low toxicity, low immunogenicity and strong biocompatibility.

[0045] 3. The engineered extracellular vesicles provided by the present invention are of great significance for basic research on placental function and tumors / cancer, as well as for the clinical diagnosis and treatment of placental diseases and tumors / cancer. On the one hand, drugs can be loaded into these engineered extracellular vesicles to intervene in the placenta in normal physiological conditions and pathological conditions, achieving purposes such as contraception and palliative treatment, respectively. They can also be delivered to tumor / cancer tissue lesions for therapeutic purposes. On the other hand, contrast agents can be loaded into these engineered extracellular vesicles to enable imaging observation of the entire placenta and tumors / cancer, achieving diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 is a plasmid map and a schematic diagram of fusion protein design;

[0047] Figure 2 is a schematic diagram of the construction process and the monoclonal cell line after eGFP sorting;

[0048] Figure 3 shows the morphology of extracellular vesicles detected by scanning electron microscopy;

[0049] FIG4 shows the particle size of extracellular vesicles detected by NTA;

[0050] Figure 5 shows the expression of extracellular vesicle molecular markers detected by Western Blot;

[0051] FIG6 is a diagram showing the distribution of extracellular vesicles in mouse uterine tissue detected by a small animal imaging system;

[0052] FIG7 is a diagram showing the distribution of extracellular vesicles in various organs and tissues of mice detected by a small animal imaging system;

[0053] FIG8 is a diagram showing the distribution of extracellular vesicles in mouse fetus and placenta detected by a small animal imaging instrument;

[0054] FIG9 is an immunofluorescence analysis of the enrichment distribution of extracellular vesicles and CK7 in the mouse placenta trophoblast. DETAILED DESCRIPTION

[0055] The present invention will be further explained below with reference to the accompanying drawings and examples. However, it should be noted that the following examples are only used to explain the present invention and are not used to limit the present invention. All technical solutions similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in the present examples, the operation shall be carried out in accordance with the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments is not specified, they are all conventional reagents and consumables that can be obtained commercially.

[0056] Example 1: Plasmid vector construction

[0057] 1. Primer design and PCR cloning of target fragments. The specific steps are as follows:

[0058] The primer sequences used are as follows:

[0059] F1 (SEQ ID NO. 8):

[0060] R1 (SEQ ID NO.9):

[0061] F2 (SEQ ID NO. 10):

[0062] R2 (SEQ ID NO.11):

[0063] F3 (SEQ ID NO. 12):

[0064] R3 (SEQ ID NO. 13):

[0065] Note: Lowercase letters represent protected bases, bold italics represent the recognition sites of AarⅠ, and underlined letters represent the sticky ends formed after enzyme digestion.

[0066] Use the above three pairs of primers to amplify three DNA fragments respectively according to the systems and conditions in Tables 1 and 2 below.

[0067] Table 1 Amplification system

[0068] Table 2 Amplification conditions

[0069] 2. PCR product electrophoresis detection and target fragment gel excision recovery, the specific steps are as follows:

[0070] A. After the PCR reaction system is completed, add 10 μL of 6× loading buffer to the reaction system to terminate the reaction;

[0071] B. The reaction products were subjected to agarose gel electrophoresis;

[0072] C. Recover the target fragment by gel excision.

[0073] 3. GoldenGate reaction to construct the intermediate vector. The specific steps are as follows:

[0074] A. Extraction of pDown-Aar Ⅰ-ccdB-Cm-Aar Ⅰ backbone plasmid;

[0075] B. The backbone plasmid and the PCR product obtained in (2) were subjected to a GoldenGate reaction according to the system and parameters shown in Table 3 and the GoldenGate reaction procedure shown in Table 4.

[0076] Table 3 GoldenGate reaction system and parameters

[0077] Table 4 GoldenGate reaction program

[0078] C. After the GoldenGate reaction is complete, use a portion of the reaction product to transform UltraStable competent cells. Randomly select 3-6 individual colonies and rinse them in a sterile 0.2mL EP tube. Use 1μL as a template for colony PCR, and the remaining volume as the inoculum for plasmid DNA extraction. The colony PCR reaction system is shown in Table 5 below, and the colony PCR reaction procedure is shown in Table 6 below.

[0079] Table 5 PCR reaction system of colonies

[0080] Table 6 Colony PCR reaction procedure

[0081] D. After colony PCR is complete, add 6× loading buffer and perform electrophoresis. Use DNA Ladder to select clones that can amplify the target length. Inoculate into LB medium and culture overnight at 37°C and 250 rpm. Then extract small plasmid DNA and send it for sequencing. Use Sequencher software to compare the returned sequencing results with the standard sequence. The entry vector with the correct sequence can proceed to the next LR reaction to construct the final vector.

[0082] 4. LR reaction, transformation and identification of final vector positive clones. The specific steps are as follows:

[0083] (1) Perform LR reaction. The LR reaction system is shown in Table 7 below.

[0084] Table 7 LR reaction system

[0085] The LR reaction conditions were as follows: incubation at 25°C for 3 h. After the reaction was complete, 1 μL of proteinase K was added and incubated at 37°C for 15 min to terminate the reaction.

[0086] (2) The LR reaction product is transformed into competent cells.

[0087] Take 2 μL of LR reaction product to transform competent cells. Refer to the transformation steps of GoldenGate reaction product. Finally, spread the revived bacterial liquid on LB plate containing Amp antibiotic and culture it upside down at 37℃ overnight.

[0088] (3) Colony PCR

[0089] Randomly pick 3-6 single colonies and perform the colony PCR operation according to the previous identification of intermediate vector single clones. Finally, perform agarose gel electrophoresis and pick colonies that can amplify DNA bands of the target band length according to the DNA ladder. Inoculate and culture bacteria and extract plasmid DNA.

[0090] (4) Enzyme digestion identification

[0091] Use SnapGene software to open the vector map and determine the enzyme digestion scheme. Then, use NEB restriction enzymes to digest the plasmid DNA of the final vector. After the digestion reaction, perform agarose gel electrophoresis and analyze with reference to the DNA ladder. The vector that only cuts DNA bands of a specific length is the correct final vector. Figure 1 shows a plasmid map and fusion protein design schematic. The amino acid sequence of ofCSbps in Example 1 of the present invention is SEQ ID NO. 1.

[0092] Example 2: Lentivirus packaging and concentration

[0093] 1. One day before transfection, transfer HEK293T cells into a 10 cm culture dish so that the cells can reach about 60-70% confluency the next day.

[0094] 2. Take two clean sterile centrifuge tubes and add 750 μL of DMEM culture medium without antibiotics and serum to each tube. Then add 15 μg of mixed plasmid (psPAX2: pMD2.G: target plasmid = 3:1:4) to one tube and mix gently with a pipette. Add 30 μL of Lipo293 to the other tube. TM Add the transfection reagent to the culture medium containing DNA and mix it with the pipette. TM In the culture medium of transfection reagent, gently invert the centrifuge tube or gently blow with a gun to mix, and let it stand at room temperature for 15 minutes.

[0095] 3. Add the plasmid-culture medium mixed suspension obtained in step 2 evenly to the entire culture dish, and then gently mix.

[0096] 4. After 6 hours of culture, remove the old culture medium and add complete culture medium (without double antibody) to continue culture. After 48 hours of culture, collect the viral supernatant and add 2-3 mL of complete culture medium.

[0097] 6. After 72 h, collect the viral supernatant and centrifuge at 500 g for 10 min.

[0098] 7. Transfer the clear supernatant to a sterile container and mix 1 volume of Lenti-X concentrate with 3 volumes of clear supernatant by gentle inversion.

[0099] 8. Place the mixture at 4°C overnight and centrifuge it at 1500g for 45 minutes at 4°C the next day. After centrifugation, gray-white particles will be visible.

[0100] 9. Carefully and gently remove the supernatant and gently resuspend the pellet to 1 / 10 to 1 / 100 of the original volume in complete culture medium.

[0101] 10. Titrate the sample or store the sample in aliquots at -80°C.

[0102] Example 3: Lentivirus infection and eGFP-positive cell sorting

[0103] 1. One day before infection, transfer HEK293F cells into a 24-well plate;

[0104] 2. Add 20 μL of virus solution and 2 μg of Polybrene to 1 mL of SMM 293-TII medium. Green fluorescence will be visible after three days.

[0105] 3. Collect polyclonal cells and sort eGFP-positive / PI (propidium iodide)-negative single cells into 96-well cell culture plates using flow cytometry. Three weeks later, eGFP-positive monoclonal cell lines were observed under a microscope, as shown in FIG2 .

[0106] 4. Transfer them into 24-well plates, 12-well plates, and 6-well plates at a time to preserve the seeds and expand the culture.

[0107] Example 4: Extracellular vesicle extraction and identification

[0108] (1) Collect the cell supernatant and centrifuge at 300 g for 10 min at 4°C to remove the cells;

[0109] (2) Centrifugation at 2000 g for 20 min at 4°C to remove dead cells;

[0110] (3) Centrifugation at 10,000 g for 30 min at 4°C to remove cell debris;

[0111] (4) Filter with a 0.22 μm filter to remove impurities and bacteria;

[0112] (5) Ultracentrifuge at 120,000 g for 120 min, discard the supernatant, and wash once with PBS; Ultracentrifuge at 120,000 g for 60 min, discard the supernatant, precipitate extracellular vesicles, resuspend in PBS, quantify with BCA, and aliquot at 50 μg / tube and store in a -80°C refrigerator;

[0113] (6) Extracellular vesicle morphology detection: As shown in Figure 3, under a transmission electron microscope, extracellular vesicles can be seen to have a saucer-like or hemispherical morphology with one side concave;

[0114] (7) Extracellular vesicle size detection: As shown in Figure 4, the analysis results of the nanoparticle tracking analyzer showed that the average size was 160.4 nm;

[0115] (8) Detection of extracellular vesicle molecular markers: Cell and extracellular vesicle proteins were extracted and subjected to Western blot analysis at a sample load of 30 μg / well. As shown in Figure 5, compared with cells, CD81, ALIX, and TSG101 were highly expressed in extracellular vesicles, while Calnexin was negative. In addition, GFP expression was positive in HEK293F-ofCSbps-Lamp2b-eGFP cells and extracellular vesicles, indicating that the engineered cells were successfully constructed and the engineered extracellular vesicles secreted by them could be continuously obtained.

[0116] Example 5: Placenta-targeted validation of engineered extracellular vesicles

[0117] Pregnant C57BL / 6J mice (E12.5-15.5 days) were purchased. The experiment was divided into four groups: no treatment (NT), tail vein injection of DiR dye group (DiR), tail vein injection of HEK293F-Lamp2b-eGFP secreted extracellular vesicles group (L-EVs), and tail vein injection of HEK293F-ofCSbps-Lamp2b-eGFP secreted extracellular vesicles group (ofCSbps-EVs). Before tail vein injection, extracellular vesicles were stained with DiR dye (Invitrogen, D12731) as follows:

[0118] 1. Thaw the aliquoted extracellular vesicles (50 μg / tube) and add DiR dye at a working concentration of 10 μM.

[0119] 2. Wrap in tin foil and incubate at 37°C for 2 hours in the dark;

[0120] 3. Centrifuge at 15000g for 10 min and discard the supernatant;

[0121] 4. Add 100 μL of saline to resuspend the extracellular vesicles.

[0122] Twenty-four hours after tail vein injection of extracellular vesicles (50 μg / mouse), mice were sacrificed and the heart, liver, spleen, lung, kidney, pancreas, brain, and uterus (placenta) were isolated. Placental and fetal tissues were imaged using a small animal in vivo optical imaging system (Caliper Spectrum IVIS) as follows:

[0123] 1. Turn on the Caliper Spectrum IVIS system.

[0124] 2. Click Acquisition and select Auto-save to, and choose to automatically save the address when taking a photo.

[0125] 3. Click Imaging Wizard on the operation panel to set the shooting mode.

[0126] 4. Select Fluorescence and click Next to proceed to the fluorescence imaging setup. In the probes section, select Input EX / EM. You can enter the excitation and emission wavelengths for the probes. The excitation and emission wavelengths for the DiR dye are 754 / 778 nm. Once selected, the software will automatically generate the corresponding spectrum. The blue dashed box indicates the wavelength band to be scanned. Then click Next.

[0127] 5. Adjust the exposure time, bin value and aperture size in the Exposure Parameter column, adjust the lens height in the Fieldview column, and adjust the image focus in the Focus column. Click Next to return to the photo taking page. Click the Acquire Sequence button

[0128] 6. Click the Acquire Sequence button to acquire the image.

[0129] 7. ROI Tools quantitative analysis toolbar was used for image quantitative analysis. The results are shown in Figures 6, 7, and 8. Compared with the untreated group, DiR injection group, and L-EVs injection group, ofCSbps-EVs were significantly enriched in the placental tissue after injection.

[0130] After imaging, the tissues were fixed in 4% paraformaldehyde for subsequent tissue sectioning.

[0131] Example 6: Fluorescence staining of tissue sections

[0132] 1. Frozen sectioning steps: tissue fixation - dehydration in 30% sucrose at 4°C overnight - OCT embedding - cryostat sectioning;

[0133] 2. Immunofluorescence staining steps: air-dry the sections - fix with cold acetone - wash with PBS buffer - block - incubate with primary antibody overnight - wash with PBS buffer - incubate with secondary antibody at room temperature for 2 h - wash with PBS - stain with DAPI - seal the sections - observe and record under a fluorescence microscope. As shown in Figure 9, GFP-positive signals were present only in the placenta of the ofCSbps-Exos injection group, and GFP was co-localized with CK7 (a molecular marker of trophoblast cells).

[0134] Finally, it should be noted that the above embodiments are merely illustrative of the principles and performance of the present invention and are not intended to limit the present invention. Anyone skilled in the art may modify or optimize the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or optimizations accomplished by those skilled in the art without departing from the spirit and technical concepts disclosed herein shall be encompassed by the claims of the present invention.

Claims

1. An engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells, It is characterized in that The engineered extracellular vesicles are obtained by collecting and isolating the supernatant of cells stably overexpressing a fusion protein plasmid vector containing ofCSbps; Among them, the sequence of ofCSbps is selected from one or more polypeptide combination sequences in SEQ ID NO.1-7, or a derivative sequence with one polypeptide sequence in SEQ ID NO.1-7 as the backbone or a derivative sequence with multiple polypeptide combination sequences as the backbone.

2. The engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells according to claim 1, It is characterized in that The fusion protein plasmid vector containing ofCSbps contains a protein for expression and localization on the surface of extracellular vesicles; Preferably, the protein used for extracellular vesicle surface expression and localization is selected from one of Lamp2b, CD9, CD63, CD47, CD81, APMAP, TSPAN14, TSG101, Alix, Flotillin-1, Syntenin-1, and HSP70.

3. The engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells according to claim 1, It is characterized in that The fusion protein plasmid vector containing ofCSbps contains proteins used for cell transfection efficiency detection, screening, purification and tracing; Preferably, the protein used for cell transfection efficiency detection, screening, purification and tracing is selected from one of eGFP, GFP, eYFP, mRFP1, mCherry, Luciferase, 6*His, Flag, GST and c-Myc.

4. The engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells according to claim 1, It is characterized in that The cell is selected from a cell line, a primary cell, an organoid cell, a single cell, or a living cell of an animal or plant; Preferably, the cell line is HEK293F cells.

5. The engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells according to claim 1, It is characterized in that The transfection method is one of lentiviral infection, liposome transfection, calcium phosphate co-precipitation or electroporation.

6. The engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells according to claim 1, It is characterized in that The size of the extracellular vesicle is 30-200 nm, the shape of the extracellular vesicle is saucer-like or hemispherical with one side concave, and the surface of the extracellular vesicle contains ofCSbps or a peptidomimetic of ofCSbps.

7. A method for preparing engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells according to any one of claims 1 to 6, It is characterized in that The following steps are involved: S1. Construct a fusion protein plasmid vector containing ofCSbps; S2, infecting the plasmid vector obtained in step S1 into cells via lentivirus, and screening to obtain protein-positive cell monoclonal cell lines for cell transfection efficiency detection, screening, purification and tracing; S3. Collect the cell supernatant obtained in step S2, and obtain engineered extracellular vesicles by separation.

8. The preparation method according to claim 7, It is characterized in that The screening method is one of flow cytometry or limiting dilution; the separation method is one of ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, tangential flow filtration or immunoaffinity capture.

9. Use of an engineered extracellular vesicle targeting oncofetal chondroitin sulfate-positive cells as claimed in any one of claims 1 to 6 in the preparation of a drug or preparation for the research, diagnosis, treatment or prevention of tumors, placental-derived physiological or pathological diseases, or uterine-derived physiological or pathological diseases.

10. The use according to claim 9, It is characterized in that The engineered extracellular vesicles are used as delivery vehicles to load components for studying, detecting, preventing or treating tumors, placental-derived physiological or pathological diseases, and uterine-derived physiological or pathological diseases; Preferably, the component is selected from one of polypeptides, proteins, lipids, carbohydrates, RNA, DNA or small molecule compounds; Preferably, the placental physiological or pathological disease is one of early miscarriage, recurrent miscarriage, ectopic pregnancy, placental abruption, intrauterine fetal growth restriction, gestational hypertension, preeclampsia, premature birth, gestational diabetes, placenta accreta disease, and gestational trophoblastic disease; Preferably, the uterine-derived physiological or pathological disease is one of endometriosis, endometrial adenomyosis, intrauterine adhesions, uterine myoma, and uterine fibroids; Preferably, the loading method is one of genetic engineering, chemical modification, electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion, and surfactant.

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