Preparation method and application of targeted carcinoembryonic chondroitin sulfate positive cell engineered extracellular vesicle

Abstract

The invention discloses a preparation method and application of a targeted carcinoembryonic chondroitin sulfate positive cell engineered extracellular vesicle, and belongs to the technical field of biomedicine. The invention provides a targeting carcinoembryonic chondroitin sulfate positive cell engineered extracellular vesicle. The targeting carcinoembryonic chondroitin sulfate positive cell engineered extracellular vesicle is obtained by collecting stable overexpression cell supernatant containing ofCSbps protein and separating; the sequence of the ofCSbps is selected from one or more combined polypeptide sequences in SEQ ID NO.1-7, or a derivative sequence taking one polypeptide sequence in SEQ ID NO.1-7 as a skeleton or a derivative sequence taking more combined polypeptide sequences as the skeleton. The engineered extracellular vesicle disclosed by the invention has the characteristic of targeting an ofCS positive cell. As the extracellular vesicles have nanoscale lipid inclusion structures, the extracellular vesicles are suitable for fundamental research and clinical diagnosis and treatment of various diseases such as human and animal placenta-derived physiology / pathology, tumors / cancers and the like.

Description

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-structured vesicles released by cells. Due to their different diameters and occurrence modes, they are divided into exosomes (diameter 30-150nm), microvesicles (diameter 100-1000nm), apoptotic bodies (diameter 100-5000nm), oncosomes (diameter 1-10μm), etc. Extracellular vesicles have a nanoscale lipid inclusion structure, which contains a variety of proteins, nucleic acids and metabolites, regulating the biological functions of target cells to achieve intercellular communication.

[0003] Extracellular vesicles have many characteristics such as barrier penetration, good biocompatibility, and low immunogenicity, making them ideal drug delivery carriers. The engineered extracellular vesicles secreted by cells obtained by engineering have the advantages of tissue and organ targeting.

[0004] Oncofetal chondroitin sulfate ofCS was reported by Ali Salanti's research group at the University of Copenhagen in Denmark in 2015 in the journal Cancer Cell. It was found that a common class of glycosaminoglycan molecules exists in most human cancer cells and placental trophoblast cells. In addition, through the study of malaria, it was known that the Plasmodium-derived protein VAR2CSA binds to ofCS, and there are several shortest binding peptides (ofCS-Binding Peptides, ofCSbps) ofCS in VAR2CSA. Recombinant VAR2CSA protein or synthetic ofCS-BPs can be used as probes to achieve the diagnosis and treatment of placental diseases and cancer.

[0005] 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. In order 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 by genetic engineering and collected the secreted extracellular vesicles. The engineered extracellular vesicles were successfully targeted and enriched in placental trophoblast cells. Summary of the invention

[0006] 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, the diagnosis and treatment 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 human and animal placental physiology / pathology and tumors / cancer.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] On the one hand, the present invention provides an engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells, wherein the engineered extracellular vesicle is obtained by collecting and separating the supernatant of cells stably overexpressing a fusion protein plasmid vector containing ofCSbps;

[0009] The sequence of ofCSbps is selected from one or more of the combined polypeptide sequences in SEQ ID NO.1-7, or a derived sequence with one of the polypeptide sequences in SEQ ID NO.1-7 as the backbone or a derived sequence with multiple combined polypeptide sequences as the backbone. The sequences SEQ ID NO.1-7 are all the shortest binding polypeptides with several segments of ofCS in VAR2CSA.

[0010] The engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells, the fusion protein plasmid vector containing ofCSbps contains a protein for expression and localization on the surface of extracellular vesicles;

[0011] 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.

[0012] 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;

[0013] 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.

[0014] The engineered extracellular vesicles targeting oncofetal chondroitin sulfate-positive cells, wherein the cells are selected from one of cell lines, primary cells, organoid cells, single cells, and living cells of animals and plants;

[0015] Preferably, the cell line is HEK293F cells.

[0016] The engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells are transfected by a lentiviral infection method, a liposome transfection method, a calcium phosphate coprecipitation method or an electrotransfection method.

[0017] 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 a depression on one side, and the surface of the extracellular vesicles contains ofCSbps or a peptidomimetic of ofCSbps.

[0018] In a second aspect, the present invention provides a method for preparing engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells as described in any one of the above, comprising the following steps:

[0019] S1. Construct a fusion protein plasmid vector containing ofCSbps;

[0020] 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;

[0021] S3. Collect the cell supernatant obtained in step S2, and obtain engineered extracellular vesicles by separation.

[0022] The preparation method 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.

[0023] The use of any of the engineered extracellular vesicles targeting carcinoembryonic chondroitin sulfate-positive cells in the preparation of drugs or preparations for the research, diagnosis, treatment or prevention of tumors, placental-derived physiological or pathological diseases, and uterine-derived physiological or pathological diseases.

[0024] The application, the engineered extracellular vesicles are used as delivery carriers to load components for studying, detecting, preventing or treating tumors, placental-derived physiological or pathological diseases, and uterine-derived physiological or pathological diseases;

[0025] Preferably, the component is selected from one of polypeptides, proteins, lipids, carbohydrates, RNA, DNA or small molecule compounds;

[0026] 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;

[0027] Preferably, the uterine-derived physiological or pathological disease is one of endometriosis, endometrial adenomyosis, intrauterine adhesions, uterine myoma, and uterine fibroids;

[0028] Preferably, the loading method is one of genetic engineering, chemical modification, electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion, and surfactant.

[0029] The specific construction process of the fusion protein plasmid vector containing ofCSbps in step S1 is as follows:

[0030] (1) Primer design and PCR cloning of target fragments;

[0031] (2) Gel excision and recovery of target fragments;

[0032] (3) GoldenGate reaction to construct intermediate vector and sequence identification;

[0033] (4) LR reaction, transformation and identification of final vector positive clones.

[0034] When ultracentrifugation is used to separate and obtain extracellular vesicles in step S3, the specific operation process is as follows:

[0035] (1) Collect the cell supernatant, centrifuge at 300 g for 10 min at 4°C, and remove the cells;

[0036] (2) Centrifugation at 2000 g for 20 min at 4°C to remove dead cells;

[0037] (3) Centrifugation at 10,000 g for 30 min at 4°C to remove cell debris;

[0038] (4) 0.22 μm filter to remove impurities and bacteria;

[0039] (5) Centrifuge at 120,000 g for 120 min, discard the supernatant, and wash once with PBS;

[0040] (6) Centrifuge at 120,000 g for 60 min, discard the supernatant, precipitate the extracellular vesicles, resuspend in PBS, quantify with BCA, and aliquot at 50 μg / tube and store in a -80°C refrigerator.

[0041] Compared with the prior art, the method of the present invention has the following beneficial effects:

[0042] 1. The steps of the present invention are simple and the preparation cost is relatively low.

[0043] 2. Compared with the 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.

[0044] 3. The engineered extracellular vesicles provided by the present invention are of great significance to the basic research on placental function and tumor / cancer, as well as the clinical diagnosis and treatment of placental diseases and tumor / cancer. On the one hand, drugs can be loaded into the engineered extracellular vesicles to intervene in the placenta in normal physiological state and the placenta in pathological state, respectively, to achieve the purposes of contraception and relief treatment. Targeted delivery is carried out for tumor / cancer tissue lesions to achieve the purpose of treatment. On the other hand, contrast agents can be loaded into the engineered extracellular vesicles to achieve whole-body imaging observation of the placenta and tumor / cancer to achieve the purpose of diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Design schematics for plasmid maps and fusion proteins;

[0046] Figure 2 Schematic diagram of the construction process and monoclonal cell lines after eGFP sorting;

[0047] Figure 3 To examine the morphology of extracellular vesicles by scanning electron microscopy;

[0048] Figure 4 NTA was used to detect the particle size of extracellular vesicles;

[0049] Figure 5Western Blot was used to detect the expression of extracellular vesicle molecular markers;

[0050] Figure 6 The distribution map of extracellular vesicles in mouse uterine tissue detected by small animal imaging;

[0051] Figure 7 The distribution map of extracellular vesicles in various organs and tissues of mice detected by small animal imaging instrument;

[0052] Figure 8 Mapping the distribution of extracellular vesicles in mouse fetuses and placentas for small animal imagers;

[0053] Fig. 9 This is an immunofluorescence analysis of the enrichment and distribution of extracellular vesicles and CK7 in the mouse placental trophoblast. DETAILED DESCRIPTION

[0054] The present invention will be further explained below in conjunction with the accompanying drawings and examples. However, it should be noted that the following examples are only used to explain the present invention, and cannot be used to limit the present invention, and all technical solutions that are the same as and similar to the present invention are within the scope of protection of the present invention. If no specific technology or conditions are specified in this embodiment, the operation is carried out according to the conventional technical methods and instrument instructions in the art; if the manufacturer of the reagents or instruments used is not specified, they are all conventional reagents and consumables that can be purchased commercially.

[0055] Example 1: Plasmid vector construction

[0056] 1. Primer design and PCR cloning of target fragments. The specific steps are as follows:

[0057] The primer sequences used are as follows:

[0058] F1 (SEQ ID NO.8):

[0059] R1 (SEQ ID NO.9):

[0060]

[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 are protection bases, bold italics are the recognition sites of AarⅠ, and underlined letters are the sticky ends that will be formed after enzyme digestion.

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

[0067] Table 1 Amplification system

[0068]

[0069] Table 2 Amplification conditions

[0070]

[0071] 2. PCR product electrophoresis detection and target fragment gel excision recovery, the specific steps are:

[0072] A. After the PCR reaction system is completed, add 10 μL of 6× loading buffer to the reaction system to terminate the reaction;

[0073] B. The reaction products were subjected to agarose gel electrophoresis;

[0074] C. Recover the target fragment by gel excision.

[0075] 3. GoldenGate reaction to construct the intermediate vector, the specific steps are:

[0076] A. Extraction of pDown-AarⅠ-ccdB-Cm-AarⅠ backbone plasmid;

[0077] B. The backbone plasmid and the PCR product obtained in (2) were subjected to GoldenGate reaction according to the system and parameters shown in Table 3 and the GoldenGate reaction procedure shown in Table 4.

[0078] Table 3 GoldenGate reaction system and parameters

[0079]

[0080] Table 4 GoldenGate reaction program

[0081]

[0082] C. After the GoldenGate reaction is completed, part of the reaction product is directly used to transform UltraStable competent cells. Randomly pick 3-6 single colonies, rinse the bacteria in a sterile 0.2mL sterile EP tube, take 1μL as a template for colony PCR, and use the rest as a strain for inoculating bacteria to extract plasmid DNA. The colony PCR reaction system is shown in Table 5 below, and the colony PCR reaction procedure is shown in Table 6 below.

[0083] Table 5 PCR reaction system of colonies

[0084] template 1μL dNTP Mixture (2.5mM) 0.8μL Sequencing primer-F (10 μM) 0.5μL Sequencing primer-R (10 μM) 0.5μL 10×Buffer 1μL Taq DNA Polymerase 0.5μL <![CDATA[ddH2O]]> Up to 10 μL

[0085] Table 6 Colony PCR reaction procedure

[0086]

[0087]

[0088] D. After the colony PCR is completed, add 6× loading buffer and then perform electrophoresis. Refer to the DNA Ladder to select the clones that can amplify the target length, inoculate them into LB medium and culture them at 37℃ 250rpm overnight, then extract the small extract 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 sequencing can enter the next LR reaction to construct the final vector.

[0089] 4. LR reaction, transformation and final vector positive clone identification. The specific steps are as follows:

[0090] (1) Carry out LR reaction. The LR reaction system is shown in Table 7 below.

[0091] Table 7 LR reaction system

[0092] pUp-CMV 10-20ng Step 3: The vector obtained 10-20ng pLV[Exp] backbone vector 50-100ng LR clonase 1μL TE buffer up to 5μL

[0093] The LR reaction conditions were: incubate at 25°C for 3 h. After the reaction was completed, 1 μL of proteinase K was added and incubated at 37°C for 15 min to terminate the reaction.

[0094] (2) The LR reaction product is transformed into competent cells.

[0095] Take 2 μL of LR reaction product to transform competent cells. The operation refers to the transformation steps of GoldenGate reaction product. Finally, spread the revived bacterial solution on LB plate containing Amp antibiotic and culture it upside down at 37°C overnight.

[0096] (3) Colony PCR

[0097] Randomly pick 3-6 single colonies and operate according to the colony PCR for identifying the intermediate vector monoclone. Finally, perform agarose gel electrophoresis and pick the colonies that can amplify the DNA band of the target band length according to the DNA ladder. Inoculate and culture the bacteria and extract the plasmid DNA.

[0098] (4) Enzyme digestion identification

[0099] Use SnapGene software to open the vector map, determine the restriction scheme, and then use NEB's restriction endonuclease to digest the plasmid DNA of the final vector. After the restriction reaction is completed, perform agarose gel electrophoresis and refer to DNA Ladder for analysis. The vector that can only cut out a DNA band of a specific length is the correct final vector. Figure 1 It is a plasmid map and a schematic diagram of fusion protein design. The amino acid sequence of ofCSbps in Example 1 of the present invention is SEQ ID NO.1.

[0100] Example 2: Lentivirus packaging and concentration

[0101] 1. One day before transfection, transfer HEK293T cells into a 10 cm culture dish so that the cells can reach about 60-70% on the next day.

[0102] 2. Take two clean sterile centrifuge tubes, 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 it gently with a gun; add 30 μL of Lipo293 to the other tube. TM Gently pipette the transfection reagent to mix well. Gently add the culture solution containing DNA to the solution containing Lipo293 TM In the culture medium of the 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.

[0103] 3. Add the plasmid-culture medium mixed suspension obtained in 2 evenly to the entire culture dish, and then gently mix.

[0104] 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 virus supernatant once and add 2-3mL of complete culture medium.

[0105] 6. After 72 h, collect the viral supernatant and centrifuge at 500 g for 10 min.

[0106] 7. Transfer the clear supernatant to a sterile container and mix 1 volume of Lenti-X concentrate with 3 volumes of the clear supernatant by gentle inversion.

[0107] 8. Place the mixture at 4°C overnight and centrifuge it at 1500g for 45 min at 4°C the next day. Off-white particles will be visible after centrifugation.

[0108] 9. Carefully and gently remove the supernatant and gently resuspend the pellet to 1 / 10 to 1 / 100 of the original volume with complete medium.

[0109] 10. Titrate the sample or store the sample in aliquots at -80°C.

[0110] Example 3: Lentivirus infection and eGFP-positive cell sorting

[0111] 1. One day before infection, transfer HEK293F cells into 24-well plates;

[0112] 2. Add 20 μL of virus solution and 2 ug Polybrene to 1 mL of SMM 293-TII medium. Green fluorescence can be seen after three days.

[0113] 3. Collect polyclonal cells and sort eGFP-positive / PI (propidium iodide)-negative single cells into 96-well cell culture plates using flow cytometry. Observe under a microscope after 3 weeks. Figure 2 As shown, eGFP-positive monoclonal cell lines were observed.

[0114] 4. Transfer it into 24-well plates, 12-well plates, and 6-well plates at a time to preserve the seeds and expand the culture.

[0115] Example 4: Extracellular vesicle extraction and identification

[0116] (1) Collect the cell supernatant, centrifuge at 300 g for 10 min at 4°C, and remove the cells;

[0117] (2) Centrifugation at 2000 g for 20 min at 4°C to remove dead cells;

[0118] (3) Centrifugation at 10,000 g for 30 min at 4°C to remove cell debris;

[0119] (4) 0.22 μm filter to remove impurities and bacteria;

[0120] (5) Centrifuge at 120,000 g for 120 min, discard the supernatant, and wash once with PBS; centrifuge at 120,000 g for 60 min, discard the supernatant, precipitate extracellular vesicles, resuspend in PBS, quantify by BCA, and aliquot at 50 μg / tube and store in a -80°C refrigerator;

[0121] (6) Extracellular vesicle morphology detection: Figure 3 As shown, under transmission electron microscopy, the extracellular vesicles were found to have a saucer-like or hemispherical morphology with one side concave;

[0122] (7) Extracellular vesicle size detection: Figure 4 As shown, the analysis results of the nanoparticle tracking analyzer showed that the average was 160.4 nm;

[0123] (8) Detection of extracellular vesicle molecular markers: Cells and extracellular vesicle proteins were extracted and subjected to Western blot detection at a loading volume of 30 μg / well. Figure 5 As shown, compared with cells, CD81, ALIX and TSG101 were highly expressed in extracellular vesicles, while Calnexin was negative. In addition, GFP was positive in HEK293F-ofCSbps-Lamp2b-eGFP cells and extracellular vesicles, indicating that the engineered cells were successfully constructed and their secreted engineered extracellular vesicles could be obtained sustainably.

[0124] Example 5: Placenta-targeted validation of engineered extracellular vesicles

[0125] Pregnant C57BL / 6J mice (E12.5-15.5 days) were purchased. The experiment was divided into four groups: non-treatment group (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, the extracellular vesicles were stained with DiR dye (Invitrogen, D12731), and the specific method was as follows:

[0126] 1. Thaw the aliquoted extracellular vesicles (50 μg / tube) and add DiR dye. The working concentration of DiR is 10 μM.

[0127] 2. Wrap in tin foil and incubate at 37°C for 2 hours in dark;

[0128] 3. Centrifuge at 15000g for 10 min and discard the supernatant;

[0129] 4. Add 100 μL of saline to resuspend the extracellular vesicles.

[0130] Mice were killed 24 hours after tail vein injection of extracellular vesicles (50 μg / mouse), and heart, liver, spleen, lung, kidney, pancreas, brain and uterus (placenta), placenta and fetal tissues were separated. Fresh tissues were imaged and detected using a small animal in vivo optical imaging system (Caliper SpectrumIVIS), and the specific steps were as follows:

[0131] 1. Turn on the Caliper Spectrum IVIS system.

[0132] 2. Click Acquisition and select Auto-save to, and choose to automatically save the address when taking a photo.

[0133] 3. Click Imaging Wizard on the operation panel to set the shooting mode.

[0134] 4. Select Fluorescence and click Next to enter the fluorescence imaging settings. Select InputEX / EM in the probes selection column, and you can enter the excitation and emission wavelengths of the probes. The excitation and emission wavelengths of DiR dye are 754 / 778nm. After selection, the software will automatically give the corresponding spectrum. The blue dotted box is the band to be scanned. Then click Next.

[0135] 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 imaging focus in the Focus column. Click Next to return to the photo taking page. Click the AcquireSequence button

[0136] 6. Click the Acquire Sequence button to acquire the image.

[0137] 7. Use the ROI Tools quantitative analysis toolbar to perform quantitative analysis of the image. The results are as follows: Figure 6 , 7 As shown in Figures 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.

[0138] After imaging, the tissues were fixed in 4% paraformaldehyde for subsequent tissue sectioning.

[0139] Example 6: Fluorescence staining of tissue sections

[0140] 1. Frozen sectioning steps: tissue fixation - 30% sucrose dehydration at 4°C overnight - OCT embedding - cryostat sectioning;

[0141] 2. Immunofluorescence staining steps: air-dry the slices - 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 hours - wash with PBS - stain with DAPI - seal the slices - observe and record under a fluorescence microscope, such as Fig. 9 As shown, 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).

[0142] Finally, it should be noted that the above embodiments are only illustrative of the principles and performance of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology may modify or optimize the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or optimizations performed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. An engineered extracellular vesicle targeting carcinoembryonic chondroitin sulfate-positive cells, 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 carcinoembryonic chondroitin sulfate-positive cells according to claim 1, 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, 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, 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 carcinoembryonic chondroitin sulfate-positive cells according to claim 1, 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, 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, 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, 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, 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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