Preparation method for uterus-targeting engineered extracellular vesicle and use of same

Abstract

A preparation method for a uterus-targeting engineered extracellular vesicle and the use of same, belonging to the technical field of biomedicines. The uterus-targeting engineered extracellular vesicle of the present invention is obtained by collecting a supernatant of cells that stably overexpress Lamp2b protein and separating same, the amino acid sequence of the Lamp2b being selected from one amino acid sequence of or a combination of more than one amino acid sequence of SEQ ID NO. 1-7, or a derivative sequence taking one amino acid sequence of SEQ ID NO. 1-7 as a backbone, or a derivative sequence taking a combination of more than one amino acid sequence of SEQ ID NO. 1-7 as a backbone, or a homologous sequence of one amino acid sequence of or a combination of more than one amino acid sequence of SEQ ID NO. 1-7 in different species. The extracellular vesicle of the present invention has the characteristic of targeting the uterus and can deliver clinical diagnosis and treatment related molecules to the uterus, and therefore is suitable for use in physiological function research / research and diagnosis and treatment for intervention of uterus-derived diseases for humans and animals.

Description

Preparation method and application of uterus-targeted engineered extracellular vesicles Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to a preparation method and application of uterine-targeted engineered extracellular vesicles. 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, can deliver drugs to specific organs, tissues, or lesions.

[0004] The uterus is a vital organ for the development of life. Uterine dysfunction can lead to a variety of conditions, including uterine infertility, abnormal uterine bleeding, endometriosis, and endometrial cancer, severely compromising women's reproductive needs and health. Therefore, uterine-targeted delivery systems are urgently needed in both basic research and clinical diagnosis and treatment.

[0005] Lamp2b is a surface molecule on extracellular vesicles (EVs). Overexpressing Lamp2b in cells is a common strategy for obtaining engineered EVs. Lamp2 is a natural ligand for Galectin-1, which is highly expressed in human and mouse endometrial tissue. Based on this, the present invention provides a method for preparing uterine-targeted engineered EVs.

[0006] Summary of the Invention

[0007] In response to the problems existing in the above-mentioned prior art, the object of the present invention is to design and provide a method for preparing engineered extracellular vesicles targeting the uterus. The present invention utilizes a lentiviral vector system to stably overexpress Lamp2b-eGFP in HEK293F cells, collects the cell supernatant, and obtains extracellular vesicles by ultracentrifugation. The engineered extracellular vesicles have uterine targeting. The extracellular vesicles of the present invention have the characteristic of targeting the uterus. Because the extracellular vesicles have a nanoscale lipid inclusion structure, diagnostic and therapeutic molecules that have been clinically applied or are in preclinical research can be delivered to the uterus. The present invention is suitable for basic research and clinical diagnosis and treatment applications in the study / intervention of physiological functions of humans and animals and uterine-derived diseases.

[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 the uterus, wherein the engineered extracellular vesicle targeting the uterus is obtained by collecting and isolating the supernatant of cells stably overexpressing the protein Lamp2b; the function of the protein Lamp2b is to target the uterus.

[0010] The amino acid sequence of Lamp2b is selected from one or more combined amino acid sequences of SEQ ID NOs. 1-7, or a derivative sequence with one or more combined amino acid sequences of SEQ ID NOs. 1-7 as a backbone, or a derivative sequence with one or more combined amino acid sequences of SEQ ID NOs. 1-7 as a backbone, or a homologous sequence of one or more combined amino acid sequences of SEQ ID NOs. 1-7 in different species.

[0011] The uterus-targeted engineered extracellular vesicles are obtained by collecting and isolating the cell supernatant that stably overexpresses a fusion protein containing both protein Lamp2b and a protein used for cell transfection and tracing.

[0012] The engineered extracellular vesicles targeting the uterus, the cells are selected from one of cell lines, primary cells, organoid cells, single cells, and living animal and plant cells; preferably, the cell line is HEK293F cells.

[0013] The uterus-targeted engineered extracellular vesicle has a shape of a saucer or a hemispherical shape with one side concave, and a size of 30-200 nm.

[0014] The engineered extracellular vesicles targeting the uterus, the protein used for cell transfection and tracing is selected from one of eGFP, GFP, eYFP, mRFP1, mCherry, Luciferase, 6*His, Flag, GST, and c-Myc.

[0015] In a second aspect, the present invention provides a method for preparing a uterine-targeted engineered extracellular vesicle as described in any one of the preceding claims, comprising the following steps:

[0016] S1. Constructing a protein expression plasmid vector containing protein Lamp2b, or a fusion protein expression plasmid vector containing protein Lamp2b and a protein for cell transfection and tracing;

[0017] S2. Transfecting cells with a plasmid vector expressing Lamp2b protein, or a plasmid vector expressing a fusion protein containing both Lamp2b protein and a protein for cell transfection and tracing, and obtaining monoclonal antibody cell lines through screening, which are then further cultured;

[0018] S3. Collect the cell supernatant obtained from the expanded culture to obtain engineered extracellular vesicles.

[0019] The preparation method, the method for obtaining the engineered extracellular vesicles is selected from one of ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, tangential flow filtration, and immunoaffinity capture;

[0020] The transfection method in step S2 is selected from one of lentiviral infection, liposome transfection, calcium phosphate co-precipitation, and electroporation;

[0021] The method for screening positive cells in step S2 is selected from flow cytometry or limiting dilution method.

[0022] In a third aspect, the present invention provides the use of any one of the uterus-targeted engineered extracellular vesicles in the preparation of drugs or preparations for intervention, diagnosis and treatment of uterine physiological functions or uterine-related diseases;

[0023] Preferably, the uterine-derived disease is selected from one of uterine-derived infertility, abnormal uterine bleeding, endometriosis, endometrial adenomyosis, intrauterine adhesions, uterine fibroids, uterine fibroids, and endometrial cancer.

[0024] In a fourth aspect, the present invention provides a use of any one of the uterus-targeting engineered extracellular vesicles in the preparation of a nano drug delivery carrier;

[0025] The uterus-targeted engineered extracellular vesicles are used to load components for detecting, preventing or treating tumors, placental-derived physiological or pathological diseases, and uterine-derived physiological or pathological diseases;

[0026] Preferably, the component is one of polypeptide, protein, lipid, carbohydrate, RNA, DNA or small molecule compound.

[0027] In the application, the method for loading components into the extracellular vesicles is selected from one of genetic engineering, chemical modification, electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion, and surfactant.

[0028] Among them, when the fusion protein expression plasmid vector containing the protein for extracellular vesicle surface expression and localization and the protein for cell transfection and tracing in step S1 is a Lamp2b-eGFP fusion protein expression plasmid vector, the specific construction process of the Lamp2b-eGFP fusion protein expression plasmid vector is:

[0029] (1) Primer design and PCR cloning of target fragments;

[0030] (2) Gel excision and recovery of target fragments;

[0031] (3) GoldenGate reaction to construct the intermediate vector;

[0032] (4) Sequencing and identification of intermediate vectors;

[0033] (5) LR reaction, transformation and identification of final vector-positive clones.

[0034] Wherein, when the engineered extracellular vesicles are obtained in step S3 by separation using ultracentrifugation, the ultracentrifugation method is used to obtain the extracellular vesicles, and the specific operation process is as follows:

[0035] (1) Collect the cell supernatant and centrifuge at 300 g for 10 min at 4°C to 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) Filter with a 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, and precipitate the extracellular vesicles. Resuspend in PBS and quantify with BCA. Aliquot at 50 μg / tube and store in a -80°C refrigerator.

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

[0042] The engineered extracellular vesicles provided by the present invention are of great significance for the study of uterine function and the diagnosis and treatment of uterine diseases. On the one hand, drugs can be loaded into these engineered extracellular vesicles to intervene in the uterus in normal physiological and pathological states, achieving purposes such as contraception and palliative treatment. On the other hand, contrast agents can be loaded into these engineered extracellular vesicles to enable imaging observation of the uterus for diagnostic purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 is a plasmid map obtained in Example 1;

[0044] FIG2 is a diagram of a HEK293F-Lamp2b-eGFP overexpressing monoclonal cell line after eGFP sorting in Example 3;

[0045] FIG3 is a scanning electron microscopy image of extracellular vesicles in Example 4;

[0046] FIG4 is a graph showing the results of extracellular vesicle nanoparticle tracking analysis (NTA) in Example 4;

[0047] FIG5 is the Western blot analysis results of extracellular vesicle molecular markers in Example 4;

[0048] FIG6 is the mouse uterus targeting result of Example 5;

[0049] FIG7 shows the targeting results of various tissues and organs of mice in Example 5;

[0050] FIG8 is the fluorescence co-staining result of extracellular vesicles and Galectin-1 mouse uterus in Example 6. DETAILED DESCRIPTION

[0051] The present invention will be further described 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 intended to limit the present invention. All technical solutions similar to the present invention are within the scope of protection of the present invention. Specific techniques or conditions are not specified in this embodiment, and the operation is carried out in accordance with conventional technical methods and instrument instructions in the art; the reagents or instruments used, for which the manufacturer is not specified, are all conventional reagents and consumables that can be purchased commercially.

[0052] Example 1: Plasmid vector construction

[0053] 1. Primer design and PCR cloning of target fragments. The specific steps are as follows:

[0054] F1 (SEQ ID NO.8): atcgCACCTGCATCGTAACCATTTCAGACCATGGCACTGTG

[0055] R1 (SEQ ID NO.9): atcgCACCTGCATCGCAGAGTCTGATATCCAGCATAACTTTTTC

[0056] F2 (SEQ ID NO.10): atcgCACCTGCATCGTCTGTCTGGTGGCGGAGGCTCGG

[0057] R2 (SEQ ID NO.11): atcgCACCTGCATCGGGGTTCAGGCACCGGGCTTGCGG

[0058] 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.

[0059] Use the three pairs of primers above to amplify three DNA fragments respectively according to the system in Table 1 and the conditions in Table 2 below.

[0060] Table 1 Amplification system

[0061] Table 2 Amplification conditions

[0062] 2. PCR product electrophoresis detection and target fragment gel excision recovery, the specific steps are as follows:

[0063] (1) After the PCR reaction system is completed, add 10 μL of 6× loading buffer to the reaction system to terminate the reaction;

[0064] (2) The reaction products were subjected to agarose gel electrophoresis;

[0065] (3) Recover the target fragment by cutting the gel.

[0066] 3. GoldenGate reaction to construct the intermediate vector. The specific steps are as follows:

[0067] (1) Extraction of pDown-AarⅠ-ccdB-Cm-AarⅠ backbone plasmid;

[0068] (2) The backbone plasmid and the PCR product obtained in (2) were subjected to a GoldenGate reaction according to the system in Table 3 and the parameters in Table 4 below.

[0069] Table 3 GoldenGate reaction system is

[0070] Table 4 GoldenGate reaction program

[0071] (3) After the GoldenGate reaction is completed, a portion of the reaction product is directly used to transform UltraStable competent cells.

[0072] Randomly pick 3-6 single colonies, rinse the bacteria in a sterile 0.2 mL EP tube, take 1 μL as the template for colony PCR, and use the rest as the inoculation bacteria to extract plasmid DNA. The colony PCR reaction system and procedure are shown in Tables 5 and 6.

[0073] Table 5 Colony PCR reaction system

[0074] Table 6 Colony PCR reaction procedure

[0075] (4) After the colony PCR is completed, 6× loading buffer is added and then electrophoresis is performed. The clones that can amplify the target length are selected by referring to the DNA ladder and inoculated into LB medium and cultured at 37°C 250 rpm overnight. The small-plasmid DNA is then extracted and sent for sequencing. The returned sequencing results and the standard sequence are compared using Sequencher software. The entry vector with the correct sequencing can enter the next step of LR reaction to construct the final vector.

[0076] 4. LR reaction, transformation and identification of final vector positive clones. The specific steps are as follows:

[0077] (1) LR reaction. The LR reaction system is shown in Table 7.

[0078] Table 7 LR reaction system

[0079] 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.

[0080] (2) LR reaction products are transformed into competent cells

[0081] 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.

[0082] (3) Colony PCR

[0083] 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 DNA Ladder. Inoculate and culture bacteria and extract plasmid DNA.

[0084] (4) Enzyme digestion identification

[0085] 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 is completed, perform agarose gel electrophoresis and analyze with reference to DNALadder. The vector that can only cut out DNA bands of a specific length is the correct final vector. The plasmid map is shown in Figure 1. The amino acid sequence of Lamp2b in this Example 1 is SEQ ID NO. 1.

[0086] Example 2: Lentivirus packaging and concentration

[0087] 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.

[0088] 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.

[0089] 3. Add the plasmid-culture medium mixed suspension obtained in step 2 evenly to the entire culture dish, and then gently mix.

[0090] 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.

[0091] 6. After 72 h, the viral supernatant was collected and centrifuged at 500 g for 10 min.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 10. Titrate the sample or store the sample in aliquots at -80°C.

[0096] Example 3: Lentivirus infection and eGFP-positive cell sorting

[0097] 1. One day before infection, transfer HEK293F cells into a 24-well plate;

[0098] 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.

[0099] 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 .

[0100] 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.

[0101] Figure 2 shows a picture of the HEK293F-Lamp2b-eGFP overexpressing monoclonal cell line after eGFP sorting.

[0102] Example 4: Extracellular vesicle extraction and identification

[0103] The specific steps are as follows:

[0104] (1) Collect the cell supernatant and centrifuge at 300 g for 10 min at 4°C to remove the cells;

[0105] (2) Centrifugation at 2000 g for 20 min at 4°C to remove dead cells;

[0106] (3) Centrifugation at 10,000 g for 30 min at 4°C to remove cell debris;

[0107] (4) Filter with a 0.22 μm filter to remove impurities and bacteria;

[0108] (5) Centrifuge at 120,000 g for 120 min, discard the supernatant, and wash once with PBS;

[0109] Ultracentrifuge at 120,000 g for 60 min, discard the supernatant, and precipitate extracellular vesicles. Resuspend in PBS and quantify with BCA, then aliquot at 50 μg / tube and store in a -80°C freezer.

[0110] (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;

[0111] (7) Extracellular vesicle size detection: As shown in Figure 4, the analysis results of the nanoparticle tracking analyzer showed that the average size was 167.7 nm;

[0112] (8) Detection of extracellular vesicle molecular markers: Proteins from cells (HEK293F and HEK293F-Lamp2b-eGFP) and extracellular vesicles (secreted by HEK293F and HEK293F-Lamp2b-eGFP) were extracted and subjected to Western blot analysis at a sample loading of 30 μg / well. As shown in Figure 5, compared with cells, CD81, ALIX, and TSG101 were highly expressed in extracellular vesicles, while Calnexin expression was negative. In addition, GFP expression was positive in HEK293F-Lamp2b-eGFP cells and extracellular vesicles, indicating that the engineered cells were successfully constructed and that the engineered extracellular vesicles secreted by them could be continuously obtained.

[0113] Example 5: Uterine targeting validation of engineered extracellular vesicles

[0114] Eight-week-old female C57BL / 6J mice were purchased. The experiment was divided into four groups: no treatment (NT), tail vein injection of DiR dye (DiR), tail vein injection of HEK293F-derived extracellular vesicles (F-EVs), and tail vein injection of HEK293F-Lamp2b-eGFP-derived extracellular vesicles (L-EVs). Before tail vein injection, extracellular vesicles were stained with DiR dye (Invitrogen, D12731) as follows:

[0115] 1. Thaw the aliquoted extracellular vesicles (50 μg / tube) and add DiR dye at a working concentration of 10 μM.

[0116] 2. Wrap in tin foil and incubate at 37°C for 2 hours in the dark;

[0117] 3. Centrifuge at 15000g for 10 min and discard the supernatant;

[0118] 4. Add 100 μL of saline to resuspend the extracellular vesicles.

[0119] Twenty-four hours after tail vein injection of extracellular vesicles (50 μg / mouse), mice were sacrificed, and heart, liver, spleen, lung, kidney, pancreas, brain, and uterine tissues were isolated. Fresh tissues were imaged using a small animal in vivo optical imaging system (Caliper Spectrum IVIS) as follows:

[0120] 1. Turn on the Caliper Spectrum IVIS system.

[0121] 2. Click Acquisition and select Auto-save to, and choose to automatically save the address when taking a photo.

[0122] 3. Click Imaging Wizard on the operation panel to set the shooting mode.

[0123] 4. Select Fluorescence and click Next to proceed to the fluorescence imaging setup. In the probes selection column, 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.

[0124] 5. Adjust the exposure time, bin value, and aperture size in the Exposure Parameter column, adjust the lens height in the Field View column, and adjust the image focus in the Focus column. Click Next to return to the photo capture page. Click the Acquire Sequence button

[0125] 6. Click the Acquire Sequence button to acquire images. The results are shown in Figures 6 and 7. Compared with the NT group, DiR group, and F-EVs group, L-EVs were significantly enriched in the uterus after injection.

[0126] 7. After imaging, the tissue was fixed in 4% paraformaldehyde for subsequent tissue sectioning.

[0127] Example 6: Fluorescence staining of tissue sections

[0128] 1. Frozen sectioning steps: tissue fixation - dehydration in 30% sucrose at 4°C overnight - OCT embedding - cryostat sectioning;

[0129] 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 hours - wash with PBS - stain with DAPI - seal the sections - observe and record under a fluorescence microscope. As shown in Figure 8, GFP and Gelectin-1 co-localize in mouse uterine tissue.

[0130] 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. A uterus-targeted engineered extracellular vesicle, It is characterized in that The uterus-targeted engineered extracellular vesicles are obtained by collecting and isolating the supernatant of cells that stably overexpress the protein Lamp2b; Among them, the amino acid sequence of Lamp2b is selected from one or more combined amino acid sequences of SEQ ID NO.1-7, or a derivative sequence with one amino acid sequence of SEQ ID NO.1-7 as the skeleton or a derivative sequence with multiple combined amino acid sequences as the skeleton, or a homologous sequence of one or more combined amino acid sequences of SEQ ID NO.1-7 in different species.

2. The uterus-targeted engineered extracellular vesicle according to claim 1, It is characterized in that The uterus-targeted engineered extracellular vesicles are obtained by collecting and isolating the cell supernatant that stably overexpresses a fusion protein containing both protein Lamp2b and a protein used for cell transfection and tracing.

3. The uterus-targeted engineered extracellular vesicle according to claim 1, It is characterized in that The cell is selected from the group consisting of a cell line, a primary cell, an organoid cell, a single cell, and a living cell of an animal or plant; preferably, the cell line is a HEK293F cell.

4. The uterus-targeted engineered extracellular vesicle according to claim 1, It is characterized in that The shape of the engineered extracellular vesicle targeting the uterus is saucer-like or hemispherical with one side concave, and the size of the engineered extracellular vesicle targeting the uterus is 30-200 nm.

5. The uterus-targeted engineered extracellular vesicle according to claim 2, It is characterized in that The protein used for cell transfection and tracing is selected from one of eGFP, GFP, eYFP, mRFP1, mCherry, Luciferase, 6*His, Flag, GST, and c-Myc.

6. A method for preparing a uterus-targeted engineered extracellular vesicle according to any one of claims 1 to 5, It is characterized in that The following steps are involved: S1. constructing a protein expression plasmid vector containing protein Lamp2b, or a fusion protein expression plasmid vector containing protein Lamp2b and a protein for cell transfection and tracing; S2. transfecting a protein expression plasmid vector containing protein Lamp2b, or a fusion protein expression plasmid vector containing protein Lamp2b and a protein used for cell transfection and tracing, into cells, obtaining a monoclonal antibody cell line through screening, and further expanding the culture; S3. Collect the cell supernatant obtained from the expanded culture to obtain engineered extracellular vesicles.

7. The preparation method according to claim 6, It is characterized in that The method for obtaining the engineered extracellular vesicles is selected from one of ultracentrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, tangential flow filtration, and immunoaffinity capture; The transfection method in step S2 is selected from one of lentiviral infection, liposome transfection, calcium phosphate co-precipitation, and electroporation; The screening method in step S2 is selected from flow cytometry or limiting dilution method.

8. Use of the uterus-targeted engineered extracellular vesicles according to any one of claims 1 to 5 in the preparation of drugs or preparations for intervention, diagnosis and treatment of uterine physiological functions or uterine-related diseases; Preferably, the uterine-derived related disease is selected from one of uterine-derived infertility, abnormal uterine bleeding, endometriosis, endometrial adenomyosis, intrauterine adhesions, uterine fibroids, uterine fibroids, and endometrial cancer.

9. Use of the uterus-targeted engineered extracellular vesicles according to any one of claims 1 to 5 in the preparation of nano drug delivery carriers; The uterus-targeted engineered extracellular vesicles are used to load components for detecting, preventing or treating tumors, placental-derived physiological or pathological diseases, and uterine-derived physiological or pathological diseases; Preferably, the component is one of polypeptide, protein, lipid, carbohydrate, RNA, DNA or small molecule compound.

10. The use according to claim 9, It is characterized in that The method for loading components into the extracellular vesicles is selected from one of genetic engineering, chemical modification, electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion, and surfactant.

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