Extracellular vesicle targeting ovary-associated tissues, preparation method therefor, and use thereof

Abstract

An extracellular vesicle targeting ovary-associated tissues, a preparation method therefor, and a use thereof, relating to the technical field of biomedicine. Provided is a nano-carrier targeting ovary-associated tissues, the surface of the nano-carrier is modified with an ovary-targeting peptide, and the ovary-targeting peptide is IR18 or a polypeptide fragment having 70% or higher homology to the IR18, wherein an amino acid sequence of the IR18 is as shown in SEQ ID NO. 1, and a nucleotide sequence of the IR18 is as shown in SEQ ID NO. 6. An efficient loading and delivery system is provided for physiological and pathological research, diagnosis, and treatment of the ovary-associated tissues. Due to nano-structural characteristics of the extracellular vesicle, suitable diagnostic reagents or research / treatment substances can be delivered to corresponding tissue sites, thereby achieving the effects of the extracellular vesicle as a research, diagnosis or treatment tool.

Description

Extracellular vesicle targeting ovarian-related tissues, preparation method and application thereof Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a method for preparing engineered extracellular vesicles targeting tissues such as the ovary. Background Art

[0002] Primordial follicles are the basic functional units of female reproduction, and their formation, activation, and development are key to reproductive capacity. When follicular development is blocked, such as when primordial follicles fail to activate to form primary follicles or when primary follicles fail to form dominant follicles, premature ovarian failure can occur. Depletion of the primordial follicle pool in perimenopausal or chemotherapy patients can also lead to ovarian dysfunction. The main treatments for premature ovarian failure include hormone replacement therapy, immunomodulation, in vitro activation of primordial follicles, and stem cell therapy. However, no method can target the ovaries non-invasively. Current clinical treatments have developed primordial follicle activators to treat ovarian diseases, but after entering the human body, the drugs diffuse to various organs, fail to target the ovaries, and are quickly cleared by the body. Extracellular vesicles are nanovesicles derived from cell membranes. Due to their advantages such as good biocompatibility, circulatory stability, and low immunogenicity, they have become ideal candidates for drug and gene delivery vectors in the treatment of various diseases.

[0003] Endometriosis, also known as endometriosis, is an estrogen-dependent disease characterized by the presence of endometrial tissue (glands and stroma) outside the uterine corpus. The pathogenesis of endometriosis remains unclear, with theories ranging from the "retrograde menstrual flow" implantation theory to the metaplastic theory and the eutopic endometrial determinism theory. The "retrograde menstrual flow" implantation theory is the currently recognized primary cause of abdominal endometriosis. It states that endometrial tissue fragments during menstruation enter the abdominal cavity through the fallopian tubes and implant and grow, forming ectopic tissue. These living endometrial tissue fragments possess the abilities to invade, migrate, adhere, and form angiogenesis, driven by the combined effects of multiple environmental factors, including hormonal and immune factors. Endometriosis is a highly recurrent and difficult-to-cured disease, with existing treatments struggling to effectively and specifically eradicate the lesions. Therefore, the search for new diagnostic methods and the development of targeted, effective drugs are crucial.

[0004] Gliomas and other malignant tumors within the brain are extremely dangerous, accounting for 44% of all intracranial tumors and showing an increasing incidence rate year by year. While diagnostic and treatment technologies for gliomas have continued to advance in recent years, the basic treatment regimen is still "surgery + chemotherapy + radiotherapy." Due to their high malignancy and aggressive growth, only a subset of patients see improved survival after surgery. The blood-brain barrier prevents most anticancer drugs from entering the brain, limiting the range of drugs available for chemotherapy of gliomas. Drugs that can enter the brain often have poor efficacy due to toxic side effects or drug resistance. Therefore, breaking through the blood-brain barrier to achieve targeted treatment of gliomas and minimizing or reversing tumor drug resistance are fundamental solutions for improving the effectiveness of chemotherapy for gliomas.

[0005] The skin is the body's largest organ and the primary barrier against external aggression. As the body's first line of defense and largest organ, it constantly participates in its functions, and any abnormalities can be reflected on the skin's surface. With the continuous development of human society and the ever-changing natural environment on which we depend for survival, the causes of skin diseases are becoming increasingly diverse, leading to an annual increase in the global incidence of skin diseases. The WHO predicts that skin diseases will be the most common medical condition in human history in the 21st century, especially hair loss, alopecia areata, ringworm, rashes, intractable sores, viral dermatitis, and atopic dermatitis, which are expected to become more severe and prevalent.

[0006] Summary of the Invention

[0007] To address the aforementioned challenges in the prior art, the present invention aims to provide a method for preparing engineered extracellular vesicles (EVs) that target ovarian tissues, enabling precise targeted treatment of ovarian and other diseases. In addition to targeting ovarian tissue, the engineered EVs of the present invention can also specifically target uterine tissue, brain tissue, and skin tissue.

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

[0009] In one aspect, the present invention provides a nanocarrier targeting ovarian-related tissues, wherein the surface of the nanocarrier targeting ovarian-related tissues is modified with an ovarian-targeting peptide (referred to as I peptide);

[0010] The ovary targeting peptide is IR18 or a polypeptide fragment with 70% or more homology to IR18;

[0011] Wherein, the amino acid sequence of IR18 is shown in SEQ ID NO.1;

[0012] The amino acid sequence of the polypeptide fragment is shown in any one of SEQ ID NO. 2-5;

[0013] Preferably, the nucleotide sequence of IR18 is shown as SEQ ID NO.6;

[0014] Preferably, the nucleotide sequence of the polypeptide fragment is shown in any one of SEQ ID NOs. 7-10.

[0015] The nanocarrier targeting ovarian-related tissues is an extracellular vesicle;

[0016] Preferably, the nanocarrier is one of the extracellular vesicles secreted by organs, tissues or cells of different sources, including extracellular vesicles secreted by blood, saliva, urine, amniotic fluid, milk, etc.;

[0017] More preferably, the nanocarrier is an extracellular vesicle secreted by human umbilical cord mesenchymal stem cells.

[0018] The modification method of the nanocarrier targeting ovary-related tissues is genetic engineering.

[0019] The nanocarrier targeting ovarian-related tissues comprises a protein or gene capable of being expressed on the surface of an extracellular vesicle membrane, and a fluorescent marker protein for emitting a fluorescent signal for identification and tracing.

[0020] In a second aspect, a method for preparing a nanocarrier targeting ovarian-related tissues according to any one of the items provided herein, wherein the nanocarrier is an extracellular vesicle, comprises the following steps:

[0021] (1) preparing an expression vector containing an ovarian targeting peptide, and using a lentiviral plasmid to infect human umbilical cord mesenchymal stem cells to obtain cells stably expressing the ovarian targeting peptide-protein or gene-fluorescent marker protein;

[0022] The protein or gene has the ability to be expressed on the surface of the extracellular vesicle membrane;

[0023] (2) collecting the supernatant of cells overexpressing ovarian-targeted peptide-protein or gene-fluorescent-labeled protein;

[0024] (3) filtering, purifying, and concentrating the cell culture supernatant by tangential flow ultrafiltration;

[0025] (4) The purified and concentrated culture supernatant is subjected to ultracentrifugation, and the precipitate is extracted to obtain engineered extracellular vesicles targeting ovarian-related tissues.

[0026] The specific steps include the following:

[0027] (1) Collect the cell supernatant and centrifuge at 1000 g for 10 min at 4°C to remove cell debris;

[0028] (2) filtering the cell culture supernatant by tangential flow ultrafiltration to purify and concentrate the culture supernatant volume;

[0029] (3) The concentrated culture supernatant was collected and centrifuged at 10,000 g for 30 min at 4°C to further remove cellular impurities;

[0030] (4) Centrifuge at 100,000 g for 60 min, discard the supernatant, and wash once with PBS;

[0031] (5) Centrifuge at 100,000 g for 60 min, discard the supernatant, and resuspend the pellet in PBS to obtain the extracellular vesicle solution.

[0032] In the preparation method, the protein or gene in step (1) is selected from one or more of lamp2b, CD9, CD63 or CD47.

[0033] The lentiviral plasmid infection method is one of gene editing or chemical modification;

[0034] The fluorescent marker protein is selected from one or more of GFP, eGFP, eYFP, mRFP1 or mCherry.

[0035] The fluorescent marker protein can be used for tracing and identification in later cell transfection efficiency detection or screening through fluorescent signals.

[0036] In a third aspect, the present invention provides a use of any one of the nanocarriers targeting ovarian-related tissues in the preparation of a drug or preparation, or a biological / non-biological scaffold, or a detection reagent or tool for studying, detecting, preventing, or treating ovarian-derived, uterine-derived, brain-derived, or skin-derived diseases;

[0037] Preferably, the ovarian-derived disease is selected from one of premature ovarian failure, polycystic ovary syndrome, functional menstrual irregularity, perimenopause or menopausal syndrome, ovarian insufficiency or ovarian tumor;

[0038] The uterine-derived disease is selected from the group consisting of thin endometrium, endometriosis, endometrial cancer, endometrial adenomyosis, intrauterine adhesions, abnormal intrauterine bleeding, uterine fibroids, or uterine fibroids;

[0039] The brain-related disease is selected from depression, anxiety, glioma, cerebral infarction, cerebral hemorrhage, cerebral atrophy and dementia, cerebral palsy, Parkinson's disease or brain trauma;

[0040] The skin-derived disease is selected from one of alopecia, alopecia areata, tinea, acne, dermatitis, folliculitis, rash, intractable ulcer, viral dermatitis or allergic dermatitis.

[0041] The nanocarrier is used as a delivery vehicle to carry components for studying, detecting, preventing or treating ovarian-derived, uterine-derived, brain-derived or skin-derived diseases;

[0042] The component is selected from one or more of polypeptides, proteins, lipids, carbohydrates, RNA, DNA or small molecules;

[0043] The loading method is selected from one or more of electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion or surfactant.

[0044] The use of the nanocarrier targeting ovary-related tissues in the preparation of detection reagents, drugs or tools targeting the ovary, uterus, brain or skin.

[0045] The application of the nanocarrier targeting ovarian-related tissues in the preparation of biological / non-biological scaffolds such as cells and tissues.

[0046] Extracellular vesicles are loaded with drugs via electroporation, which specifically includes the following steps:

[0047] a. Mix extracellular vesicles and drugs in a certain ratio and transfer them into the electroporation cuvette;

[0048] b. Set the various parameters of the electroporation mode and introduction mode of the electroporator, including pulse voltage, pulse time, pulse interval length, pulse number and polarity;

[0049] c. After electroporation, aspirate the mixture and transfer to a 37°C environment for incubation for 30 minutes to ensure complete recovery of the extracellular vesicle plasma membrane;

[0050] d. removing excess drug from the extracellular vesicle solution by ultracentrifugation;

[0051] e. The drug loading in the extracellular vesicles was detected using an ultraviolet spectrophotometer.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] 1. Based on tangential flow ultrafiltration, the cell culture supernatant is filtered and concentrated to remove a large number of solute molecules. Ultimately, 3-5L of culture supernatant is concentrated to about 100mL, greatly reducing the number of ultracentrifugation times and obtaining high-purity and high-concentration extracellular vesicles.

[0054] 2. The present invention modifies ovarian-targeted peptides onto extracellular vesicles derived from mesenchymal stem cells through genetic engineering, thereby achieving precise treatment of ovarian diseases under non-invasive conditions.

[0055] 3. The present invention can avoid the mental and physical damage caused by surgical trauma to patients. By injecting extracellular vesicles, the active molecules in stem cells and ovarian therapeutic drugs are delivered to target cells to play a role in repairing ovarian function. It provides new research ideas and methods for solving follicular development problems such as premature ovarian failure, and is of great significance for improving the pregnancy rate of assisted reproductive technology and achieving rebalancing of the population structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 shows the genome sequencing of human umbilical cord mesenchymal stem cells after lentiviral infection;

[0057] FIG2 is a characterization diagram of the morphology, particle size and marker proteins of engineered extracellular vesicles detected by TEM, NTA and Western Blot;

[0058] FIG3 is a graph showing the results of UV spectrophotometry for detecting drug loading in engineered extracellular vesicles;

[0059] FIG4 is a fluorescence image of cells phagocytizing extracellular vesicles detected by fluorescence microscopy;

[0060] Figure 5 shows the distribution of extracellular vesicles in different tissues of mice detected by small animal imaging, where A is a sample taken 8 hours after tail vein injection of extracellular vesicles, and B is a sample taken 24 hours after tail vein injection of extracellular vesicles;

[0061] Figure 6 shows the enrichment of extracellular vesicles in different parts of mice, where red fluorescence is the enrichment signal of extracellular vesicles. DETAILED DESCRIPTION

[0062] In order to better understand the content of the present invention, the following examples describe specific aspects of the present invention to explain the present invention and provide a description of the method of the present invention to those skilled in the art, but the protection content of the present invention is not limited to the following examples.

[0063] Example 1: Construction of target gene expression vector

[0064] The fusion protein sequence IR18-Lamp2b-GFP (Fusion sequence, FS) was designed using software and inserted into a lentiviral plasmid to obtain a lentiviral recombinant plasmid capable of expressing the fusion protein. The amino acid sequence of IR18 is shown in SEQ ID NO. 1, and the nucleotide sequence of IR18 is shown in SEQ ID NO. 6.

[0065] Example 2: Construction of engineered cells

[0066] 1. Virus packaging:

[0067] (1) To package the lentiviral recombinant plasmid using the tool cell 293T, first ensure that the cell confluence reaches 90-95% on the day of transfection and replace the culture medium with fresh one;

[0068] (2) Add packaging mixture, serum-free medium, and Lip8000 to a centrifuge tube, where the ratio of packaging mixture is main plasmid: PSPAX2:PMD2G = 4:3:1, and mix gently by pipetting;

[0069] (3) The plasmid vector contains a GFP fluorescent marker. 24 h after transfection, the transfection efficiency can be detected by observing fluorescent cells under a fluorescence microscope;

[0070] (4) 48 h after transfection, more than 90% of the cells were transfected by observing the fluorescent cells under a fluorescence microscope. At this time, the supernatant containing the virus can be collected.

[0071] 2. Virus Extraction:

[0072] (1) Centrifuge the collected virus-containing supernatant at 500 g to remove cell debris and collect the supernatant;

[0073] (2) Lenti-XTM and viral supernatant were mixed at a ratio of 1:3 and incubated at 4°C overnight;

[0074] (3) Centrifugation at 1500 g, 4°C for 45 min;

[0075] (4) Discard the supernatant, add 100 μL of DPBS to resuspend, aliquot, and store in a -80°C refrigerator. Avoid repeated freezing and thawing.

[0076] 3. Viral transfection:

[0077] (1) Cultivating umbilical cord-derived mesenchymal stem cells. When the cell density is greater than 50%, slowly adding the virus and Polybrene to the cells;

[0078] (2) After 24 h of culture, a certain amount of culture medium was added without changing the medium;

[0079] (3) After culturing for 48 h / 72 h, fluorescent cells were observed under a fluorescence microscope;

[0080] (4) After the cells have grown to full size, they are passaged and Puro antibiotics are added after they have attached to the wall. This is continued for one week to select cells expressing the fusion protein.

[0081] (5) The transfection efficiency of engineered cells was detected by flow cytometry.

[0082] Figure 1 shows the genome sequencing results of human umbilical cord mesenchymal stem cells following lentiviral infection. To verify expression of the lentiviral recombinant plasmid in umbilical cord mesenchymal stem cells, transfected cells were digested and harvested, and DNA was extracted for genomic sequencing. Cellular expression of the fusion protein sequence was observed, indicating successful integration into the host cell genome.

[0083] Example 3: Extraction and characterization of extracellular vesicles

[0084] (1) Collect the cell supernatant and centrifuge at 1000 g for 10 min at 4°C to remove cell debris;

[0085] (2) filtering the cell culture supernatant by tangential flow ultrafiltration to concentrate the volume of the culture supernatant;

[0086] (3) The concentrated culture supernatant was collected and centrifuged at 10,000 g for 30 min at 4°C to further remove cellular impurities;

[0087] (4) Centrifuge at 100,000 g for 60 min, discard the supernatant, and wash once with PBS;

[0088] (5) Ultracentrifuge at 100,000 g for 60 min, discard the supernatant, and resuspend the pellet in PBS to obtain the extracellular vesicle solution;

[0089] (6) Detect the morphological characteristics of extracellular vesicles by transmission electron microscopy (TEM);

[0090] (7) Detecting the size of extracellular vesicles by nanoparticle tracking analysis (NTA);

[0091] (8) Detect extracellular vesicle surface markers by Western Blot.

[0092] Figure 2 shows the morphology, particle size, and marker proteins of engineered extracellular vesicles as determined by TEM, NTA, and Western Blot analysis. Transmission electron microscopy (TEM) analysis revealed that exosomes secreted by human umbilical cord mesenchymal stem cells (HUMSCs) exhibited disc-shaped or elliptical vesicle-like structures, with diameters approximately 100 nm. Nanoparticle tracking analysis (NTA) revealed that the particle size of HUCMSC exosomes in solution was concentrated at 129.1 nm, consistent with morphological observations. The narrow particle size distribution indicated uniform particle size. Western Blot analysis revealed that exosomes extracted from the cell supernatant expressed specific membrane proteins, including ALIX, CD63, and CD9. These proteins were expressed at loading levels of 2, 4, 6, 8, and 10 μg.

[0093] Example 4: Extracellular vesicle drug loading

[0094] (1) Extracellular vesicles and drugs are mixed in a certain ratio and transferred into the electroporation cup;

[0095] (2) Setting various parameters of the electroporation mode and introduction mode of the electroporator, including pulse voltage, pulse time, pulse interval length, pulse number and polarity;

[0096] (3) After electroporation, the mixture was aspirated and transferred to a 37°C environment for incubation for 30 min to ensure that the extracellular vesicle plasma membrane was completely restored;

[0097] (4) removing excess drugs from the extracellular vesicle solution by ultracentrifugation;

[0098] (5) Use UV spectrophotometer to detect the drug loading in extracellular vesicles.

[0099] Figure 3 shows the results of UV spectrophotometry analysis of the drug loading of engineered extracellular vesicles. Fluorescence spectrophotometry was used to measure the absorption peaks of the short peptide at different concentrations, and a calibration curve was created to determine the drug loading. The drug loading of FITC-G3 / G5 was 72.8% and 75.8%, respectively.

[0100] Example 5: Cell phagocytosis of extracellular vesicles

[0101] (1) To ensure that extracellular vesicles can enter the cells and exert their effects, SKOV3 cells were cultured in a serum-free state;

[0102] (2) adding extracellular vesicles stained with DIL dye for co-culture;

[0103] (3) After 24 h, red fluorescent extracellular vesicles were distributed around the cell nucleus, indicating that the extracellular vesicles entered the cell.

[0104] Figure 4 shows fluorescence microscopy images of cells engulfing extracellular vesicles. Cells were seeded into 12-well plates. After attachment, DIL-labeled exosomes were added to the cell culture medium. After 24 hours of incubation, the cell nuclei were stained with DAPI. Under fluorescence microscopy, blue-labeled nuclei were observed, while red-fluorescing exosomes were distributed around the nuclei, indicating that the exosomes have entered the cells and are capable of cellular uptake.

[0105] Example 6: Detection of the ability of extracellular vesicles to target ovarian-related tissues

[0106] (1) Extracellular vesicle labeling: extracellular vesicles are labeled with the lipophilic dye DIR;

[0107] (2) Extracellular vesicle purification: Use the Exosome spin column to remove excess dye from extracellular vesicles;

[0108] (3) Extracellular vesicle targeting ability: Adult C57BL / 6 mice were injected with engineered extracellular vesicles that can target the ovary, DIR dye, and PBS via the tail vein. After 8 hours, the distribution of extracellular vesicles in various tissues of the mice was observed using a small animal imaging device to determine the ability of extracellular vesicles to target the ovary.

[0109] Figure 5 shows the distribution of extracellular vesicles in different mouse tissues detected by a small animal imaging system. A shows the sample taken 8 hours after the tail vein injection of extracellular vesicles, and B shows the sample taken 24 hours after the tail vein injection of extracellular vesicles. To verify the ovarian targeting of genetically modified engineered exosomes, DIR dye-labeled exosomes were injected into mice via the tail vein, and the mice were observed by in vivo fluorescence imaging using a small animal imaging system. 8 hours and 24 hours after the tail vein injection of exosomes, the mice were sacrificed and dissected, and the various organs of the mice were removed for in vitro imaging. In the figure, NT represents the control group, DIR represents the dye group, and FS-EV represents the engineered exosome group. The results showed that FS-EVs could target the ovaries and brain tissues 8 hours after tail vein injection (Figure 5A); 24 hours after injection, FS-EVs could specifically target the uterus (Figure 5B).

[0110] Example 7: Enrichment of extracellular vesicles in different parts of mice

[0111] (1) DIL-labeled engineered exosomes were injected into mice via the tail vein;

[0112] (2) After 24 h, the mice were sacrificed and their ovaries, brains, uteri, and skin tissues were collected;

[0113] (3) Frozen sections of the above tissues;

[0114] (4) The distribution of extracellular vesicles in various tissues was observed using laser confocal microscopy.

[0115] Figure 6 shows the distribution of extracellular vesicles in various mouse tissues, with red fluorescence representing the enrichment signal of extracellular vesicles. Specifically, they are enriched in different cells of ovarian follicles at different stages, such as granulosa cells, theca cells, and stromal cells. They can cross the blood-brain barrier and target the brain, where they are enriched in different brain cells, with strong positive signals in the dentate gyrus and third ventricular region of the hippocampus. Fluorescence is also enriched in fibers within the ventricles, further indicating that they can enter the cerebrospinal fluid. They are enriched in the epidermis, dermis, and subcutaneous tissue of skin tissue, including epithelial cells, hair follicle cells, sebaceous gland cells, muscle cells, stromal cells, and adipocytes in the subcutaneous tissue. They are enriched in different cells of uterine tissue, with strong positive signals in glandular epithelial cells, stromal cells, and muscle cells.

[0116] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A nanocarrier targeting ovarian-related tissues, characterized in that: The surface of the nanocarrier targeting ovarian-related tissues is modified with an ovarian-targeting peptide; The ovary targeting peptide is IR18 or a polypeptide fragment with 70% or more homology to IR18; Wherein, the amino acid sequence of IR18 is shown in SEQ ID NO.1; The amino acid sequence of the polypeptide fragment is shown in any one of SEQ ID NO. 2-5; Preferably, the nucleotide sequence of IR18 is shown as SEQ ID NO.6; Preferably, the nucleotide sequence of the polypeptide fragment is shown in any one of SEQ ID NOs. 7-10.

2. The nanocarrier targeting ovarian-related tissues according to claim 1, wherein: The nanocarrier is an extracellular vesicle.

3. The nanocarrier targeting ovarian-related tissues according to claim 1, wherein: The nanocarrier is a type of extracellular vesicle secreted by organs, tissues or cells from different sources.

4. The nanocarrier targeting ovarian-related tissues according to claim 1, wherein: The nanocarrier is an extracellular vesicle secreted by human umbilical cord mesenchymal stem cells.

5. The nanocarrier targeting ovarian-related tissues according to claim 1, wherein: The modification method is genetic engineering.

6. The nanocarrier targeting ovarian-related tissues according to claim 1, wherein: The surface of the nanocarrier is modified with a protein or gene capable of being expressed on the surface of an extracellular vesicle membrane, and a fluorescent marker protein for emitting a fluorescent signal for identification and tracing.

7. A method for preparing a nanocarrier targeting ovarian-related tissues according to any one of claims 1 to 6, characterized in that: When the nanocarrier is an extracellular vesicle, the method comprises the following steps: (1) preparing an expression vector containing an ovarian targeting peptide, and using a lentiviral plasmid to infect human umbilical cord mesenchymal stem cells to obtain cells stably expressing the ovarian targeting peptide-protein or gene-fluorescent marker protein; The protein or gene has the ability to be expressed on the surface of the extracellular vesicle membrane; (2) collecting the supernatant of cells overexpressing ovarian-targeted peptide-protein or gene-fluorescent-labeled protein; (3) filtering, purifying, and concentrating the cell culture supernatant by tangential flow ultrafiltration; (4) The purified and concentrated culture supernatant is subjected to ultracentrifugation, and the precipitate is extracted to obtain engineered extracellular vesicles targeting ovarian-related tissues.

8. The preparation method according to claim 7, wherein The protein or gene in step (1) is selected from one or more of lamp2b, CD9, CD63 or CD47; The lentiviral plasmid infection method is one of gene editing or chemical modification; The fluorescent marker protein is selected from one or more of GFP, eGFP, eYFP, mRFP1 or mCherry.

9. Use of a nanocarrier targeting ovarian-related tissues according to any one of claims 1 to 6 in the preparation of a drug or preparation, or a biological / non-biological scaffold, or a detection reagent or tool for studying, detecting, preventing or treating ovarian, uterine, brain or skin diseases; Preferably, the ovarian-derived disease is selected from one of premature ovarian failure, polycystic ovary syndrome, functional menstrual irregularity, perimenopause or menopausal syndrome, ovarian insufficiency or ovarian tumor; The uterine-derived disease is selected from the group consisting of thin endometrium, endometriosis, endometrial cancer, endometrial adenomyosis, intrauterine adhesions, abnormal intrauterine bleeding, uterine fibroids, or uterine fibroids; The brain-related disease is selected from depression, anxiety, glioma, cerebral infarction, cerebral hemorrhage, cerebral atrophy and dementia, cerebral palsy, Parkinson's disease or brain trauma; The skin-derived disease is selected from one of alopecia, alopecia areata, tinea, acne, dermatitis, folliculitis, rash, intractable ulcer, viral dermatitis or allergic dermatitis.

10. The use according to claim 9, characterized in that The nanocarrier is used as a delivery vehicle to carry components for studying, detecting, preventing or treating ovarian-derived, uterine-derived, brain-derived or skin-derived diseases; The component is selected from one or more of polypeptides, proteins, lipids, carbohydrates, RNA, DNA or small molecules; The loading method is selected from one or more of electroporation, liposome transfection, co-incubation, ultrasound, freeze-thaw cycle, extrusion or surfactant.

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