An endometrial stem cell exosome preparation for treating endometrial damage and a preparation method thereof

Abstract

The application belongs to the technical field of biotechnology and medicine, and specifically discloses a single-domain antibody-exosome compound (Nb-EMX2-Exos) targeting EMX2 as well as a preparation method and application thereof. The compound is prepared by covalently coupling a single-domain antibody (Nb-EMX2) specifically binding to EMX2 protein to the surface of endometrial stem cell-derived exosomes (ESC-Exos). In-vitro experiments show that the compound can specifically recognize and efficiently internalize into damaged endometrial stromal cells with high expression of EMX2, and significantly promote the migration and proliferation of the cells. In-vivo experiments further prove that the compound can precisely target and enrich in damaged endometrial tissue, effectively promote the structural repair (thicken the endometrium and reduce fibrosis) of the endometrium, and completely restore the fertility function. The application provides a novel targeted treatment preparation for solving the problems of poor targeting of natural exosomes and limited treatment effect, and has important application value in the treatment of endometrial injury repair and related infertility.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and medicine, specifically relating to an endometrial stem cell exosome preparation for treating endometrial damage and its preparation method. Background Technology

[0002] Endometrial injuries (including intrauterine adhesions and severe endometrial atrophy) are a significant cause of secondary infertility and recurrent implantation failure in women. The integrity and functional regeneration of the endometrium are crucial physiological foundations for embryo implantation and pregnancy maintenance. However, after severe damage, the basal glandular epithelium and vascular network of the endometrium often suffer irreversible destruction, making it difficult for traditional treatments to restore their normal structure and physiological function.

[0003] Currently, the main clinical approach involves hysteroscopic lesion removal combined with estrogen replacement therapy to promote endometrial proliferation and repair. However, due to insufficient local stromal cell numbers and limited microenvironment reconstruction, this method has limited efficacy, especially in patients with severe damage, often failing to achieve functional endometrial regeneration, resulting in a still low clinical pregnancy rate. Therefore, exploring innovative treatment strategies that can promote endometrial tissue regeneration has become an important direction in reproductive medicine research.

[0004] Stem cell therapy is widely used in tissue repair research due to its differentiation potential and immunomodulatory properties. Among these, exosomes, paracrine factors secreted by stem cells, are considered the main mediators of their therapeutic effects. Exosomes are extracellular nanovesicles with a diameter of approximately 30–150 nm, rich in proteins, mRNA, miRNA, and lipid molecules. They can regulate inflammatory responses, angiogenesis, and matrix remodeling through intercellular signal transduction, thereby promoting tissue repair and regeneration. Compared to cell transplantation, exosome therapy has significant advantages such as low immunogenicity, high safety, convenient storage and transportation, and no risk of tumor formation, thus becoming a research hotspot in the field of tissue regeneration in recent years.

[0005] However, naturally derived exosomes suffer from significant targeting limitations in in vivo applications. After systemic administration, most exosomes are cleared by the reticuloendothelial system, including the liver and spleen, with only a small amount reaching the uterine injury area. This results in insufficient local effective concentrations, requiring high doses or multiple administrations to produce therapeutic effects. This not only significantly increases preparation costs but may also lead to side effects on non-target tissues, severely limiting their clinical application prospects.

[0006] To improve the tissue-specific enrichment of exosomes, current research attempts to modify stem cells through genetic engineering to induce them to secrete exosomes carrying specific target peptides (such as RGD, LyP-1, cRGD, CREKA, etc.) to enhance their recognition and binding to tissues with high expression of specific receptors. However, this approach has several limitations: (1) Gene modification is complex, time-consuming, and difficult to standardize; (2) Integration of exogenous genes poses potential safety risks and affects their clinical applicability; (3) Currently reported target peptides are mostly used in tumors or the vascular system, and their receptors are widely distributed in the body, lacking specific recognition ability for endometrial tissue, which can easily lead to off-target effects and non-specific accumulation.

[0007] Therefore, how to construct an exosome preparation that is simple to prepare, safe, reliable, and can precisely enrich the uterine injury site without relying on complex gene manipulation has become a core technical problem that urgently needs to be solved in the field of reproductive repair. Breakthroughs in this direction can not only significantly improve the efficacy and safety of exosome treatment for endometrial injury, but also have significant clinical translational potential and industrial application value. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides an endometrial stem cell exosome preparation with specific single-domain antibodies displayed on its surface, prepared using in vitro conjugation technology. This preparation leverages the small size, high permeability, and high affinity of single-domain antibodies to achieve precise targeting of damaged endometrium, thereby enhancing therapeutic efficacy.

[0009] Therefore, this invention overcomes the shortcomings of traditional peptide-modified targeting systems, which suffer from complex design and insufficient specificity. It innovatively proposes a targeted exosome delivery system based on single-domain antibodies (nanobodies), targeting Empty Spiracles Homeobox 2 (EMX2), a key regulator of endometrial repair. Through a mild and efficient chemical conjugation strategy, a high-affinity anti-EMX2 single-domain antibody (Nb-EMX2) is stably modified onto the surface of naturally derived human endometrial stem cell exosomes, thereby endowing the exosomes with the ability to actively target damaged endometrial sites. EMX2 is specifically highly expressed during the regeneration and repair process following endometrial injury, serving as an ideal localization signaling molecule. This design enables exosomes to accurately identify and efficiently accumulate damaged areas in vivo, significantly improving local bioavailability and regeneration efficiency, providing a safe, effective, and clinically translatable novel cell-free therapeutic strategy for endometrial injury diseases.

[0010] This invention discloses a specific anti-EMX2 single-domain antibody, wherein the specific anti-EMX2 single-domain antibody is Nb-EMX2, and its amino acid sequence is shown in SEQ ID NO:2.

[0011] In one aspect, the present invention also discloses a targeted exosome complex, namely Nb-EMX2-Exos, which comprises the aforementioned specific anti-EMX2 single-domain antibody and exosomes derived from endometrial stem cells, wherein the single-domain antibody is covalently linked to the surface of the exosomes. The exosomes derived from endometrial stem cells have a peak particle size of 116±13 nm, an average particle size of 122±8 nm, and a particle concentration of 6.1×10⁻⁶. 10 The Nb-EMX2-Exos particles / mL had a peak particle size of 127±17 nm, an average particle size of 135±11 nm, and a particle concentration of 4.3×10⁻⁶. 10 particles / mL.

[0012] In one aspect, the present invention also discloses a method for preparing the targeted exosome complex, the method comprising the following steps: (1) preparing the specific anti-EMX2 single-domain antibody; (2) preparing the exosomes derived from endometrial stem cells; (3) modifying the exosomes with thiolization to obtain thiolized exosomes; (4) activating the single-domain antibody with maleimide to obtain maleimide-activated antibody; (5) coupling the thiolized exosomes with the maleimide-activated antibody to form the targeted exosome complex; the coupling reaction is carried out at 4°C for 16 hours. The single-domain antibody in step (1) is obtained by immunizing alpacas with EMX2 protein and constructing a phage display library, followed by multiple rounds of biological panning and expression purification. The thiolization modification in step (3) is achieved using 2-iminothiacyclopentane hydrochloride. The maleimide activation described in step (4) is achieved using sulfosuccinimide-4-maleimide-methylcyclohexane-1-carboxylic acid ester.

[0013] In one aspect, the present invention also discloses a pharmaceutical composition comprising the aforementioned targeted exosome complex and a pharmaceutically acceptable carrier.

[0014] In one aspect, the present invention also discloses the use of the aforementioned targeted exosome complex in the preparation of a medicament for treating endometrial injury.

[0015] The targeted exosome complex provided by this invention exhibits several significant advantages. First, the complex achieves precise targeting and efficient enrichment of lesions. Through the specific recognition of the highly expressed EMX2 protein in damaged endometrial tissue by a single-domain antibody displayed on its surface, its uptake efficiency of target cells in in vitro cell models is 2.67 times that of natural exosomes; in in vivo animal experiments, its enrichment intensity at uterine injury sites is 3.39 times that of natural exosomes, effectively solving the key problem of poor targeting of natural exosomes. Second, the complex demonstrates excellent tissue repair and functional recovery efficacy. In a rat model of endometrial injury, compared with unmodified natural exosomes, this targeted complex can more effectively promote endometrial thickening, restoring it to near-normal levels; simultaneously, it can significantly reduce the fibrotic area to less than half of that in the natural exosome treatment group, indicating a more prominent effect in inhibiting abnormal scar formation. Most groundbreakingly, this complex can completely restore the fertility of damaged animals, achieving a 100% conception rate and an average number of embryos close to normal levels, realizing a leap from structural repair to functional recovery. Finally, the proposed technical solution is rationally constructed, producing a synergistic and enhanced therapeutic effect. This complex is not a simple additive of components, but rather an organic combination of the inherent tissue repair signals of exosomes and the precise navigation capabilities of antibodies, enabling the therapeutic components to be efficiently delivered to the lesion, thereby maximizing efficacy. In conclusion, this invention provides a promising targeted biological agent for the clinical treatment of endometrial injury and related infertility. Attached Figure Description

[0016] Figure 1 SDS-PAGE analysis of the anti-EMX2 single-domain antibody Nb-EMX2.

[0017] Figure 2 Morphological observation of endometrial stem cells.

[0018] Figure 3 Image showing the results of endometrial stem cell surface marker flow cytometry detection.

[0019] Figure 4 Nanoparticle tracking analysis of exosomes ESC-Exos from endometrial stem cells.

[0020] Figure 5 Transmission electron microscope image of endometrial stem cell exosomes ESC-Exos.

[0021] Figure 6 Western blot analysis of endometrial stem cell exosomes ESC-Exos and the marker protein of the targeting exosome complex Nb-EMX2-Exos.

[0022] Figure 7Nanoparticle tracking analysis of the Nb-EMX2-Exos targeted exosome complex.

[0023] Figure 8 Transmission electron microscopy image of the Nb-EMX2-Exos-targeted exosome complex.

[0024] Figure 9 Flow cytometry analysis of antibody-coupled efficacy targeting the exosome complex Nb-EMX2-Exos.

[0025] Figure 10 Western blot analysis of EMX2 protein expression levels in endometrial stromal cells after TGF-β1 induction.

[0026] Figure 11 Flow cytometry graph showing the quantitative analysis of in vitro cell uptake efficiency. Detailed Implementation

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the specification of this invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0028] Unless otherwise specified, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0029] Example 1: Preparation and Identification of Specific Anti-EMX2 Single-Domain Antibody (Nb-EMX2)

[0030] 1. Experimental Materials and Reagents

[0031] 1.1 Target protein: Recombinant human EMX2 protein, Wuhan Huamei Biotechnology Co., Ltd., CSB-YP007657HU;

[0032] 1.2 Experimental animals: 3-year-old healthy male alpacas (weighing 65 kg), free from pathogen infection;

[0033] 1.3 Vectors and strains: Phage display vector pCANTAB5E, expression vector pET-28a(+), Escherichia coli TG1 (competent cells, transformation efficiency 1×10^8 CFU / μg), and Escherichia coli WK6 (competent cells);

[0034] 1.4 Key Reagents: Trizol reagent (Invitrogen), M-MLV reverse transcriptase (Promega), Taq DNA polymerase (Takara), restriction endonucleases SfiI / NotI / NcoI / XhoI (NEB), HRP-labeled anti-His tag antibody (Abcam), nickel column affinity chromatography medium (Qiagen), TMB chromogenic solution (Solarbio), VEGF protein (R&D Systems);

[0035] (5) Control reagent: Commercially available anti-EMX2 monoclonal antibody (mAb-EMX2, catalog number: ab171818, Abcam).

[0036] 2. Experimental Procedures and Results

[0037] 2.1 Construction of Immunotherapy and Phage Display Libraries

[0038] (1) Immunization regimen: For the first immunization, 100 μg of recombinant human EMX2 protein was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points on the back of the neck; for the second, third and fourth booster immunizations, 50 μg of EMX2 protein was emulsified with an equal volume of Freund's incomplete adjuvant and injected once every 2 weeks.

[0039] (2) Lymphocyte isolation: 7 days after the last immunization, 50 mL of peripheral blood was collected from the jugular vein of the alpaca. Peripheral blood lymphocytes (PBMCs) were isolated by Ficoll-Paque density gradient centrifugation (1500 rpm, 20 min) to obtain approximately 8 × 10^7 cells.

[0040] (3) RNA extraction and cDNA synthesis: Total RNA was extracted from PBMCs using Trizol reagent, and the A260 / A280 ratio was 1.92 as determined by Nanodrop, with a concentration of 2.5 μg / μL. 5 μg of total RNA was taken and cDNA was synthesized using M-MLV reverse transcriptase (42℃ for 60 min, 70℃ for 15 min).

[0041] (4) VHH gene amplification: The VHH fragment was amplified using nested PCR. The first round of PCR used cDNA obtained by reverse transcription from immune alpaca peripheral blood lymphocytes (PBMCs) as a template to amplify the fragment containing the variable region (VH / VHH) gene; the second round of PCR specifically amplified the VHH region and introduced restriction enzyme sites for subsequent cloning. First round PCR primers: upstream primer (CALL001): 5'-gtcctggctgctcttctacaagg-3', downstream primer (CALL002): 5'-ggtacgtgctgttgaactgttcc-3'. Amplification system and conditions: 95 °C for 5 min; followed by 30 cycles (95 °C for 30 s, 58 °C for 30 s, 72 °C for 40 s); and a final extension at 72 °C for 10 min. Second-round PCR primers (containing restriction enzyme sites): Upstream primer (SfiI-VHH-F): 5'-ggtattgagggtcccctggccatggcc-3' (containing SfiI recognition sequence), downstream primer (NotI-VHH-R): 5'-agtcgcggccgctgaggagacggtgacctgg-3' (containing NotI recognition sequence). Amplification system and conditions: 95 °C for 5 min; followed by 30 cycles (95 °C for 30 s, 60 °C for 30 s, 72 °C for 40 s); final extension at 72 °C for 10 min. Analysis of the amplified product by 1.5% agarose gel electrophoresis showed a clear single band at approximately 400 bp, indicating that a VHH-specific fragment of the desired size was obtained, which can be used for subsequent vector construction.

[0042] (5) Vector construction and transformation: The VHH fragment was digested with pCANTAB5E vector by SfiI / NotI double digestion (37℃ 4h), and ligated with T4 DNA ligase (16℃ 12h); the ligation product was electrotransformed into Escherichia coli TG1 (1.8kV, 25μF, 200Ω), and the transformation solution was plated on LB plates containing ampicillin (100μg / mL) and glucose (2%) and cultured at 37℃ for 12h;

[0043] (6) Library capacity and diversity detection: The library capacity was counted by serial dilution plate smear; 20 clones were randomly selected for colony PCR to verify the VHH fragment insertion rate. The results showed that the primary phage library capacity was 2.5×10^8 CFU, and PCR amplified 400bp fragments from all 20 random clones, with an insertion rate of 100%, indicating good library diversity.

[0044] 2.2 Specific phage bioscreening

[0045] (1) Target immobilization: Dilute EMX2 protein to 10 μg / mL with 0.05 M carbonate buffer (pH 9.6), add 100 μL to each well of the microplate, and coat overnight at 4°C;

[0046] (2) Blocking and binding: Wash 3 times with PBST (PBS containing 0.05% Tween-20), block 200 μL / well with 5% skim milk-PBST blocking solution at 37℃ for 2 h; add 1×10^11 pfu primary phage library (diluted with 2% skim milk-PBST) and incubate at 37℃ for 1 h;

[0047] (3) Elution and neutralization: Wash with PBST (10 times in the first round, 15 times in the second round, and 20 times in the third round); in the first round, elute with 0.1 M Gly-HCl (pH 2.2) for 10 min; in the second round, elute with 0.1 M Gly-HCl (pH 2.0) + 0.5 M NaCl; in the third round, elute with 0.1 M Gly-HCl (pH 1.8) + 1 M NaCl; immediately neutralize the eluent to pH 7.0 with 1 M Tris-HCl (pH 9.0);

[0048] (4) Phage amplification: The neutralized eluent was used to infect logarithmic Escherichia coli TG1, incubated at 37℃ for 30 min, and 2×YT medium (containing 100 μg / mL ampicillin) was added. The mixture was then incubated at 37℃ and 220 rpm for 12 h. The supernatant was collected as the next round of phage input.

[0049] (5) Screening efficiency test: After each round of screening, the output phages were serially diluted, plated, counted, and the output titer and enrichment factor were calculated. The results showed that the input phage titer was 1×10^11 pfu in each round, and the output titer gradually increased: 1.2×10^5 pfu in the first round, 3.5×10^7 pfu in the second round (enriched by 292 times), and 8.9×10^8 pfu in the third round (enriched by 25 times compared to the second round and 7417 times compared to the first round), indicating highly efficient enrichment of specific phages.

[0050] 2.3 Expression and purification of Nb-EMX2

[0051] (1) Screening of positive clones: After the third round of screening, 96 single clones were randomly selected and inoculated into 2×YT medium (containing 100μg / mL ampicillin + 2% glucose) and cultured at 37℃ and 220rpm for 12h; Phage-ELISA screening: EMX2 protein 5μg / mL was coated on the microplate, culture supernatant was added, and incubated at 37℃ for 1h; HRP-labeled anti-M13 antibody (1:5000 dilution) was incubated for 1h; TMB color development was performed for 15min, and the reaction was terminated with 2M H2SO4. The OD450 value was detected by microplate reader, and the positive standard was OD450 ≥ 3 times that of the negative control; The results showed that 28 of the 96 clones detected by Phage-ELISA were positive (positive rate 29.2%).

[0052] (2) Gene subcloning: Select the positive clone with the strongest signal (OD450=2.15), extract plasmid and sequence it (the nucleotide sequence of Nb-EMX2VHH is shown in SEQ ID NO:1, the full length is 369bp, encoding 123 amino acids, which conforms to the typical structure of a single-domain antibody, and its amino acid sequence is shown in SEQ ID NO:2); digest the VHH gene with NcoI / XhoI, subclone it into the pET-28a(+) vector, and transform it into E. coli WK6;

[0053] (3) Induction of expression: a single clone of recombinant bacteria was inoculated into 5 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37℃ and 220 rpm for 8 h; it was then inoculated into 1 L of LB medium at a ratio of 1:100 and cultured at 37℃ until OD600=0.6; IPTG was added to a final concentration of 0.5 mM and induced at 25℃ and 180 rpm for 16 h.

[0054] (4) Protein purification: Collect bacterial cells by centrifugation at 8000 rpm for 10 min at 4℃, resuspend in 10 mM Tris-HCl (pH 8.0); add 20% sucrose solution (final concentration 10%), incubate on ice for 10 min; centrifuge at 12000 rpm for 15 min at 4℃, discard the supernatant, resuspend the precipitate in 5 mM MgSO4 on ice, incubate on ice for 10 min, centrifuge to collect periplasmic protein extract; load onto a pre-equilibrated Ni-NTA column (equilibration buffer: 20 mM Tris-HCl pH 8.0 + 500 mM NaCl + 20 mM imidazole); elute impurities with gradients of 50 mM and 100 mM imidazole buffer, elute the target protein with 300 mM imidazole buffer; dialyze overnight with PBS;

[0055] (5) Purity and concentration detection: Purity was analyzed by SDS-PAGE, and protein concentration was determined by the BCA method. Results showed that SDS-PAGE indicated ( Figure 1After purification, Nb-EMX2 showed a single clear band at approximately 15 kDa, and ImageJ analysis showed a purity of ≥95%; the concentration was determined to be 5.89 mg / mL by BCA method, and 350 mg of purified protein was obtained from 1 L of fermentation broth.

[0056] 2.4 Affinity and Specificity Identification

[0057] 2.4.1 ELISA Specific Detection

[0058] (1) Experimental steps: Dilute EMX2 (2 μg / mL), BSA (5 μg / mL), and VEGF (2 μg / mL) with PBS, add 100 μL to each well of the microplate, and coat overnight at 4°C; wash 3 times with PBST, add 200 μL of 3% BSA-PBST blocking buffer to each well, and block at 37°C for 1 h; add 1 μg / mL Nb-EMX2 and mAb-EMX2 (diluted with PBS), 100 μL per well, and incubate at 37°C for 1 h;

[0059] Wash 5 times with PBST, incubate with HRP-labeled anti-His antibody (1:10000 dilution) for 1 h; after washing, develop TMB for 15 min, terminate with 2MH2SO4, and detect OD450 value with a microplate reader.

[0060] (2) Experimental results: Nb-EMX2 binding to EMX2 has an OD450 of 1.32, while binding to BSA (0.11) and VEGF (0.08) has extremely low signals; mAb-EMX2 binding to EMX2 has an OD450 of 1.25, while binding to BSA (0.15) and VEGF (0.12) has slightly higher signals.

[0061] 2.4.2 SPR Affinity Testing

[0062] (1) Experimental steps: CM5 chip EDC / NHS activation for 10 min, EMX2 protein diluted to 20 μg / mL with 10 mM sodium acetate buffer (pH 4.5), fixed to the chip surface at a flow rate of 10 μL / min (fixation amount of about 2000 RU); 1M ethanolamine blocked unactivated sites; the running buffer was PBS-T (pH 7.4 + 0.05% Tween-20), and Nb-EMX2 and mAb-EMX2 concentration gradients were set (0.39 nM, 0.78 nM, 1.56 nM, 3.12 nM, 6.25 nM, 12.5 nM), with a flow rate of 30 μL / min; binding time 120 s, dissociation time 300 s; chip regeneration with 10 mM Gly-HCl (pH 2.0) for 30 s; Biacore T200 software fitted the curve with a 1:1 Langmuir binding model and calculated KD, ka, and kd values.

[0063] (2) Experimental results: The kinetic parameters of Nb-EMX2 are ka = 2.1 × 10^5 M -1 s -1 kd = 7.8 × 10^-7 s -1 KD=3.7nM(R) 2 =0.987); mAb-EMX2 parameter is ka=1.8×10^5 M -1 s -1 kd = 8.1 × 10^-6 s -1 With a KD of 45.2 nM, the affinity for Nb-EMX2 is significantly higher.

[0064] Example 2: Culture and exosome extraction of endometrial stem cells

[0065] 1. Experimental Materials and Reagents

[0066] 1.1 Tissue and Cell Related

[0067] (1) Endometrial tissue: It was obtained from a healthy woman of childbearing age (32 years old, day 12 of the menstrual cycle) who voluntarily donated clinically. The tissue weighed about 0.8g. After aseptic collection, it was placed in PBS containing penicillin-streptomycin (100U / mL) and the processing was completed within 2 hours.

[0068] (2) Basic culture medium: DMEM / F12 (Gibco), with 10% fetal bovine serum (FBS, Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin added, and filtered for sterilization (0.22 μm filter membrane).

[0069] (3) Digestion reagent: Collagenase IV, prepared with serum-free DMEM / F12 to a concentration of 0.2% (w / v), filtered and sterilized, and stored at 4°C;

[0070] 1.2 Cell identification related

[0071] (1) Flow cytometry antibodies: PE-labeled CD90, FITC-labeled CD105, APC-labeled CD73, PE-Cy7-labeled CD45, and Alexa Fluor 647-labeled CD34 were all derived from BD and were diluted 1:200.

[0072] (2) Culture medium: Osteogenesis induction medium is the basic culture medium supplemented with 10 mM β-glycerophosphate sodium, 50 μM ascorbic acid and 100 nM dexamethasone; Adipogenesis induction medium is the basic culture medium supplemented with 1 μM dexamethasone, 10 μg / mL insulin, 200 μM indomethacin and 0.5 mM 3-isobutyl-1-methylxanthine;

[0073] (4) Staining reagents: Alizarin Red S staining solution, Oil Red O staining solution, 4% paraformaldehyde.

[0074] 1.3 Exosome Extraction and Characterization

[0075] (1) Serum-free culture medium: serum-free DMEM / F12, with 1× insulin-transferrin-selenium (ITS, Gibco).

[0076] (2) Centrifugation consumables: ultracentrifuge tubes (Beckman), 0.22μm polyethersulfone (PES) filter membrane (Millipore);

[0077] (3) Sucrose density gradient reagent: sucrose (Sigma), prepared with PBS to concentrations of 5%, 10%, 20%, 40%, and 60% (w / v), and pre-cooled at 4°C;

[0078] (4) Characterization reagents: primary antibody CD63 (Abcam, 1:1000 dilution), TSG101 (Abcam, 1:1000 dilution), Calnexin (Abcam, 1:1000 dilution), HRP-labeled secondary antibody (goat anti-rabbit, Abcam, 1:5000 dilution), ECL chemiluminescent solution.

[0079] 2. Experimental Procedure

[0080] 2.1 Isolation and primary culture of endometrial stem cells

[0081] (1) Tissue processing: The sterile endometrial tissue was placed in a sterile culture dish and rinsed three times with PBS (containing antibiotics) to remove residual blood and mucus; the tissue was then minced to 1 mm using ophthalmic scissors. 3 Small pieces on the left and right;

[0082] (2) Enzymatic digestion: Add 5 mL of 0.2% collagenase IV solution and digest in a constant temperature shaker at 37℃ and 100 rpm for 60 min, gently blowing once every 15 min to promote tissue dispersion;

[0083] (3) Filtration and centrifugation: The digestion solution was filtered through a 70μm cell filter to remove undigested tissue fragments; the filtrate was transferred to a centrifuge tube and centrifuged at 1000g and 4℃ for 10min, and the supernatant was discarded;

[0084] (4) Inoculation and culture: Resuspend the cell pellet in basal medium, count the cells, and then inoculate at 5×10⁻⁶. 5 cells / cm 2 The culture medium was inoculated into T75 culture flasks at a density of 37°C, 5% CO2, and saturated humidity incubator;

[0085] (5) Medium change and passage: Change the medium for the first time after 48 h of culture to remove non-adherent cells; change the medium once every 2-3 days thereafter; when the cell confluence reaches 80%-90%, passage is performed: aspirate the culture medium, wash twice with PBS, add 3 mL of 0.25% trypsin-EDTA, and incubate at 37℃ for 3 min; after observing the cells become round and detached under a microscope, add 5 mL of basal culture medium to stop digestion; pipette to prepare a single-cell suspension, centrifuge at 1000g for 5 min, and passage to a new culture flask at a ratio of 1:3, and label it as P1 generation; take P3 generation cells for subsequent identification and exosome extraction.

[0086] 2.2 Identification of endometrial stem cells

[0087] 2.2.1 Flow cytometry detection of surface markers

[0088] (1) Cell preparation: Collect P3 generation logarithmic phase cells, wash twice with PBS, centrifuge at 1000g for 5min, and discard the supernatant; resuspend the cells in PBS and adjust the concentration to 1×10⁻⁶. 6 cells / mL;

[0089] (2) Antibody incubation: Add 500 μL of cell suspension to flow cytometry tubes, add 5 μL of the corresponding fluorescently labeled antibody (1:200 dilution) to each tube, and set up blank control tubes (without antibody); incubate at 4℃ in the dark for 30 min;

[0090] (3) Washing and detection: After incubation, add 3 mL PBS, centrifuge at 1000 g for 5 min, and discard the supernatant; repeat washing twice, and finally resuspend the cells in 500 μL PBS; detect by flow cytometry and analyze the proportion of positive cells using FlowJo software.

[0091] 2.2.2 Identification of Multidirectional Differentiation Potential

[0092] (1) Osteogenic differentiation: P3 generation cells were differentiated at a rate of 2×10⁻⁶. 4 cells / cm 2 The cells were seeded into 6-well plates. When the cell confluence reached 70%, the medium was replaced with osteogenic induction medium. The medium was changed every 3 days and the induction was continued for 21 days. After induction, the medium was aspirated and the cells were washed twice with PBS. The cells were fixed with 4% paraformaldehyde at room temperature for 30 min. The cells were washed three times with PBS, and Alizarin Red S staining solution was added. The cells were stained at room temperature for 20 min. The cells were washed three times with distilled water to remove residual staining solution. The formation of calcium nodules was observed under a microscope and photographed.

[0093] (2) Adipogenic differentiation: P3 generation cells were differentiated at a rate of 3 × 10⁻⁶. 4 cells / cm 2The cells were seeded into 6-well plates and, when the cell confluence reached 100% (24 hours after confluence), the medium was replaced with lipid-inducing medium. The medium was changed every 3 days and the induction was continued for 21 days. After induction, the cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 30 minutes. The cells were washed three times with PBS, and Oil Red O staining solution was added and stained at room temperature for 15 minutes. The cells were rinsed once with 60% isopropanol and twice with distilled water. The formation of lipid droplets was observed under a microscope and photographed.

[0094] 2.3 Extraction and purification of exosomes

[0095] (1) Conditioned medium collection: P3 generation endometrial stem cells were seeded into T175 culture flasks. When the cell confluence reached 80%, the basal medium was discarded and the cells were washed twice with PBS. 30 mL of serum-free medium was added and the cells were cultured for another 48 h. The culture supernatant (conditioned medium) was collected and labeled as “initial supernatant”.

[0096] (2) Differential centrifugation pretreatment:

[0097] Step 1: Centrifuge at 300g, 4℃ for 10 minutes to remove cell pellet;

[0098] Step 2: Take the supernatant, centrifuge at 2000g and 4℃ for 20 minutes to remove cell debris;

[0099] Step 3: Take the supernatant and filter it through a 0.22μm PES membrane to remove large vesicles and impurities;

[0100] (3) Crude extraction by ultracentrifugation: The filtered supernatant was transferred to a pre-cooled ultracentrifuge tube and centrifuged at 100,000g and 4℃ for 70 min; the supernatant was discarded and the precipitate was resuspended in 1 mL of pre-cooled PBS to obtain crude exosome extract;

[0101] (4) Sucrose density gradient purification:

[0102] Preparation of sucrose density gradient: Add 2 mL each of 60%, 40%, 20%, 10%, and 5% sucrose solutions sequentially from bottom to top into an ultracentrifuge tube to form a continuous density gradient;

[0103] Sample loading and centrifugation: Slowly spread 1 mL of crude exosome extract on the top layer of a sucrose gradient and centrifuge at 100,000 g and 4 °C for 16 h;

[0104] Component collection: After centrifugation, use a sterile syringe to collect 1 mL of each component from the bottom to the top of the tube, for a total of 10 tubes; use a BCA protein quantification kit to detect the protein concentration of each component, and collect the components corresponding to the peak protein concentration (tubes 4-6, corresponding to sucrose concentrations of 20%-40%).

[0105] (5) Washing and resuspension: The collected target fraction was diluted with 5 mL of pre-cooled PBS, centrifuged at 100000 g and 4 °C for 70 min, and the supernatant was discarded; the precipitate was resuspended in 200 μL of pre-cooled PBS and stored at -80 °C for later use, which is the purified endometrial stem cell exosome (ESC-Exos).

[0106] 2.4 Characterization of exosomes

[0107] 2.4.1 Nanoparticle Tracking Analysis (NTA): Take 20 μL of purified ESC-Exos, dilute it 100 times with sterile PBS, and vortex for 30 s to ensure uniform dispersion; turn on ZetaView PMX 110 and calibrate the instrument with 100 nm standard nanoparticles (Malvern); set the detection parameters: temperature 25℃, detection time 60 s / frame, for a total of 11 frames; inject the diluted sample into the detection cell, and the instrument automatically collects the particle movement trajectory. Analyze the particle size distribution, peak particle size, average particle size, and particle concentration using NTA software.

[0108] 2.4.2 Transmission Electron Microscopy (TEM) Observation: Take 10 μL of ESC-Exos suspension and drop it onto the surface of a copper mesh (200 mesh, carbon support film). Let it stand at room temperature for 5 min to allow the exosomes to be adsorbed onto the copper mesh. Use filter paper to absorb excess liquid, add 10 μL of 2% phosphotungstic acid (pH 7.0) staining solution, and negatively stain at room temperature for 5 min. Use filter paper to absorb the staining solution, and let the copper mesh air dry at room temperature for 30 min. Place the copper mesh into a JEM-1400Plus transmission electron microscope with an accelerating voltage of 80 kV, observe the morphology of exosomes at different magnifications (×20000, ×50000), and take representative images.

[0109] 2.4.3 Western Blot detection of marker proteins: Take 50 μL of ESC-Exos suspension, add 10 μL of 6×SDS loading buffer, boil in water for 10 min to lyse exosomes and denature proteins; determine protein concentration using the BCA method, and adjust the loading volume to 20 μg / lane; prepare 12%... SDS-PAGE gel was loaded with samples and electrophoresed at 80V for 30 min (stacking gel), followed by 120V for 90 min (separating gel). After electrophoresis, proteins were transferred to a PVDF membrane using a wet transfer method, and transferred at 100V for 90 min. The PVDF membrane was blocked with 5% skim milk blocking buffer at room temperature for 2 h. The membrane was washed three times with TBST (10 min each time). Primary antibodies CD63, TSG101, and Calnexin (diluted 1:1000) were added and incubated overnight at 4°C. The membrane was washed three times with TBST, and HRP-labeled secondary antibody (diluted 1:5000) was added and incubated at room temperature for 1 h. The membrane was washed three times with TBST, and ECL chemiluminescence solution was added. The band images were acquired using a ChemiDoc XRS+ imaging system, and the band gray values ​​were analyzed using ImageJ software.

[0110] 3. Experimental Results

[0111] 3.1 Results of culture and morphological observation of endometrial stem cells ( Figure 2 ): 24 hours after primary culture (P0), a small number of cells can be observed adhering to the wall, appearing as round or short spindle-shaped cells. After 48 hours, the number of adherent cells increases and their shape gradually changes to long spindle-shaped cells. After about 7 days, the cell confluence reaches 80%, and the first passage can be performed. After passage to P3, the cell morphology is uniform, showing typical fibroblast-like characteristics. The cells are arranged regularly and grow in a whorl or radial pattern, with no obvious contamination from other cells. The cell proliferation activity is good, and the cells can be passaged once every 3-4 days.

[0112] 3.2 Identification results of endometrial stem cells

[0113] 3.2.1 Flow cytometry results of surface markers showed that endometrial stem cells highly expressed positive markers of mesenchymal stem cells, with positive rates of CD90 (98.8%), CD105 (97.5%), and CD73 (99.3%), all higher than 95%, consistent with the expression characteristics of mesenchymal stem cell surface markers. Meanwhile, the positive rates of hematopoietic stem cell negative markers CD45 and CD34 were 1.5% and 0.7%, respectively, both below 2%, indicating no hematopoietic stem cell contamination. Furthermore, flow cytometry histograms showed a significant rightward shift of the fluorescence signal peaks of the positive markers, showing a significant difference from the blank control, while the signal peaks of the negative markers largely overlapped with the blank control. Figure 3 ).

[0114] 3.2.2 The results of multi-lineage differentiation potential identification showed that after 21 days of culture in osteogenic induction medium, Alizarin Red S staining revealed a large number of uniformly sized (50-100 μm in diameter) and widely distributed red calcium nodules, proving that they have osteogenic differentiation ability; after 21 days of culture in adipogenic induction medium, Oil Red O staining revealed a large number of plump red round lipid droplets in the cytoplasm, and some lipid droplets fused into large lipid droplets, indicating that they also have adipogenic differentiation ability.

[0115] 3.3 Results of exosome extraction and purification: After purification by differential centrifugation combined with sucrose density gradient, the protein concentration of the exosomes was 1.8 mg / mL according to BCA quantitative analysis. The results of sucrose density gradient component analysis showed that the protein concentration peak was concentrated in tubes 4-6 (corresponding to sucrose concentration of 20%-40%), which is the typical buoyancy density range of exosomes (1.13-1.19 g / mL), confirming that high-purity exosomes have been successfully isolated.

[0116] 3.4 Characterization results of exosomes

[0117] 3.4.1 NTA analysis showed that the exosomes exhibited a unimodal particle size distribution with a dispersion index (PDI) of 0.18 ± 0.03, indicating good particle size uniformity. The peak particle size was 116 ± 13 nm, the average particle size was 122 ± 8 nm, and the particle concentration was 6.1 × 10⁻⁶. 10 The particle size distribution was 80-150 nm, and the particle trajectories in the NTA spectrum were clear with no obvious aggregation. This perfectly matches the typical particle size characteristics of exosomes. Figure 4 ).

[0118] 3.4.2 TEM observations revealed numerous well-formed vesicle structures in the field of view, exhibiting typical saucer-like, biconcave disc-like, or cup-like shapes. The vesicles had clear boundaries and a double-membrane structure, with diameters primarily concentrated between 90-140 nm, consistent with NTA detection results. No significant large-sized impurities or aggregates were observed. Figure 5 ).

[0119] 3.4.3 Western blot results showed that the exosome positive marker proteins CD63 and TSG101 both exhibited clear and specific bands, with gray values ​​of 0.82±0.06 and 0.75±0.05, respectively, indicating stable expression levels. However, the endoplasmic reticulum protein (exosome negative marker) Calnexin showed no obvious band, with a gray value of only 0.05±0.01, indicating that the extracted exosomes were free of endoplasmic reticulum contamination and had good purity. Figure 6 ).

[0120] Example 3: Preparation and characterization of a targeted exosome formulation (Nb-EMX2-Exos)

[0121] 1. Experimental Materials and Reagents

[0122] 1.1 Core Raw Materials

[0123] (1) Endometrial stem cell exosomes (ESC-Exos): prepared in Example 2;

[0124] (2) Anti-EMX2 single-domain antibody (Nb-EMX2): prepared in Example 1;

[0125] (3) Auxiliary reagents: Traut's Reagent (2-iminothiacyclopentane hydrochloride), sulfonyl-SMCC (sulfosuccinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester), PBS buffer (pH 7.4, containing 1 mM EDTA);

[0126] 1.2 Characterization Reagents

[0127] (1) Flow cytometry antibody: Alexa Fluor 488-labeled anti-VHH single-domain antibody (self-prepared, titer 1:500 dilution);

[0128] (2) Western Blot reagents: Same as in Example 2 (CD63, TSG101, Calnexin primary antibody and HRP secondary antibody);

[0129] 2. Experimental Procedure

[0130] 2.1 Preparation of Targeted Exosome Formulation (Nb-EMX2-Exos)

[0131] 2.1.1 Exosome thiolation modification

[0132] (1) Sample pretreatment: Take 200 μL of ESC-Exos (containing 360 μg of protein, concentration 6.1 × 10⁻⁶) 10 (particles / mL), diluted to 500 μL with PBS buffer containing 1 mM EDTA to ensure that the system is free of free thiol interference;

[0133] (2) Thiolization reaction: Add Traut's Reagent (10 μL 50 mM stock solution) to a final concentration of 1 mM, gently vortex to mix, and incubate at room temperature (25°C) in the dark for 1 hour;

[0134] (3) Ultrafiltration purification: Transfer the reaction solution to a 100kDa ultrafiltration centrifuge tube, centrifuge at 10000g and 4℃ for 15min, and discard the filtrate; resuspend the precipitate in 500μL with PBS buffer containing 1mM EDTA, repeat ultrafiltration washing 3 times to remove unreacted Traut's Reagent, and collect thiolized exosomes (Exos-SH).

[0135] 2.1.2 Nb-EMX2 maleimide activation

[0136] (1) Antibody pretreatment: Take 100 μL of Nb-EMX2 (containing 120 μg of protein, the concentration was adjusted to 1.2 mg / mL) and dilute it to 200 μL with EDTA-free PBS buffer;

[0137] (2) Activation reaction: Calculated as 20 times molar excess, add sulfonyl-SMCC (molar ratio of Nb-EMX2 to sulfonyl-SMCC 1:20, i.e., add 18 μL of 10 mM stock solution), and incubate at room temperature in the dark for 30 min;

[0138] (3) Desalting and purification: Equilibrate the PD-10 desalting column with PBS buffer without EDTA (elution volume 10 mL), load the activation reaction solution onto the column bed, elute with PBS buffer, collect 1 mL of eluent from each tube, for a total of 10 tubes; determine the protein concentration of each tube by BCA method, collect the fraction corresponding to the peak protein concentration (tubes 4-6), obtain maleimide-activated Nb-EMX2 (Nb-Mal), and adjust the concentration to 0.5 mg / mL.

[0139] 2.1.3 Thiol-maleimide coupling reaction

[0140] (1) Construction of the conjugation system: According to the ratio of exosome protein to antibody protein of 3:1 (i.e., Exos-SH 360μg, Nb-Mal 120μg), 500μL of thiolized exosomes and 240μL of activated antibody were mixed, and PBS buffer containing 1mM EDTA was added to 1mL and gently pipetted to mix.

[0141] (2) Low temperature coupling: The mixture was placed in a constant temperature rotary apparatus at 4℃ (20 rpm) and rotated in the dark for 16 hours;

[0142] (3) Washing and purification: Transfer the reaction solution to a 100kDa ultrafiltration centrifuge tube, centrifuge at 10000g and 4℃ for 15min, and discard the filtrate; resuspend the precipitate in PBS buffer (pH 7.4) to 1mL, repeat ultrafiltration washing 3 times to remove uncoupled free Nb-EMX2; collect the final precipitate, resuspend it in 200μL PBS to obtain the targeted exosome preparation Nb-EMX2-Exos, and store it at -80℃ for later use.

[0143] 2.2 Characterization of targeted exosome formulations

[0144] 2.2.1 NTA detection of particle size and concentration: Take 20 μL of Nb-EMX2-Exos, dilute it 100 times with sterile PBS, and vortex for 30 s to ensure uniform dispersion; as in Example 2, calibrate ZetaView PMX 110 with 100 nm standard nanoparticles, and set the detection parameters (temperature 25℃, detection time 60 s / frame, acquisition 11 frames); after injecting the sample, the instrument automatically collects the particle motion trajectory, analyzes the particle size distribution, peak particle size, average particle size, dispersion index (PDI) and particle concentration, and compares it with the data of natural ESC-Exos.

[0145] 2.2.2 TEM Observation Morphology: Take 10 μL of Nb-EMX2-Exos suspension and drop it onto the surface of a 200-mesh carbon support film copper mesh. Let it stand at room temperature for 5 min. Absorb excess liquid with filter paper, add 10 μL of 2% phosphotungstic acid (pH 7.0) staining solution, and negatively stain at room temperature for 5 min. Absorb the staining solution with filter paper and let the copper mesh air dry at room temperature for 30 min. Place the copper mesh into a JEM-1400Plus transmission electron microscope, accelerate at 80 kV, and observe the morphology at ×20000 and ×50000 magnifications. Take representative images and compare the morphological differences of natural ESC-Exos.

[0146] 2.2.3 Western Blot detection of exosome biomarkers: 50 μL each of Nb-EMX2-Exos and natural ESC-Exos were added to 10 μL of 6×SDS loading buffer and incubated in boiling water for 10 min; protein concentration was determined by BCA method, and the loading volume was adjusted to 20 μg / lane; the process was the same as in Example 2 (12% SDS-PAGE gel, constant voltage electrophoresis, wet transfer, 5% skim milk blocking, primary antibody incubation at 4℃ overnight, secondary antibody incubation at room temperature for 1 h); ECL chemiluminescence solution was used for development, bands were acquired using the ChemiDoc XRS+ imaging system, and the gray values ​​of the bands of CD63, TSG101, and Calnexin were analyzed using ImageJ software to compare the expression differences between the two groups of biomarkers.

[0147] 2.2.4 Flow cytometry analysis of coupling efficiency: Nb-EMX2-Exos and native ESC-Exos were taken and their concentrations adjusted to 2×10⁻⁶ with PBS. 10For each particle / mL sample, add 200 μL of each sample to a flow cytometry tube; add 5 μL of Alexa Fluor 488-labeled anti-VHH antibody (1:500 dilution) to each tube, and do not add antibody to the blank control tube; incubate at 4°C in the dark for 30 min; add 3 mL of PBS, centrifuge at 10000g for 10 min, and discard the supernatant; repeat the washing twice, and finally resuspend the sample in 500 μL of PBS; detect by flow cytometry, excitation wavelength 488 nm, and collect green fluorescence signal; analyze the mean fluorescence intensity (MFI) of the two groups using FlowJo software.

[0148] 3. Experimental Results

[0149] 3.1 NTA analysis results showed that the particle size of Nb-EMX2-Exos exhibited a unimodal distribution with a dispersion index (PDI) of 0.21±0.04, indicating good particle size uniformity. The peak particle size was 127±17 nm, the average particle size was 135±11 nm, and the particle concentration reached 4.3×10⁻⁶. 10 particles / mL; compared with natural ESC-Exos (peak particle size 116±13nm, average particle size 122±8nm, concentration 6.1×10⁻⁶). 10 Compared to the previous method, there was no statistically significant difference in particle size (P>0.05). Although the concentration decreased slightly due to a small loss during ultrafiltration washing, it remained at a high level, confirming that the coupling reaction did not significantly change the physical dispersion characteristics of exosomes. Figure 7 ).

[0150] 3.2 TEM observation showed that Nb-EMX2-Exos retained the typical saucer-like and biconcave disc-like structure of exosomes, with clear vesicle boundaries, intact double membrane structure, and no rupture or aggregation. Its diameter was mainly concentrated in the 100-160 nm range, consistent with NTA detection results, and showed no significant difference in morphology from natural ESC-Exos, indicating that the coupling process did not disrupt the structural integrity of the exosomes. Figure 8 ).

[0151] 3.3 Western Blot results showed that the Nb-EMX2-Exos group exhibited clear and specific bands for the exosome positive markers CD63 and TSG101, with gray values ​​of 0.79±0.07 and 0.72±0.06, respectively, which were not significantly different from the natural ESC-Exos group (CD63 gray value 0.82±0.06, TSG101 gray value 0.75±0.05). However, the negative marker Calnexin showed no obvious bands in either group (gray values ​​<0.06), indicating that after Nb-EMX2 conjugation, the characteristic markers of exosomes were expressed stably, without the introduction of endoplasmic reticulum contamination, maintaining the inherent protein expression characteristics of exosomes. Figure 6 ).

[0152] 3.4 Flow cytometry analysis of coupling efficiency results showed that ( Figure 9 The MFI of the blank control tube was 326±45, the MFI of the natural ESC-Exos group was 1245±167, and the MFI of the Nb-EMX2-Exos group was 15892±1358, which means that the fluorescence signal of Nb-EMX2-Exos was 12.8 times that of natural ESC-Exos. The flow cytometry histogram showed that the fluorescence signal peak of the Nb-EMX2-Exos group was significantly shifted to the right, which was significantly different from that of the natural ESC-Exos group, confirming that Nb-EMX2 was efficiently coupled to the surface of exosomes.

[0153] Example 4: In vitro targeting and functional verification

[0154] 1. Experimental Materials and Reagents

[0155] 1.1 Cells and Core Raw Materials

[0156] (1) Human endometrial stromal cells (hESCs): qualified (no mycoplasma contamination, expressing the endometrial stromal cell marker Vimentin);

[0157] (2) Targeted / Natural Exosomes: Nb-EMX2-Exos (prepared in Example 3, concentration 4.3 × 10⁻⁶) 10 particles / mL), ESC-Exos (prepared in Example 2, concentration 6.1 × 10⁻⁶) 10 (particles / mL)

[0158] (3) Damage induction reagents: recombinant human TGF-β1 (10 ng / mL stock solution), H2O2 (100 mM stock solution).

[0159] 1.2 Experimental Reagents

[0160] (1) Cell culture medium: DMEM / F12 (Gibco) + 10% FBS (Gibco) + 1% antibiotics;

[0161] (2) Reagents: DiI cell membrane dye (1 mg / mL stock solution), DAPI staining solution (1 μg / mL working solution); CCK-8 kit; EMX2 primary antibody (Abcam, 1:1000 dilution), HRP-labeled secondary antibody (goat anti-rabbit, Abcam, 1:5000 dilution); PBS buffer (pH 7.4), trypsin-EDTA.

[0162] 2. Experimental Procedure

[0163] 2.1 Cell pretreatment and validation of EMX2 upregulation

[0164] (1) hESCs culture: hESCs were inoculated into T75 culture flasks and cultured in DMEM / F12 medium containing 10% FBS. The medium was changed every 2-3 days and passaged to P3-P5 generation for experiments.

[0165] (2) EMX2 up-regulation: Take P4 instead of hESCs, and adjust according to 5×10 5 Cells were seeded into 6-well plates and cultured for 24 hours until adherence. The medium was then replaced with medium containing 10 ng / mL TGF-β1, and cultured for another 24 hours to construct a damage-mimicking cell model with high EMX2 expression (hESCs-EMX2). + The control group (untreated hESCs) was cultured in a medium without TGF-β1.

[0166] (3) EMX2 upregulation verification (Western Blot): Two groups of cells were collected, total protein was extracted, and after BCA quantification, samples were loaded (20 μg / lane), followed by 10% SDS-PAGE electrophoresis, membrane transfer, and blocking. EMX2 primary antibody (incubated overnight at 4℃) and secondary antibody (incubated at room temperature for 1 h) were added. ECL was used for imaging, and the gray values ​​of the bands were analyzed using ImageJ to calculate hESCs-EMX2. + Relative expression level of EMX2 in the group.

[0167] 2.2 Cellular uptake assay (DiI labeling method)

[0168] 2.2.1 DiI-labeled exosomes: Take 100 μL each of Nb-EMX2-Exos and ESC-Exos (containing 4.3 × 10⁻⁶ ppm of each). 9 particles and 6.1×10 9 Add DiI dye to the particles to a final concentration of 5 μM, gently vortex to mix, and incubate at 37°C in the dark for 30 min. Wash three times with a 100 kDa ultrafiltration centrifuge tube (10000 g, 4°C, 15 min) to remove free DiI dye, and resuspend in PBS to 100 μL to obtain DiI-Nb-EMX2-Exos and DiI-ESC-Exos, which can be used immediately.

[0169] 2.2.2 Cell co-incubation and detection

[0170] (1) Take hESCs-EMX2 + Cells, at 2×10 5 Cells / wells were seeded into 6-well plates (for flow cytometry) and confocal culture dishes (for imaging) and cultured for 24 hours to adhere to the plates;

[0171] (2) Group processing:

[0172] PBS group: Add 100 μL of PBS;

[0173] Natural Exos group: 100 μL DiI-ESC-Exos was added (final concentration 4.3 × 10⁻⁶). 7 (particles / mL, consistent with the number of particles in the target group);

[0174] Nb-EMX2-Exos group: 100 μL of DiI-Nb-EMX2-Exos was added (final concentration 4.3 × 10⁻⁶). 7 (particles / mL)

[0175] (3) Co-incubation: Incubate at 37℃ and 5% CO2 for 4 hours;

[0176] (4) Flow cytometry detection: Collect cells from 6-well plates, wash twice with PBS, digest with 0.25% trypsin, centrifuge at 1000g for 5 min, and resuspend in PBS to 500 μL; detect by flow cytometry, and analyze the percentage of DiI positive cells (n=4) using FlowJo software.

[0177] (5) Confocal microscopy observation: Cells in confocal culture dishes were washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 15 min, incubated with DAPI staining solution at room temperature for 5 min, and washed three times with PBS. The cells were observed and photographed using a laser confocal microscope (excitation wavelength: DiI 543 nm, DAPI 405 nm), and the intracellular DiI fluorescence intensity was analyzed using ImageJ.

[0178] 2.3 Scratch healing experiment (verification of migration-promoting function)

[0179] (1) Construction of cell damage model: hESCs were prepared at a ratio of 3×10 5 Cells were seeded into 6-well plates and cultured for 24 hours until adherence. Then, the medium was replaced with a medium containing 200 μM H2O2 and incubated at 37°C for 2 hours to induce cell damage.

[0180] (2) Scratch preparation: Discard the H2O2 culture medium, wash twice with PBS, and use a sterile 200μL pipette tip to draw straight lines perpendicular to the bottom of the well (3 parallel scratches per well). Wash gently twice with PBS to remove exfoliated cells.

[0181] (3) Group processing:

[0182] PBS group: Add 2 mL of serum-free DMEM / F12 + 100 μL of PBS;

[0183] Natural Exos group: Add 2 mL of serum-free DMEM / F12 + 100 μL of ESC-Exos (final concentration 2.1 × 10⁻⁶) 7(particles / mL)

[0184] Nb-EMX2-Exos group: Add 2 mL of serum-free DMEM / F12 + 100 μL of Nb-EMX2-Exos (final concentration 2.1 × 10⁻⁶). 7 (particles / mL)

[0185] (4) Photographing and healing rate calculation: At 0h, an inverted microscope (10× objective lens) was used to take three field-of-view photos at the same position of each well scratch; after culturing at 37℃ and 5% CO2 for 24h, the same position was photographed again; the scratch width was measured using ImageJ software and the healing rate was calculated: Healing rate (%) = [(initial scratch width - 24h scratch width) / initial scratch width] × 100% (n=6).

[0186] 2.4 CCK-8 cell proliferation experiment

[0187] (1) Cell seeding and damage induction: hESCs were seeded at a rate of 5 × 10⁻⁶. 3 Cells / wells were seeded into 96-well plates, cultured for 24 h, and then treated with 200 μM H2O2 for 2 h to construct a damage model.

[0188] (2) Group treatment: Similar to the scratch healing experiment, each group was set up with 6 replicates, and the corresponding reagents were added and cultured for a longer period of time;

[0189] (3) Absorbance detection: After culturing for 24h and 48h, 10μL of CCK-8 reagent was added to each well and incubated at 37℃ for 2h; the absorbance value at OD450nm was detected by microplate reader, and the cell proliferation activity was evaluated with PBS group as control (n=6).

[0190] (4) Data correction: Subtract the OD value of blank wells (medium culture + CCK-8 only) to ensure the accuracy of the results.

[0191] 3. Experimental Results

[0192] 3.1 EMX2 Upgrade Verification Results

[0193] Western blot analysis showed that ( Figure 10 hESCs-EMX2 after TGF-β1 treatment + In the treatment group, the gray value of the EMX2 protein band was 0.87±0.09, which was significantly higher than that in the untreated group (0.12±0.03), and the relative expression level increased by 7.25 times (P<0.001), confirming that TGF-β1 successfully induced the upregulation of EMX2 expression in hESCs, and the damage simulation cell model was successfully constructed.

[0194] 3.2 Results of Cell Uptake Experiment

[0195] Flow cytometry ( Figure 11 Quantitative analysis showed that the percentage of DiI-positive cells in the PBS group was 2.5±0.5% (background signal), in the natural Exos group it was 26.8±3.5%, and in the Nb-EMX2-Exos group it reached 71.4±6.2%. Statistical analysis showed that the uptake efficiency of the Nb-EMX2-Exos group was significantly higher than that of the natural Exos group (P<0.001), which was 2.67 times higher.

[0196] Confocal microscopy revealed that DAPI staining showed blue cell nuclei and red DiI-labeled exosomes; the PBS group showed almost no red fluorescence, the natural Exos group showed a few scattered red signals in cells, while the Nb-EMX2-Exos group showed significantly enhanced red fluorescence intensity, concentrated in the cytoplasm, consistent with flow cytometry results, confirming that the targeted agent can be activated by hESCs-EMX2. + Highly efficient and specific uptake.

[0197] 3.3 Results of scratch healing experiment

[0198] At 0 h, there was no significant difference in scratch width among the three groups (mean width 450±20 μm); after 24 h, the scratch healing rate in the PBS group was 29.5±4.1%, and the scratch was still relatively wide (remaining width 318±25 μm); the healing rate in the natural Exos group was 52.1±5.3%, and the scratch width narrowed to 216±22 μm; the healing rate in the Nb-EMX2-Exos group reached 80.3±7.5%, and the scratch was basically closed (remaining width 88±15 μm); the intergroup comparison showed that the healing rate of the Nb-EMX2-Exos group was significantly higher than that of the natural Exos group (P<0.01) and the PBS group (P<0.001), confirming that the targeted agent has a better ability to promote cell migration.

[0199] 3.4 Results of CCK-8 cell proliferation experiment

[0200] At 24 h: the OD450 values ​​were 0.32±0.05 in the PBS group, 0.48±0.06 in the natural Exos group, and 0.69±0.08 in the Nb-EMX2-Exos group; the proliferation activity of the Nb-EMX2-Exos group was significantly higher than that of the other two groups (P<0.01).

[0201] At 48 h: the OD450 values ​​were 0.41±0.06 in the PBS group, 0.65±0.07 in the natural Exos group, and 0.94±0.09 in the Nb-EMX2-Exos group; the proliferation activity of the Nb-EMX2-Exos group continued to increase, being 2.29 times that of the PBS group and 1.45 times that of the natural Exos group (P<0.001).

[0202] The results showed that the proliferative effect of the targeted formulation was more significant and superior to that of natural exosomes as the culture time was extended.

[0203] 4. Results Summary: This experiment systematically verified the in vitro targeting and functional activity of Nb-EMX2-Exos by constructing a damaged endometrial stromal cell model with high EMX2 expression. The core conclusions are as follows:

[0204] 4.1 Targeted approach significantly improves uptake efficiency: TGF-β1 successfully induced hESCs to upregulate EMX2 expression (relative expression level increased by 7.25 times), while Nb-EMX2-Exos, with the help of the anti-EMX2 single-domain antibody on its surface, achieved specific recognition of target cells, with an uptake efficiency of 71.4±6.2%, which is 2.67 times that of natural exosomes. Flow cytometry and confocal microscopy experiments have confirmed this targeting advantage.

[0205] 4.2 Functional activity is superior to natural exosomes in all aspects: In the scratch healing experiment, the healing rate of Nb-EMX2-Exos group reached 80.3±7.5% at 24h, which was significantly higher than that of natural exosomes (52.1±5.3%); CCK-8 experiment showed that its proliferation activity (OD450=0.94±0.09) at 48h was 1.45 times that of natural exosomes, indicating that efficient uptake directly translates into better migration and proliferation promotion effects;

[0206] 4.3 Clear mechanism and outstanding application potential: The targeted formulation exhibits excellent repair potential in the damaged endometrial cell model through the pathway of "EMX2 specific binding - efficient cellular uptake - enhanced repair function", which solves the problems of poor targeting and low efficiency of natural exosomes.

[0207] In summary, this experiment demonstrates that Nb-EMX2-Exos not only possesses highly efficient targeting recognition capabilities but also significantly promotes the migration and proliferation of damaged endometrial stromal cells. This provides solid in vitro data support for subsequent in vivo endometrial injury repair experiments and clinical translation studies, and has significant application value.

[0208] Example 5: Evaluation of in vivo targeting and therapeutic efficacy

[0209] 1. Experimental Materials and Reagents

[0210] 1.1 Experimental animals: Female SD rats, 8 weeks old, weighing 200-220g (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.); Male SD rats, 10 weeks old, weighing 250-280g, used for fertility assessment, purchased from the same batch.

[0211] 1.2 Core Raw Materials and Reagents

[0212] (1) Targeted / Natural Exosomes: Nb-EMX2-Exos (prepared in Example 3, concentration 4.3 × 10⁻⁶) 10 particles / mL), ESC-Exos (prepared in Example 2, concentration 6.1 × 10⁻⁶) 10 (particles / mL)

[0213] (2) Labeling reagents: DIR near-infrared fluorescent dye (5 mg / mL stock solution), sterile physiological saline (0.9% NaCl);

[0214] (3) Histological reagents: 4% paraformaldehyde, paraffin embedding kit, H&E staining kit, Masson trichrome staining kit;

[0215] (4) Anesthesia and surgical reagents: 10% chloral hydrate (3 mL / kg body weight), iodine, alcohol swabs, sterile surgical instrument pack; PBS buffer (pH 7.4), disposable sterile syringes (1 mL, 5 mL).

[0216] 2. Experimental Procedure

[0217] 2.1 Establishment of a rat model of mechanical curettage of the uterine lining

[0218] (1) Preoperative preparation: Female SD rats were acclimatized for 1 week (ambient temperature 22±2℃, humidity 50±5%, 12h light-dark cycle, free access to food and water); fasting for 12h and water restriction for 4h before surgery;

[0219] (2) Anesthesia and fixation: 10% chloral hydrate was injected intraperitoneally at 3 mL / kg body weight to anesthetize rats. After the corneal reflex disappeared, the rats were fixed supine on a sterile operating table. After the hair on the abdomen was removed, the rats were disinfected with povidone-iodine three times and then with alcohol twice.

[0220] (3) Surgical procedure: Make a 1cm longitudinal incision 1cm to the left of the midline of the abdomen, and separate the skin, subcutaneous fat and abdominal muscles layer by layer to expose the uterus; cut the mesentery of the uterus with sterile ophthalmic scissors and free the bilateral uterine horns; use a No. 4 sterile curette to insert into the uterine horn and scrape the endometrium repeatedly from the fundus to the cervix 3 times until the endometrial surface is rough and there is a small amount of bleeding (simulating clinical mechanical injury).

[0221] (4) Postoperative care: The abdominal cavity was rinsed with saline, and the abdominal muscles, subcutaneous tissue and skin were sutured layer by layer. The incision was disinfected with povidone-iodine. The rats were placed in a warm environment for recovery. For the first 3 days after the operation, penicillin (50,000 U / rat) was injected into the peritoneum daily to prevent infection. The rats were then fed in a routine manner.

[0222] 2.2 Experimental grouping and drug administration

[0223] (1) Grouping: After successful model establishment, rats were randomly divided into 3 groups (n=6 / group), and a normal control group (no model established, n=3) was set up:

[0224] PBS group: 100 μL of sterile PBS was injected via the tail vein;

[0225] Natural Exos group: 100 μL of ESC-Exos (containing 4.3 × 10⁻⁶) was injected via tail vein. 9 (particles, consistent with the number of particles in the target group);

[0226] Nb-EMX2-Exos group: 100 μL of Nb-EMX2-Exos (containing 4.3 × 10⁻⁶) was injected via the tail vein. 9 particles);

[0227] (2) Timing of administration: Administer the drug on the 3rd day after surgery, once a week for 4 consecutive weeks (4 times in total). The normal control group was not given the drug and was fed in a routine manner.

[0228] 2.3 In vivo targeted imaging (small animal in vivo imaging)

[0229] (1) Exosome DIR labeling: Take 200 μL each of Nb-EMX2-Exos and ESC-Exos (containing 8.6 × 10⁻⁶ ppm each). 9 Particles), add DIR dye to a final concentration of 10 μM, incubate at 37 °C in the dark for 40 min; wash 3 times in a 100 kDa ultrafiltration centrifuge tube (10000 g, 4 °C for 15 min) to remove free dye, and resuspend in sterile physiological saline to 200 μL to obtain DIR-Nb-EMX2-Exos and DIR-ESC-Exos;

[0230] (2) Imaging grouping and injection: Eight more model rats were randomly divided into two groups (n=4 / group), and each group was injected with 100 μL of DIR-ESC-Exos or DIR-Nb-EMX2-Exos (containing 4.3×10⁻⁶ ppm) via the tail vein. 9 particles);

[0231] (3) Imaging detection: 24 hours after injection, rats were anesthetized with 10% chloral hydrate and placed in a small animal in vivo imaging system. The parameters were set as follows: excitation wavelength 745nm, emission wavelength 820nm, exposure time 30s, and whole-body fluorescence images were collected.

[0232] (4) Analysis: The uterine region (region of interest, ROI) was delineated using Living Image software, and the average fluorescence intensity (Average Radiance, unit: photosns / s / cm) of this region was measured. 2 / sr×108 The targeted enrichment effects of the two groups were compared.

[0233] 2.4 Histological assessment (28 days post-treatment)

[0234] (1) Sample collection: On the 28th day after the end of administration, rats were anesthetized with 10% chloral hydrate and euthanized by cervical dislocation; the bilateral uteruses were quickly dissected and separated, rinsed with PBS, and the surrounding connective tissue was removed;

[0235] (2) Fixation and embedding: Left uterine tissue was taken and fixed in 4% paraformaldehyde at room temperature for 24 h; gradient dehydration (70%, 80%, 90%, 95%, 100% ethanol), clearing (xylene), paraffin embedding, and 5 μm continuous sections were prepared.

[0236] (3) H&E staining and endometrial thickness measurement: The sections were dewaxed to water, stained with hematoxylin for 5 min, differentiated with hydrochloric acid ethanol for 30 s, stained with eosin for 2 min, dehydrated in a gradient, cleared, and mounted; observed under an inverted microscope (20× objective lens), 10 complete endometrial fields were randomly selected from each section, and the vertical distance from the endometrial cavity epithelium to the myometrium junction was measured using ImageJ software. The average value was taken as the endometrial thickness of the rat.

[0237] (4) Masson staining and calculation of fibrosis area: Dewax the sections to water, stain with hematoxylin for 10 min, differentiate with hydrochloric acid ethanol for 1 min, stain with Ponceau S and acid fuchsin for 10 min, treat with phosphomolybdic acid solution for 5 min, stain with aniline blue for 5 min, dehydrate in gradient, clear, and mount; observe under an inverted microscope (20× objective lens), randomly select 5 fields of view for each section, analyze the ratio of the area of ​​blue fibrosis area to the total area of ​​the field of view using ImageJ software, and calculate the percentage of fibrosis area.

[0238] 2.5 Fertility Assessment

[0239] (1) Co-cage experiment: On the 28th day after the drug administration ended, the three groups of treated rats (n=6 / group) were co-caged with healthy male SD rats (female-to-male ratio 2:1), and the normal control group (n=3) were co-caged simultaneously;

[0240] (2) Observation of conception: After the rats were put into the same cage, the formation of vaginal plugs was observed every day and the conception time was recorded. Four weeks after the rats were put into the same cage, the pregnant rats were sacrificed by cervical dislocation, the uterus was dissected and separated, and the number of embryos in each rat was counted.

[0241] (3) Statistical analysis: Calculate the pregnancy rate (number of pregnant rats / total number of rats in the group × 100%) and the average number of embryos (total number of embryos of all pregnant rats in the group / number of pregnant rats) for each group.

[0242] 3. Experimental Results

[0243] 3.1 In vivo targeted imaging results

[0244] Fluorescence distribution: 24 hours after injection, strong near-infrared fluorescence signals appeared in the uterine region of rats in the Nb-EMX2-Exos group. The fluorescence was concentrated in the damaged uterine area, while the fluorescence signals in other organs (such as liver and kidney) were weak. The fluorescence signal in the uterine region of rats in the natural Exos group was weaker, and the fluorescence distribution throughout the body was more dispersed. Obvious non-specific enrichment was observed in the liver and kidney.

[0245] Quantitative analysis: The average fluorescence intensity in the uterine region of the Nb-EMX2-Exos group was 6.1 ± 0.8 × 10⁻⁶. 8 The photons / s / cm² / sr ratio was significantly higher than that of the natural Exos group (1.8 ± 0.4 × 10⁻⁶). 8 photons / s / cm 2 / sr (P<0.001), which is 3.39 times higher, confirming that Nb-EMX2-Exos can efficiently target and accumulate in damaged uterine tissue, demonstrating significant advantages in targeted delivery.

[0246] 3.2 Histological repair results

[0247] 3.2.1 Endometrial thickness: The endometrial thickness in the normal control group was 510±35μm; in the PBS group it was only 190±18μm, with significantly thinner endometrium, sparse glands, and disordered structure; the endometrial thickness in the natural Exos group was 320±30μm, significantly thicker than the PBS group (P<0.01), but still lower than the normal control group; the endometrial thickness in the Nb-EMX2-Exos group reached 472±41μm, significantly higher than the PBS group (P<0.001) and the natural Exos group (P<0.01), close to the level of the normal control group, with dense and regularly arranged glandular structure and intact epithelial cells, confirming that the targeted agent can effectively promote the structural repair of the endometrium.

[0248] 3.2.2 Uterine wall fibrosis area: The percentage of uterine wall fibrosis area in the normal control group was 5.2±0.8%; in the PBS group it was as high as 37.2±3.2%, with a large amount of blue fibrotic tissue deposited at the junction of the myometrium and endometrium; the percentage of fibrosis area in the natural Exos group was 22.8±2.5%, which was significantly lower than that in the PBS group (P<0.01); the percentage of fibrosis area in the Nb-EMX2-Exos group was only 11.5±1.7%, which was significantly lower than that in the PBS group (P<0.001) and the natural Exos group (P<0.01), with a significant reduction in fibrotic tissue and loose tissue arrangement, confirming that the targeted agent can effectively inhibit uterine fibrosis and improve the tissue microenvironment.

[0249] 3.3 Fertility recovery results

[0250] Pregnancy rate: None of the 6 rats in the PBS group became pregnant, with a pregnancy rate of 0%; 3 rats in the natural Exos group became pregnant, with a pregnancy rate of 50.0%; all 6 rats in the Nb-EMX2-Exos group became pregnant, with a pregnancy rate of 100%, which was consistent with the normal control group (100%).

[0251] Mean number of embryos: The mean number of embryos in the natural Exos group was 5.5±1.1, which was significantly lower than that in the normal control group (12.3±1.5); the mean number of embryos in the Nb-EMX2-Exos group was 10.8±1.4, which was significantly higher than that in the natural Exos group (P<0.01) and close to that in the normal control group. This confirms that the targeted agent can not only repair the endometrial structure, but also completely restore its fertility.

[0252] 4. Results Summary: This experiment systematically verified the in vivo targeting and therapeutic effect of Nb-EMX2-Exos using an SD rat endometrial mechanical curettage model. The core conclusions are as follows:

[0253] 4.1 Significant in vivo targeted enrichment effect: DIR near-infrared imaging confirmed that Nb-EMX2-Exos achieves efficient targeted delivery by specifically binding to EMX2 highly expressed in damaged uterine tissue through surface single-domain antibody. The fluorescence intensity in the uterine region is 3.39 times that of natural exosomes, effectively solving the problems of dispersed distribution and poor targeting of natural exosomes in vivo.

[0254] 4.2 Outstanding advantages in structural repair: 28 days after treatment, the endometrial thickness in the Nb-EMX2-Exos group reached 472±41μm, which was 47.5% higher than that in the natural exosome group, and the percentage of fibrosis area decreased to 11.5±1.7%, which was only 50.4% of that in the natural exosome group. This indicates that the targeted agent can more effectively promote endometrial thickening, inhibit fibrosis, and restore normal tissue structure.

[0255] 4.3 Complete and stable functional recovery: Fertility assessment showed that the Nb-EMX2-Exos group had a 100% pregnancy rate and an average number of embryos of 10.8±1.4, which was completely restored to the level of normal rats. In contrast, the natural exosome group had a pregnancy rate of only 50% and a significantly lower number of embryos. This confirms that the therapeutic effect of the targeted agent has been extended from structural repair to functional recovery, and has the core value for clinical translation.

[0256] In summary, Nb-EMX2-Exos achieves a closed-loop therapeutic effect of "precise targeted enrichment - efficient structural repair - complete functional recovery" in vivo, which is significantly superior to natural exosomes. It provides a novel targeted formulation for the clinical treatment of infertility related to endometrial damage and has significant clinical application prospects.

[0257] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A specific anti-EMX2 single-domain antibody, characterized in that, The specific anti-EMX2 single-domain antibody is Nb-EMX2, and its amino acid sequence is shown in SEQ ID NO:

2.

2. A targeted exosome complex, characterized in that, The complex is Nb-EMX2-Exos, which comprises the specific anti-EMX2 single-domain antibody of claim 1 and exosomes derived from endometrial stem cells, wherein the single-domain antibody is covalently linked to the surface of the exosomes.

3. The targeted exosome complex according to claim 2, characterized in that, The peak particle size of the exosomes derived from endometrial stem cells was 116±13 nm, the average particle size was 122±8 nm, and the particle concentration was 6.1×10⁻⁶. 10 particles / mL.

4. The targeted exosome complex according to claim 2, characterized in that, The Nb-EMX2-Exos particles had a peak size of 127±17 nm, an average particle size of 135±11 nm, and a particle concentration of 4.3×10⁻⁶. 10 particles / mL.

5. A method for preparing the targeted exosome complex as described in claim 2, characterized in that, The method includes the following steps: (1) Prepare the specific anti-EMX2 single-domain antibody as described in claim 1; (2) Preparation of exosomes derived from the endometrial stem cells; (3) The exosomes are modified by thiolation to obtain thiolated exosomes; (4) The single-domain antibody is activated with maleimide to obtain maleimide-activated antibody; (5) The thiolized exosomes are coupled with the maleimide-activated antibody to form the targeted exosome complex; the coupling reaction is carried out at 4°C for 16 hours.

6. The preparation method according to claim 5, characterized in that, The single-domain antibody described in step (1) was obtained by immunizing alpacas with EMX2 protein and constructing a phage display library, followed by multiple rounds of biological panning and expression purification.

7. The preparation method according to claim 5, characterized in that, The thiolization modification described in step (3) is achieved using 2-iminothionecyclopentane hydrochloride.

8. The preparation method according to claim 5, characterized in that, The maleimide activation described in step (4) is achieved using sulfosuccinimide-4-maleimide-methylcyclohexane-1-carboxylic acid ester.

9. A pharmaceutical composition, characterized in that, The composition comprises a targeted exosome complex as described in claim 2 and a pharmaceutically acceptable carrier.

10. Use of the targeted exosome complex as described in claim 2 in the preparation of a medicament for treating endometrial injury.

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