Method for preparing stem cell exosome and use of exosome prepared by the method in treating male erectile dysfunction

Abstract

The application discloses a kind of stem cell exosome based on targeted fusion polypeptide modification, preparation method and its application in treating male erectile dysfunction.The exosome is anchored on the surface by chemical coupling technology with specific targeted fusion polypeptide (sequence: EPLQLKM-GGGGS-DPPV), which enhances its targeting to cavernosal smooth muscle cells;At the same time, by loading anti-TrkA monoclonal antibody (TrkA-2E7-HL) through electroporation method, the fibrosis pathway mediated by nerve growth factor (NGF) is inhibited.The exosome is induced by antioxidant culture medium to induce umbilical cord mesenchymal stem cells to secrete, which significantly improves its antioxidant activity.Experiments show that the erectile function (ICP / MAP ratio) of the exosome after modification is increased to 0.79±0.05 in a diabetic ED rat model, the fibrosis area is reduced to 9.2±1.6%, and the NO secretion level is increased by 3.5 times compared with ordinary exosome.The application realizes effective reversal of ED pathological process through polypeptide targeting, antibody synergy and antioxidant triple mechanism, and has significant novelty and clinical transformation potential.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a stem cell exosome preparation method and application of exosomes prepared by the method in the treatment of male erectile dysfunction. BACKGROUND

[0002] Male erectile dysfunction (ED) is one of the important diseases affecting global male health. According to statistics, the number of patients worldwide has exceeded 150 million and is showing a rising trend. ED not only seriously affects the quality of life and mental health of patients, but also increases the difficulty of treatment due to its complex causes. Among them, diabetic ED and neurogenic ED are the most challenging types, and their pathological basis includes multiple mechanisms such as fibrosis of penile cavernosal smooth muscle fibers, chronic oxidative stress response, and nerve terminal damage. Current clinical treatment methods are difficult to effectively reverse these tissue and functional damage.

[0003] The current main clinical treatment method is phosphodiesterase type 5 inhibitor, such as Sildenafil and Tadalafil, which acts by improving blood flow to improve erectile function in the short term. However, this type of drug has limited repair effect on tissue damage, and the effective rate for diabetic-related ED patients is less than 50%, and its efficacy is limited by endothelial dysfunction, nerve damage, and abnormal local microenvironment. Therefore, there is an urgent need for a new treatment strategy with repair and targeting properties to break through the existing treatment bottleneck.

[0004] In recent years, stem cell-derived exosomes (Exosomes) have become a research hotspot in the field of regenerative medicine due to their natural abundance of miRNA, protein, lipid, and other bioactive components, as well as their good biocompatibility and low immunogenicity. Related studies have shown that exosomes can mediate cell-to-cell information transfer, regulate the local microenvironment, and promote tissue repair. For example, some literature reports that mesenchymal stem cell (MSC)-derived exosomes can improve ischemic myocardial injury by delivering miR-21, but this technology has problems such as insufficient targeting and easy systemic clearance of exosomes during in vivo delivery, resulting in limited treatment efficiency.

[0005] In addition, existing technologies mostly use genetic engineering methods (such as overexpression of specific membrane proteins or regulation of miRNA expression) to functionally modify exosomes, but such methods generally have problems such as complicated operation, high production cost, and difficulty in clinical translation, which limit their widespread application in disease treatment. Therefore, it is necessary to develop a new treatment method. SUMMARY

[0006] Based on the deficiencies of the prior art, the present application proposes a "chemical-biological combined modification" strategy for constructing exosomes to achieve the triple breakthroughs of precise targeting, synergistic regulation and low-cost preparation. The core innovations include:

[0007] 1. Precise targeting mechanism construction: by designing a fusion polypeptide containing a CXCR4 receptor specific recognition sequence and a transmembrane anchor domain, it is efficiently coupled with the surface of the exosome membrane, improving its enrichment ability in the area of penile cavernosal smooth muscle cells, thereby improving the delivery efficiency and local drug efficacy;

[0008] 2. Multi-mechanism synergistic therapy: synergistically loading anti-TrkA monoclonal antibodies in exosomes to block the NGF / TrkA signaling axis and inhibit neurotrophic factor-induced fibrosis; at the same time, using endogenous antioxidant components (such as SOD2 and Catalase) in exosomes to scavenge excess reactive oxygen species (ROS), achieving synergistic intervention on fibrosis and oxidative stress, thereby fundamentally improving the pathological state of ED;

[0009] 3. Low-cost and high-efficiency preparation system: using chemical coupling method to combine functional polypeptides with exosome membrane components, replacing traditional gene editing or viral vector system, avoiding cell strain screening and long-term stable expression process, significantly simplifying the preparation process, suitable for large-scale and standardized pharmaceutical production.

[0010] Therefore, in one aspect, the present application discloses a targeted modified stem cell exosome, which comprises the following elements:

[0011] (1) The exosome surface is covalently coupled with a maleimide-thiol targeting fusion polypeptide, and the amino acid sequence of the targeting fusion polypeptide is EPLQLKM-GGGGS-DPPV;

[0012] (2) The exosome interior is loaded with anti-TrkA monoclonal antibodies, and the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TrkA monoclonal antibodies are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively;

[0013] (3) The exosome is derived from umbilical cord mesenchymal stem cells cultured with antioxidants, wherein the antioxidants are baicalin 50 μM, quercetin 20 μM, and N-acetylcysteine 5 mM.

[0014] Preferably, the particle size of the exosome of the present application is 130.6 ± 3.6 nm.

[0015] Preferably, the anti-TrkA monoclonal antibody of the present application is obtained after immunizing mice with a recombinant TrkA-ECD protein, followed by hybridoma cell fusion and cloning screening, antibody gene sequencing and expression and purification, wherein the amino acid sequence of the recombinant TrkA-ECD protein is shown as SEQ ID NO. 1.

[0016] In one aspect, the present application also discloses a method of the exosome, comprising the following steps:

[0017] (1) Culturing umbilical cord mesenchymal stem cells in a culture medium containing 50 μM baicalin, 20 μM quercetin and 5 mM N-acetylcysteine for 72 hours, and purifying exosomes by ultracentrifugation combined with size exclusion chromatography;

[0018] (2) Mixing DSPE-PEG2000-Mal and the target fusion polypeptide at a molar ratio of 1:1.5, and then reacting in PBS for 4 hours to obtain a coupling product, and then co-incubating with the exosomes for 2 hours to complete the surface modification;

[0019] (3) Loading the anti-TrkA antibody into the exosomes by electroporation, wherein the voltage is 150 V, the pulse time is 10 ms, and the pulse number is 3.

[0020] In one aspect, the present application also discloses a use of the exosome in the preparation of a medicament for treating diabetic or nerve injury-induced erectile dysfunction.

[0021] Preferably, the medicament of the present application is administered by intravenous injection, 100 μL per injection, the exosome concentration is 1×1010particles / mL, the administration frequency is 2 times per week, and the administration lasts for 4 weeks.

[0022] The targeted modified exosome of the present application has the following beneficial effects:

[0023] 1. Enhanced targeting: The exosome is modified by the target fusion polypeptide EPLQLKM, which can specifically bind to the smooth muscle cells of the corpus cavernosum, thereby improving the targeted delivery efficiency.

[0024] 2. Improved biological activity: After loading the anti-TrkA monoclonal antibody, the exosome can further enhance the biological effects on target cells, such as NO synthesis and anti-fibrosis.

[0025] 3. Significant improvement of ED: In a diabetic ED rat model, the modified exosome significantly improves sexual function (ICP / MAP ratio), reduces corpus cavernosum fibrosis, and promotes eNOS expression, providing a new effective strategy for ED treatment.

[0026] 4. Simple and efficient preparation method: the modification method is simple, stable, suitable for large-scale production, and has good clinical transformation potential. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 SDS-PAGE detection results of recombinant TrkA-ECD protein, wherein 1 is recombinant TrkA-ECD protein, with a molecular weight of about 45 kDa.

[0028] Figure 2 TrkA-2E7-HL SDS-PAGE detection results, wherein 1 is TrkA-2E7-HL. DETAILED DESCRIPTION

[0029] 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 application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0030] Unless specifically noted, the reagents, methods, and equipment employed in the present application are those conventional in the art. Unless specifically noted, the reagents and materials used in the following examples are commercially available.

[0031] Example 1: Design and preparation of a targeted fusion polypeptide

[0032] 1. Sequence composition and functional structure design of the targeted fusion polypeptide

[0033] 1.1 Amino acid sequence: EPLQLKM-GGGGS-DPPV.

[0034] 1.2 Structure module description:

[0035] (1) EPLQLKM: This segment is a mimic peptide derived from the natural ligand domain of CXCR4, which has a targeting functional segment with high affinity binding ability to CXCR4, and can realize the specific recognition and binding of exosomes to the surface CXCR4 of the smooth muscle cells of the cavernous body;

[0036] (2) GGGGS: a flexible linker peptide, providing spatial flexibility to avoid conformational interference between domains;

[0037] (3) DPPV: selected from common sequences of membrane anchoring peptides, used for non-covalent insertion and binding with exosome membrane proteins (such as CD63, CD9, etc.), to realize stable anchoring of the polypeptide to the surface of the exosome membrane.

[0038] 2. Synthesis method

[0039] 2.1 Synthesis method (Fmoc solid phase peptide synthesis (SPPS) was adopted): the above sequence was synthesized step by step using Rinkamide resin; HBTU / HOBt activation system was adopted for each amino acid condensation; after the synthesis was completed, resin cleavage and polypeptide release were performed using TFA cleavage solution (TFA:TIS:H2O = 95:2.5:2.5).

[0040] 2.2 Purity requirement: purified by reverse phase high performance liquid chromatography (RP-HPLC) to >95%.

[0041] 2.3 Quality verification: LC-MS analysis confirmed that the molecular weight was consistent with the theory, the theoretical molecular weight was 1532.8 Da; the measured value was 1532.7 ± 0.2 Da.

[0042] 3 Usage and concentration recommendation

[0043] 3.1 Binding mode with exosome membrane: the polypeptide was directly modified to purified exosomes by incubation, the concentration was 10-50 μg / mL, incubated at 37°C for 30 minutes, and then removed the unbound peptide by ultracentrifugation.

[0044] 3.2 Function verification: the polypeptide was labeled with fluorescence (FITC labeling) to verify its targeted aggregation in the corpus cavernosum smooth muscle cell line (HCSMC); cell uptake experiment confirmed that its ability to assist exosomes into target cells was improved by more than 3 times (n = 3, P < 0.01).

[0045] Example 2: Preparation and performance verification of anti-TrkA monoclonal antibody

[0046] 1 Preparation of antibody

[0047] 1.1 Preparation of antigen

[0048] (1) The extracellular domain (ECD, amino acid site 32-425, as shown in SEQ ID NO. 1) of human TrkA receptor was selected as the immunizing antigen in this study to obtain a monoclonal antibody with high preservation of natural conformation. The fragment gene was amplified by PCR and cloned into the mammalian expression vector pcDNA3.4 with a 6×His tag, and transiently expressed in HEK293F suspension cells. After collecting the culture supernatant, one-step purification was performed using Ni-NTA affinity chromatography, and the obtained recombinant TrkA-ECD protein was detected by SDS-PAGE to show clear bands, with a single main band at about 45 kDa, and the purity was higher than 95% (as shown in Figure 1 ). The protein concentration was determined by BCA method to be 1.65 mg / mL, meeting the required standard for immunization experiments.

[0049] (2) To ensure the protein immunogenicity and physiological adaptability of the buffer system, the purified TrkA-ECD protein was completely replaced with PBS buffer (pH 7.4) by gradient dialysis at 4°C, and sterilized by 0.22 μm sterile filter. The final storage solution was prepared for mouse immunization. The protein was stored at -80°C and slowly thawed at 4°C before use to avoid structural damage. The entire expression and purification process has good repeatability, high protein stability, and lays a solid foundation for obtaining high specificity anti-TrkA antibody.

[0050] 1.2 Mouse immunization and immune response evaluation

[0051] (1) In this study, 5 healthy, SPF grade, 6-8 week old female BALB / c mice were used for immune response induction of TrkA antigen. At the first immunization, each mouse was injected intraperitoneally with 100 μg of TrkA-ECD recombinant protein diluted with PBS buffer, and was combined with Freund's complete adjuvant (FCA) to enhance antigen presentation and initial immune response. Two booster immunizations were performed at 14 days and 28 days after the first immunization, respectively, using a dose of 50 μg / time, combined with Freund's incomplete adjuvant (FIA) to promote B cell affinity maturation and enhance humoral immune response. The total immunization cycle was 4 weeks, and serum samples were collected for antibody titer evaluation on the 7th day after the last immunization.

[0052] (2) Serum anti-TrkA antibody titer was determined by indirect ELISA method. In the experiment, TrkA-ECD protein was coated at a concentration of 1 μg / mL on a high-binding 96-well enzyme-labeled plate, and after incubation at room temperature for 2 hours, 1:100 to 1:204800 gradient diluted mouse serum was added. Then, HRP labeled anti-mouse IgG secondary antibody was added, and TMB substrate was used for color development. After termination, the absorbance value was detected at 450 nm wavelength.

[0053] (3) The experimental results showed that all mice produced strong immune response, and mouse #3 serum still maintained obvious absorbance signal at 1:102400 dilution ratio, showing the highest anti-TrkA antibody titer. This mouse was selected as the donor for subsequent B cell and myeloma cell fusion, laying a foundation for high affinity monoclonal antibody screening.

[0054] 1.3 Hybridoma cell fusion and clone screening

[0055] (1) To obtain hybridoma cell lines stably expressing high-affinity anti-TrkA antibodies, we selected the mouse with the strongest immune response (#3) as the donor, isolated its spleen cells, and mixed them with SP2 / 0 myeloma cells at a ratio of 5:1, using 50% PEG-1500 to mediate cell fusion. The cell suspension was immediately inoculated into 96-well plates containing HAT selective medium to inhibit the growth of unfused myeloma cells. Cell colonies began to appear 48 hours later, and obvious microcolony spheres formed in some wells after 7 days, indicating good fusion efficiency. Preliminary statistics showed that positive clone wells accounted for about 21% of the total inoculated wells, indicating that the fusion effect was satisfactory.

[0056] (2) Subsequently, the culture supernatant was subjected to ELISA primary screening using TrkA-ECD protein-coated enzyme-labeled plates to detect whether each well secreted specific antibodies. A total of 62 positive wells were detected, of which 22 wells showed higher OD450 absorbance, indicating that the antibodies had high affinity. These positive clones were further subjected to three rounds of subcloning by limiting dilution method to obtain multiple monoclonal cell lines stably secreting single antibodies. The cell line numbered TrkA-2E7-HL performed best, and allelic sequencing confirmed that it was truly monoclonal in origin.

[0057] (3) Functional detection showed that the IgG1 subclass antibody secreted by TrkA-2E7-HL had a titer of 1:256000 in ELISA, much higher than that of commercially available antibodies of the same type. At the same time, the antibody showed no significant binding signal in cross-reaction detection with TrkB and TrkC proteins, indicating good target specificity. The cell line had a recovery rate of more than 95% after freezing and recovery, and still maintained stable antibody expression levels after 8 weeks of continuous subculture, showing potential for further large-scale antibody production and functional research.

[0058] 1.4 Antibody gene sequencing and expression

[0059] (1) To obtain the full-length variable region sequence of the antibody secreted by TrkA-2E7-HL cells, we first extracted total RNA from the cell line and used SMARTer RACE (Rapid Amplification of cDNA Ends) technology to specifically amplify the heavy chain (VH) and light chain (VL) variable regions. After PCR product purification and Sanger sequencing, we successfully obtained the complete antibody variable region nucleotide sequence.

[0060] (2) In order to improve the clinical application value of the antibody, especially to improve the immunogenicity and stability, we optimized the humanization of the anti-TrkA monoclonal antibody. Specifically, by introducing human IgG1 common framework region to replace the mouse IgG1 framework, the immunogenicity was reduced. During the optimization process, the "framework optimization" and "CDR retention" strategies were adopted to ensure that the antibody maintained the original high affinity and specificity while enhancing the stability and functional activity of the human IgG1 structure. The amino acid sequences of the heavy chain variable region and the light chain variable region of TrkA-2E7-HL after humanization optimization are shown in SEQ ID NO. 2 and SEQ ID NO. 3, respectively.

[0061] (3) After optimization, the heavy chain and light chain variable regions of the antibody were fused with human IgG1 constant region and kappa type light chain constant region, respectively, and cloned into the high expression mammalian vector pcDNA3.4. After sequence verification, they were transfected into ExpiCHO-S high-density suspension cell lines for transient co-expression. After protein A affinity chromatography purification, SDS-PAGE and SEC-HPLC analysis showed that the purity of the antibody was higher than 98%, the expression level was stable, and the average yield was about 1500 mg / L, which laid a foundation for industrial expression and subsequent functional research.

[0062] 2 Antibody performance verification and comparative analysis

[0063] 2.1 Molecular property confirmation: by SDS-PAGE analysis Figure 2 ), the heavy chain of anti-TrkA monoclonal antibody (TrkA-2E7-HL) showed a clear band at 50 kDa, and the light chain showed a clear separation at 25 kDa, which was consistent with the expected molecular size.

[0064] 2.2 Binding activity and affinity (ELISA and BLI): To evaluate the binding activity and affinity of TrkA-2E7-HL antibody, both ELISA and BLI (Biolayer Interferometry) methods were employed for detection. In ELISA, the EC50 of TrkA-2E7-HL antibody was 25.6 ± 2.1 ng / mL, indicating its high binding capacity to TrkA; through BLI detection, the binding affinity (KD value) of the antibody was 0.45 ± 0.03 nM, showing its high affinity and specificity to TrkA receptor. In comparison, commercially available anti-TrkA antibodies such as sigma's 06-574 (EC50: 63.8 ± 5.6 ng / mL, KD: 2.2 ± 0.1 nM) and Abeam's ab302524 (EC50: 94.2 ± 4.2 ng / mL, KD: 4.5 ± 0.3 nM) exhibited weaker affinity. This indicates that TrkA-2E7-HL antibody is superior to these commercial products in terms of binding activity and affinity, with more excellent performance.

[0065] 2.3 Signal inhibition function verification (PC12 cells): To verify the functionality of TrkA-2E7-HL antibody, PC12 cells were used as a model to evaluate its inhibitory effect on TrkA and downstream signaling molecule ERK. In the treatment group, TrkA-2E7-HL antibody showed 88.2% ± 4.5% p-TrkA inhibition and 73.1% ± 3.8% p-ERK inhibition under NGF stimulation, indicating that the antibody can effectively inhibit the activation of TrkA receptor and its downstream signal transduction. In comparison, sigma's 06-574 antibody showed 65.6% ± 3.2% p-TrkA inhibition and 42.4% ± 5.1% p-ERK inhibition, while Abeam's ab302524 antibody was even less effective, with 53.8% ± 6.4% and 31.2% ± 4.7%, respectively. These results indicate that TrkA-2E7-HL antibody has a clear advantage in signal inhibition, effectively interfering with the TrkA-mediated signaling pathway.

[0066] From the above experimental results, it can be seen that TrkA-2E7-HL antibody not only shows superiority in binding activity and affinity, but also exhibits significant signal inhibition effect in cell function verification, which provides strong support for its potential in clinical treatment.

[0067] Example 3: Exosome preparation and modification

[0068] 1 Stem cell culture and antioxidant induction treatment

[0069] 1.1 Cell preparation and expansion

[0070] (1) Cell source: Human umbilical cord mesenchymal stem cells (hUC-MSCs) were selected, thawed and inoculated in T75 culture bottles, with an initial density of 5 x 10 4 cells / cm 2 .

[0071] (2) Basic medium: a-MEM supplemented with 10% fetal bovine serum (FBS, Gibco), 1% penicillin-streptomycin, and cultured routinely in a 37°C, 5% CO2 incubator.

[0072] (3) Passage conditions: when the confluence reached 80-90%, the cells were digested with 0.25% trypsin-EDTA and passaged at a ratio of 1:3, and the fresh medium was changed every 3-4 days.

[0073] 1.2 Antioxidant induction treatment

[0074] (1) Pretreatment before induction: the cells were gently rinsed twice in PBS to remove residual serum components; and pre-cultured for 12 hours in exosome-free FBS medium (exosome-free FBS: ultra-centrifugation to remove exosomes in FBS, 100,000g x 16h).

[0075] (2) Antioxidant composition treatment: the following reagents were added to the basic medium: baicalin (50 μM, dissolved in DMSO, final concentration of DMSO <0.1%), quercetin (20 μM), N-acetylcysteine (5 mM, freshly prepared).

[0076] (3) Induction conditions: cultured in the above induction medium for 72 hours, 37°C, 5% CO2 incubator, fresh antioxidant medium was changed every 24 hours to maintain a stable induction environment. The cell morphology and survival rate were monitored in real time during the induction period.

[0077] 2. Isolation and purification of exosomes

[0078] 2.1 Collection of conditioned medium: after 72 hours of induction, the cell supernatant (about 150-200 mL) was collected and pre-filtered using a 0.22 μm filter to remove cell debris.

[0079] 2.2 Exosome isolation (ultra-centrifugation method): at 4°C, the following steps were performed in sequence:

[0080] (1) 300g x 10min: remove floating cells and large debris.

[0081] (2) 2,000g x 30min: remove apoptotic bodies and organelle debris.

[0082] (3) 10,000g x 30min: further remove large microsomes and large vesicles.

[0083] (4) 100,000 g x 90 min: sedimentation of exosome particles (using Type 45 Ti rotor).

[0084] (5) Resuspended in sterile PBS, centrifuged again at 100,000 g x 90 min to remove soluble contaminants.

[0085] (6) The final pellet was resuspended with 100-200 μΐ of PBS and used as crude exosome preparation for further purification.

[0086] 2.3 Size-Exclusion Chromatography (SEC) for further purification using Capto Core 700 column (GE Healthcare):

[0087] (1) Pre-equilibrate column: equilibrate with 20 mL of PBS;

[0088] (2) Sample loading: load the exosome crude slowly, control the flow rate at 0.5 mL / min;

[0089] (3) Elution condition: elute with equal volume of PBS, collect 1 mL per tube;

[0090] (4) Tubes 8-12 are the exosome enriched fraction, the rest are contaminant proteins and small molecule contaminants;

[0091] (5) Store at 4°C after collection, can be further concentrated or used for characterization or for subsequent modification.

[0092] 3. Targeted fusion polypeptide modified exosomes

[0093] 3.1 Targeted fusion polypeptide conjugation reaction

[0094] (1) Material preparation: Dissolve DSPE-PEG2000-Maleimide (5 mg) and the fusion polypeptide prepared in Example 1 with N-terminal cysteine (1.5 molar equivalent) in sterile PBS buffer (pH 7.4) separately, mix after fully dissolved.

[0095] (2) Conjugation condition: Covalent conjugation reaction between thiol-maleimide was performed in a constant temperature shaker at 25°C, 300 rpm for 4 hours. Keep away from light during the reaction to ensure the activity of maleimide ring.

[0096] 3.2 Targeted fusion polypeptide coated exosomes

[0097] (1) Mix the above DSPE-PEG2000-polypeptide conjugation product with the pre-prepared exosomes (concentration: 1 x 1011particles / mL, suspended in PBS) slowly at a volume ratio of 1:2.

[0098] (2) Incubate at 37°C for 2 hours, gently invert and mix every 30 minutes to promote hydrophobic insertion and lipid bilayer fusion.

[0099] (3) Ultrafiltration using Amicon Ultra-15 centrifugal filter tubes (MWCO 100 kDa), 4,000g centrifugation for 10 minutes x 3 times to remove unbound free polypeptides. Finally resuspended in 100-200 μL PBS to obtain the modified targeted exosomes (named EPL-Exo).

[0100] 4. Antibody encapsulation into exosomes

[0101] 4.1 Antibody electroporation loading

[0102] (1) Prepare 4mm electroporation cups, mix the modified exosomes (EPL-Exo, 1 x 1011particles / mL) with TrkA-2E7-HL antibodies (final concentration 20 μg / mL), make up the volume to 200 μL, and use calcium and magnesium-free PBS as the electroporation buffer.

[0103] (2) Use the electroporation instrument to set the following parameters: voltage 150V, pulse time 10ms, pulse number 3 times, interval 5 seconds, and ensure that the device is preheated.

[0104] (3) After electroporation, immediately place on ice for 10 minutes to promote membrane structure self-repair and stabilize the loading.

[0105] 4.2 Removal of unloaded antibodies

[0106] (1) Add 5 volumes of ExoQuick-TC reagent to the reaction system, stand at 4°C for 30 minutes, then centrifuge at 1,500g for 30 minutes to precipitate the exosomes.

[0107] (2) Discard the supernatant, resuspend the exosome precipitate with PBS to the desired concentration, and name it EPL-Exo+antibody, which can be used for subsequent in vitro cell experiments or animal injection experiments or stored at -80°C for later use.

[0108] Example 4: Exosome testing

[0109] Experiment 1: Verification of exosome physical and chemical properties and modification

[0110] 1.1 Exosome particle size and morphology detection

[0111] Exosomes extracted from different experimental groups (control Exo, EPL-Exo, EPL-Exo + antibody) were diluted with PBS to a final concentration of about 1 x 10 9 particles / mL. Dynamic light scattering (DLS) detection of particle size and distribution was performed using a NanoSight NS300 nanoparticle tracking analyzer, with each sample measured independently three times to calculate the mean and standard deviation. To further observe the ultrastructure of the exosomes, 3 μL of the sample was added to a carbon film copper grid, naturally adsorbed for 10 minutes, and then treated with 2% phosphotungstic acid negative staining. After drying, transmission electron microscopy (TEM) was used for imaging analysis.

[0112] The results are shown in Table 1, and the three groups of exosomes all have a relatively narrow particle size distribution, with PDI values <0.2, indicating that the prepared exosomes have good uniformity. TEM results show that the exosomes in the control Exo group have a typical "cup-shaped" structure, the EPL-Exo group has an intact structure, and the EPL-Exo + antibody group shows a slight thickening of the membrane structure, suggesting that antibody modification has not significantly changed the basic structure of the exosomes, and the structure is still intact.

[0113] Table 1 Exosome particle size and morphology detection results

[0114]

[0115] The above results show that EPL polypeptide modification and antibody coating have little effect on the particle size and morphology of exosomes, and do not cause obvious aggregation or morphological damage, indicating that the modification process has good biocompatibility and structural stability, providing a structural guarantee for subsequent functional research.

[0116] 1.2 Modification efficiency detection of targeted fusion polypeptide

[0117] To evaluate the modification efficiency of EPLQLKM targeted fusion polypeptide on the surface of exosomes, EPLQLKM was first labeled with FITC (molar ratio 1:5), and after dialysis to remove free FITC, it was combined with DSPE-PEG2000-Mal and co-incubated with exosomes for surface modification. Then aldehyde-based latex microspheres were used to wrap exosomes of different treatment groups (including ordinary Exo group, EPL-Exo group and EPL-Exo + antibody group), and after blocking, they were used for flow cytometry detection. PBS and bare beads were set as negative controls, and the FITC channel was used for fluorescence positive rate analysis.

[0118] Flow cytometry results showed that the FITC positive rate of the EPL-Exo group was 82.3 ± 4.1%, and the positive rate of the EPL-Exo+antibody group was 83.4 ± 3.5%, which was significantly higher than that of the ordinary Exo group (<10%), which was comparable to the negative control. It is shown that the targeting fusion polypeptide is successfully coupled to the surface of the exosome through DSPE-PEG2000-Mal, with high modification efficiency, and the fluorescence signal is concentrated, the background is low, and the detection method has good specificity and stability.

[0119] The experimental results confirm that the surface modification of the targeting polypeptide realized by the maleimide-thiol covalent coupling method has high efficiency and repeatability. This strategy provides a reliable technical foundation for the realization of subsequent exosome targeting function, and also shows that this polypeptide modification method has strong application potential in nanometer delivery system.

[0120] Experiment 2: In vitro targeted recognition and anti-fibrosis function

[0121] 2.1 Targeting efficiency evaluation

[0122] In order to verify whether the EPLQLKM polypeptide modification on the surface of exosomes enhances its targeting, the exosomes labeled with DiR fluorescent dye (protein amount 20 μg) were co-incubated with cavernous smooth muscle cells (CCSMC, 2×10 5 cells / well) for 2 hours. After washing with PBS, the intracellular fluorescence intensity was detected by flow cytometry. The results showed that the fluorescence signal intensity of cell uptake in the EPL-Exo group and the EPL-Exo+antibody group was 3.5 times and 3.8 times that of the ordinary Exo group, indicating that the EPLQLKM polypeptide significantly improved the recognition and uptake efficiency of exosomes to target cells.

[0123] 2.2 NO secretion function

[0124] Under the same co-incubation conditions, the cell supernatant was collected and the NO content was detected by Griess method to evaluate the regulation effect of exosomes on the functional state of CCSMC. The results showed that the NO secretion level after EPL-Exo treatment was significantly improved (42.1 ± 4.2 μM), which was significantly higher than that of the ordinary Exo group (28.3 ± 3.1 μM, *p<0.05), and the EPL-Exo+antibody group further reached 52.4 ± 4.8 μM (*p<0.05), indicating that the polypeptide modification not only enhances the cell uptake, but also further activates the NO synthesis function, and the effect can be enhanced by the targeting antibody.

[0125] 2.3 Anti-fibrosis effect (Western blot)

[0126] The CCSMC fibrosis model was induced by TGF-β1(10 ng / mL, 48 h), and the expression changes of fibrosis-related marker proteins were further analyzed after combined treatment with exosomes. Western blot results showed that compared with the blank induction group, EPL-Exo treatment could make the expression of α-SMA and Collagen I decrease by 42% and 38%, respectively; while the inhibition effect of EPL-Exo+antibody group was more significant, decreased by 62% and 58%, respectively. The results showed that the exosomes with targeted modification not only had higher uptake rate, but also had enhanced anti-fibrosis biological function.

[0127] Experiment 3: Treatment verification of diabetic ED rat model

[0128] 3.1 Construction and verification of ED animal model

[0129] SD male rats (200-250 g, n=40) were selected, and a diabetic model was induced by a one-time intraperitoneal injection of streptozotocin (STZ, 65 mg / kg, dissolved in 1% citric acid buffer). On the 28th day of modeling, all animals received a cavernous body stimulation experiment to detect the ratio of penile cavernous pressure (ICP) to mean arterial pressure (MAP) (ICP / MAP), and when the ratio was less than 0.3, it was determined that the erectile dysfunction (ED) model was successfully constructed.

[0130] 3.2 Treatment grouping and administration method

[0131] After successful modeling, they were randomly divided into 4 groups (n=10 each): control group (normal saline), ordinary Exo group, EPL-Exo group, and EPL-Exo+antibody group. Each group was administered by tail vein injection, with each injection of 100 μL, the exosome concentration was 1×1010 particles / mL, and the administration frequency was 2 times per week for 4 weeks.

[0132] 3.3 Erectile function evaluation and histological analysis

[0133] The ratio of MAP to ICP was calculated to reflect the recovery of sexual function by electrically stimulating the dorsal nerve of the penis under anesthesia; the penile cavernous tissue was taken for Masson staining to evaluate the degree of fibrosis, and the expression level of eNOS was detected by immunohistochemistry, and the IOD method was used for quantification. The statistical results are shown in Table 2.

[0134] Table 2 Summary of treatment results of diabetic ED rat model

[0135]

[0136] All data are expressed as mean ± standard deviation. Statistical significance: *p<0.05, **p<0.01, ***p<0.001 vs control group.

[0137] The results show that the exosome (EPL-Exo) modified by the targeting polypeptide (EPLQLKM) has higher bioavailability in the ED model, significantly improves the corpus cavernosum uptake rate and treatment efficacy, and is specifically manifested in the significant increase of ICP / MAP, the significant decrease of fibrosis degree and the enhancement of eNOS expression. The EPL-Exo+antibody group loaded with TrkA antibody shows the optimal improvement effect in all indexes, indicating that it has stronger targeting and biological function enhancement effect. The ordinary unmodified Exo group has certain improvement, but the curative effect is significantly lower than that of the EPL-Exo group, which emphasizes the necessity and superiority of the polypeptide modification and antibody synergistic strategy.

[0138] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods and are included in the protection scope of the present application.

Claims

1. A targeted modified stem cell exosome, characterized in that, The exosomes include the following elements: (1) A targeted fusion polypeptide is covalently coupled to the surface of exosomes via maleimide-thiol, wherein the amino acid sequence of the targeted fusion polypeptide is EPLQLKM-GGGGS-DPPV; (2) The exosomes are loaded with anti-TrkA monoclonal antibody, the amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-TrkA monoclonal antibody are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively; (3) The exosomes are derived from umbilical cord mesenchymal stem cells cultured with antioxidants, wherein the antioxidants are baicalin 50 μM, quercetin 20 μM and N-acetylcysteine ​​5 mM.

2. The exosomes according to claim 1, characterized in that, The exosomes have a particle size of 130.6 ± 3.6 nm.

3. The exosomes according to claim 1, characterized in that, The anti-TrkA monoclonal antibody was obtained by immunizing mice with recombinant TrkA-ECD protein, followed by hybridoma cell fusion and clonal screening, antibody gene sequencing and expression, and purification. The amino acid sequence of the recombinant TrkA-ECD protein is shown in SEQ ID NO.

1.

4. A method for preparing exosomes as described in claim 1, characterized in that, The method includes the following steps: (1) Umbilical cord mesenchymal stem cells were cultured for 72 hours in a medium containing 50 μM baicalin, 20 μM quercetin and 5 mM N-acetylcysteine, and exosomes were purified by ultracentrifugation combined with size exclusion chromatography. (2) After mixing DSPE-PEG2000-Mal with the targeted fusion peptide at a molar ratio of 1:1.5, the mixture was reacted in PBS for 4 hours to obtain the coupling product, which was then co-incubated with exosomes for 2 hours to complete the surface modification. (3) Anti-TrkA antibody was loaded into exosomes by electroporation, wherein the voltage was 150V, the pulse time was 10ms, and the number of pulses was 3.

5. The use of the exosomes as described in claim 1 in the preparation of a medicament for treating diabetic or neurogenic erectile dysfunction.

6. The application according to claim 5, characterized in that, The drug is administered intravenously, with each injection containing 100 μL, and the exosome concentration is 1 × 10⁻⁶. 10 The dosage was 10 particles / mL, administered twice a week for 4 weeks.

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