Functional exosome as well as preparation method and application thereof

Abstract

The invention discloses a functional exosome as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The exosome highly expresses miR-124-3p and miR-10a-5p, and the miR-124-3p can inhibit degradation of an extracellular matrix and promote remodeling of the extracellular matrix by inhibiting signal molecules such as ROCK1 and the like; and the latter can inhibit a proinflammatory factor receptor (IL6R) in a targeted manner so as to inhibit local inflammation. According to the dual synergistic effect of matrix remodeling and anti-inflammatory regulation, accurate intervention is carried out on core pathological links (matrix degradation and inflammation) of intervertebral disc degeneration, and the treatment effect which is difficult to achieve by a single therapy is achieved. According to the exosome, Syntenin-1 and IL-1RA are taken as dual molecular targets for screening functional exosome subgroups, and the specific exosome subgroups with dual functional potentials of'tissue repair 'and'immune regulation' are accurately screened from a complex exosome group derived from fibroblasts through a double-antibody immunoaffinity method.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically relating to a functional exosome, its preparation method, and its application. Background Technology

[0002] Exosomes are extracellular vesicles derived from intracellular multivesicular bodies, with a diameter of approximately 30–150 nanometers. Their formation depends on the endosome pathway, and they are widely present in bodily fluids such as blood and urine, and secreted by various cells including immune cells, mesenchymal stem cells, and tumor cells. These vesicles are rich in bioactive molecules such as proteins, nucleic acids, and lipids, and express characteristic marker proteins such as CD9, CD63, and CD81 on their surface. With their stable lipid bilayer structure and tissue-targeting capabilities, they serve as key carriers mediating intercellular communication: they can participate in physiological and pathological processes such as immune regulation, tissue repair, and tumor progression by delivering functional substances. Based on these characteristics, exosomes have shown significant application value in the biomedical field, including as liquid biopsy biomarkers for disease diagnosis, as drug delivery systems as natural targeting carriers, as mesenchymal stem cell-derived exosome therapy as an alternative to cell therapy, and as a novel vaccine platform for tumor immunotherapy.

[0003] Existing techniques for separating and purifying exosomes include: (1) Ultrafiltration, which uses ultrafiltration membranes with different molecular weight cutoffs (usually 10-100 kDa) to allow small molecular weight proteins, salt ions and other impurities in the solution to pass through the membrane by centrifugation or pressurization, while larger exosomes (30-150 nm) are trapped and concentrated on the membrane, thus achieving separation. This process does not depend on specific antibodies and is a physical separation method; (2) Immunoaffinity capture, which uses CD9, CD63 and CD81 antibodies coupled to a solid support (such as magnetic beads) to capture marker proteins on the surface of exosomes; (3) Centrifugation, which uses centrifugal force generated by different rotation speeds to gradually separate exosomes from complex biological samples (such as cell supernatant, plasma and urine) based on the differences in particle size, density and sedimentation coefficient. Among these methods, ultrafiltration separates only by size and cannot distinguish impurities such as protein polymers and lipoprotein particles that are similar in size to exosomes. These contaminants are concentrated together, resulting in low purity of the final product, which can seriously interfere with downstream functional analysis, proteomics, or nucleic acid sequencing results. Immunoaffinity magnetic beads currently use universal antibodies and cannot screen for specific exosomes, such as those expressing IL-1RA and Syntenin-1. Ultracentrifugation cannot effectively distinguish impurities that are similar in size and density to exosomes, such as protein polymers, lipoproteins (especially low-density lipoproteins), and viral particles. The recovery rate is low and unstable, and it is highly dependent on equipment such as ultracentrifuges.

[0004] The main clinical manifestations of lumbar disc degeneration are varying degrees of low back pain and limited lumbar function. It can also present with lower limb pain, weakness, numbness, intermittent claudication, urinary and fecal incontinence, and sexual dysfunction, indicating neurological impairment. Patients with long-term chronic low back pain may also experience anxiety or depression, severely impacting their work and quality of life. The choice of treatment requires comprehensive consideration of the patient's age, weight, the degree and stability of disc degeneration, and the presence of neurological impairment. A stepwise treatment strategy is adopted for different stages of lumbar disc degeneration. The vast majority of patients begin with basic conservative treatment, including lifestyle interventions such as health education and core muscle exercises, combined with physical therapy (e.g., traction, massage) and drug therapy (primarily nonsteroidal anti-inflammatory drugs, with epidural steroid injections used in severe cases). If conservative treatment is ineffective, minimally invasive interventional treatments are used, such as percutaneous endoscopic discectomy or radiofrequency ablation, to directly relieve nerve compression. For patients with severe symptoms or neurological dysfunction, destructive discectomy and surgical fusion of the two vertebrae above and below are ultimately performed. Although there are many treatment options for lumbar disc degeneration, current treatment strategies aim to manage symptoms, primarily relieving pain. Conservative treatment cannot slow the progression of degeneration, and the long-term benefits to patients are unclear, resulting in poor quality of life. Furthermore, current treatments for lumbar disc degeneration have some limitations. Conservative treatments such as physical therapy and drug therapy have limited effectiveness and are difficult to fundamentally reverse the degenerative process; drug therapy can provide temporary and intermittent relief from lower back pain, but its long-term efficacy is unstable and is often accompanied by adverse reactions; surgical treatment can relieve symptoms, but it has problems such as large trauma, long recovery period, high cost, and risk of recurrence. Moreover, current surgical methods mainly target symptom relief and cannot fundamentally treat the intervertebral disc itself. Summary of the Invention

[0005] 1. Purpose of the invention Firstly, the purpose of this application is to provide a functional exosome and its application, which can effectively intervene in the imbalance of the local inflammatory microenvironment and the metabolic disorder of the extracellular matrix, thereby achieving the dual therapeutic goals of anti-inflammatory and tissue regeneration in intervertebral disc degeneration.

[0006] Secondly, the purpose of this application is to provide a method for preparing the first aspect of functional exosomes.

[0007] 2. Technical Solution To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In one aspect, this application provides an exosome that highly expresses miR-124-3p (miRBase Database: MIMAT0000422) and miR-10a-5p (miRBase Database: MIMAT0000253). As a further explanation of this application, miR-124-3p can inhibit the degradation of extracellular matrix and promote extracellular matrix remodeling by inhibiting signaling molecules such as ROCK1; while miR-10a-5p can target and inhibit the pro-inflammatory factor receptor (IL6R) to suppress local inflammation.

[0008] Furthermore, the exosomes also highly express IL-1RA (NCBI GENE ID: 3557) and Syntenin-1 (NCBI GENE ID: 6386). As further explanation of this application, Syntenin-1 regulates exosome biogenesis and specific miRNA loading. The exosome subsets that highly express Syntenin-1 specifically enrich functional miRNAs such as miR-124-3p and miR-10a-5p. These miRNAs can be effectively delivered to nucleus pulposus cells, promoting extracellular matrix synthesis and remodeling by inhibiting signaling pathways such as ROCK1. At the same time, exosomes that highly express IL-1RA and carry miR-10a-5p can efficiently neutralize IL-1-mediated inflammatory signals, effectively improving the inflammatory microenvironment of intervertebral disc degeneration. This dual synergistic effect of "matrix remodeling + anti-inflammatory regulation" precisely intervenes in the core pathological links of intervertebral disc degeneration (matrix degradation and inflammation), achieving therapeutic effects that are difficult to achieve with single therapies.

[0009] Furthermore, the exosomes mentioned above are derived from human fibroblasts.

[0010] Secondly, this application provides a method for preparing exosomes according to the first aspect, the method comprising the following steps: S1, culture cells and induce cells to secrete exosomes; S2, after induction culture, the supernatant was collected, and exosomes were screened using anti-Syntenin-1 antibody and anti-IL-1RA antibody.

[0011] Furthermore, in S2 above, the exosome screening process includes ultrafiltration, in which a molecular weight cutoff of 100 kDa is retained, resulting in a concentrated and highly pure exosome suspension. As a further explanation of this application, antibody screening (immunoaffinity) ensures the specific capture of the target exosome subset, while the ultrafiltration step efficiently removes unbound impurities and small molecule components in the elution buffer, achieving exosome concentration. This method overcomes the shortcomings of ultracentrifugation, such as poor purity, numerous impurities, and unstable recovery rates, and also avoids the problems of harsh elution conditions and damage to exosome integrity that may exist in traditional immunoaffinity methods, ultimately obtaining highly pure, highly active, and highly concentrated therapeutic exosomes.

[0012] Furthermore, in S1 above, the cell is a fibroblast. Even further, in S1 above, the cell is a human fibroblast.

[0013] Furthermore, in S1 above, inducing cells to secrete exosomes includes stimulating cells with Y-27632 dihydrochloride and / or tunicamycin to induce them to secrete exosomes.

[0014] Furthermore, in S1 above, the final concentration of Y-27632 dihydrochloride stimulating cells is 1 μM, and the final concentration of tunicamycin stimulating cells is 0.1 μM.

[0015] Furthermore, in S2 above, the supernatant pretreatment is also included before screening with anti-Syntenin-1 antibody and anti-IL-1RA antibody. Specifically, the supernatant is centrifuged at 2,000×g at 4°C for 20-30 minutes to thoroughly remove intact cells and cell debris.

[0016] Further, in S2 above, the screening using anti-Syntenin-1 antibody and anti-IL-1RA antibody includes: mixing the supernatant or pretreated supernatant with superparamagnetic microspheres modified with anti-Syntenin-1 antibody and superparamagnetic microspheres modified with anti-IL-1RA antibody, and gently incubating in a binding buffer containing calcium ions at room temperature for 30-60 minutes; after incubation, performing magnetic separation to obtain the magnetic microsphere-exosome complex; and washing and eluting to obtain an eluent containing exosomes.

[0017] Furthermore, the ratio of the number of superparamagnetic microspheres with surface-modified anti-Syntenin-1 antibody to the number of superparamagnetic microspheres with surface-modified anti-IL-1RA antibody is 1:1.

[0018] Furthermore, the washing process uses a washing buffer, which is PBS or a PBS solution containing 0.01%~0.05% (v / v) Tween-20. This condition can effectively remove non-specific adsorbed proteins without disrupting the calcium-dependent binding between T cell immunoglobulins and mucin domain protein 4 (TIM4) and exosomes.

[0019] Furthermore, the above elution includes: directly adding an elution buffer containing EGTA to the magnetic microsphere-exosome complex for elution, and collecting the eluent.

[0020] Furthermore, the preparation methods of the superparamagnetic microspheres with surface-modified anti-Syntenin-1 antibody and the superparamagnetic microspheres with surface-modified anti-IL-1RA antibody include the following steps: (a) Take a suspension of carboxyl-modified superparamagnetic microspheres; (b) Add a freshly prepared mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in MES buffer and activate at room temperature; (c) Magnetic separation, removal of supernatant, and rapid washing twice with PBS buffer at pH 7.4 to obtain activated microspheres; (d) Mix the activated microspheres with anti-human Syntenin-1 antibody or anti-human IL-1RA antibody in a binding buffer containing calcium ions and incubate gently by rotating at room temperature; (e) Magnetic separation, remove the supernatant, add 1 M Tris-HCl pH 8.0 solution, and block at room temperature for 1 hour; (f) Wash and resuspend with PBS buffer containing 1 mM CaCl2 and 0.01% ProClin 300 to obtain superparamagnetic microspheres with surface modified with anti-Syntenin-1 antibody or superparamagnetic microspheres with surface modified with anti-IL-1RA antibody.

[0021] Thirdly, this application provides the application of the above-mentioned exosomes in the preparation of drugs for treating intervertebral disc degeneration; as a further explanation of this application, using functionalized exosomes derived from fibroblasts as therapeutic agents avoids the immune rejection, ethical controversies and tumorigenesis risks associated with direct cell transplantation, providing a safer, more stable and easily standardized "cell-free therapy" approach for the treatment of intervertebral disc degeneration.

[0022] Fourthly, this application provides a pharmaceutical composition comprising the above-mentioned exosomes and a pharmaceutically acceptable carrier.

[0023] 3. Beneficial effects Compared with the prior art, the advantages of this application are as follows: (1) The present application provides a functional exosome that highly expresses miR-124-3p and miR-10a-5p. miR-124-3p can inhibit the degradation of extracellular matrix and promote extracellular matrix remodeling by inhibiting signaling molecules such as ROCK1; while miR-10a-5p can target and inhibit the pro-inflammatory factor receptor (IL6R) to inhibit local inflammation. Furthermore, this exosome also highly expresses IL-1RA and Syntenin-1. On the one hand, due to its high expression of Syntenin-1, this exosome subset specifically enriches functional miRNAs such as miR-124-3p and miR-10a-5p. These miRNAs can be effectively delivered to nucleus pulposus cells, promoting extracellular matrix synthesis and remodeling by inhibiting signaling pathways such as ROCK1. On the other hand, due to its high expression of IL-1RA and carrying miR-10a-5p, this exosome can efficiently neutralize IL-1-mediated inflammatory signals, effectively improving the inflammatory microenvironment of intervertebral disc degeneration. This dual synergistic effect of "matrix remodeling + anti-inflammatory regulation" precisely intervenes in the core pathological links of intervertebral disc degeneration (matrix degradation and inflammation), achieving therapeutic effects that are difficult to achieve with single therapies. Exosomes isolated by existing technologies (such as ultracentrifugation and universal biomarker immunoaffinity assays) are complex mixtures and lack functional targeting.

[0024] (2) The present application provides a method for preparing functional exosomes, which uses Syntenin-1 (which regulates exosome biogenesis and specific miRNA loading) and IL-1RA (a key anti-inflammatory factor) as dual molecular targets for screening functional exosome subsets. Under hypoxic conditions, fibroblasts are stimulated with small molecule inducers (such as Y-27632 and tunicamycin) to promote the secretion of supernatant rich in target exosome subsets. Immunoaffinity capture is performed using a solid-phase carrier (magnetic beads) coupled with anti-Syntenin-1 antibody and anti-IL-1RA antibody, and combined with ultrafiltration concentration technology to form a highly efficient and high-purity separation process. The screened exosomes are specifically enriched with miRNAs (miR-124-3p and miR-10a-5p) with matrix remodeling and anti-inflammatory functions and IL-1RA protein with inhibitory inflammatory pathways.

[0025] (3) The method for preparing functional exosomes provided in this application, through a double antibody immunoaffinity method, can precisely screen out specific exosome subpopulations with dual functional potential of "tissue repair" and "immune regulation" from a complex exosome population derived from fibroblasts. This realizes the transformation from separation based on physical properties to fine screening based on specific functions. The engineered exosomes obtained have synergistic therapeutic effects: the exosomes enriched by this method no longer have a single function.

[0026] (4) This application provides a method for preparing functional exosomes that combines dual-target immunoaffinity assay with ultrafiltration concentration. The immunoaffinity step ensures the specific capture of the target exosome subsets, while the subsequent ultrafiltration step efficiently removes unbound impurities and small molecule components in the elution buffer, and achieves exosome concentration. This method overcomes the disadvantages of high purity, many impurities, and unstable recovery rate of ultracentrifugation, and also avoids the problems of harsh elution conditions and damage to the integrity of exosomes that may exist in traditional immunoaffinity assays, ultimately obtaining a high-purity, high-activity, and high-concentration therapeutic exosome preparation.

[0027] (5) The application of exosomes provided in this application in the preparation of drugs for treating intervertebral disc degeneration. Animal experimental results show that, compared with exosomes isolated by traditional methods, exosomes prepared by the method of this application can significantly better maintain the intervertebral disc height (DHI value close to the blank control group) when treating rat caudal intervertebral disc degeneration model, proving that it has a clear therapeutic effect in vivo that is superior to the prior art in delaying or repairing intervertebral disc degeneration. At the same time, using functionalized exosomes derived from fibroblasts as therapeutic agents avoids the immune rejection, ethical controversies and tumorigenesis risks brought about by direct cell transplantation, providing a safer, more stable and easier-to-standardize production and storage of a new "cell-free therapy" for the treatment of intervertebral disc degeneration. Attached Figure Description

[0028] Figure 1 These are the results of nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) analysis of exosomes.

[0029] Figure 2 This is the result of real-time quantitative PCR (RT-qPCR) detection of miRNA expression in exosomes.

[0030] Figure 3 The results are obtained by fluorescence in situ hybridization (FISH) detection of miR-124-3p expression in exosomes.

[0031] Figure 4 These are the results of Western blot (WB) analysis of the expression levels of IL-1RA and Syntenin-1 in exosomes.

[0032] Figure 5 This is an X-ray image of a rat during exosome therapy for intervertebral disc degeneration.

[0033] Figure 6 This is the result of DHI calculation during exosome therapy for intervertebral disc degeneration.

[0034] Figure 7 This is a schematic diagram of the parameters used in the DHI calculation. Detailed Implementation

[0035] The present application will be further described below with reference to specific embodiments.

[0036] It should be noted that terms such as "upper", "lower", "left", "right", and "middle" used in this specification are only for clarity of description and are not intended to limit the scope of implementation. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered as within the scope of this application.

[0037] 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 pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0038] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0039] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable. As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof. Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0040] As used in this application, fibroblasts are the most common cells in connective tissue. During embryonic development, the vast majority of fibroblasts originate from the mesoderm. Fibroblasts are widely distributed in human connective tissues, such as skin, tendons, and ligaments. Fibroblasts are one of the main cell types in the skin, responsible for maintaining its structure and function. Fibroblasts produce and maintain collagen fibers in tendons and ligaments, giving them mechanical strength and elasticity. After tissue injury, fibroblasts are activated and proliferate rapidly, migrating to the wound site and producing large amounts of temporary extracellular matrix (such as collagen III), forming granulation tissue. Subsequently, some fibroblasts differentiate into myofibroblasts, expressing α-smooth muscle actin, promoting wound closure through contraction. They synthesize and secrete collagen (mainly type I and type III), elastin, fibronectin, laminin, and various proteoglycans, which together constitute the extracellular matrix structure supporting cells. Fibroblasts have wide applications in basic research, regenerative medicine, and clinical treatment. They can be used in conjunction with keratinocytes to construct tissue-engineered skin for the treatment of burns and chronic ulcers, for the treatment of tendinopathy, and to promote collagen regeneration and structural repair through injection.

[0041] As used in this application, Syntenin-1 is an important adaptor protein that plays a core organizer role in the biogenesis and secretion of extracellular vesicles. Its main function is to connect the endocytic pathway with the formation of multivesicular bodies (MVBs) and ultimately affect the release of exosomes. Syntenin-1 participates in regulating the loading of specific cargoes in exosomes, such as miRNAs, to perform important functions.

[0042] As used in this application, interleukin-1 receptor antagonists (IL-1RAs) are an important member of the interleukin-1 (IL-1) family, encoded by the IL1RN gene, and play a crucial role in regulating inflammatory responses and immune responses. IL-1RAs can bind to the IL-1 receptor IL-1R1 without activating the receptor, thereby preventing pro-inflammatory cytokines such as IL-1α and IL-1β from binding to their receptors, inhibiting the activation of downstream inflammatory signaling pathways, and exerting an anti-inflammatory effect. In various autoinflammatory diseases, the deficiency or dysfunction of IL-1RAs is closely related to the pathogenesis of the disease. For example, IL-1RA-deficient mice exhibit severe systemic inflammatory responses, including arteritis, arthritis, and psoriasis-like skin lesions. These findings indicate that IL-1RAs play an indispensable role in maintaining immune homeostasis and suppressing excessive inflammatory responses.

[0043] The reagents used in this application are as follows: High-glucose DMEM medium is Gibco TMBASIC DMEM (High Glucose, Pyruvate), purchased from ThermoFisher SCIENTIFIC, part number C11995500BT.

[0044] Fetal bovine serum is Gibco TM Fetal bovine serum, purchased from Thermo Fisher Scientific, catalog number 10091148.

[0045] Phosphate-buffered saline (PBS) was purchased from Thermo Fisher Scientific, catalog number 10010023.

[0046] Y-27632 dihydrochloride, CAS number 129830-38-2, purchased from MedChemExpress, catalog number HY-10583. Y-27632 dihydrochloride is an orally active, ATP-competitive ROCK (Rho-kinase) inhibitor (ROCK-I K). i =220 nM; ROCK-II K i =300 nM).

[0047] Tunicamycin, CAS number 11089-65-9, was purchased from MedChemExpress, catalog number HY-A0098.

[0048] Anti-human Syntenin-1 antibody, purchased from Abcam, catalog number ab19903.

[0049] Anti-human IL-1RA antibody, purchased from R&D Systems, catalog number MAB280.

[0050] Carboxyl-modified superparamagnetic microspheres (Dynabeads) TM MyOne TM Carboxylic acid, purchased from Thermo Fisher Scientific, item number 65011.

[0051] Calcium-binding buffer solution: 1 mM CaCl2 + 1 mM Tris-HCl + 1% BSA, adjusted to pH 7.4.

[0052] CaCl2, CAS number 10043-52-4, purchased from Sigma-Aldrich, product number C4901.

[0053] 1 mM Tris-HCl, purchased from Sangon Biotech (Shanghai) Co., Ltd., product number B548127.

[0054] Bovine serum albumin (BSA), purchased from Sigma-Aldrich, catalog number A2153.

[0055] Tween-20 was purchased from Sangon Biotech (Shanghai) Co., Ltd., product number A100777-0500.

[0056] Elution buffer containing EGTA: 5 mM EGTA (Sigma, E4378) + 20 mM Tris-HCl + 0.25 M sucrose (Sigma, S7903) + 1% BSA, adjusted to pH 7.4.

[0057] EGTA, CAS number 67-42-5, purchased from Sigma-Aldrich, item number E4378.

[0058] Sucrose, CAS number 57-50-1, purchased from Sigma-Aldrich, product number S7903.

[0059] MES, CAS number 4432-31-9, purchased from Sigma-Aldrich, item number M3671.

[0060] 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) (Thermo Fisher Scientific, PG82079).

[0061] N-hydroxysuccinimide (NHS) (Thermo Fisher Scientific, 24500).

[0062] ProClin TM 300, purchased from Sigma-Aldrich, item number 48912-U.

[0063] Example 1 This embodiment provides a method for preparing exosomes.

[0064] Specifically, it includes: (1) Culture of fibroblasts Frozen human fibroblasts, passage P3, were rapidly thawed in a 37°C water bath and then cultured at a rate of 15,000 cells / cm³. 2 The cells were seeded into culture dishes at a certain density and cultured at 37°C and 5% CO2 for about 4 days. The human fibroblast culture medium was high-glucose DMEM (with 10% fetal bovine serum added by volume).

[0065] (2) Inducing exosome secretion Once the cell confluence reaches 50%–60%, discard the culture medium and wash twice with PBS. Add serum-free, high-glucose DMEM (with a final concentration of 1 μM Y-27632 dihydrochloride and a final concentration of 0.1 μM tunicamycin). Place the cells in a hypoxic incubator (5% CO2 and 2% O2, 37°C) for adherent culture. After 24 hours of continuous culture, directly aspirate the culture medium from the cell culture container to obtain the cell culture supernatant, which is used for exosome isolation.

[0066] (3) Isolation of exosomes The cell culture supernatant was centrifuged at 2,000×g for 20 minutes at 4°C to thoroughly remove intact cells and cell debris. The supernatant was then collected for later use.

[0067] In a reaction tube, the supernatant collected after centrifugation was mixed with pre-prepared superparamagnetic microspheres modified with anti-Syntenin-1 antibody and anti-IL-1RA antibody (superparamagnetic microspheres modified with anti-Syntenin-1 antibody and superparamagnetic microspheres modified with anti-IL-1RA antibody were pre-mixed at a 1:1 ratio) and gently incubated in a binding buffer containing calcium ions at room temperature for 30-60 minutes.

[0068] After incubation, the reaction tube was placed on a magnetic separator for magnetic separation. Once the magnetic microsphere-exosome complex was completely aggregated on the tube wall, the supernatant was carefully removed. Subsequently, under magnetic adsorption, the complex was washed multiple times with washing buffer (PBS or PBS solution containing 0.01%–0.05% (v / v) Tween-20) to remove non-specifically adsorbed impurities. This condition effectively removes non-specifically adsorbed proteins without disrupting the calcium-dependent binding between T-cell immunoglobulins and mucin domain protein 4 (TIM4) and exosomes. Elution buffer containing EGTA was added directly to the magnetic microsphere-exosome complex, and the eluent was collected. The eluent was then concentrated and replaced with buffer using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa. Specifically, the eluent was added to the upper chamber of the ultrafiltration centrifuge tube and centrifuged at 1000 × 10⁻⁶. g After centrifuging for 10 minutes, discard the liquid in the lower chamber. Then, add the eluent to be concentrated to the upper chamber until all the eluent is added. Centrifuge to concentrate the liquid in the upper chamber to about 1 mL. Then, add 10 mL of PBS buffer to the upper chamber and continue centrifuging until the liquid in the upper chamber is about 1 mL. Gently pipette the liquid in the upper chamber and gently blow the filter membrane of the upper chamber of the ultrafiltration centrifuge tube. Then, remove the liquid from the upper chamber to obtain a concentrated and highly pure exosome suspension. Aliquot and store at -80°C for later use.

[0069] In this embodiment, the method for preparing superparamagnetic microspheres with surface modifications of anti-Syntenin-1 antibody and anti-IL-1RA antibody includes the following steps: (a) Take 100 μL of carboxyl-modified superparamagnetic microsphere suspension and wash twice with MES (2-morpholinoethanesulfonic acid) buffer at pH 6.0; (b) Add a freshly prepared mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) in MES buffer to a final concentration of 50 mM and 25 mM, respectively, and activate at room temperature for 30 minutes. (c) Magnetic separation, removal of supernatant, and rapid washing twice with PBS buffer at pH 7.4 to obtain activated microspheres; (d) The activated microspheres were mixed with 50 μg of anti-human Syntenin-1 antibody or anti-human IL-1RA antibody in a binding buffer containing calcium ions and gently rotated and incubated at room temperature for 2 hours. (e) Magnetic separation, remove the supernatant, add 1 M Tris-HCl pH 8.0 solution, and block at room temperature for 1 hour; (f) Finally, wash and resuspend with PBS buffer containing 1 mM CaCl2 and 0.05% ProClin 300 to obtain superparamagnetic microspheres with anti-Syntenin-1 antibody or superparamagnetic microspheres with anti-IL-1RA antibody, and store at 4°C for later use.

[0070] Comparative Example 1 This embodiment provides a method for preparing exosomes by ultracentrifugation.

[0071] Frozen human fibroblasts, passage P3, were rapidly thawed in a 37°C water bath and then cultured at a rate of 15,000 cells / cm³. 2 The cells were seeded into culture dishes at a density of 37°C and 5% CO2 for about 4 days. The human fibroblast culture medium was high-glucose DMEM (with 10% fetal bovine serum added by volume).

[0072] Once the cell confluence reaches 50%–60%, discard the culture medium, rinse twice with PBS, add serum-free high-glucose DMEM, and continue culturing at 37°C and 5% CO2 for 24 hours. Collect the cell culture supernatant.

[0073] Centrifuge the collected supernatant at 300×g for 10 minutes to remove cell debris and non-adherent cells, and collect the supernatant again. Then, centrifuge the supernatant at 2000×g for 10 minutes to remove larger cell debris and impurities, and collect the supernatant again. Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000×g for 70 minutes to separate the exosomes, which will precipitate at the bottom of the tube. Carefully remove the supernatant, add an appropriate amount of PBS buffer (pH 7.4) to resuspend the precipitate, and centrifuge again at 100,000×g for 70 minutes to further purify the exosomes. Finally, carefully remove the supernatant, add an appropriate amount of PBS buffer or other suitable preservation solution to resuspend the exosomes, and obtain the purified exosome sample, which can be aliquoted and stored at -80℃ for subsequent experimental applications.

[0074] Comparative Example 2 This embodiment provides a method for preparing exosomes using conventional magnetic beads.

[0075] In this comparative example, anti-CD63 magnetic beads (Thermo Fisher SCIENTIFIC, 10606D) were used for exosome capture.

[0076] Frozen human fibroblasts, passage P3, were rapidly thawed in a 37°C water bath and then cultured at a rate of 15,000 cells / cm³. 2 The cells were seeded into culture dishes at a density of 37°C and 5% CO2 for about 4 days. The human fibroblast culture medium was high-glucose DMEM (with 10% fetal bovine serum added by volume).

[0077] Once the cell confluence reaches 50%–60%, discard the culture medium, rinse twice with PBS, add serum-free high-glucose DMEM, and continue culturing at 37°C and 5% CO2 for 24 hours. Collect the cell culture supernatant.

[0078] After harvesting the supernatant from the cell culture medium, first... g Centrifuge for 10 minutes to remove cell debris and non-adherent cells, and collect the supernatant.

[0079] Following the instructions for the magnetic beads, remove them from the storage solution and wash them 2-3 times with PBS buffer. After each centrifugation, use a magnetic bead separator to remove the supernatant under magnetic field. Then, resuspend the magnetic beads in PBS buffer to a concentration of 1-5 mg / ml. Add the pretreated supernatant to a centrifuge tube containing the magnetic beads and incubate at 4°C for 1-2 hours, gently shaking the tube during this time to promote the binding of the magnetic beads to the exosomes.

[0080] After incubation, place the centrifuge tube in the magnetic field of the magnetic bead separator to allow the magnetic beads to adhere to the tube wall. Carefully aspirate the supernatant. Wash the magnetic beads 3-5 times with PBS buffer, aspirating the supernatant after each wash to remove impurities. Finally, resuspend the magnetic beads and exosomes in an appropriate amount of PBS buffer or other preservation solution to obtain a purified exosome sample, which can be aliquoted and stored at -80°C.

[0081] Example 2 This embodiment provides characterization of the exosomes prepared in Example 1, Comparative Example 1, and Comparative Example 2.

[0082] In this embodiment, the exosomes prepared in Example 1 are labeled as MB-IP(NEW); the exosomes prepared in Comparative Example 1 are labeled as UC; and the exosomes prepared in Comparative Example 2 are labeled as MB-IP.

[0083] (1) Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) of exosomes Nanoparticle tracking analysis (NTA): Extracted exosome samples were diluted with PBS at a ratio of 1:100 (volume) to ensure that the number of particles per field of view was within the instrument's optimal detection range (20–100 particles / frame). Detection was performed using a Malvern Panalytical NanoSight NS300 instrument (or a similar device). Refer to the manufacturer's instruction manual for specific operating parameter settings.

[0084] Transmission electron microscopy (TEM) analysis: 10 μL of exosome suspension was dropped onto a copper grid supported by a carbon film and allowed to stand at room temperature for 5–10 minutes for adsorption. Excess liquid was blotted away with filter paper, and then negatively stained with 2% phosphotungstic acid aqueous solution (pH 7.0) (Sigma, P4006) for 1–2 minutes. Excess staining was blotted away again with filter paper, and the sample was allowed to air dry at room temperature. The prepared sample was observed and imaged using a Hitachi HT7800 transmission electron microscope (or similar equipment) at an accelerating voltage of 80 kV or 100 kV. Specific operating parameter settings should be referred to the manufacturer's instructions.

[0085] Results analysis: The results are as follows Figure 1 As shown, the exosome particles prepared in Example 1 are relatively uniform in size, mainly concentrated between 50 and 200 nm, and have fewer impurities; the exosome particles prepared in Comparative Example 1 have a diameter between 50 and 400 nm, and more impurities and a less clear background can be seen under transmission electron microscopy.

[0086] (2) Real-time quantitative PCR (RT-qPCR) detection of miRNA in exosomes Total RNA was extracted from isolated exosomes (exosome suspension frozen at -80°C) using the miRNeasy Mini Kit (Qiagen, 217004). Reverse transcription was performed using stem-loop primers, wherein: miR-124-3p: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGGGCAT-3' (SEQ IDNO.1); miR-10a-5p: 5'-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCACAA-3' (SEQ ID NO. 2).

[0087] Real-time quantitative PCR was performed using qPCR premix (catalog number: LS2062, Promega), and all reactions were performed in triplicate. U6 snRNA was used as the internal reference gene for miRNA, and a 2... -ΔΔCt The relative expression level is calculated using the following method: qPCR primers for miR-124-3p: Forward: 5'-TAAGGCACGCGGTG-3' (SEQ ID NO.3); qPCR primers for miR-10a-5p: Forward: 5'-TACCCTGTAGATCCGAAT-3' (SEQ ID NO.4); Universal reverse primer: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC-3' (SEQ ID NO.5).

[0088] Results analysis: The results are as follows Figure 2 As shown, compared with the ultracentrifugation group, the expression levels of miR-124-3p and miR-10a-5p in the conventional magnetic bead separation group did not change significantly. However, the exosomes obtained by the separation method used in this application showed significantly high expression of miR-124-3p and miR-10a-5p. Specifically, miR-124-3p can inhibit the degradation of extracellular matrix and promote extracellular matrix remodeling by inhibiting signaling molecules such as ROCK1; while miR-10a-5p can target and inhibit the pro-inflammatory factor receptor (IL6R) to suppress local inflammation.

[0089] (3) Fluorescence in situ hybridization (FISH) was used to detect the expression of miR-124-3p in exosomes. First, human nucleus pulposus cells were cultured in 24-well plates. When the cell confluence reached 40%–50%, 2–4 μg / mL of exosome sample (PKH67 labeled using the PKH67 Green Fluorescent Cell Linker Mini Kit, purchased from Sigma-Aldrich) was added. The cells were cultured for another 24 hours. The supernatant was then discarded, and the cells were gently washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10–15 minutes. After fixation, the paraformaldehyde was washed away with PBS, and the cell membrane was permeated with 0.1%–0.5% Triton X-100 solution for 5–10 minutes to increase probe permeability.

[0090] Subsequently, prehybridization was performed according to the kit instructions (Thermo Fisher SCIENTIFIC, F32956): an appropriate amount of probe-free prehybridization solution was added, and the cells were incubated in a humidified chamber at 37°C for 1 hour to reduce nonspecific binding. Next, the prehybridization solution was removed, and hybridization solution containing the Cy3-labeled miR-124-3p probe (Cy3-5'-GGCATTCACCGCGTGCCTTA-3', SEQ ID NO.6) was added directly, ensuring complete coverage of the cells. The slides were placed in a preheated humidified chamber and incubated at 37°C in the dark for 12 hours. After hybridization, the cells were washed sequentially with buffers of different concentrations (usually at a temperature slightly higher than the hybridization temperature) to thoroughly remove unbound and nonspecifically bound probes.

[0091] Finally, the nuclei were stained and mounted using mounting medium containing DAPI, and the images were observed and acquired under a fluorescence microscope: red fluorescence (miR-124-3p), green fluorescence (PKH67-labeled exosomes) were observed through specific fluorescence channels, and the cell nuclei could be observed by observing the blue fluorescence of DAPI. The expression level of miR-124-3p was analyzed based on the intensity of the fluorescence signal and its intracellular localization.

[0092] Results analysis: The results are as follows Figure 3 As shown, exosomes isolated by ultracentrifugation (UC) and conventional magnetic bead-IP (MB-IP) methods can be taken up into cells, but very few exosomes contain miR-124-3p; while the exosomes isolated in this application contain a large amount of miR-124-3p, which is consistent with the results of RT-qPCR.

[0093] (4) Western blot (WB) was used to detect the expression levels of IL-1RA and Syntenin-1 in exosomes. Take 20–40 μg of exosome sample, mix with SDS-PAGE loading buffer, and heat at 95–100°C for 5–10 minutes to fully denature the protein. Load the denatured protein sample and pre-stained protein molecular weight standards together for SDS-PAGE gel electrophoresis. After electrophoresis, transfer the protein from the gel to a PVDF membrane using a wet transfer method. After transfer, block with TBST solution containing 5% skim milk at room temperature for 1 hour. Place the blocked membrane in a primary antibody (Syntenin antibody; IL1RA antibody) dilution buffer and incubate overnight at 4°C with gentle shaking. The next day, wash the membrane three times with TBST to remove unbound primary antibody, and then incubate with the corresponding HRP-labeled secondary antibodies (goat anti-mouse: Abcam, ab6789; goat anti-rabbit: Abcam, ab6721) at room temperature for 1 hour. After thorough washing again, ECL chemiluminescence reagent (Thermo, 32106) was added to the membrane, and the membrane was exposed and images were acquired in a chemiluminescence imaging system.

[0094] Results analysis: The results are as follows Figure 4 As shown, the contents of IL-RA and Syntenin-1 in exosomes isolated by ultracentrifugation (UC) and conventional magnetic bead method (MB-IP) are significantly lower than those in the exosomes isolated in this application.

[0095] Example 3 This embodiment provides the application of exosomes prepared in Example 1, Comparative Example 1, and Comparative Example 2 in the treatment of intervertebral disc degeneration.

[0096] (1) Preparation of a rat intervertebral disc degeneration model All animals were acclimatized in the animal laboratory for one week. Rats were anesthetized by intramuscular injection of a mixture of acetaminophen 50, atropine sulfate, and xylazine hydrochloride at a dose of 1.5 mL / kg. Whole-body images of the rats were taken using a dual-energy X-ray absorptiometry (DXA) system to locate the Co7 / 8 intervertebral disc in the tail. The tail area was disinfected with iodine solution. A longitudinal incision of approximately 0.5 cm was made in the tail to expose the Co7 / 8 intervertebral disc. A semi-transverse puncture was performed on the tail vertebra using a 20G micro-puncture device to obtain a rat model of intervertebral disc degeneration.

[0097] (2) Exosome therapy The rat intervertebral disc degeneration model was divided into 4 groups, with 5 rats in each group, as follows: G1, Sham Group G2, MB-IP (NEW) for the isolation of exosomes G3, MB-IP separation of exosomes G4, UC isolates exosomes In the Sham group, the Co7 / 8 intervertebral discs of the rat caudal vertebrae were exposed and then sutured. The other groups were given the drug (referred to as day 0), and 3 μg of exosomes were injected into each intervertebral disc.

[0098] X-ray examinations of the coccygeal intervertebral discs were performed on days 14, 28, and 84, and the Disc Height Index (DHI) was calculated. The DHI calculation method is as follows: the height of the intervertebral discs and the lateral and mid-vertebral bodies at the superior and inferior margins of each group were measured, and the result was calculated using the following formula: DHI = (A + B + C) × 2 / (D + E + F + G + H + I). Figure 7 As shown, where: A and C are the heights of the intervertebral disc on both sides, B is the height of the intervertebral disc in the middle; D, G, F and I are the heights of the upper and lower edge vertebral bodies on both sides, and E and H are the heights of the upper and lower edge vertebral bodies in the middle.

[0099] Results analysis: The results are as follows Figure 5 and Figure 6 As shown, compared with the UC (ultracentrifugation to separate exosomes) group, the DHI of the exosomes isolated in this application (G2 group) was significantly increased, and the DHI value was close to that of the blank control group (G1 group), indicating that the exosomes isolated in this application have a good therapeutic effect in the coccygeal intervertebral disc degeneration model.

Claims

1. A functional exosome, characterized in that, The exosomes highly express miR-124-3p and miR-10a-5p.

2. The functional exosomes according to claim 1, characterized in that, The exosomes also highly express IL-1RA and Syntenin-1.

3. A method for preparing functional exosomes, characterized in that, The method includes the following steps: S1, culture cells and induce cells to secrete exosomes; S2, after induction culture, the supernatant was collected, and exosomes were screened using anti-Syntenin-1 antibody and anti-IL-1RA antibody.

4. The method for preparing functional exosomes according to claim 3, characterized in that, The exosome screening process also includes ultrafiltration, in which the molecular weight cutoff is 100 kDa.

5. The method for preparing functional exosomes according to claim 3 or 4, characterized in that, In S1, The cells are fibroblasts; and / or Inducing cell secretion of exosomes includes stimulating cells with Y-27632 dihydrochloride and / or tunicamycin to induce exosome secretion.

6. The method for preparing functional exosomes according to claim 5, characterized in that, In S2, the screening using anti-Syntenin-1 antibody and anti-IL-1RA antibody includes: The supernatant or pretreated supernatant was mixed with superparamagnetic microspheres modified with anti-Syntenin-1 antibody and superparamagnetic microspheres modified with anti-IL-1RA antibody, and gently incubated in a calcium-containing binding buffer at room temperature for 30-60 minutes. After incubation, magnetic separation was performed to obtain the magnetic microsphere-exosome complex. After washing and elution, an exosome-containing eluent was obtained.

7. The method for preparing functional exosomes according to claim 6, characterized in that, The washing process uses a washing buffer, which is PBS or a PBS solution containing 0.01% to 0.05% (v / v) Tween-20; and / or The elution process includes: adding an elution buffer containing EGTA to the magnetic microsphere-exosome complex for elution, and collecting the eluent.

8. The method for preparing functional exosomes according to claim 6 or 7, characterized in that, The preparation method of the superparamagnetic microspheres with surface modifications of anti-Syntenin-1 antibody and anti-IL-1RA antibody includes the following steps: (a) Take a suspension of carboxyl-modified superparamagnetic microspheres; (b) Add a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide prepared with MES buffer and activate at room temperature; (c) Magnetic separation, removal of supernatant, and rapid washing twice with PBS buffer to obtain activated microspheres; (d) Mix the activated microspheres with anti-human Syntenin-1 antibody or anti-human IL-1RA antibody in a binding buffer containing calcium ions and incubate gently by rotating at room temperature; (e) Magnetic separation, remove the supernatant, add 1 M Tris-HCl solution, and block at room temperature for 1 hour; (f) Wash and resuspend with PBS buffer containing 1 mM CaCl2 and 0.01% ProClin 300 to obtain superparamagnetic microspheres with surface modified with anti-Syntenin-1 antibody or superparamagnetic microspheres with surface modified with anti-IL-1RA antibody.

9. The use of the functional exosomes according to claim 1 or 2 in the preparation of drugs for treating intervertebral disc degeneration.

10. A pharmaceutical composition comprising the functional exosomes of claim 1 or 2, and a pharmaceutically acceptable carrier.

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