Method for isolating cardiomyocyte-derived small extracellular vesicles from ex vivo plasma and applications thereof

Abstract

The present application relates to a method for isolating cardiomyocyte-derived small extracellular vesicles from an ex vivo plasma sample and its in vitro application. The method comprises: (1) providing a ligand capable of specifically binding to gamma-sarcoglycan protein SGCG; (2) isolating total small extracellular vesicles from an ex vivo plasma sample; (3) co-incubating the ligand with the total small extracellular vesicles obtained in step (2) to form a ligand-vesicle complex containing the ligand and cardiomyocyte-derived small extracellular vesicles that specifically bind to the surface expressing SGCG; (4) binding the ligand-vesicle complex formed in step (3) to a solid phase carrier, thereby separating the cardiomyocyte-derived small extracellular vesicles expressing SGCG on the surface of the ligand-vesicle complex from the mixture; (5) washing and / or eluting the ligand-vesicle complex bound to the solid phase carrier in step (4) to obtain enriched cardiomyocyte-derived small extracellular vesicles.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and in vitro detection technology, and specifically relates to a method for specifically isolating small extracellular vesicles derived from cardiomyocytes from complex biological samples such as isolated plasma. Background Technology

[0002] Heart disease is a leading cause of death and disability worldwide. Current clinical diagnosis relies primarily on biomarkers (such as cardiac troponin) and imaging studies (such as echocardiography). However, these methods have inherent limitations: the release of biomarkers is delayed, making it difficult to capture early molecular changes in chronic pathological processes; and imaging studies typically only detect problems after significant alterations in cardiac structure have occurred. Therefore, there is an urgent need in clinical and basic research for novel in vitro detection tools and molecular marker research methods that can reflect the real-time pathophysiological state of cardiomyocytes earlier and more specifically.

[0003] Small extracellular vesicles (sEVs) are nanoscale vesicles actively secreted by cells, carrying molecular information such as proteins, nucleic acids, and lipids from their parent cells, and are hailed as ideal targets for "liquid biopsies." Theoretically, cardiomyocyte-derived sEVs can dynamically reflect the health and disease status of cardiomyocytes. However, plasma sEVs are a highly heterogeneous mixture, originating from platelets, immune cells, endothelial cells, and various parenchymal cells. Current technologies, such as ultracentrifugation and size exclusion chromatography, can only separate total sEVs, not distinguish their cellular origin. This fundamental technical challenge of signal confounding means that any molecular changes resolved from circulating sEVs cannot be definitively attributed to cardiomyocytes, severely hindering the translational application of cardiac sEVs in precision cardiovascular medicine.

[0004] Comparative document 1 (“Circulating small extracellular vesicles as blood-based biomarkers of muscle health in aging nonhuman primates”, Mishra S et al, Geroscience. 47(3):3709-3723, 2025 Jun) discloses a method for isolating skeletal muscle-derived small extracellular vesicles from serum. This method involves incubating serum sEVs with biotin-labeled SGCA (alpha-caryogamin) or MuSK antibodies, followed by incubation with streptavidin-conjugated magnetic beads. Elution and magnetic separation are then performed to prepare sEVSKM (see the “sEV isolation” section of the methods). This method uses alpha-caryogamin SGCA as a skeletal muscle-specific sEV surface marker. Total serum sEVs are incubated with biotin-labeled anti-SGCA antibodies, then bound to streptavidin-conjugated magnetic beads, followed by elution and magnetic separation to prepare skeletal muscle-derived sEVs. Comparative document 1 validates the purity and specificity of the isolated sEVs and uses them for research on biomarkers of muscle aging.

[0005] Therefore, there is an urgent need in this field to develop a method that can specifically isolate and enrich cardiomyocyte-derived sEV subsets from the total plasma sEV pool, thereby providing pure materials with clearly defined cell origins for the early diagnosis, mechanism research, and drug development of heart diseases. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the technical defects of the prior art in that it is impossible to specifically isolate small extracellular vesicles (sEVs) of cardiomyocytes from complex biological samples such as isolated plasma. The invention provides an immunoaffinity separation method based on cardiomyocyte-specific surface markers to achieve efficient and specific capture and enrichment of cardiomyocyte-derived sEV subsets from total sEV mixtures, and the method and application of isolating small extracellular vesicles of cardiomyocytes from isolated plasma.

[0007] The first technical solution of the present invention is the method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma, characterized by comprising the following steps: (1) Provide ligands that can specifically bind to γ-sarcosin SGCG; (2) Isolate total small extracellular vesicles from isolated plasma samples; (3) The ligand is co-incubated with the total small extracellular vesicles obtained in step (2) to form a ligand-vesicle complex containing the ligand and small extracellular vesicles derived from cardiomyocytes expressing SGCG on their surface. (4) The ligand-vesicle complex formed in step (3) is combined with a solid support to separate the ligand-vesicle complex from the mixture; (5) The ligand-vesicle complex bound to the solid support in step (4) is washed and / or eluted to obtain enriched small extracellular vesicles derived from cardiomyocytes.

[0008] This independent claim achieves the specific isolation of cardiomyocyte-derived sEVs through the following technical features: Using ligands that specifically bind to γ-sarcosin SGCG is key to achieving specific recognition of cardiomyocyte-derived sEVs.

[0009] The method steps are characterized by the following steps: separation of total sEVs, incubation of ligands and ligand-vesicle complexes, binding with a solid support, washing and elution, which constitute a complete separation process.

[0010] Product-specific features: Ultimately, it yields enriched small extracellular vesicles derived from cardiomyocytes.

[0011] Preferably, the ligand is selected from antibodies, antibody fragments, nucleic acid aptamers, peptide aptamers, or small molecule ligands.

[0012] Preferably, the ligand is an antibody against SGCG.

[0013] Preferably, the anti-SGCG antibody is a biotin-labeled antibody; and the solid-phase carrier is microspheres coated with streptavidin or neutral avidin. The solid-phase carrier can be a microsphere or a plate-like carrier.

[0014] Preferably, the separation of total small extracellular vesicles in step (2) is performed using ultracentrifugation combined with density gradient centrifugation. The separation of total small extracellular vesicles from plasma samples is performed using ultracentrifugation combined with density gradient centrifugation. Gradient centrifugation is used to obtain high-purity total sEVs, reducing the non-specific background of subsequent immune capture. Preferably, the γ-sarcosinin SGCG, as a specific surface marker of small extracellular vesicles derived from cardiomyocytes, is obtained through screening using a method including the following steps: (1.1) Construct transgenic animals that specifically express the L274G mutant of methionine-tRNA synthetase in cardiomyocytes; (1.2) The transgenic animals were given L-azidoleucine to label the cardiomyocyte neoplasm; (1.3) The heart tissue and extracellular vesicles of heart-derived small cells of the transgenic animal were isolated and identified by mass spectrometry analysis of the proteins labeled with L-azidoleucine. (1.4) Cross-analysis was performed on labeled proteins in cardiac tissue and cardiac-derived small extracellular vesicles (sEVs) to screen for SGCG, a membrane protein expressed in both and located on the vesicle surface, as the specific surface marker. This screening method first constructs a transgenic animal model that specifically expresses the methionine-tRNA synthetase L274G mutant (MetRSL274G) in cardiomyocytes; then, L-azoleucine (ANL) is administered to this model to specifically label the newly generated cardiomyocyte protein; next, cardiac tissue and cardiac-derived small extracellular vesicles are isolated, and ANL-labeled proteins are enriched using click chemistry and identified by mass spectrometry; finally, cross-analysis of the two sets of mass spectrometry data is performed to screen for membrane proteins that are highly expressed in both cardiac tissue and its secreted sEVs and located on the vesicle surface, ultimately identifying γ-sarcosin (SGCG) as the ideal target. This screening method not only ensures the high specificity of the marker but also provides a valuable strategy for the discovery of sEV markers from other cell sources.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Achieved specific isolation of cardiomyocyte-derived sEVs: For the first time, SGCG obtained through rigorous in vivo screening was used as a specific marker to successfully separate cardiomyocyte-derived sEVs from complex plasma sEV mixtures, solving the long-standing bottleneck problem of signal confounding and lack of traceability in this field, and opening up new avenues for liquid biopsy and mechanism research in cardiovascular diseases.

[0016] (2) High purity and good activity of the separated products: Through the efficient synergy between SGCG antibody and biotin-streptavidin amplification system, high affinity capture and low background washing of ligand-vesicle complexes were achieved, which significantly improved the enrichment purity and integrity of target sEVs.

[0017] (3) Complete analytical tools are provided: This invention not only provides a separation method, but also provides the resulting high-purity cardiomyocyte-derived sEV product, as well as a kit containing the core capture component and / or downstream detection reagents. These products provide a reliable technical basis and tools for discovering and validating heart disease-specific molecular biomarkers and conducting in vitro molecular typing studies.

[0018] (4) The biomarker screening method is universal: The biomarker screening method of “transgenic animal model + non-classical amino acid marker + proteomics cross-analysis” is highly innovative and scalable, and can be used to discover sEV-specific biomarkers from other tissue or cell sources. Attached Figure Description

[0019] Figure 1A This invention relates to mice that specifically express mutant methionine-tRNA synthetase (MetRS*) in cardiomyocytes.CM A schematic diagram of the breeding strategy for MetRS* mice; Figure 1B This invention is a Western blot detection method. CM Schematic diagram showing the results of GFP expression abundance in the hearts of MetRS* mice and control mice; Figure 1C This invention is a Western blot detection method. CM A schematic diagram showing the results of GFP expression abundance in various tissues and organs of MetRS* mice; Figure 1D-1 This invention relates to immunofluorescence detection. CM Schematic diagram of GFP expression results in heart tissue of MetRS* control mice; Figure 1D-2 This invention relates to immunofluorescence detection. CM Schematic diagram showing the results of GFP expression in MetRS* mouse heart tissue; Figure 1D-3 This invention relates to immunofluorescence detection. CM Schematic diagram of α-actinin expression results in MetRS* control mouse cardiac tissue; Figure 1D-4 This invention relates to immunofluorescence detection. CM Schematic diagram showing the results of α-actinin expression in MetRS* mouse cardiac tissue; Figure 1D-5 This invention relates to immunofluorescence detection. CM Schematic diagram of the results of DAPI staining in the heart tissue of MetRS* control mice; Figure 1D-6 This invention relates to immunofluorescence detection. CM Schematic diagram of the results of DAPI staining in MetRS* mouse heart tissue; Figure 1D-7 This invention relates to immunofluorescence detection. CM Schematic diagram of the combined results of GFP, α-actinin and DAPI staining in heart tissue of MetRS* control mice; Figure 1D-8 This invention relates to immunofluorescence detection. CM Schematic diagram of the combined results of GFP, α-actinin and DAPI staining in MetRS* mouse heart tissue; Figures 1D-1 to 1D-8 This invention relates to immunofluorescence detection. CM Schematic diagram showing the results of GFP expression abundance in the heart tissues of MetRS* mice and their control mice; Figure 1E This invention is for CMA schematic diagram illustrating the strategy for administering L-azoleucine (ANL) to MetRS* mice and their control mice; Figure 1F-1 This invention uses BONCAT technology for detection. CM A schematic diagram showing the results of ANL incorporation in the myocardial tissue of MetRS* mice and their control mice. Figure 1F-2 This invention uses BONCAT technology for detection. CM A schematic diagram showing the total amount of protein loaded into the myocardial tissue of MetRS* mice and their control mice, indicating the incorporation of ANL protein into the myocardial tissue. Figures 1F-1 to 1F-2 This invention uses BONCAT technology for detection. CM Figure 1 shows the results of ANL incorporation in MetRS* mice and their control mice. Figure 1G This invention uses BONCAT technology for detection. CM Figure 1 shows the results of ANL incorporation in different organs of MetRS* mice and their control mice. Figure 1H-1 This invention relates to immunofluorescence detection. CM Schematic diagram of ANL incorporation in the myocardial tissue of MetRS* control mice; Figure 1H-2 This invention relates to immunofluorescence detection. CM Schematic diagram of ANL incorporation in the myocardial tissue of MetRS* mice; Figure 1H-3 This invention relates to immunofluorescence detection. CM Schematic diagram of GFP expression in the myocardial tissue of MetRS* control mice; Figure 1H-4 This invention relates to immunofluorescence detection. CM Schematic diagram of GFP expression in the myocardial tissue of MetRS* mice; Figure 1H-5 This invention relates to immunofluorescence detection. CM Schematic diagram of the results of DAPI staining in the myocardial tissue of MetRS* control mice; Figure 1H-6 This invention relates to immunofluorescence detection. CM Schematic diagram of the results of DAPI staining in the myocardial tissue of MetRS* mice; Figure 1H-7 This invention relates to immunofluorescence detection. CM A schematic diagram showing the combined results of ANL incorporation, GFP expression, and DAPI staining in the myocardial tissue of MetRS* control mice; Figure 1H-8 This invention relates to immunofluorescence detection. CMA schematic diagram showing the combined results of ANL incorporation, GFP expression, and DAPI staining in the myocardial tissue of MetRS* mice; Figures 1H-1 to 1H-8 This invention relates to immunofluorescence detection. CM A schematic diagram showing the results of ANL incorporation in the myocardial tissue of MetRS* mice and their control mice. Figure 1A Figure 1H shows a schematic diagram of the construction and validation of a cardiomyocyte-specific transgenic mouse model; Figure 2A This is a schematic flowchart of the cardiac sEV extraction method of the present invention; Figure 2B This is a schematic diagram of the expression of sEV markers in each layer of cardiac sEVs after iodixanol density gradient centrifugation using Western Blot according to the present invention. Figure 2C This is a representative result of the nanoparticle tracking analysis (NTA) method used in this invention to detect the size distribution and concentration of sEVs in the heart. Figure 2D These are representative images of cardiac sEV morphology observed by transmission electron microscopy (TEM) according to the present invention; Figures 2A to 2D A schematic diagram showing the isolation and identification of small extracellular vesicles derived from the heart; Figure 3A This is a schematic diagram of the expression of sEV markers in each layer of plasma after density gradient centrifugation using Western Blot according to the present invention. Figure 3B This is a representative result diagram of the NTA detection of plasma sEV particle size distribution according to the present invention; Figure 3C These are representative images of plasma sEV morphology observed by TEM according to this invention; Figures 3A to 3C A schematic diagram showing the isolation and identification of plasma-derived small extracellular vesicles; Figure 4A-1 This invention uses BONCAT technology for detection. CM A schematic diagram of ANL incorporation in the cardiac sEV protein of MetRS* mice and their control mice. Figure 4A-2 This invention uses BONCAT technology for detection. CM A schematic diagram of ANL incorporation in the heart sEV protein of MetRS* mice and control mice and the total protein loading amount. Figures 4A-1 to 4A-2 This invention uses BONCAT technology for detection. CM A schematic diagram of ANL incorporation in the heart sEV of MetRS* mice; Figure 4BThis invention is a Venn diagram showing the quantity and overlap of ANL-labeled proteins and total proteins in cardiac sEVs. Figure 4C-1 This is a schematic diagram showing the results of molecular functional enrichment analysis of ANL-labeled proteins in cardiac sEVs according to the present invention. Figure 4C-2 This is a schematic diagram showing the results of the biological process enrichment analysis of ANL-labeled proteins in cardiac sEVs according to the present invention. Figure 4C-3 This is a schematic diagram showing the results of the cellular component enrichment analysis of ANL-marked proteins in cardiac sEVs according to the present invention. Figures 4C-1 to 4C-3 This is a graph showing the results of GO enrichment analysis of ANL-labeled proteins in cardiac sEVs according to this invention. Figures 4A to 4C show schematic diagrams of the analysis of ANL marker proteins in cardiomyocyte-derived sEVs; Figure 5A This is a flowchart of the strategy for screening specific surface markers of cardiomyocyte-derived sEVs according to the present invention; Figure 5B This is a schematic diagram illustrating the Western blot detection of the expression specificity of the candidate protein SGCG in various organ tissues of mice according to the present invention; Figure 5C This is a schematic diagram of the Western blot detection of SGCG distribution in different density gradient layers of the cardiac sEV according to the present invention; Figure 5D This is a schematic diagram of the Western blot detection of SGCG distribution in different density gradient layers of plasma sEVs according to the present invention; Figure 5E This is a schematic diagram illustrating the co-enrichment of myocardial-specific proteins (such as cTNT) and sEV markers (such as CD63) in mouse plasma sEV enriched with SGCG antibody, as verified by Western blot in this invention. Figure 5F This is a schematic diagram illustrating the co-enrichment of myocardial-specific proteins and sEV markers in human plasma sEVs enriched using SGCG antibodies, as verified by Western blot analysis of this invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments, following the structure of technical problem-technical feature-beneficial effect.

[0021] definition: 1. Unless otherwise specified, the term "plasma" as used in this invention refers to "in vitro plasma sample," the processing and detection of which are carried out in a non-in vivo environment.

[0022] 2. Unless otherwise specified, the term "ligand-vesicle complex" as used in this invention refers to a complex formed by the binding of a ligand that specifically binds to SGCG to small extracellular vesicles derived from cardiomyocytes that express SGCG on their surface.

[0023] Furthermore, although the embodiments of the present invention have been described in detail using isolated plasma as an example, those skilled in the art should understand that the separation system and principle based on SGCG ligands of the present invention are also reasonably applicable to the in vitro enrichment and scientific research of other isolated biological samples containing extracellular vesicles (such as isolated serum, cell culture supernatant, etc.).

[0024] Example 1: Construction and validation of a cardiomyocyte-specific transgenic mouse model The technical problem to be solved in this embodiment is: how to obtain an animal model that can specifically label new cardiomyocyte proteins so as to screen for specific biomarkers of cardiomyocyte-derived sEVs.

[0025] The technical features adopted include: Transgenic mice expressing the methionine-tRNA synthetase L274G mutant (MetRSL274G) specifically for cardiomyocytes were constructed. CM MetRS* mice. Specifically, stopfl-MetRS* mice (JAX Lab, strain number 028071) were mated with Myh6-Cre mice (Cyagen Biosciences, strain number C001041) to obtain offspring. CM MetRS* mice. These mice specifically express the MetRS*L274G mutant and green fluorescent protein (GFP) only in cardiomyocytes. Figure 1A (As shown).

[0026] The expression of GFP in cardiac tissue was detected by Western blot to verify the genotype. The results showed that only... CM MetRS* mice have strong GFP signals in their hearts (e.g., Figure 1B As shown). Western blot analysis of organ-specific components showed that GFP was highly expressed only in the heart, and not in other tissues (e.g., Figure 1C As shown). Immunofluorescence staining also confirmed the specific localization of GFP in cardiomyocytes (e.g., Figure 1D-2 , Figure 1D-4 , Figure 1D-6 and Figure 1D-8As shown), while the control mice of CMMetRS* did not have the corresponding GFP signal (as shown). Figure 1D-1 , Figure 1D-3 , Figure 1D-5 and Figure 1D-7 (As shown).

[0027] All mice were housed in an SPF-grade environment with free access to water and food, 12-hour light and dark cycles, a room temperature of 22-24℃, and a humidity of 40-70%.

[0028] The beneficial effects obtained through the above technical features are: a cardiomyocyte-specific MetRS*L274G expression mouse model was successfully constructed. This model can specifically introduce the non-classical amino acid ANL into cardiomyocytes, providing an ideal tool for subsequent labeling and screening of cardiomyocyte-specific proteins.

[0029] Example 2: Using ANL to metabolically label new cardiac cell proteins The technical problem to be solved in this embodiment is: how to specifically label the newly generated proteins of cardiomyocytes at the in vivo level so as to identify which proteins are highly expressed in cardiomyocytes and their secreted sEVs by mass spectrometry.

[0030] The technical features adopted include: ANL dosing regimen: Dissolve L-azoleucine (ANL) in PBS to prepare a 200 mM solution, and adjust the pH to 7.4. CM MetRS* mice and control MetRS* mice were first fed a low-methionine diet for 12 hours, followed by intraperitoneal injection of ANL solution (10 mL / kg) every 12 hours for 3 consecutive days. Six hours after the last injection, the mice were sacrificed, and tissue samples were collected (e.g., ...). Figure 1E (As shown).

[0031] BONCAT assay: Proteins extracted from cardiac tissue were subjected to click chemistry with biotin-PEG4-azide. Western blot and HRP-streptavidin assays showed that only in patients injected with ANL... CM A large amount of biotin-labeled protein was detected in the heart of MetRS* mice. Figure 1F-1 and Figure 1F-2 ) 。 Organ-specific BONCAT analysis confirmed that ANL-labeled proteins were mainly enriched in CM The heart of MetRS* mice showed no significant markers in other organs (e.g., Figure 1G (As shown).

[0032] FUNCAT assay: Fluorescent click chemistry was performed on frozen sections of ANL-labeled cardiac tissue. Fluorescence microscopy revealed that only the sections injected with ANL showed positive fluorescence. CMStrong red fluorescence (AF555) was observed in MetRS* mouse cardiomyocytes (e.g. Figure 1H-1 , Figure 1H-3 , Figure 1H-5 and Figure 1H-7 (As shown).

[0033] The beneficial effects obtained through the above technical features are: the specific incorporation of ANL into the new proteins of cardiomyocytes has been successfully achieved, and the incorporated proteins can be enriched and detected by click chemistry, laying a solid foundation for subsequent screening of cardiomyocyte-specific proteins by mass spectrometry.

[0034] Example 3: Isolation and Identification of Extracellular Vesicles from Heart and Plasma Cells The technical problem to be solved in this embodiment is: how to isolate high-purity, morphologically intact small extracellular vesicles from heart tissue and plasma as materials for subsequent screening and verification of biomarkers.

[0035] The technical features adopted include: Heart-derived sEVs: Fresh mouse hearts were minced and digested with 1 mg / mL type II collagenase and 80 U / mL DNase I for 30 minutes. The digest was filtered through a 70 μm filter, then centrifuged multiple times to remove cell debris, large vesicles, and mitochondria, followed by filtration through a 0.22 μm filter. Crude sEVs were precipitated by ultracentrifugation (120,000 × g, 4 h). The crude sEVs were resuspended and subjected to density gradient centrifugation with iodixanol (12%, 18%, 24%, 30%, and 36%), and the different fractions were collected in layers. Figure 2A ,2B).

[0036] Plasma sEV separation: Collect isolated blood samples into test tubes containing EDTA. Centrifuge twice at 3000×g for 15 minutes each time. Collect the plasma components, dilute 20-fold in pre-cooled PBS, centrifuge to remove large vesicles, and filter through a 0.22μm filter membrane. The remaining ultracentrifugation steps are the same as those for cardiac sEV separation (e.g., ...). Figure 2A , Figure 3A (As shown).

[0037] Particle size distribution and concentration were detected by NTA (e.g., Figure 2C , Figure 3B As shown), morphology was observed through TEM (e.g. Figure 2D , Figure 3C (As shown).

[0038] The beneficial effects obtained through the above technical features are: sEVs with a particle size of about 100nm and typical morphology were successfully obtained with high purity, which can meet the requirements of subsequent proteomics and validation experiments.

[0039] Example 4: Mass spectrometry identification and analysis of ANL-labeled proteins in cardiomyocyte-derived sEVs The technical problem to be solved in this embodiment is: how to identify proteins that are specifically expressed in cardiomyocytes and may serve as surface markers of sEVs from cardiomyocyte-derived sEVs.

[0040] The technical features adopted include: Enrichment and mass spectrometry detection of ANL-labeled peptides: [The text abruptly ends here, likely due to an incomplete sentence or missing information.] CM MetRS* mouse heart tissue proteins and cardiac sEV proteins were subjected to click reactions with DADPS biotinyne probes. After enzymatic digestion, ANL-labeled peptides were enriched using streptavidin agarose gels, eluted, and identified by HPLC-MS / MS. Mass spectrometry data were retrieved from the UniProt mouse database using MaxQuant software, with ANL modification set as a variable modification.

[0041] Data Analysis and Initial Biomarker Screening: BONCAT Results Show Efficient ANL Incorporation CM MetRS* mouse cardiac sEV protein (e.g. Figure 4A-1 , Figure 4A-2 (As shown), analysis of the mass spectrometry results indicates that... CM A large number of ANL-labeled proteins were identified in the myocardial sEVs of MetRS* mice, accounting for 8.1% of the entire cardiac sEV proteome (e.g., ...). Figure 4B (As shown). GO analysis of ANL-labeled proteins in cardiac sEVs revealed that these proteins were enriched in extracellular vesicles, membrane structures, and other cellular components (such as...). Figure 4C-1 , Figure 4C-2 and Figure 4C-3 (As shown).

[0042] ANL-labeled proteins in cardiac tissue were cross-referenced with those in cardiac sEVs to screen for candidate molecules that were highly expressed in both and predicted to be membrane proteins or membrane-associated proteins. Through literature review and bioinformatics analysis, γ-sarcosin (SGCG) was preliminarily identified as a highly myocardial-specific candidate biomarker (e.g.,...). Figure 5A (As shown).

[0043] The beneficial effects obtained through the above technical features are: SGCG was successfully screened as a potential specific biomarker for cardiomyocyte-derived sEVs, providing a target for subsequent validation and application.

[0044] Example 5: Validation of SGCG as a specific biomarker for cardiomyocyte-derived sEVs The technical problem to be solved in this embodiment is to verify whether SGCG is indeed specifically highly expressed in myocardial tissue and whether it exists on the surface of sEVs.

[0045] The technical features adopted include: Tissue-specific expression validation: Total protein was extracted from mouse organs including heart, brain, lung, liver, spleen, kidney, and skeletal muscle, and the expression level of SGCG was detected by Western blot. Results showed that SGCG was highly expressed only in heart tissue, weakly expressed in skeletal muscle, and almost not expressed in other tissues (e.g., Figure 5B (As shown).

[0046] Distribution validation in sEVs: Density gradient centrifugation was performed on cardiac and plasma sEVs to detect the distribution of SGCG and the universal sEV markers CD9 and CD63 by Western blot. The results showed that SGCG signals were mainly enriched in density layers co-localized with sEV markers (such as CD63, CD9, and TSG101), indicating that SGCG does indeed exist in sEVs and can be enriched in specific sEV subpopulations (such as...). Figure 5C , Figure 5D (As shown).

[0047] The beneficial effects obtained through the above technical features are: verifying the specific high expression of SGCG in cardiac tissue and confirming its presence in specific density layers of cardiac sEVs and plasma sEVs, further supporting its feasibility as a marker of cardiomyocyte-derived sEVs.

[0048] Example 6: Isolation of cardiomyocyte-derived sEVs using SGCG-based immunoaffinity assay The technical problem to be solved in this embodiment is: how to use SGCG antibody to specifically isolate cardiomyocyte-derived sEVs from total plasma sEVs and verify their enrichment efficiency.

[0049] 6.1 Materials and Methods: Antibody conjugation: Take an appropriate amount of Protein A / G agarose beads, wash three times with PBS, and resuspend in an equal volume of PBS. Add SGCG antibody (2-4 μL antibody per 350 μg total protein) and negative control IgG according to the ratio, and incubate gently with shaking at 4°C for 2 hours to allow the antibody to bind to the agarose beads. Centrifuge at 500×g for 30 seconds at 4°C, discard the supernatant, and wash three times with washing buffer to remove unbound antibody.

[0050] Immune capture: Take the total sEV (approximately 350 μg protein) from isolated human or mouse plasma in Example 3, add the above-mentioned antibody-conjugated agarose beads, and incubate overnight with gentle shaking at 4°C.

[0051] Washing and elution: Centrifuge at 500×g for 30 seconds at 4°C and discard the supernatant. Wash the precipitate three times with washing buffer to completely remove non-specifically bound proteins. Add 5×SDS loading buffer to the precipitate and heat at 100°C for 10 minutes to elute and denature the proteins from the agarose beads.

[0052] Enrichment efficiency verification: Samples were subjected to Western blot, and after transfer to a membrane, the presence of the target protein in the "IP product" was detected using antibodies against the target protein (ACTN2, Alix, CD63, cTNT, SGCG, CD81, CD9); simultaneously, the negative control IgG group was tested to exclude non-specific binding (e.g., ...). Figure 5E , Figure 5F (As shown).

[0053] 6.2 Results: Western blot results show (e.g.) Figure 5E , Figure 5F As shown in the figure, after immunocapture using SGCG antibody, the resulting product (i.e., cardiomyocyte-derived sEVs obtained after elution of the ligand-vesicle complex bound to the solid-phase carrier) showed significant enrichment of cardiomyocyte-specific proteins (such as cTNT) and universal sEV markers (such as CD63 and CD9). Conversely, no obvious target protein signal was detected in the group using the negative control IgG. This result fully demonstrates that the method of the present invention can efficiently and specifically isolate cardiomyocyte-derived sEV subsets from total plasma sEVs.

[0054] The beneficial effects obtained through the above technical features are: SGCG antibody was successfully used to specifically enrich cardiomyocyte-derived sEVs from total plasma sEVs. The enriched products showed significant enrichment of cardiomyocyte-specific proteins and sEV markers, confirming the high efficiency and specificity of the method.

[0055] Example 7: In vitro enrichment and detection kit containing SGCG capture system The technical problem to be solved in this embodiment is: how to convert the separation method of the present invention into a reagent kit product that can be used in clinical or scientific research.

[0056] The technical features employed include: constructing a kit for isolating and / or analyzing cardiomyocyte-derived sEVs, comprising the following components: (1) Reagents used to separate total sEVs from plasma samples, such as ultracentrifuge tubes and density gradient media; (2) Biotin-labeled anti-SGCG antibodies; including small extracellular vesicles derived from cardiomyocytes; A ligand that can specifically bind to γ-sarcosin (SGCG); the ligand is a biotin-labeled anti-SGCG antibody; (3) Streptavidin-coated solid-phase carriers, such as magnetic beads or ELISA plates; reagents for separating total small extracellular vesicles from plasma samples; (4) Washing buffer and elution buffer; (5) Optional, detection reagents for detecting sEV contents (such as heart disease-related miRNAs or proteins), such as PCR primers or specific antibodies; detection reagents for detecting nucleic acid or protein molecules in the small extracellular vesicles; the detection reagents include primers for detecting myocardial injury-related miRNAs or antibodies for detecting myocardial-specific proteins. The beneficial effects obtained through the above technical features are: providing a complete kit for research or in vitro sample analysis purposes; specifically enriching myocardial cell-derived sEVs and combining them with downstream molecular detection to assess the physiological or pathological state of myocardial cells; and having the advantages of simple operation and high standardization.

[0057] Example 8: A kit containing a small extracellular vesicle for isolating cardiomyocyte-derived vesicles It contains a ligand that specifically binds to γ-sarcosin SGCG, and a solid-phase carrier.

[0058] The ligand is a biotin-labeled anti-SGCG antibody, and the solid-phase carrier is streptavidin-coated microspheres; it also contains reagents for separating total small extracellular vesicles from plasma samples.

[0059] Example 9: A method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma (Comprehensive Example) This embodiment integrates all the aforementioned technical features and provides a complete method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma, including the following steps: 1. Screening and confirmation of SGCG biomarkers: Following the methods described in Examples 1-4 and Example 6, γ-sarcosin SGCG was screened and confirmed as a specific surface biomarker for cardiomyocyte-derived sEVs; 2. Provide ligand: Provide a ligand that can specifically bind to SGCG. In this embodiment, a biotin-labeled anti-SGCG monoclonal antibody is preferred. 3. Isolation of total small extracellular vesicles: Collect in vitro plasma samples and use ultracentrifugation combined with iodixanol density gradient centrifugation to separate high-purity total small extracellular vesicles; 4. Formation of ligand-vesicle complex: The biotin-labeled anti-SGCG antibody prepared in step 2 is co-incubated with the total small extracellular vesicles separated in step 3, so that the antibody specifically binds to sEVs derived from cardiomyocytes expressing SGCG on their surface, forming an "antibody-vesicle complex". 5. Capture the complex: Mix the incubation system from step 4 with streptavidin-coated magnetic beads (solid carrier) and incubate; the "antibody-vesicle complex" is bound to the magnetic beads by the high affinity of biotin and streptavidin, and the complex can be separated from the mixture by a magnetic rack. 6. Washing and elution: Wash the magnetic beads multiple times with washing buffer to remove unbound or non-specifically adsorbed substances; then add elution buffer or protein loading buffer to elute the "antibody-vesicle complex" bound to the magnetic beads, and finally obtain enriched and purified small extracellular vesicles derived from cardiomyocytes. 7. Downstream Analysis: The obtained cardiomyocyte-derived sEVs can be further analyzed using Western blotting and nucleic acid detection (such as miRNA qPCR). The three-part argument regarding technical issues, methods, and effects: The technical problem addressed by this invention is that existing technologies cannot specifically isolate cardiomyocyte-derived sEVs from a mixture of total sEVs in plasma, resulting in mixed signals and making it impossible to perform molecular analysis with a clear cell origin.

[0060] To solve this technical problem, the technical means adopted by the present invention are as follows: First, we screened and validated SGCG as a specific surface marker for cardiomyocyte-derived sEVs using innovative in vivo labeling and proteomics methods. Secondly, based on this biomarker, an immunoaffinity separation method consisting of "total sEV pre-separation - SGCG ligand specific binding - solid-phase affinity capture" was constructed.

[0061] Through the above-mentioned technical means, the technical effect achieved by the present invention is that it successfully achieves the specific and high-purity separation of sEVs derived from cardiomyocytes from plasma, completely solving the problem of signal mixing, and providing key analytical materials with clear sources for the precise diagnosis and mechanism research of cardiovascular diseases.

[0062] Demonstration of the synergistic effect and system effect of technical features The various technical features of this invention are not simply superimposed, but rather achieve a systematic technical effect through synergistic action. First, the synergy between biomarker screening and capture methods: the SGCG antigen obtained through rigorous in vivo screening methods possesses natural cardiomyocyte specificity and membrane expression stability, which is the foundation for the success of all subsequent steps. Second, the synergy between ligand and solid-phase carrier: biotin-labeled anti-SGCG antibody is selected as the ligand and binds to a streptavidin-coated solid-phase carrier (such as magnetic beads), forming a high-affinity, high-specificity capture system. The strong stability of the biotin-streptavidin bond ensures that the ligand-vesicle complex remains firmly bound even under harsh washing conditions. Finally, the synergy between pretreatment and specific capture: total sEVs are first separated from plasma using ultracentrifugation combined with density gradient centrifugation, effectively removing free proteins and other large particulate impurities from the plasma. This provides a low-background, high-purity starting material for subsequent specific immunocapture, greatly reducing non-specific adsorption and improving the overall purity and separation efficiency of the final product.

[0063] Creative discussion of the overall technical solution This invention has outstanding substantive features and significant progress compared with the prior art (including prior art document 1).

[0064] The closest prior art (“Circulating small extracellular vesicles as blood-based biomarkers of muscle health in aging nonhuman primates”, Mishra S etal, Geroscience. 47(3):3709-3723, 2025 Jun) discloses a method for isolating skeletal muscle-derived sEVs from serum using α-caryogamic acid (SGCA). The present invention differs from this prior art in that: (1) Different target proteins: This invention uses γ-caryogaprotein SGCG as a specific marker for isolating sEVs derived from cardiomyocytes; prior art 1 uses α-caryogaprotein SGCA as a marker for isolating sEVs derived from skeletal muscle.

[0065] (2) The ligand-vesicle complexes originate from different tissues: This invention aims to isolate sEVs derived from cardiomyocytes; prior art 1 aims to isolate sEVs derived from skeletal muscle.

[0066] (3) Different screening methods for biomarkers: This invention uses an innovative in vivo labeling method to screen for SGCG, including constructing transgenic mice that specifically express the MetRS*L274G mutant in cardiomyocytes. ANL-labeled cardiomyocyte progenitor protein was administered, and mass spectrometry analysis and cross-comparison were performed on ANL-labeled proteins in cardiac tissue and cardiac sEVs to ultimately screen SGCG as a specific biomarker for cardiomyocyte-derived sEVs. Comparative document 1 directly used the known muscle biomarker SGCA without disclosing any systematic screening methods.

[0067] Based on the above distinguishing features, the technical problem to be solved by the present invention is: how to specifically isolate sEVs derived from cardiomyocytes from plasma.

[0068] Analysis of the non-obviousness of this invention relative to prior art document 1: The technical problem posed is creative: There has long been a need in this field to isolate tissue-specific sEVs from plasma, but the isolation of cardiomyocyte-derived sEVs has remained unachieved. While prior art document 1 achieves the isolation of skeletal muscle sEVs, skeletal muscle and cardiomyocytes differ significantly in developmental origin, physiological function, and molecular expression profiles. Directly transferring the skeletal muscle sEV isolation method to cardiomyocytes requires first identifying a biomarker that is specifically and stably expressed in cardiomyocyte-derived sEVs; this technical challenge is not self-evident.

[0069] The non-obviousness of technical means: (1) Non-obviousness of biomarker selection: Although SGCA and SGCG belong to the same carotenoid family, members of this family exhibit tissue-specific distribution. SGCA is primarily highly expressed in skeletal muscle, while SGCG shows a different expression pattern in cardiac muscle. Those skilled in the art cannot directly deduce from the information disclosed in Prior Art Document 1 regarding the use of SGCA for skeletal muscle sEV isolation that SGCG can be used for cardiomyocyte sEV isolation. The expression and function of different subunits of the carotenoid complex vary in different muscle types, requiring experimental verification to determine suitable biomarkers.

[0070] (2) Inventiveness of the Biomarker Screening Method: The biomarker screening method of "transgenic animal model + non-classical amino acid labeling + proteomics cross-analysis" adopted in this invention is a key technical innovation. This method can accurately label neovascular proteins in cardiomyocytes and screen for membrane proteins that are truly highly expressed in both cardiomyocytes and their secreted sEVs by comparing the protein expression profiles of cardiac tissue and cardiac sEVs. This systematic screening method is not disclosed in prior art document 1 and is not a conventional technique in this field.

[0071] (3) Overall Innovation of the Separation Method: This invention constructs a complete technical chain of "total sEV pre-separation - SGCG ligand specific binding - solid-phase affinity capture," with each step working synergistically to achieve the technical effect of efficiently and specifically separating cardiomyocyte-derived sEVs from complex plasma samples. Although comparative document 1 also uses a similar immune capture framework, its ligand-vesicle complexes and markers are different, and it does not involve pretreatment steps such as density gradient centrifugation purification of total sEVs.

[0072] For those skilled in the art, simply transferring known methods for isolating sEVs from skeletal muscle to cardiac muscle is not a direct success, as it requires validation and confirmation of a biomarker that is specifically and stably expressed in cardiomyocyte-derived sEVs.

[0073] The unpredictability of technical effects: This invention, through the aforementioned technical means, achieves for the first time the specific isolation of cardiomyocyte-derived sEVs from plasma, solving the long-standing "bottleneck" problems of signal confounding and lack of traceability in the field. This technical effect is unpredictable by existing technologies. Specifically: This invention confirms that SGCG is specifically highly expressed in cardiac tissue and exists in specific density layers of cardiac-derived sEVs and plasma sEVs, indicating that SGCG can serve as a reliable marker for cardiomyocyte-derived sEVs.

[0074] Cardiac cell-derived sEVs were successfully enriched from total plasma sEVs using immunoaffinity capture with SGCG antibodies. The enriched products showed significant enrichment of myocardial-specific proteins and sEV markers, demonstrating the high efficiency and specificity of this method.

[0075] While comparison document 1 achieved the separation of skeletal muscle sEVs, it failed to provide any insights or technical guidance regarding the separation of cardiomyocyte sEVs.

[0076] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be covered by the claims of the present invention.

Claims

1. A method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma, characterized in that, Includes the following steps: (1) Provide ligands that can specifically bind to γ-sarcosin SGCG; (2) Isolate total small extracellular vesicles from isolated plasma samples; (3) The ligand is co-incubated with the total small extracellular vesicles obtained in step (2) to form a ligand-vesicle complex containing the ligand and small extracellular vesicles derived from cardiomyocytes expressing SGCG on their surface. (4) The ligand-vesicle complex formed in step (3) is combined with a solid support to separate the ligand-vesicle complex from the mixture; (5) The ligand-vesicle complex bound to the solid support in step (4) is washed and / or eluted to obtain enriched small extracellular vesicles derived from cardiomyocytes.

2. The method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma according to claim 1, characterized in that, The ligand is selected from antibodies, antibody fragments, nucleic acid aptamers, peptide aptamers, or small molecule ligands.

3. The method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma according to claim 2, characterized in that, The ligand is an antibody against SGCG.

4. The method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma according to claim 3, characterized in that, The anti-SGCG antibody is a biotin-labeled antibody; and the solid-phase carrier is microspheres coated with streptavidin or neutral avidin.

5. The method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma according to claim 1, characterized in that, The separation of total small extracellular vesicles in step (2) was performed using ultracentrifugation combined with density gradient centrifugation.

6. The method for isolating small extracellular vesicles derived from cardiomyocytes from isolated plasma according to claim 1, characterized in that, The γ-sarcosin SGCG, as a specific surface marker of small extracellular vesicles derived from cardiomyocytes, was obtained through screening using a method including the following steps: (1.1) Construct transgenic animals that specifically express the L274G mutant of methionine-tRNA synthetase in cardiomyocytes; (1.2) The transgenic animals were given L-azidoleucine to label the cardiomyocyte neoplasm; (1.3) The heart tissue and extracellular vesicles of heart-derived small cells of the transgenic animal were isolated and identified by mass spectrometry analysis of the proteins labeled with L-azidoleucine. (1.4) Cross-analysis was performed on labeled proteins in cardiac tissue and small extracellular vesicles derived from the heart to screen out the membrane protein SGCG, which is expressed in both and located on the surface of the vesicles, as the specific surface marker.

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