Membrane source specific extracellular vesicle labeling method and application thereof

Abstract

The invention discloses a membrane source specific extracellular vesicle marking method and application thereof. The membrane source specific extracellular vesicle labeling method comprises the following steps: mixing and incubating a cell to be labeled and a metabolic sugar precursor containing an azide group, so that the azide group is integrated onto a sugar complex of the cell through a glycosylation pathway, incubating the cell with a first click probe, and continuing to culture to obtain the membrane source specific extracellular vesicle. And collecting and extracting the extracellular vesicles, carrying out second click chemical labeling on the surfaces of the extracellular vesicles, and carrying out signal detection and grouping analysis after labeling. According to the method disclosed by the invention, time sequence marking of'closing / marking a cell outer side site at first and then marking a new vesicle exposed site 'can be realized in the same system, endocytosis mismarking and background interference are reduced, and the method can be used for vesicle subgroup change monitoring, disease diagnosis, curative effect evaluation and drug screening related to drug treatment / drug resistance.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biotechnology, cell biology and biomedical engineering, and relates to a membrane-derived specific extracellular vesicle labeling method and its application. Background Technology

[0002] Extracellular vesicles are phospholipid bilayer vesicles actively secreted by cells. They are widely distributed in body fluids and cell culture supernatants, carrying bioactive molecules such as proteins, nucleic acids, and lipids. They mediate intercellular communication and participate in processes such as immune regulation, tumor metastasis, pathogen transmission, and drug resistance. Extracellular vesicles exhibit significant heterogeneity, and different subpopulations may show systemic differences in their biogenic pathways, membrane component origins, and functional effects.

[0003] The heterogeneity of extracellular vesicle membrane origin is closely related to its biogenic pathway: vesicles formed directly from budding of the plasma membrane are usually rich in plasma membrane-related membrane components; vesicles released from the fusion of endosome multivesicles with the plasma membrane typically carry more membrane components related to the endosome / intramolecular membrane system. Different pathological conditions (e.g., tumor microenvironment and therapeutic stress) can alter the secretory mechanisms and membrane composition of extracellular vesicles, leading to differential functions in drug resistance transmission, immune regulation, angiogenesis, and metabolic reprogramming. Therefore, distinguishable labeling of extracellular vesicles according to their membrane origin and conducting time-resolved analysis will help elucidate the mechanisms and improve the explanatory power of extracellular vesicles as biomarkers.

[0004] In existing technologies, membrane dyes or whole-cell labeling methods can usually only label all vesicles in general, making it difficult to distinguish different membrane-derived subpopulations; immunolabeling based on surface protein markers cannot directly map the origin and biogenetic pathway of extracellular vesicle membrane components, and the markers are not strictly specific; some bioorthogonal labeling strategies are commonly used for overall labeling of cell surface or extracellular vesicles, but it is difficult to simultaneously achieve temporal differentiation between "plasma membrane-derived subpopulations" and "endomembrane system-derived subpopulations" in the same system, and probe endocytosis during live cell labeling can easily lead to mislabeling of the endomembrane system and increased background.

[0005] Therefore, there is an urgent need for a technology that can selectively close lateral sites and relabel newly exposed vesicle sites under living cell conditions to support the differentiation and quantitative analysis of vesicle subpopulations from different membrane sources. Summary of the Invention

[0006] To address the limitations of existing technologies in distinguishing extracellular vesicle membrane origins and in performing temporal differentiation and quantification, this invention provides a membrane-origin-specific extracellular vesicle labeling method and its application. Through a combination of "first-stage lateral site closure / labeling and second-stage labeling of newly exposed vesicle sites," the method achieves differentiation and quantification of vesicle subpopulations from different membrane origins.

[0007] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a membrane-derived specific extracellular vesicle labeling method, the method comprising: mixing and incubating cells to be labeled with a metabolic sugar precursor containing an azide group, allowing the azide group to integrate into the glycocomplex of the cells via glycosylation; incubating the cells with a first click probe; continuing to culture; collecting and extracting extracellular vesicles and performing a second click chemical labeling on the surface of the extracellular vesicles; and performing signal detection and cluster analysis after labeling; wherein the first click probe is a strained cycloalkyne compound that is membrane-impermeable or low-permeability and carries a first fluorescent group.

[0008] The method of this invention enables sequential labeling within the same system, involving "first sealing / labeling extracellular sites, then labeling newly exposed vesicle sites," reducing endocytosis mislabeling and background interference. It can be used for monitoring changes in vesicle subsets related to drug treatment / resistance, disease diagnosis, efficacy evaluation, and drug screening. A schematic diagram of the method of this invention is shown below. Figure 1 As shown.

[0009] As a preferred technical solution, the extracellular vesicle labeling method of the present invention includes the following steps: (a) The cells or tissues to be labeled are incubated with azide-containing metabolic sugar precursors, so that the azide groups are integrated into glycosylated molecules of the cell membrane and intracellular membrane system via sugar metabolism and glycosylation pathways. (b) When the cell fusion rate reaches 60%-70% (e.g., 60%, 65% or 70%), a first click probe is added to the outer side of the cell for low-temperature incubation. The first click probe undergoes a copper ion-independent bioorthogonal click reaction with the azide group exposed on the outer side of the cell membrane, thereby closing the outer site and introducing a first detectable label to obtain label 1. (c) Continue culturing to induce cell secretion of extracellular vesicles, collect the culture supernatant and extract the extracellular vesicles; (d) The extracellular vesicles are mixed with the second click probe and incubated to allow them to undergo a bioorthogonal click reaction with the azide groups on the surface of the extracellular vesicles that were not blocked in step (b) and introduce a second detectable label to obtain label 2; (e) Extracellular vesicle subpopulations from different membrane sources were distinguished and quantified by detecting the signals of marker 1 and marker 2 on extracellular vesicles using fluorescence detection.

[0010] Preferably, the cells in step (a) include any one of tumor cells, immune cells, stem cells, nerve cells, or cells derived from a body fluid sample.

[0011] Preferably, the metabolic sugar precursor in step (a) comprises any one or a combination of at least two of tetraacetyl N-azidoacetylmannosamine (AC4ManNAz), tetraacetyl N-azidoacetylgalactosamine (AC4GalNAz), or tetraacetyl N-azidoacetylglucosamine (AC4GlcNAz).

[0012] Preferably, the final concentration of the metabolic sugar precursor in step (a) is 10-100 μM, for example, it can be 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM.

[0013] Preferably, the incubation temperature in step (a) is 35-38°C, for example, 35°C, 36°C, 37°C or 38°C; the incubation time is 12-72 h, for example, 12 h, 24 h, 36 h, 48 h, 60 h or 72 h.

[0014] Preferably, the temperature of the low-temperature incubation in step (b) is 0-10°C, for example, 0°C, 2°C, 5°C, 8°C or 10°C; the time of the low-temperature incubation is 5-30 min, for example, 5 min, 10 min, 20 min, 25 min or 30 min.

[0015] Preferably, in step (b), the first click probe is a strained cycloalkyne compound with a first fluorescent group that is impermeable or poorly permeable to the membrane.

[0016] Preferably, the first click probe is a probe with a negatively charged group and is water-soluble.

[0017] Preferably, the negatively charged group includes any one of a sulfonic acid group, a carboxylic acid group, a phosphate group, or a phosphonic acid group.

[0018] Preferably, the first fluorescent group includes any one of cy3, cy5, cy5.5, cy7, FITC, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, DyLight 550, DyLight 650, ATTO 550, or ATTO 647N.

[0019] Preferably, the final concentration of the first click probe is 5-50 μM, for example, it can be 5 μM, 10 μM, 20 μM, 30 μM, 40 μM or 50 μM.

[0020] Preferably, the bioorthogonal click reaction is a strain-promoted cycloalkyne-azide cycloaddition reaction.

[0021] Preferably, the culture time for continued culture in step (c) is 4-48 h, for example, it can be 4 h, 8 h, 12 h, 24 h or 48 h.

[0022] Preferably, the extraction of extracellular vesicles in step (c) includes extraction using any one or a combination of at least two of differential centrifugation, ultracentrifugation, density gradient centrifugation, or size exclusion chromatography.

[0023] In one embodiment of the present invention, the differential centrifugation includes: removing cells at 200-500×g for 5-15 min; removing cell debris at 1000-3000×g for 15-25 min; removing large particles at 8000-20000×g for 25-35 min; filtration at 0.22 μm; ultracentrifugation at 80000-200000×g for 60-80 min to precipitate extracellular vesicles; resuspending in PBS and washing again at 80000-200000×g for 60-80 min; and finally resuspending in PBS and quantifying the protein content or particle number.

[0024] Preferably, in step (d), the second click probe is a strained cycloalkyne compound with a second fluorescent group that is spectrally distinguishable from the first fluorescent group.

[0025] Preferably, the second fluorescent group includes any one of cy3, cy5, cy5.5, cy7, FITC, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, DyLight 550, DyLight 650, ATTO 550, or ATTO 647N.

[0026] Preferably, the incubation temperature in step (d) is 25-37°C, for example, 25°C, 30°C, 32°C or 37°C; and the incubation time is 5-60 min, for example, 5 min, 10 min, 30 min or 60 min.

[0027] In this invention, the reaction buffer for the incubation reaction between extracellular vesicles and the second click probe is PBS buffer or serum-free culture medium with pH 7.2-7.4 (e.g., pH 7.2, pH 7.3 or pH 7.4). After the reaction, the free probe is removed by ultracentrifugation, size exclusion chromatography or ultrafiltration.

[0028] Preferably, the fluorescence detection in step (e) includes detection using any one or a combination of at least two of the following: confocal microscopy, nanoflow cytometry, flow cytometry, fluorescent nanoparticle tracking, or single-particle fluorescence tracking.

[0029] The second marker can be distinguished from the first marker in the spectrum or detection channel. For example, the first marker is DBCO-cy5, and the second marker is DBCO-FITC or DBCO-cy3.

[0030] Preferably, the criteria for differentiation in step (e) are as follows: vesicles that are positive for label 1 and negative for label 2 are identified as a subpopulation of vesicles rich in plasma membrane origin; vesicles that are positive for label 2 and negative for label 1 are identified as a subpopulation of vesicles rich in endometrial origin; and vesicles that are positive for both label 1 and label 2 are identified as a subpopulation of mixed origin or membrane component exchange related.

[0031] In this invention, the double negative markers 1 and 2 represent the background / unlabeled group, i.e., the particle group that has not been effectively metabolized or effectively click-labeled.

[0032] Further verification can be achieved by combining the results of detection of classic extracellular vesicle markers (such as CD63, CD81, TSG101, ALIX, etc.), particle size distribution, electron microscopy morphology, etc.

[0033] In a second aspect, the present invention provides a kit for implementing the membrane-derived specific extracellular vesicle labeling method described in the first aspect, the kit comprising: any one or a combination of at least two of the following: an azide-containing metabolic sugar precursor, a first click probe, a second click probe, and an extracellular vesicle extraction or purification component.

[0034] Preferably, the first click probe is a strained cycloalkyne compound with a first fluorescent group that is impermeable or poorly permeable to the membrane.

[0035] Preferably, the second click probe is a strained cycloalkyne compound with a second fluorescent group that is spectrally distinguishable from the first fluorescent group.

[0036] Preferably, the final concentration of the first click probe is 5-50 μM, for example, it can be 5 μM, 10 μM, 20 μM, 30 μM, 40 μM or 50 μM.

[0037] Preferably, the final concentration of the second click probe is 1-50 μM, for example, it can be 1 μM, 10 μM, 20 μM, 30 μM, 40 μM or 50 μM.

[0038] The labeling method of this invention has high specificity. Bioorthogonality can reduce the influence of non-specific reactions. The reaction between azide and DBCO can be completed at low temperature within tens of minutes, thereby achieving efficient labeling of the outer side of the cell membrane. The low temperature and the membrane-impermeable probe have a low cellular uptake background, and their binding with azide on the cell membrane can be completed efficiently at low temperature. Therefore, the resulting blank cells have a low fluorescence background, which can achieve good cell membrane labeling specificity.

[0039] Thirdly, the present invention provides the application of the membrane-derived specific extracellular vesicle labeling method described in the first aspect or the kit for distinguishing different membrane-derived extracellular vesicle subpopulations described in the second aspect in the monitoring of changes in extracellular vesicle subpopulations.

[0040] Fourthly, the present invention provides the application of the membrane-derived specific extracellular vesicle labeling method described in the first aspect or the kit for distinguishing different membrane-derived extracellular vesicle subpopulations described in the second aspect in the preparation of products for tumor drug resistance mechanism research, drug resistance transmission monitoring, disease diagnosis, efficacy evaluation or drug screening.

[0041] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention adopts a two-stage strategy of “first sealing / marking the outer sites and then marking the newly exposed sites of vesicles” to achieve the differentiation and quantification of vesicle subpopulations from different membrane sources in the same system; (2) The first click of this invention is performed under low temperature conditions and a membrane-impermeable or low-permeability probe is used, which can reduce the mislabeling of the inner membrane and background interference caused by probe endocytosis; (3) By collecting supernatant at different times (e.g., 4 h, 8 h, 12 h, 24 h, 48 h), the present invention can obtain the release kinetic information of different labeled subgroups, which can be used for mechanism research and efficacy evaluation, etc. (4) This invention can be used to monitor changes in vesicle subpopulations under drug treatment conditions, providing technical support for research on drug resistance-related mechanisms, efficacy evaluation and drug screening. Attached Figure Description

[0042] Figure 1 This is a schematic diagram of the two-stage labeling process for membrane-derived specific extracellular vesicles in this invention; Figure 2 To verify at the cellular level that the first click probe achieves blockage only at sites on the outer side of the cell membrane; and to verify the sufficient blockage at the outer site by incubating with the second probe after one blockage, the results are shown in the figure. Figure 3 Curves showing the proportion of extracellular vesicle release subsets at different time points; Figure 4 The results of flow cytometry analysis of labeled extracellular vesicles are shown in the figure. Figure 5 This is a schematic diagram showing the changes in the proportion of two extracellular vesicle subsets in a drug treatment model. Figure 6 Typical transmission electron microscopy image of the extracted extracellular vesicles; Figure 7This is a schematic diagram of two-dimensional scatter plots of double-labeled extracellular vesicles. With the fluorescence intensity of the FITC channel as the vertical axis and the fluorescence intensity of the APC channel as the horizontal axis, the events are divided into four quadrants under a uniform threshold condition. This is used to demonstrate that the method of the present invention can achieve distinguishable grouping and statistical analysis of extracellular vesicle subpopulations. Detailed Implementation

[0043] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0044] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0045] Example 1 This embodiment provides a membrane-derived specific extracellular vesicle labeling method, as detailed below: (1) Metabolic sugar labeling The test cells (SNU449 cells) were prepared at a ratio of 2 × 10⁻⁶. 5 The cells were seeded at a density of 5 mL of 1640 medium per well in culture dishes. After 6 h of cell adhesion, the metabolic sugar precursor Ac4ManNAz was added to the medium to a final concentration of 10 μM. The cells were then incubated at 37 °C in a 5% CO2 incubator for 72 h. An equal volume of DMSO was added to the control group.

[0046] (2) Click to close and introduce marker 1 for the first time. Discard the culture medium and wash twice with PBS. Incubate the cells at 4°C, add the first click probe DBCO-Sulfo-cy5 to a final concentration of 10 μM, and incubate at 4°C for 15 min. After the reaction, wash five times with cold PBS to remove the free probe.

[0047] (3) Extracellular vesicle secretion culture and supernatant collection The culture was then replaced with serum-free medium and cultured for another 24 h. The culture supernatant was collected and its volume was recorded. The cell count and protein content were also recorded for subsequent normalization.

[0048] (4) Extraction of extracellular vesicles The supernatant was subjected to differential centrifugation at 4℃: 300×g for 10 min, 2000×g for 10 min, and 10000×g for 30 min. The supernatant was then filtered through a 0.22 μm filter membrane. Extracellular vesicles were then precipitated by ultracentrifugation at 100000×g for 70 min, resuspended in PBS, and washed again by ultracentrifugation at 100000×g for 70 min. Finally, the extracellular vesicle sample was obtained by resuspending in PBS.

[0049] (5) Second click labeling on the surface of extracellular vesicles introduces a second label. Add the second click probe DBCO-FITC to the extracellular vesicle resuspension obtained in step (4) to a final concentration of 10 μM, and incubate at room temperature for 30 min. After the reaction, remove the free probe by ultrafiltration to obtain the extracellular vesicles with secondary labeling.

[0050] (6) Detection and cluster analysis The first and second label signals were detected by flow cytometry, and a two-dimensional scatter plot was output for cluster statistics: first label positive / second label negative group, second label positive / first label negative group, double positive group and double negative group.

[0051] Example 2 This embodiment illustrates that the method of the present invention can achieve complete sealing of the outer membrane with the first click.

[0052] After completing steps (1) and (2) of Example 1, the vesicle secretion step was not performed. The following verification was performed directly: Live cells that had undergone DBCO-Sulfo-Cy5 blocking were kept at 37°C, and the verification probe DBCO-FITC was added at a final concentration of 15 μM. The cells were incubated for 15 min; washed 5 times with PBS; and Cy5 and FITC signals were acquired using a confocal fluorescence microscope. Figure 2 As shown, the blocked group displays the signal of the blocked probe Cy5 but not the signal of the verification probe FITC, while the unblocked group displays the signal of the verification probe FITC. This indicates that the method of the present invention can effectively label the outer side of the cell membrane and does not contain unlabeled azide groups.

[0053] Example 3 This embodiment illustrates that the method of the present invention can be used for time-resolved monitoring of extracellular vesicle subset release.

[0054] After completing steps (1) and (2) according to Example 1, the culture medium was changed and the supernatant was collected at 4 h, 8 h, 12 h, 24 h, and 48 h, respectively. Extracellular vesicles were extracted from the supernatant at each time point according to step (4) of Example 1, and a second click-marking was performed according to step (5) of Example 1. Finally, the proportion of extracellular vesicles in different subpopulations at each time point was detected and statistically analyzed according to step (6) of Example 1, thereby obtaining the release curve of the proportion of extracellular vesicle subpopulations changing with time, such as... Figure 3 As shown, the proportion of the endometrial subpopulation increases continuously over time, while the proportion of the plasma membrane subpopulation shows a phased peak characteristic. This indicates that the method of the present invention can achieve time-resolved monitoring of extracellular vesicle subpopulations of different membrane origins and reveal the dynamic changes in the relative contributions of different subpopulations over time.

[0055] Example 4 This embodiment illustrates that the method of the present invention can verify extracellular vesicle subpopulations from different membrane sources using flow cytometry dual-channel signals.

[0056] The experimental group underwent vesicle extraction and secondary labeling with DBCO-FITC as described in Example 1. The control group consisted of extracellular vesicles secreted by cells with an equal amount of DMSO solvent (but not azide sugar molecules). The control group also underwent the labeling process described in Example 1. Flow cytometry was then used to analyze the results of both the experimental and control groups. Figure 4 As shown, the experimental group exhibited a distinguishable distribution in both the APC (plasma membrane labeling probe) and FITC (intrinsic membrane system labeling probe) channels compared to the DMSO control group, demonstrating that this method can generate dual-channel detectable signals at the vesicle level and achieve cluster statistics.

[0057] Example 5 This embodiment illustrates that the method of the present invention can monitor changes in vesicle subsets under drug treatment conditions and can be used as a quantitative readout for drug response assessment and screening.

[0058] In the vesicle secretion stage of step (3) of Example 1, different concentrations of doxorubicin were added to the cells (gradient concentrations were set according to the sensitivity of different cell lines to doxorubicin; in this example, 0.1 μM, 0.2 μM, 0.4 μM and the group without doxorubicin were used as control groups). After culturing for 12 h, the supernatant was collected and vesicles were extracted according to Example 1 and subjected to secondary DBCO-FITC labeling. Flow cytometry was used to statistically analyze the changes in the proportion of each vesicle subset under different drug concentrations, such as... Figure 5 As shown, the proportion of Label 2 positive (endometrial system-related) vesicle subsets increases in a dose-dependent manner with increasing doxorubicin concentration, indicating that the present invention can provide stable and quantifiable vesicle subset readouts under drug-induced conditions. Based on this readout, the following potential applications can be realized: (1) Used for drug response assessment: The proportion of label 2 positive vesicle subsets (or its fold change relative to the control group) is used as the evaluation index to compare different drug concentrations, duration of action or treatment conditions; (2) Used for drug screening / condition optimization: Under the same culture and extraction conditions, the changes in vesicle subpopulations of candidate drugs or combined treatments are compared horizontally to screen for drugs or treatments that cause significant changes in the target subpopulation. (3) Used for drug resistance-related risk warning: When a characteristic pattern of sustained increase in the marker 2 positive vesicle subset appears under drug stress, it can serve as a basis for further verification of drug resistance-related mechanisms or subsequent testing.

[0059] Therefore, this invention can not only distinguish and quantify the vesicle membrane-derived subpopulations, but also output comparable and repeatable quantitative indicators in drug processing scenarios, thus possessing practical application value.

[0060] Test Example 1 This test case is used to perform basic characterization of the extracellular vesicle samples obtained in Example 1, as detailed below: (1) Particle concentration and particle size distribution were determined using a resistive pulse sensing (RPS) method. As shown in Table 1, the average particle size of the extracellular vesicle sample obtained in Example 1 was 81 nm, and the concentration was 1.86 × 10⁻⁶. 11 Particles / mL. The sample particle size distribution is dispersed (Span value > 0.8), mainly concentrated in the 50 nm-90 nm range, with a proportion greater than 70%. Particles are also distributed in the 90 nm-150 nm and 150 nm-270 nm ranges, with proportions of 21.74% and 4.35%, respectively.

[0061] Table 1 (2) The morphology of the obtained extracellular vesicle samples was observed using transmission electron microscopy. For example... Figure 6 As shown, extracellular vesicles exhibit a typical cup-shaped structure under transmission electron microscopy, with a size between 50 nm and 160 nm, consistent with the RPS test results.

[0062] The above characterization verified that the sample was an extracellular vesicle enriched component.

[0063] Test Example 2 This test case is used to determine the clustering of the dual-labeled extracellular vesicles obtained in Example 1, as detailed below: (1) Blank unlabeled control samples, single-labeled samples, and double-labeled samples were set up, wherein: the blank control was a control without the introduction of reaction sites (i.e., only the solvent DMSO was added to the control); the single-labeled samples contained only label 2 (DBCO-FITC, FITC channel) or only label 1 (DBCO-Sulfo-Cy5, APC channel); the double-labeled samples contained both label 1 and label 2. The positive threshold and four-quadrant division were determined by the scatter distribution of the blank control in the FITC and APC channels to ensure that both channels were in the low background range under the condition of no reaction sites.

[0064] (2) Each sample was serially diluted during collection, and the dilution intervals in which the number of events and the dilution factor were linearly related were selected for detection; (3) Output a two-dimensional scatter plot with the FITC channel as the vertical axis and the APC channel as the horizontal axis, and calculate the proportion of the four quadrants, such as Figure 7 As shown, the blank control samples (unlabeled group) were mainly distributed in the double-negative quadrant (LL 99.60%), with only very low background in the other quadrants (UL 0.36%, LR 0.04%, UR 0.00%), indicating that the background signal was low and channel crosstalk was negligible after the threshold was set. The FITC single-positive samples (FITC-labeled group) mainly entered the FITC single-positive quadrant (UL 14.08%), while the APC-related quadrant was close to the background (UR 0.03%, LR 0.02%), indicating that the label 2 signal could be stably identified under the given threshold and had very low interference with the APC channel. The APC single-positive samples (CY5-labeled group) mainly entered the APC single-positive quadrant (LR 12.28%), while the FITC-related quadrant was close to the background (UR 0.28%, UL 0.13%), indicating that the label 1 signal could be stably identified and had little interference with the FITC channel. The dual-labeled samples simultaneously showed FITC single positivity (UL 4.48%), APC single positivity (LR 3.07%), and double positivity (UR 7.31%), indicating that under the same detection platform and the same threshold rule, the method of the present invention can divide extracellular vesicle samples into at least three subgroups with distinguishable labeling characteristics (label 1 rich cluster, label 2 rich cluster, and dual-labeled cluster), thereby achieving quantitative statistical and comparative analysis of subgroup proportions.

[0065] Through a combination of blank / single-label / double-label controls, this test case verifies that the threshold setting of this method can maintain the background at an extremely low level, and that the two-channel signals are distinguishable with low crosstalk, while also obtaining stable four-quadrant cluster statistics. Therefore, the method of this invention can achieve the clustering and quantitative output of extracellular vesicle subpopulations under conventional flow cytometry detection conditions, and has operability and practical application value.

[0066] In summary, the method of the present invention can achieve temporal labeling of "first blocking / labeling extracellular sites and then labeling newly exposed vesicle sites" within the same system, reducing endocytosis mislabeling and background interference. It can be used for monitoring changes in vesicle subpopulations related to drug treatment / resistance, disease diagnosis, efficacy evaluation, and drug screening.

[0067] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A membrane-derived, specific method for labeling extracellular vesicles, characterized in that, The membrane-derived specific extracellular vesicle labeling method includes: mixing and incubating cells to be labeled with a metabolic sugar precursor containing an azide group, allowing the azide group to integrate into the glycocomplex of the cells via glycosylation; incubating the cells with a first click probe; continuing to culture; collecting and extracting extracellular vesicles; performing a second click chemical labeling on the surface of the extracellular vesicles; and performing signal detection and cluster analysis after labeling. The first click probe is a strained cycloalkyne compound that is membrane-impermeable or low-permeability and carries a first fluorescent group.

2. The membrane-derived specific extracellular vesicle labeling method according to claim 1, characterized in that, The method includes the following steps: (a) The cells or tissues to be labeled are incubated with azide-containing metabolic sugar precursors, so that the azide groups are integrated into glycosylated molecules of the cell membrane and intracellular membrane system via sugar metabolism and glycosylation pathways. (b) When the cell fusion rate reaches 60%-70%, the first click probe is added to the outer side of the cell for low-temperature incubation. The first click probe undergoes a copper ion-independent bioorthogonal click reaction with the azide group exposed on the outer side of the cell membrane, thereby closing the outer site and introducing the first detectable label to obtain label 1. (c) Continue culturing to induce cell secretion of extracellular vesicles, collect the culture supernatant and extract the extracellular vesicles; (d) The extracellular vesicles are mixed and incubated with the second click probe, so that the second click probe undergoes a bioorthogonal click reaction with the azide groups on the surface of the extracellular vesicles that are not blocked by step (b) and introduces a second detectable label, to obtain label 2; (e) Extracellular vesicle subpopulations from different membrane sources were distinguished and quantified by detecting the signals of marker 1 and marker 2 on extracellular vesicles using fluorescence detection.

3. The membrane-derived specific extracellular vesicle labeling method according to claim 2, characterized in that, The cells mentioned in step (a) include any one of tumor cells, immune cells, stem cells, nerve cells, or cells derived from a body fluid sample; Preferably, the metabolic sugar precursor in step (a) comprises any one or a combination of at least two of tetraacetyl N-azidoacetylmannosamine, tetraacetyl N-azidoacetylgalactosamine, or tetraacetyl N-azidoacetylglucosamine; Preferably, the final concentration of the metabolic sugar precursor in step (a) is 10-100 μM; Preferably, the incubation temperature in step (a) is 35-38°C; and the incubation time is 12-72 h.

4. The membrane-derived specific extracellular vesicle labeling method according to claim 2 or 3, characterized in that, The temperature for low-temperature incubation in step (b) is 0-10℃; the incubation time is 5-30 min.

5. The membrane-derived specific extracellular vesicle labeling method according to any one of claims 2-4, characterized in that, In step (b), the first click probe is a strained cycloalkyne compound with a first fluorescent group that is impermeable or poorly permeable to the membrane. Preferably, the first click probe is a probe with a negatively charged group and water solubility; Preferably, the negatively charged group includes any one of a sulfonic acid group, a carboxylic acid group, a phosphate group, or a phosphonic acid group; Preferably, the first fluorescent group includes any one of cy3, cy5, cy5.5, cy7, FITC, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, DyLight 550, DyLight 650, ATTO 550, or ATTO 647N; Preferably, the final concentration of the first click probe is 5-50 μM; Preferably, the bioorthogonal click reaction is a strain-promoted cycloalkyne-azide cycloaddition reaction.

6. The membrane-derived specific extracellular vesicle labeling method according to any one of claims 2-5, characterized in that, The culture time for the continued culture described in step (c) is 4-48 h; Preferably, the extraction of extracellular vesicles in step (c) includes extraction using any one or a combination of at least two of differential centrifugation, ultracentrifugation, density gradient centrifugation, or size exclusion chromatography. Preferably, in step (d), the second click probe is a strained cycloalkyne compound with a second fluorescent group that is spectrally distinguishable from the first fluorescent group; Preferably, the second fluorescent group includes any one of cy3, cy5, cy5.5, cy7, FITC, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647, DyLight 550, DyLight 650, ATTO 550, or ATTO 647N; Preferably, the incubation temperature in step (d) is 25-37°C; and the incubation time is 5-60 min.

7. The membrane-derived specific extracellular vesicle labeling method according to any one of claims 2-6, characterized in that, The fluorescence detection described in step (e) includes detection using any one or a combination of at least two of the following: confocal microscopy, nanoflow cytometry, flow cytometry, fluorescent nanoparticle tracking, or single-particle fluorescence tracking. Preferably, the criteria for differentiation in step (e) are as follows: vesicles that are positive for label 1 and negative for label 2 are identified as a subpopulation of vesicles rich in plasma membrane origin; vesicles that are positive for label 2 and negative for label 1 are identified as a subpopulation of vesicles rich in endometrial origin; and vesicles that are positive for both label 1 and label 2 are identified as a subpopulation of mixed origin or membrane component exchange related.

8. A kit for implementing the membrane-derived specific extracellular vesicle labeling method according to any one of claims 1-7, characterized in that, The kit includes any one or a combination of at least two of the following: an azide-containing metabolic sugar precursor, a first click probe, a second click probe, and an extracellular vesicle extraction or purification component; Preferably, the first click probe is a strained cycloalkyne compound with a first fluorescent group that is impermeable or poorly permeable to the membrane. Preferably, the second click probe is a strained cycloalkyne compound with a second fluorescent group that is spectrally distinguishable from the first fluorescent group; Preferably, the final concentration of the first click probe is 5-50 μM; Preferably, the final concentration of the second click probe is 1-50 μM.

9. The application of the membrane-derived specific extracellular vesicle labeling method according to any one of claims 1-7 or the kit according to claim 8 for distinguishing extracellular vesicle subpopulations of different membrane origins in monitoring changes in extracellular vesicle subpopulations.

10. The membrane-derived specific extracellular vesicle labeling method of any one of claims 1-7 or the kit of claim 8 for distinguishing different membrane-derived extracellular vesicle subpopulations, used in the preparation of products for tumor drug resistance mechanism research, drug resistance transmission monitoring, disease diagnosis, efficacy evaluation, or drug screening.

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