Exosome marking method

Abstract

The invention belongs to the technical field of medical diagnosis, and provides an exosome marking method. The method comprises the following steps: culturing a mother cell for producing an exosome in a culture medium containing GlcA and Ac4GlcNAz to obtain the exosome of the mother cell; and co-incubating the labeled molecule coupled with the DBCO and the exosome suspension to obtain the labeled exosome. According to the invention, N3-HA is introduced to an exosome membrane by using non-natural glucose metabolism, the yield and physiological activity of the exosome are not affected, and the method has the advantages of high stability and high labeling efficiency. The N3-HA labeled exosome can be used for exosome tracing and targeted drug delivery, operation is simple, convenient and rapid, and fluorescence stability and reliability and drug targeting can be greatly improved. The strategy provided by the invention can become a powerful tool of exosomes in research of treatment mechanisms of various diseases, provides an effective method for targeted delivery of antitumor drugs, and has very important clinical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical diagnosis and relates to a method for labeling and tracing exosomes. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Exosomes are small extracellular vesicles of 30nm-150nm that can be secreted by most cells and are widely found in animals, plants and microorganisms. Exosomes originate from the cells of organisms and are released by the cell membrane and intracellular multivesicular bodies. They contain multiple vesicles produced by extracellular fusion with the cell membrane, and eventually form exosomes. As a communication tool between cells and between cells and the environment, exosomes participate in a variety of physiological processes. Due to their good biocompatibility, high-efficiency delivery and easy transmembrane characteristics, exosomes also have great development potential as drug delivery carriers. Exosomes exist in the body fluids of all organisms and have a wide range of biological activities. They play an important role in the clinical treatment and diagnosis of various diseases. However, the research on the specific treatment and diagnostic mechanisms of exosomes is still a difficult shackle to break through, and most of them are limited by the visualization of their behavior process.

[0004] At present, more advanced imaging systems allow us to observe even smaller vesicles, but the behavior of exosomes cannot be directly observed. A major challenge in observing, measuring, and visually tracking the behavior of exosomes is how to functionalize exosomes so that they can be firmly bound to fluorescent molecules. The current methods for functionalizing exosomes to trace them mainly include: 1. Fusion expression of fluorescent proteins with marker proteins on the exosome membrane to present the movement of exosomes in the body (CN 115161260 A). 2. Encoding lentivirus transfected cells, amino acids incorporated into the cell proteome to secrete exosomes, and covalently linked fluorescent dyes to make exosome membrane proteins fluorescent (CN 115305253 A). However, both methods have many limitations, such as poor specificity of fluorescent dyes, unstable and dim fluorescence, and therefore, it is difficult to visually observe and track the uptake of exosomes in the body.

[0005] As a natural intercellular communication carrier, exosomes have also received extensive attention in the field of drug delivery in recent years. However, the targeting of exosomes to tumors is not ideal, and the existing engineering methods are relatively complex. These processes are not only cumbersome to operate, but may also damage the structure and function of exosomes. Therefore, how to improve the targeting of exosomes while maintaining their integrity has become a key issue that needs to be solved in current research. Summary of the invention

[0006] In view of the current problems of difficulty in tracing exosomes and poor targeting to tumors, the present invention provides an exosome labeling method, which uses cell metabolic glycoengineering to label azidohyaluronic acid to prepare azidohyaluronic acid exosomes (N3-HA exosomes). Compared with traditional exosomes, the exosomes have better stability and higher labeling efficiency, and have good targeting to tumor cells.

[0007] Another object of the present invention is to provide a use of the above-mentioned exosomes in the preparation of medical test reagents and medicines.

[0008] To achieve the above purpose, the present invention adopts the following technical solution.

[0009] A method for labeling exosomes, comprising the following steps: (1) The exosome-producing mother cells are cultured in a medium containing D-glucuronic acid (GlcA) and azido-N-acetylglucosamine (Ac4GlcNAz); (2) isolating the exosomes from the mother cells in step (1); (3) Co-incubating the DBCO-coupled labeled molecule with the exosome suspension obtained in step (2) to obtain labeled exosomes.

[0010] The mass ratio of GlcA to Ac4GlcNAz is 1:2-2:1, preferably 1:1.

[0011] The final concentration of the GlcA and Ac4GlcNAz in the culture medium is 5 μg / mL-20 μg / mL; the culture time is 6h-24h.

[0012] The exosome labeling method of the present invention can select different parent cell types according to the specific application requirements of the labeled exosomes, including tumor cells (such as breast cancer, lung cancer cells, etc.) for tumor targeting and imaging, stem cells (such as mesenchymal stem cells, induced pluripotent stem cells, etc.) for tissue repair and regeneration, dendritic cells for immunotherapy, and neural cells for neural repair. By selecting appropriate parent cell types, the targeting and function of exosomes can be effectively optimized to meet the needs of different medical research and clinical treatments.

[0013] The separation method can be selected from existing methods according to the scale and purpose of the sample, such as differential centrifugation, ultrafiltration, magnetic bead separation, protein precipitation, density gradient centrifugation, micro-flow technology, immunoaffinity separation, and polymer precipitation.

[0014] In step (3), the concentration of the exosomes in the suspension is not limited; the ratio of the total protein concentration of the exosomes to the molecular mass of the coupled DBCO marker is (32-4):1; preferably, the ratio of the total protein concentration of the exosomes to the molecular mass of the coupled DBCO marker is (8-4):1; more preferably, under the above ratio, the concentration of the exosome suspension is 0.4μg / μL-0.8μg / μL.

[0015] In step (3), the labeling molecule is a fluorescent molecule or a small molecule anti-tumor drug connected to a reactive group; the molecular weight of the small molecule anti-tumor drug is not greater than 1000Da; the reactive group undergoes a bioorthogonal reaction with azidohyaluronic acid. Preferably, the reactive group is selected from a cycloalkynyl group, a cycloalkene group, a five-membered ring reactive group, an epoxy group, a difluoroalkene group, and a thioalkene group. The small molecule anti-tumor drug is selected from doxorubicin, doxorubicin, paclitaxel, cisplatin, carboplatin, 5-fluorouracil, a coumarin drug, or an acacetin drug; the fluorescent group is selected from fluorescein isothiocyanate (FITC), a cyanine dye, or a rhodamine fluorescent dye; for example, FITC, Cy-3, Cy-5, Cy-7, AF488, AF594, and AF555.

[0016] In step (3), the co-incubation conditions are preferably 37° C., 15 min-4 h.

[0017] A use of the above-mentioned labeled exosomes in the preparation of diagnostic reagents and medicines.

[0018] The present invention has the following advantages: The exosome tracing and targeted drug delivery technology of metabolic sugar engineering labeled N3-HA adopted in the present invention uses non-natural sugar metabolism to introduce N3-HA into the exosome membrane, which will not affect the production and physiological activity of the exosomes, and has the advantages of high stability and high labeling efficiency. The fluorescence intensity advantage of this functionalized exosome and its targeting effect on tumor cells were verified and clarified at the cellular level by confocal microscopy, and the visualization advantage of the behavioral process of this functionalized exosome between cells was determined by in vitro experiments. The application of exosome tracing and targeted drug delivery of metabolic sugar engineering labeled N3-HA of the present invention is not only simple and fast to operate, but also can greatly improve the stability and reliability of fluorescence and the targeting of drugs. The application strategy provided by the present invention can become a powerful tool for the study of exosomes in the treatment mechanism of various diseases, and provide an effective method for the targeted delivery of anti-tumor drugs, which has very important innovative significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Confocal micrographs of cells labeled with different sugars (A) and changes in fluorescence intensity (B); Figure 2Confocal micrographs of cells obtained at different ratios of raw materials (A) and changes in fluorescence intensity (B); Figure 3 is a transmission electron micrograph of N3-HA exosomes; Figure 4 is a Western Blot image of DBCO-AF488-labeled and unlabeled N3-HA exosome surface markers; Figure 5 The gel electrophoresis fluorescence images (A) and Coomassie brilliant blue staining images (B) of DBCO-AF488-labeled and unlabeled N3-HA exosomes; Figure 6 Confocal micrographs (A) and fluorescence intensity changes (B) of DBCO-AF488-N3-HA exosomes at different time points; Figure 7 is the distribution of DBCO-Cy5-N3-HA exosomes in mice; Figure 8 is the distribution of DBCO-Cy5-N3-HA exosomes in isolated mouse tissues (stomach and intestine); Fig. 9 The targeted distribution of DBCO-Cy5-Ac4ManNAz exosomes (A) and DBCO-Cy5-N3-HA exosomes (B) in mouse tumor tissues; In the above figures, Indicates p < 0.01; Indicates p<0.001. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0021] Example 1 Preparation of DBCO-AF488-N3-HA exosomes 1. Different sugar markers Place a cell slide in a 24-well plate, add 1640 complete medium (1 mL per well), and inoculate 4T1 cells in the 24-well plate with the medium at a volume of 5 × 10 cells per well. 5cells; after inoculation and culture for 12 hours, D-glucuronic acid (GlcA) and azido N-acetylglucosamine (Ac4GlcNAz) with a mass ratio of 1:1 were added to the cell culture wells, respectively, so that the final concentrations were 10 μg / mL, and N3-HA was marked on the cell membrane surface, as the experimental group; in addition, azidomannose (Ac4ManNAz) with a final concentration of 10 μg / mL was added to the cell culture wells, respectively, to mark azidomannose on the cell membrane surface, as the control group, and incubated at 37°C and 5% CO2 for 13 hours; N3-HA labeled cells and Ac4ManNAz labeled cells were obtained; the cells were stained with DBCO-AF488 and DAPI in turn, and the cell membrane fluorescence of the two cells was observed under a confocal inverted fluorescence microscope at 6h and 12h, respectively.

[0022] The results are as follows Figure 1 As shown in the figure, the cell membrane fluorescence after N3-HA labeling is stronger than that of Ac4ManNAz at the initial time point. Over time, the fluorescence signal of N3-HA labeling is more persistent and decays more slowly than that of Ac4ManNAz.

[0023] 2. GlcA to Ac4GlcNAz ratio Place a cell slide in a 24-well plate, add 1640 complete medium (1 mL per well), and inoculate 4T1 cells in the 24-well plate with the medium at a volume of 5 × 10 cells per well. 5 cells; after inoculation and culture for 12 hours, D-glucuronic acid (GlcA) and azido N-acetylglucosamine (Ac4GlcNAz) were added to the cell culture wells at a mass ratio of 1:1, 2:1, and 1:2, respectively, with a final concentration of 10 μg / mL, and incubated for 13 hours at 37°C and 5% CO2; the cells were stained with DBCO-AF488 and DAPI, and their localization on the cell membrane was observed under a confocal inverted fluorescence microscope.

[0024] The results are as follows Figure 2 As shown, when the ratio of GlcA: Ac4GlcNAz was 1:1, the fluorescence intensity of the cell membrane reached the highest.

[0025] 3. Preparation of N3-HA Exosomes Bone marrow of healthy adult mice was selected, and bone marrow mesenchymal stem cells (BMSCs) were isolated and cultured using standard serum-free α-MEM medium. BMSCs were treated with 10% fetal bovine serum medium, inoculated into culture bottles, and cultured at 37°C and 5% CO2 for about 3-5 days until the cells reached 80%-90% confluence. Adhesion screening was used to collect BMSCs attached to the wall by removing cells that were not attached to the wall. GlcA and Ac4GlcNAz were added to the α-MEM medium at a mass ratio of 1:1 at a concentration of 10μg / mL, and cultured for 13h. The medium was replaced with 10% exosome-free FBS in the medium and cultured in an incubator at 37°C and 5% CO2 for 24-48h. The culture fluid was collected and sterilized by filtration with a 0.22μm needle filter. The BMSCs culture fluid was collected, and cell impurities were removed by low-speed centrifugation. Then, exosomes were extracted by ultracentrifugation (100,000 g, 4°C, 1 h). The exosome samples were purified by immunoaffinity separation, and incubated with antibody-coated magnetic beads containing a mesenchymal stem cell surface marker (CD73) for 2 h to selectively enrich the exosomes by binding to the specificity of the antibody. The exosomes were separated from the antibody-coated magnetic beads, the exosome enrichment fluid was collected, and the non-specific binding substances were washed with PBS buffer to obtain N3-HA labeled exosomes. Exosomes extracted from cells cultured in DMEM medium without any treatment were used as a control.

[0026] 4. Preparation of DBCO-AF488-N3-HA Exosomes The N3-HA labeled exosomes were resuspended in PBS with pH = 7 to prepare an exosome suspension with a concentration of 0.8 μg / μL, and then aliquoted and frozen at -80°C for later use. The protein content of the exosomes was determined using a BCA protein concentration assay kit. The extracted N3-HA exosomes were incubated with DBCO-AF488 at a mass ratio of 8:1 for 45 minutes according to the total protein concentration, and ultracentrifuged at 100,000 g, 4°C, 90 minutes to remove residual dyes to obtain DBCO-AF488-N3-HA exosomes.

[0027] The morphological structure of the exosomes extracted above was observed using a transmission electron microscope (TEM). Figure 3 As shown in Figure 2, DBCO-AF488-N3-HA exosomes showed a typical "tray-like" exosome shape; Western blot was used to identify the surface marker proteins of the two exosomes, such as Figure 4 As shown, the expression of exosome characteristic marker proteins CD9 and CD81 was positive; gel fluorescence imaging and Coomassie brilliant blue method were used to detect the fluorescence and protein content of DBCO-AF488-N3-HA exosomes, as shown in Figure 5As shown in the figure, the fluorescence of DBCO-AF488-N3-HA exosomes was significantly higher than that of the control group exosomes, and the Coomassie Brilliant Blue results showed that there was no significant difference in the content of DBCO-AF488-N3-HA exosomes and the control group exosomes. The above results indicate that the metabolic labeling of N3-HA does not affect the characteristics of exosomes.

[0028] Application Example 1 Tracing effect of DBCO-AF488-N3-HA exosomes DMEM complete medium (1 mL per dish) was added to the laser confocal cell culture dish, and 4T1 cells were inoculated at a volume of 5 × 10 cells per well. 5 cells; after inoculation and culture for 12 h, the cells were stained with Hoechst 33342, and then 500 μL of DBCO-AF488-N3-HA exosomes were added for co-incubation. The cells were placed under a confocal inverted fluorescence microscope at 0 h, 3 h, and 6 h to observe the tracking of DBCO-AF488-N3-HA exosomes in vitro.

[0029] The results are as follows Figure 6 As shown, the blue signal is the DAPI staining of the cell nucleus, and the green dot signal is the tracer exosome signal. The above results show that the azidohyaluronic acid-modified fluorescent group-labeled exosomes can be used for cell tracing.

[0030] Example 2 Preparation of DBCO-Cy5-N3-HA exosomes 4T1 cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C and 5% CO2. The medium was changed once a day and the cells were observed under a microscope. When the cell confluence reached 80%, they were passaged. After 12 hours of culture, the cells were cultured in a medium containing D-glucuronic acid (GlcA) and azido-N-acetylglucosamine (Ac4GlcNAz) in a mass ratio of 1:1 and a concentration of 10 μg / mL in DMEM medium. The culture was continued for 13 hours, and the medium was replaced. The medium contained 10% exosome-free FBS and was placed in an incubator at 37°C and 5% CO2 for 24-48 hours. The culture medium was collected and sterilized by filtration with a 0.22 μm needle filter. The filtered cell culture supernatant was centrifuged at low speed for 300g for 10min to remove cells; the supernatant was centrifuged at 2000g for 10min to remove dead cells and the supernatant was centrifuged at 10000g for 20min to remove cell debris; the supernatant was prepared for ultracentrifugation and centrifuged at 100000g for 90min to obtain N3-HA exosomes. The azidohyaluronic acid-labeled exosomes were resuspended in PBS to a suspension of 0.4μg / μL, pH=7, and aliquoted and frozen at -80℃ for later use. The protein content of the exosomes was determined using a BCA protein concentration assay kit. Exosomes extracted from cells cultured in DMEM medium without any treatment were used as a control. The extracted N3-HA exosomes were incubated with Cy5-DBCO at a mass ratio of 4:1 for 15min according to the total protein concentration, ultracentrifuged at 100000g, 4℃, 90min, and the residual dye was removed to obtain DBCO-Cy5-N3-HA exosomes.

[0031] Application Example 2 In vivo tracing of DBCO-Cy5-N3-HA exosomes Normal mice (n=3) were orally gavaged with Cy5-exosomes (1.5 mg / kg), and the time-dependent in vivo biodistribution of Cy5-exosomes was evaluated by a non-invasive NIRF imaging system for 24 h. 24 h after gavage, the mice were killed and organs were collected from the mice. Then, the NIRF intensity of Cy5-exosomes in the organs was measured using IVIS Lumina Series III (PerkinElmer).

[0032] The results are as follows Figure 7 As shown, mice orally administered with DBCO-Cy5-N3-HA exosomes showed strong NIRF intensity in the gastrointestinal tract 24 h after treatment; Figure 8 The results showed that fluorescence was also concentrated in the gastrointestinal tract in the mouse isolated tissue imaging after 24 hours.

[0033] Application Example 3 Tumor-targeted application of DBCO-Cy5-N3-HA exosomes In order to verify the targeting of DBCO-Cy5-N3-HA exosomes in tumors, in vivo experiments were conducted in mice: First, 4T1 mouse breast cancer cells were orthotopically inoculated into the right mammary fat pad of female BALB / c mice to establish an orthotopic tumor-bearing model. After the tumor grew to an appropriate size (about 200-300 mm³), the DBCO-Cy5-N3-HA exosomes were administered.

[0034] The experimental group mice were orally gavaged with DBCO-Cy5-N3-HA exosomes at a dose of 1.5 mg / kg, while the control group mice were given the same dose of Ac4ManNAz-labeled exosomes (DBCO-Cy5-Ac4ManNAz exosomes). The near-infrared fluorescence imaging system (NIRF) was used to dynamically monitor the biodistribution of exosomes in mice. The characteristic fluorescence wavelength of Cy5 was used for imaging, and the distribution of exosomes in mice was regularly evaluated 24 hours after administration.

[0035] The results are as follows Fig. 9 As shown in the figure, after NIRF imaging analysis, exosomes accumulated significantly in tumor tissues 24 hours after administration, and DBCO-Cy5-N3-HA exosomes showed significant tumor targeting. The control group (DBCO-Cy5-Ac4ManNAz exosomes) was weakly localized in tumor tissues, with a significantly lower fluorescence signal than the experimental group, and was also distributed in non-tumor tissues in vivo, indicating that DBCO-Cy5-N3-HA exosomes have higher localization efficiency in tumor tissues through tumor targeting provided by sugar metabolism engineering labeling.

[0036] Example 3 Preparation of DBCO-doxorubicin-N3-HA exosomes DC2.4 cells were cultured in 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C and 5% CO2. The medium was changed once a day and the cells were observed under a microscope. When the cell confluence reached 80%, they were subcultured. After 12 hours of culture, the cells were cultured in 1640 complete medium with D-glucuronic acid (GlcA) and azido N-acetylglucosamine (Ac4GlcNAz) added to the medium at a mass ratio of 1:1 and a concentration of 10 μg / mL. The culture was continued for 13 hours, and the medium was changed. The medium contained 10% exosome-free FBS and was placed in an incubator at 37°C and 5% CO2 for 24-48 hours. The culture medium was collected and sterilized by filtration with a 0.22 μm needle filter. Larger cell debris and particles were removed by ultracentrifugation (100000g, 1h), and the supernatant was obtained as the initial enrichment solution of exosomes. The treated exosome enriched solution was input into the microfluidic chip through the inlet of the microfluidic system. The exosomes were effectively separated by a microcolumn array and a gradient electric field. After screening, the exosomes flowed out through the microchannel and were collected. The N3-HA exosomes were resuspended in PBS to a suspension of 0.6 μg / μL, pH=7, and aliquoted and frozen at -80°C for later use. The protein content of the exosomes was determined using a BCA protein concentration assay kit. Exosomes extracted from cells cultured in 1640 complete medium without any treatment were used as a control. The extracted N3-HA exosomes were incubated with DBCO-doxorubicin at a mass ratio of 6:1 for 60 minutes according to the total protein concentration, ultracentrifuged at 100,000 g, 4°C, 90 minutes, and the residual dye was removed to obtain DBCO-doxorubicin-N3-HA exosomes.

[0037] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for labeling exosomes, characterized in that: The following steps are involved: (1) Cultivating exosome-producing mother cells in a culture medium containing D-glucuronic acid and azido-N-acetylglucosamine; (2) isolating the exosomes from the mother cells in step (1); (3) Co-incubating the DBCO-coupled labeled molecule with the exosome suspension obtained in step (2) to obtain labeled exosomes.

2. The method according to claim 1, characterized in that: The mass ratio of D-glucuronic acid to azido N-acetylglucosamine is 1:2-2:1; The final concentration of D-glucuronic acid and azido N-acetylglucosamine in the culture medium is 5 μg / mL-20 μg / mL; the culture time is 6h-24h; In step (3), the ratio of total protein concentration of exosomes to molecular weight of coupled DBCO marker is (32-4):1; In step (3), the co-incubation condition is 37°C for 15 min-4 h; The concentration of exosomes in the suspension was 0.4 μg / μL-0.8 μg / μL.

3. The method according to claim 1, characterized in that The mass ratio of D-glucuronic acid to azido N-acetylglucosamine was 1:1; In step (3), the ratio of total protein concentration of exosomes to molecular weight of coupled DBCO marker is (8-4):

1.

4. The method according to claim 1, characterized in that: The mother cell is selected from a tumor cell, a stem cell, a dendritic cell or a neural cell; The separation method is selected from differential centrifugation, ultrafiltration, magnetic bead separation, protein precipitation, density gradient centrifugation, microfluidics, immunoaffinity separation or polymer precipitation.

5. The method according to claim 1, characterized in that The mother cell is selected from breast cancer cells, lung cancer cells, mesenchymal stem cells or induced pluripotent stem cells.

6. The method according to claim 1, characterized in that In step (3), the labeling molecule is a fluorescent molecule or a small molecule anti-tumor drug connected to a reactive group; the molecular weight of the small molecule anti-tumor drug is not greater than 1000 Da; and the reactive group undergoes a bioorthogonal reaction with the azide group.

7. The method according to claim 6, characterized in that The reactive group is selected from a cycloalkynyl group, a cycloalkene group, a five-membered ring reactive group, an epoxy group, a bisfluoroalkenyl group or a thiol group; The small molecule anti-tumor drug is selected from doxorubicin, doxorubicin, paclitaxel, cisplatin, carboplatin, 5-fluorouracil, coumarin drugs or acacetin drugs; The fluorescent molecule is selected from fluorescein isothiocyanate, cyanine fluorescent dyes or rhodamine fluorescent dyes.

8. The method according to claim 6, characterized in that The fluorescent group is selected from FITC, Cy-3, Cy-5, Cy-7, AF488, AF594 or AF555.

9. Exosomes prepared by the method according to any one of claims 1 to 8.

10. Use of the exosomes according to claim 9 in the preparation of diagnostic reagents and medicines.

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