A method of labeling exosomes

Abstract

The application belongs to the technical field of medical diagnosis, and provides a method for labeling exosomes: culturing mother cells for producing exosomes in a culture medium containing GlcA and Ac4GlcNAz to obtain exosomes of the mother cells; and co-incubating a labeling molecule coupled with DBCO with an exosome suspension to obtain labeled exosomes. The application uses non-natural sugar metabolism to introduce N3-HA into the exosome membrane, which does not affect the yield and physiological activity of the exosomes, and has the advantages of high stability and high labeling efficiency. The exosomes labeled with N3-HA can be used for exosome tracing and targeted drug delivery, which is not only simple and fast to operate, but also can greatly improve the stability and reliability of fluorescence and the drug targeting property. The strategy provided by the application can become a powerful tool for the research on the treatment mechanism of exosomes in various diseases, and can provide an effective method for the targeted delivery of anti-tumor drugs, which has very important clinical significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medical diagnosis, and relates to a method for labeling and tracing exosomes. BACKGROUND

[0002] The information disclosed in this background section is intended to increase an understanding of the general context of the present application and is not necessarily recognized in the prior art before the present application.

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

[0004] At present, more advanced imaging systems enable us to observe smaller vesicles, but the behavior of exosomes cannot be directly observed. One of the biggest problems in observing, measuring and visualizing the behavior of exosomes is how to functionalize exosomes to firmly bind with fluorescent molecules. The current methods for functionalizing exosomes to make them traceable mainly include: 1. fusing fluorescent proteins with marker proteins on the exosome membrane to present the movement of exosomes in vivo (CN 115161260 A). 2. encoding lentivirus to transfect cells, incorporating amino acids into cell protein components to secrete exosomes, and covalently linking 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, so it is difficult to visualize the uptake of exosomes in vivo.

[0005] Exosomes, as a natural intercellular communication carrier, 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 complex. These processes not only have cumbersome operations, but also may cause damage to the structure and function of exosomes. Therefore, how to improve the targeting of exosomes while maintaining their integrity has become a key problem to be solved in current research. SUMMARY

[0006] In view of the current problems of difficult exosome tracing and poor tumor targeting, the present application provides an exosome labeling method, which uses cell metabolic sugar engineering to label azide hyaluronic acid, and prepares N3-HA exosomes, which have better stability and higher labeling efficiency than traditional exosomes, and good targeting to tumor cells.

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

[0008] To achieve the above object, the present application adopts the following technical solution.

[0009] A method for labeling exosomes, comprising the following steps:

[0010] (1) culturing mother cells producing exosomes in a culture medium containing D-glucuronic acid (GlcA) and azide N-acetylglucosamine (Ac4GlcNAz);

[0011] (2) isolating the exosomes of the mother cells in step (1);

[0012] (3) co-incubating a labeling molecule coupled with DBCO with a suspension of the exosomes obtained in step (2) to obtain labeled exosomes.

[0013] The mass ratio of GlcA and Ac4GlcNAz is 1:2-2:1; preferably 1:1.

[0014] The final concentration of GlcA and Ac4GlcNAz in the culture medium is 5-20 μg / mL; the culture time is 6-24 h.

[0015] The exosome labeling method of the present application can select different types of mother cells according to the specific application requirements of the labeled exosomes, including tumor cells (such as breast cancer cells, 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, neural cells for neural repair, etc. By selecting appropriate types of mother cells, the targeting and function of exosomes can be effectively optimized to meet the needs of different medical research and clinical treatment.

[0016] The separation method can be selected from existing methods according to the size and purpose of the sample, such as differential centrifugation, ultrafiltration, magnetic bead separation, protein precipitation, density gradient centrifugation, microfluidic technology, immunoaffinity separation, polymer precipitation.

[0017] In step (3), the concentration of the exosomes in the suspension is not limited; the mass ratio of the total protein concentration of the exosomes to the DBCO-labeled molecule is 32-4:1; preferably, the mass ratio of the total protein concentration of the exosomes to the DBCO-labeled molecule is 8-4:1; more preferably, the concentration of the exosome suspension is 0.4 μg / μL-0.8 μg / μL under the above ratio.

[0018] In step (3), the labeling molecule is a fluorescent molecule or a small molecule antitumor drug connected to a reactive group; the molecular weight of the small molecule antitumor drug is not more than 1000 Da; the reactive group bioreacts with azide hyaluronic acid. Preferably, the reactive group is selected from a cycloalkynyl group, a cycloalkene group, a five-membered ring reactive group, an epoxy group, a difluoroalkenyl group, and a thioalkenyl group. The small molecule antitumor drug is selected from doxorubicin, doxorubicin, paclitaxel, cisplatin, carboplatin, 5-fluorouracil, coumarin drugs, or acacia drugs; the fluorescent group is selected from fluorescein isothiocyanate (FITC), cyanine dyes, or rhodamine fluorescent dyes; such as FITC, Cy-3, Cy-5, Cy-7, AF488, AF594, AF555.

[0019] In step (3), the co-incubation conditions are preferably 37℃, 15min-4h.

[0020] The application of the above-mentioned labeled exosomes in the preparation of diagnostic reagents and drugs.

[0021] The application has the following advantages:

[0022] The metabolic sugar engineering labeled N3-HA exosome tracing and targeted drug delivery technology adopted by the application introduces N3-HA into the exosome membrane using non-natural sugar metabolism, which does not affect the yield and physiological activity of the exosome, and has the advantages of high stability and high labeling efficiency. The fluorescence intensity advantage of the functionalized exosome and its targeting effect on tumor cells are verified by confocal microscopy at the cellular level, and the visualization advantage of the behavior process of the functionalized exosome between cells is determined by in vitro experiments. The application of the metabolic sugar engineering labeled N3-HA exosome tracing and targeted drug delivery not only has the advantages of simple and fast operation, but also can greatly improve the stability and reliability of fluorescence and the targeting of drugs. The application strategy provided by the application can become a powerful tool for exosome in the research of various disease treatment mechanisms, and provides an effective method for targeted delivery of antitumor drugs, which has very important innovative significance. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 is the confocal microscopic image (A) and the fluorescence intensity change (B) of the cell labeled with different sugars;

[0024] Figure 2 are confocal microscopic images (A) and fluorescence intensity changes (B) of cells obtained at different proportions of raw materials;

[0025] Figure 3 is a transmission electron microscope image of N3-HA exosomes;

[0026] Figure 4 are Western Blot images of DBCO-AF488-labeled and unlabeled N3-HA exosome surface markers;

[0027] Figure 5 are gel electrophoresis fluorescence images (A) and Coomassie blue staining images (B) of DBCO-AF488-labeled and unlabeled N3-HA exosomes;

[0028] Figure 6 are confocal microscopic images (A) and fluorescence intensity changes (B) of DBCO-AF488-N3-HA exosomes at different time points;

[0029] Figure 7 is the distribution of DBCO-Cy5-N3-HA exosomes in mice in vivo;

[0030] Figure 8 is the distribution of DBCO-Cy5-N3-HA exosomes in mouse isolated tissues (stomach, intestine);

[0031] Figure 9 are the target distribution of DBCO-Cy5-Ac4ManNAz exosomes (A) and DBCO-Cy5-N3-HA exosomes (B) in tumor tissue sites of mice;

[0032] In the above figures, represents p < 0.01; represents p < 0.001. DETAILED DESCRIPTION

[0033] The present application will be further described below in conjunction with examples and drawings, but the present application is not limited by the following examples.

[0034] Example 1 Preparation of DBCO-AF488-N3-HA exosomes

[0035] 1. Different saccharide labels

[0036] Put cell slides into a 24-well plate, add 1640 complete culture medium (1 mL per well), inoculate 4T1 cells in the 24-well plate with added culture medium, and the inoculation amount is 5 × 10 5Cells were inoculated and cultured for 12 h. D-glucuronic acid (GlcA) and N-acetylglucosamine (Ac4GlcNAz) at a mass ratio of 1:1 were added to the cell culture wells to a final concentration of 10 μg / mL for both. N3-HA was then labeled on the cell membrane surface, serving as the experimental group. Additionally, 10 μg / mL azide-mannose (Ac4ManNAz) was added to the cell culture wells to label the cell membrane surface, serving as the control group. Cells were incubated at 37℃ and 5% CO2 for 13 h to obtain N3-HA-labeled cells and Ac4ManNAz-labeled cells. Cells were then stained sequentially with DBCO-AF488 and DAPI. Cell membrane fluorescence of both cell types was observed under a confocal inverted fluorescence microscope at 6 h and 12 h.

[0037] The results are as follows Figure 1 As shown, the cell membrane fluorescence of N3-HA labeling was stronger than that of Ac4ManNAz at the initial time point. Over time, the fluorescence signal of N3-HA labeling was more persistent and decayed more slowly than that of Ac4ManNAz.

[0038] 2. Ratio of GlcA to Ac4GlcNAz

[0039] Place cell crawling smears into 24-well plates, add 1 mL of 1640 complete culture medium per well, and seed 4 T1 cells into each well at a density of 5 × 10⁴ cells. 5 Cells were inoculated and cultured for 12 h. D-glucuronic acid (GlcA) and azido-N-acetylglucosamine (Ac4GlcNAz) were added to the cell culture wells at mass ratios of 1:1, 2:1, and 1:2, respectively, to a final concentration of 10 μg / mL. The cells were incubated at 37°C and 5% CO2 for 13 h. The cells were stained with DBCO-AF488 and DAPI, and their localization on the cell membrane was observed under a confocal inverted fluorescence microscope.

[0040] The results are as follows Figure 2 As shown, the fluorescence intensity of the cell membrane reaches its maximum when the ratio of GlcA: Ac4GlcNAz is 1:1.

[0041] 3. Preparation of N3-HA exosomes

[0042] Bone marrow of healthy adult mice was selected, and standard serum-free a-MEM medium was used for the isolation and culture of bone marrow mesenchymal stem cells (BMSCs). The BMSCs were treated with 10% fetal bovine serum medium, inoculated into culture bottles, and cultured at 37°C and 5% CO2. The culture was continued for about 3-5 days until the cells reached 80%-90% confluence. The adherent screening method was used to remove non-adherent cells and collect adherent BMSCs. GlcA and Ac4GlcNAz were added to the a-MEM medium at a mass ratio of 1:1, and the concentration was 10 μg / mL. The culture was continued for 13 h, and the medium was replaced. This medium contained 10% exosome-free FBS and was placed in a 37°C, 5% CO2 incubator for continuous culture for 24-48 h. The culture solution was collected and filtered with a 0.22 μm needle filter to remove bacteria. The BMSCs culture solution was centrifuged at low speed to remove cell impurities, and then exosomes were extracted by ultracentrifugation (100,000 g, 4°C, 1 h). The exosome sample was incubated with antibody-coated magnetic beads containing mesenchymal stem cell surface markers (CD73) for 2 h, and the specific selectivity of the antibody was used to enrich exosomes. The exosomes were separated from the antibody-coated magnetic beads, and the exosome enrichment solution was collected and washed with PBS buffer to remove non-specifically bound substances to obtain N3-HA labeled exosomes. Exosomes extracted from cells cultured in untreated DMEM medium were used as controls.

[0043] 4. Preparation of DBCO-AF488-N3-HA exosomes

[0044] The N3-HA labeled exosomes were resuspended in PBS at pH 7 to prepare an exosome suspension at a concentration of 0.8 μg / μL, and were aliquoted and stored at -80°C for later use. The protein content of the exosomes was determined using a BCA protein concentration determination kit. The extracted N3-HA exosomes were co-incubated with DBCO-AF488 at a mass ratio of 8:1 for 45 min, and then ultracentrifuged at 100,000 g and 4°C for 90 min to remove residual dyes to obtain DBCO-AF488-N3-HA exosomes.

[0045] The morphology and structure of the extracted exosomes were observed by transmission electron microscopy (TEM), as shown in FIG. 1, where the DBCO-AF488-N3-HA exosomes exhibited a typical "tea tray-like" exosome shape. Figure 3 Western blot was used to identify the surface marker proteins of the two types of exosomes, as shown in FIG. 2, where the exosome marker proteins CD9 and CD81 were positively expressed. Figure 4 Gel fluorescence imaging and Coomassie brilliant blue staining were used to detect the fluorescence and protein content of the DBCO-AF488-N3-HA exosomes, as shown in FIG. 3. Figure 5As shown, the fluorescence of DBCO-AF488-N3-HA exosomes was significantly higher than that of the control exosomes. The Coomassie blue results showed that there was no significant difference in the content of DBCO-AF488-N3-HA exosomes and control exosomes. The above results show that the metabolic labeling of N3-HA does not affect the characteristics of exosomes.

[0046] Application Example 1 Tracing effect of DBCO-AF488-N3-HA exosomes

[0047] DMEM complete medium (1 mL per dish) was added to the laser confocal cell culture dish, 4T1 cells were inoculated, and the inoculation amount was 5 x 10 5 cells per well; after inoculation, the cells were cultured for 12 h, stained with Hoechst 33342, and then 500 μL of DBCO-AF488-N3-HA exosomes were added for co-incubation. The tracing of DBCO-AF488-N3-HA exosomes in vitro was observed under a confocal inverted fluorescence microscope at 0 h, 3 h, and 6 h, respectively.

[0048] The results are shown in Figure 6 , wherein the blue signal is the nucleus DAPI staining, and the green point signal is the tracing exosome signal. The above results show that the hyaluronic acid labeled exosomes modified with a fluorescent group can be used for cell tracing.

[0049] Example 2 Preparation of DBCO-Cy5-N3-HA exosomes

[0050] 4T1 cells were cultured in high glucose DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and the culture conditions were 37°C and 5% CO2 environment. The medium was changed once a day, and the cells were observed under a microscope. When the cell confluence reached 80%, the cells were passaged and cultured for 12 h. Then, the cells were cultured in a medium containing D-glucuronic acid (GlcA) and azido N-acetylglucosamine (Ac4GlcNAz) at a mass ratio of 1:1 and a concentration of 10 μg / mL. The medium was replaced after 13 h of continuous culture, and the medium contained 10% exosome-free FBS. The culture was continued in a 37°C, 5% CO2 incubator for 24-48 h. The culture medium was collected and filtered with a 0.22 μm needle filter to remove bacteria. The filtered cell culture supernatant was centrifuged at 300 g for 10 min to remove cells. The supernatant was centrifuged at 2000 g for 10 min to remove dead cells, and the supernatant was centrifuged at 10000 g for 20 min to remove cell debris. The supernatant was prepared for ultracentrifugation at 100000 g for 90 min to obtain N3-HA exosomes. The azido hyaluronic acid-labeled exosomes were resuspended in a 0.4 μg / μL suspension at pH=7, and the suspension was aliquoted and stored at -80°C for later use. The protein content of the exosomes was determined using a BCA protein concentration determination kit. The 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 15 min, and then ultracentrifuged at 100000 g at 4°C for 90 min to remove residual dye to obtain DBCO-Cy5-N3-HA exosomes.

[0051] Example 2 In vivo tracing effect of DBCO-Cy5-N3-HA exosomes

[0052] Normal mice (n=3) were orally gavaged with Cy5-exosomes (1.5 mg / kg) according to the dose, and the time-dependent in vivo biodistribution of Cy5-exosomes was evaluated by non-invasive NIRF imaging system for 24 h. After 24 h of gavage, the mice were sacrificed, and the organs were collected from the mice. Then, the NIRF intensity of Cy5-exosomes in the organs was measured using IVIS Lumina Series III (PerkinElmer) measurement.

[0053] As shown in Figure 7 The results showed that the DBCO-Cy5-N3-HA exosomes orally administered to mice exhibited strong NIRF intensity in the gastrointestinal tract 24 h after treatment. Figure 8 The results showed that the fluorescence also accumulated in the gastrointestinal tract in the tissue imaging of mice after 24 h.

[0054] Application Example 3 Tumor targeting of DBCO-Cy5-N3-HA exosomes

[0055] To verify the targeting of DBCO-Cy5-N3-HA exosomes in tumors, in vivo experiments were performed on mice: first, 4T1 mouse breast cancer cells were inoculated in 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 administration experiment of DBCO-Cy5-N3-HA exosomes was performed.

[0056] The experimental group mice were orally administered DBCO-Cy5-N3-HA exosomes at a dose of 1.5 mg / kg, and the control group mice were administered the same dose of Ac4ManNAz-labeled exosomes (DBCO-Cy5-Ac4ManNAz exosomes). The biodistribution of exosomes in mice was dynamically monitored using a near-infrared fluorescence imaging system (NIRF). Imaging was performed using the characteristic fluorescence wavelength of Cy5, and the distribution of exosomes in mice was evaluated at 24 hours after administration.

[0057] The results, as shown in Figure 9 NIRF imaging analysis showed that exosomes accumulated significantly in tumor tissue 24 hours after administration, and DBCO-Cy5-N3-HA exosomes exhibited significant tumor targeting. The control group (DBCO-Cy5-Ac4ManNAz exosomes) had weaker localization in tumor tissue, with significantly lower fluorescence signals than the experimental group, and also had distribution in non-tumor tissues in vivo, indicating that DBCO-Cy5-N3-HA exosomes had higher localization efficiency in tumor tissue through the tumor targeting provided by glycometabolic engineering labeling.

[0058] Example 3 Preparation of DBCO-doxorubicin-N3-HA exosomes

[0059] DC2.4 cells were cultured in 1640 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution, and the culture conditions were 37℃ and 5% CO2 environment. The medium was changed once a day, and the cells were observed under a microscope. When the cell confluence reached 80%, the cells were passaged and cultured for 12 h. Then, the cells were cultured in 1640 complete medium containing D-glucuronic acid (GlcA) and azido N-acetylglucosamine (Ac4GlcNAz) at a mass ratio of 1:1 and a concentration of 10 μg / mL. The cells were cultured for another 13 h, and the medium was replaced with a medium containing 10% exosome-free FBS. The cells were cultured in a 37℃, 5% CO2 incubator for 24-48 h, and the culture solution was collected and filtered through a 0.22 μm needle filter to remove bacteria. Larger cell debris and particles were removed by ultracentrifugation (100000g, 1 h), and the supernatant was used as the initial exosome-rich solution. The treated exosome-rich solution was input into the microfluidic chip through the inlet end of the microfluidic system. The exosomes were effectively separated by the microcolumn array and the 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 with a concentration of 0.6 μg / μL and a pH of 7, and were aliquoted and stored at -80℃ for later use. The protein content of the exosomes was determined using a BCA protein concentration determination 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 min, and ultracentrifugation was performed at 100000g and 4℃ for 90 min to remove residual dye, thereby obtaining DBCO-doxorubicin-N3-HA exosomes.

[0060] The above only describes the embodiments of the present application and is not intended to limit the present application. The present application can have various modifications and changes for those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the scope of the claims of the present application.

Claims

1. A method for labeling exosomes, characterized in that, Includes the following steps: (1) The exosome-producing mother cells were cultured in a medium containing D-glucuronic acid and azido-N-acetylglucosamine; (2) Separate the exosomes of the mother cells from step (1); (3) The DBCO-conjugated labeled molecules are co-incubated with the suspension of exosomes 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 concentrations of D-glucuronic acid and azido-N-acetylglucosamine in the culture medium were 5 μg / mL-20 μg / mL; the incubation time was 6 h-24 h. In step (3), the total protein concentration of exosomes is in the ratio of the molecular weight of the coupled DBCO label to (32-4):1; In step (3), the co-incubation conditions are 37℃ for 15 min to 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 is 1:1; In step (3), the ratio of the total protein concentration of exosomes to the molecular weight of the coupled DBCO label is (8-4):

1.

4. The method according to claim 1, characterized in that, The blast cells are selected from tumor cells, stem cells, dendritic cells, or nerve cells; 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 blast cells are 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 labeled molecule is a fluorescent group or a small molecule antitumor drug; the molecular weight of the small molecule antitumor drug is not greater than 1000 Da.

7. The method according to claim 6, characterized in that, The small molecule antitumor drugs are selected from doxorubicin, doxorubicin, paclitaxel, cisplatin, carboplatin, 5-fluorouracil, coumarin drugs or farnesin drugs; The fluorescent group 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. An exosome prepared by the method according to any one of claims 1-8.

10. The use of the exosomes as described in claim 9 in the preparation of diagnostic reagents and pharmaceuticals.

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