Hybrid vesicle for regulating purine metabolism as well as preparation method and application of hybrid vesicle

Abstract

The invention discloses a hybrid vesicle for regulating purine metabolism and a preparation method and application thereof.The hybrid vesicle is formed by fusing extracellular vesicles derived from macrophages and extracellular vesicles derived from endothelial cells, CD39 and CD73 are arranged on membranes of the hybrid vesicle at the same time, purine metabolism can be efficiently and stably catalyzed, and pro-inflammatory ATP is converted into ADO with the immunoregulation function; the preparation method of the hybrid vesicle is simple and suitable for mass production; the application of the hybrid vesicle is helpful for overcoming the limitation of the existing autoimmune disease treatment method, improving the accuracy and effectiveness of autoimmune disease treatment and reducing the side effect of treatment, and has important clinical application value and wide market prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of hybrid vesicle and its preparation method and application, especially to a kind of hybrid vesicle for regulating purine metabolism and its preparation method and application. BACKGROUND

[0002] Autoimmune diseases seriously threaten human health, and existing treatment methods are difficult to achieve precise intervention on immune microenvironment, and there are problems such as large side effects and unsatisfactory treatment effect. Adenosine triphosphate (ATP) released into the extracellular by damaged or necrotic cells can trigger inflammatory response and aggravate the degree of injury. Therefore, by regulating purine metabolism, converting proinflammatory ATP into immunosuppressive adenosine (ADO) has become a new idea for treating autoimmune diseases.

[0003] As nanoscale particles naturally secreted by cells, extracellular vesicles (EVs) have become a promising precise immunomodulation platform due to their inherent biocompatibility, immunological inertia and extremely low cytotoxicity. EVs also have tissue targeting ability, so they have great potential in the field of disease treatment. However, EVs from a single source have limited functions and are difficult to meet the demand for synergistic action of multiple functions in the treatment of autoimmune diseases. For example, macrophage-derived EVs (EV MC ) highly express CD39 and can catalyze extracellular proinflammatory ATP to AMP; but EV MC lack CD73, so they cannot further hydrolyze AMP to anti-inflammatory mediator ADO.

[0004] Hybrid vesicles are obtained by fusing EVs from different cell sources, which can integrate the advantages of multiple EVs. However, there is no hybrid vesicle that can efficiently and stably regulate purine metabolism, and there is no preparation method for the vesicle. SUMMARY

[0005] The first object of the present application is to provide a hybrid vesicle that can efficiently and stably regulate purine metabolism, the second object is to provide a preparation method for the hybrid vesicle, and the third object is to provide applications of the hybrid vesicle.

[0006] Technical solution: The membrane of the hybrid vesicle for regulating purine metabolism of the present application has CD39 and CD73.

[0007] Since CD39 can catalyze ATP to hydrolyze to AMP, and CD73 can catalyze AMP to hydrolyze to ADO, therefore, the hybrid vesicle with CD39 and CD73 on the membrane can efficiently catalyze purine metabolism and convert proinflammatory ATP to ADO with immunoregulatory effect.

[0008] The preparation method comprises the following steps: (1) culturing macrophages and endothelial cells respectively; (2) separating extracellular vesicles of the macrophages and extracellular vesicles of the endothelial cells respectively; and (3) co-extruding the extracellular vesicles of the macrophages and the extracellular vesicles of the endothelial cells to obtain hybrid vesicles.

[0009] Since the macrophage-derived EVs (EV MC ) highly express CD39 and the endothelial cell-derived EVs (EV EC ) highly express CD73, the hybrid vesicles formed by fusing the EV MC and the EV EC have a large amount of CD39 and CD73 at the same time and can efficiently catalyze purine metabolism.

[0010] In step (1), the endothelial cells are preferably human umbilical vein endothelial cells, rat myocardial microvascular endothelial cells or rat brain microvascular endothelial cells.

[0011] In step (3), preferably, the extracellular vesicles of the macrophages and the extracellular vesicles of the endothelial cells are subjected to protein quantification respectively before the co-extrusion; the co-extrusion is preferably achieved by filtration; the filtration is preferably performed using a polycarbonate membrane filter with a pore size of 100-200 nm; and the filtration can be performed for several times.

[0012] In step (2), the extracellular vesicles are preferably separated using differential centrifugation.

[0013] The application of the hybrid vesicles is for catalyzing ATP to generate ADO or for preparing drugs for autoimmune diseases.

[0014] Advantages: Compared with the prior art, the hybrid vesicles prepared by the method have the following advantages: 1. The hybrid vesicles are prepared from different vesicles capable of catalyzing purine metabolism, and the membranes of the hybrid vesicles have CD39 and CD73 at the same time, which can efficiently catalyze purine metabolism and convert proinflammatory ATP into ADO with immunoregulatory effect; and the hybrid vesicles have good stability and have small size fluctuation when stored in FBS or PBS for 7 days; 2. The preparation method of the hybrid vesicles is simple and suitable for mass production; and 3. The application of the hybrid vesicles helps to overcome the limitations of the existing autoimmune disease treatment methods, improve the precision and effectiveness of autoimmune disease treatment, reduce the side effects of treatment, and has important clinical application value and broad market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 A synthesis mechanism diagram of the hybrid vesicles (CALM) for regulating purine metabolism is shown in the figure. Figure 2Concentration of catalytic production of ADO for 9 kinds of extracellular vesicles with high expression of CD73; Figure 3 Concentration of catalytic production of ADO for 3 kinds of extracellular vesicles of endothelial cells over time; Figure 4 Calm (scale: 100 nm) taken by transmission electron microscope (TEM); Figure 5 Fused vesicles before and after (Fig. a, b, c are detection results of different fluorescence respectively. DiO (green) stains EV EC , DiD (red) stains EV MC . Fig. a scale: 1 μm, Fig. b, c scale: 0.5 μm) taken by laser scanning confocal fluorescence microscope; Figure 6 Particle size comparison chart of EV MC , EV EC and CALM; Figure 7 Trend chart of size of CALM over time; Figure 8 Surface charge comparison chart of EV MC , EV EC and CALM; Figure 9 CD39, CD73, CD9 protein immunoblotting chart of EV MC , EV EC and CALM; Figure 10 Efficiency comparison of catalytic production of ADO of CALM and Mix (Mix is composed of EV MC and EV EC ); Figure 11 Production of CALM catalytic ATP to generate ADO under different conditions. DETAILED DESCRIPTION

[0016] The technical solutions of the present application will be further described below in combination with the drawings.

[0017] Example 1

[0018] This embodiment provides the hybrid vesicle for regulating purine metabolism and its preparation method. The synthesis mechanism is shown as Figure 1 , and the specific preparation method is as follows: 1. Cell culture: Mouse bEnd.3 endothelial cells and mouse RAW264.7 cells were inoculated in T75 cell culture flasks, and DMEM high glucose medium (containing 10% fetal bovine serum and 1% (v / v) penicillin / streptomycin) was added, and the flasks were placed in a 37°C, 5% CO2 incubator for culture. The medium was replaced every 2-3 days, and when the cell confluence reached 80-90%, the cells were passaged or the cell culture supernatant was collected for extracellular vesicle isolation.

[0019] 2. Isolation of extracellular vesicles: The cell culture supernatant was collected, and the extracellular vesicles were isolated by differential centrifugation. First, the cells were removed by centrifugation at 500g for 10 minutes; the supernatant was transferred to a new centrifuge tube and centrifuged at 2000g for 10 minutes to remove dead cells; then the supernatant was centrifuged at 10000g for 90 minutes to remove cell debris; finally, the collected supernatant was centrifuged at 100000g for 2 hours, the supernatant was discarded, and the precipitate was resuspended in PBS to obtain macrophage-derived extracellular vesicles (EV MC ) and endothelial cell-derived extracellular vesicles (EV EC ).

[0020] 3. Vesicle fusion: The protein content of EV MC and EV EC was quantified using a BCA protein assay kit. The quantified EV MC and EV EC were mixed in a 1:1 protein mass ratio, then passed through polycarbonate membrane filters with pore sizes of 200 nm and 100 nm, respectively, and the mixture was extruded 13 times using a micro-extruder to obtain catalytic hybrid vesicles (CALM) that can regulate purine metabolism, which were aliquoted and stored at -80°C.

[0021] Example 2

[0022] This example detects the hybrid vesicles prepared in Example 1.

[0023] 1. Morphological detection: 5 μL of CALM suspension was dropped on a copper mesh, which was air-dried at room temperature for 5 minutes, then negatively stained with 1% uranyl acetate solution for 3 times, 1 minute each time, and observed under a transmission electron microscope (TEM) to observe the morphology of CALM.

[0024] The results are shown in Figure 4 , and the CALM has a typical vesicular structure with uniform size.

[0025] 2. Size and surface charge measurement: An appropriate amount of CALM suspension was taken and its hydrodynamic size and surface charge were measured using dynamic light scattering technology on ZetaPALS. Meanwhile, the CALM was placed in PBS and FBS, respectively, and the change in hydrodynamic size was measured periodically within 7 days.

[0026] Results show that the average hydrodynamic diameter of CALM is about 200 nm (see Figure 6 ), and the surface charge (zeta potential) is about -15 mV (see Figure 8 ). CALM has good stability in PBS or FBS, with small size fluctuations (see Figure 7 ).

[0027] 3. Fusion effect detection: EV MC and EV EC were cultured in a confocal dish and incubated for 24 hours under standard culture conditions (37°C, 5% CO2). After incubation, EV MC and EV EC were stained with DiD and DiO, respectively, and membrane fusion was observed by laser scanning confocal fluorescence microscopy.

[0028] Results are shown in Figure 5 . CALM has high fluorescence co-localization efficiency, high fluorescence intensity, and high area coincidence.

[0029] 4. SDS-PAGE analysis: EVs were lysed with RIPA buffer containing 1 mM PMSF protease inhibitor in an ice bath environment. After 4 minutes of lysis, the sample was shaken for 30 seconds and repeated 5 times. The lysate was centrifuged at 12000g for 5 min at 4°C, and the supernatant was collected. The protein concentration was determined using a BCA detection kit, and the sample buffer was prepared using Laemmli sample buffer to ensure that the protein content in each well was 20 µg. After denaturation of the proteins in the sample at 95°C for 10 minutes, SDS-PAGE analysis was performed in a Mini-PROTEAN® TGX stain-free™ protein gel to detect the proteins in CALM.

[0030] Results are shown in Figure 9 . CALM has CD39, CD73, and CD9.

[0031] 5、CALM's function detection_HPLC analysis of ADO production: Different concentrations of ATP (0.097, 0.39, 1.56, 6.25 mM) were incubated with different concentrations of CALM (100, 200, 300, 400 μg / mL) at 37 °C for 5 min. After the reaction, the samples were filtered through a 0.22 μm filter and the nucleotide content in the solution was determined by HPLC. The chromatographic conditions were as follows: column 250 mm x 4.6 mm C-18 BDS Multohyp 5 μm column and C-18 guard column, mobile phase A 90% methanol, mobile phase B 10 mM KH2PO4, flow rate 1.0 mL / min, column temperature 30 °C, detection wavelength 254 nm, injection volume 10 uL. The column was first flushed with mobile phase A for 8 min, then the sample was injected for detection, and the next sample was injected after flushing with mobile phase A for another 8 min. The ADO content produced by catalysis was calculated from the peak area corresponding to the standard.

[0032] As shown in Figure 11 , CALM can efficiently catalyze the metabolism of ATP to ADO.

[0033] 6、CALM's function detection_Real-time detection of ADO: The adenosine detection kit was used to determine the efficiency of CALM catalyzing ATP to produce ADO in the solution in real time. The control group was EV MC and EV EC mixed directly without being made into hybrid vesicles, denoted as Mix. After incubating a certain concentration of ATP with Mix and CALM at 37 °C for 5 min, Reaction Mix was added, and the emission intensity was detected at 590-600 nm after excitation at 530-570 nm using a microplate reader. The RFU value in the sample was calculated by the average relative fluorescence unit (RFU) value corresponding to the ADO standard, and then the adenosine content in the sample was calculated. To determine the Km and Vmax of the sample, different concentrations of ATP (0, 0.04882813, 0.09765625, 0.1953125, 0.390625, 0.78125, 1.5625, 3.125, 6.25 and 25 mM) were incubated with Mix and CALM, and the production of ADO was monitored for two minutes using the above method. The kinetic parameters were derived using the Michaelis-Menten equation.

[0034] As shown in Figure 10 , the Km value of CALM catalyzing ATP to generate ADO was 0.611 mM, and the Vmax value was 0.108 μmol / min. Compared with Mix mixed directly, the hybrid vesicle catalyzed the metabolism of ATP to ADO more efficiently.

[0035] Example 3

[0036] This example screened EVs with high CD73 enzymatic activity from 9 kinds of CD73-expressing cell EVs. The 9 kinds of cell EVs were: dendritic cell EV (EV DC ), monocyte EV (EV MO ), natural killer cell EV (EV NK ), neutrophil EV (EV NE ), peripheral blood lymphocyte EV (EV PB ), lymph node lymphocyte EV (EV LL ), human umbilical vein endothelial cell EV (EV MU ), rat myocardial microvascular endothelial cell EV (EV MM ), and brain microvascular endothelial cell EV (EV BE ). After 10 mM AMP was added to each of the 9 EVs and incubated for 15 minutes, the ADO content in the supernatant was detected to evaluate the CD73 enzymatic activity. The content of the catalytically produced purine metabolite was calculated from the peak area corresponding to the standard.

[0037] The results are shown in Figure 2 , EV MU , EV MM , and EV BE showed high ability to catalyze AMP to ADO, with high CD73 enzymatic activity. Therefore, endothelial cell EVs were selected as CD73-providing EVs to prepare hybrid vesicles with EV MC to efficiently regulate purine metabolism.

[0038] Example 4

[0039] Based on the three kinds of endothelial cell EVs screened in Example 3, this example compared their ability to catalyze ADO production. CD3 + T cells were inoculated in a 96-well culture plate and incubated under standard culture conditions (37°C, 5% CO2) for 24 hours. After 24 hours, DMEM high glucose medium containing EV MU , EV MM , and EV BE was added, respectively. Then, at 0, 3, 6, 9, 12, and 15 min time points, the cells were treated with cell lysis solution, centrifuged to obtain the supernatant, and then the ADO detection kit was used to prepare working solution, add samples, and then use the enzyme label instrument to detect the luminescence value and convert it to ADO concentration.

[0040] The results are shown in Figure 3 , EV BE had the highest efficiency and yield of catalytic ADO production.

Claims

1. A hybrid vesicle for modulating purine metabolism, characterized in that, CD39 and CD73 are on the membrane thereof.

2. A method of preparing the hybrid vesicle of claim 1, characterized by, The method comprises the following steps: (1) culturing macrophages and endothelial cells respectively; (2) isolating extracellular vesicles of the macrophages and extracellular vesicles of the endothelial cells respectively; and (3) co-extruding the extracellular vesicles of the macrophages and the extracellular vesicles of the endothelial cells to obtain hybrid vesicles.

3. The production method according to claim 2, characterized by, In step (1), the endothelial cells are human umbilical vein endothelial cells, rat myocardial microvascular endothelial cells or rat brain microvascular endothelial cells.

4. The production method according to claim 2, characterized by, In step (3), the extracellular vesicles of the macrophages and the extracellular vesicles of the endothelial cells are subjected to protein quantification respectively before the co-extrusion.

5. The preparation method according to claim 2, characterized in that, In step (3), the co-extrusion is achieved by filtration.

6. The production method according to claim 5, wherein The filtration is performed using a polycarbonate membrane filter with a pore size of 100-200 nm.

7. The preparation method according to claim 5, characterized in that, The filtration is performed for several times.

8. The preparation method according to claim 2, characterized in that, In step (2), the extracellular vesicles are isolated by using differential centrifugation.

9. Use of the hybrid vesicles of claim 1 in catalyzing ATP production ADO.

10. Use of the hybrid vesicles of claim 1 in preparing drugs for autoimmune diseases.

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