Exosome-containing medicine as well as preparation method and application thereof

Abstract

The invention provides a medicine containing exosome and a preparation method and application thereof, and belongs to the technical field of biological medicine. The medicine containing the exosome, provided by the invention, is prepared from the exosome and ampelopsin, the mass ratio of the exosome to the ampelopsin is 1: (4-6); the exosome is an umbilical cord mesenchymal stem cell exosome. The preparation method comprises the following steps: mixing an exosome and a buffer solution to obtain an exosome suspension; and mixing the exosome suspension with ampelopsin, incubating for 15-25 minutes at the temperature of 25-35 DEG C, freezing for 3-5 minutes at the temperature of-90 DEG C to-70 DEG C, unfreezing at room temperature, circularly repeating freezing and unfreezing for three times, performing ultrafiltration centrifugation, and collecting precipitate to obtain the medicine containing the exosome. The umbilical cord mesenchymal stem cell source exosome is used as an efficient cell delivery carrier of the ampelopsin, so that the cell delivery efficiency is greatly improved, and the bioavailability of the ampelopsin and the anti-tumor effect of the medicine are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a drug containing exosomes, a preparation method thereof, and an application thereof. Background Art

[0002] Exosomes are discoid multi-vesicular bodies with a diameter of 30-150 nm secreted by different types of cells. Exosomes were first discovered in sheep reticulocytes in 1983 and were named by Johnston in 1987. Exosomes exist and are distributed in various body fluids, including blood, urine, saliva, and cerebrospinal fluid, etc. Almost all types of cells can secrete exosomes under normal and pathological conditions, and there are differences in the composition and function of exosomes secreted by different cells.

[0003] Traditional Chinese medicines and their active ingredients can play an anti-tumor role through multiple components, multiple pathways, and multiple targets. However, traditional Chinese medicines have problems such as large oral doses, poor absorption, low solubility, and low bioavailability in the process of disease diagnosis and treatment. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a drug containing exosomes, a preparation method thereof, and an application thereof, so as to solve the problems of large oral doses, poor absorption, low solubility, and low bioavailability existing in the process of traditional Chinese medicines playing an anti-tumor role.

[0005] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0006] The present invention provides a drug containing exosomes, including exosomes and tengmatrine;

[0007] The mass ratio of the exosomes to tengmatrine is 1:4-6.

[0008] Preferably, the exosomes are umbilical cord mesenchymal stem cell exosomes.

[0009] The present invention also provides a preparation method of the drug, including the following steps:

[0010] Mix the exosomes and a buffer solution to obtain an exosome suspension; mix the exosome suspension and tengmatrine, incubate at a temperature of 25-35 °C for 15-25 min, then freeze at -90 to -70 °C for 3-5 min, and thaw at room temperature. After repeating the freeze-thaw cycle three times, perform ultrafiltration centrifugation, and collect the precipitate to obtain the drug containing exosomes.

[0011] Preferably, tengmatrine is dissolved in ethanol as a solvent before mixing, and the concentration of tengmatrine is 20-50 μg / mL.

[0012] Preferably, the buffer solution is PBS buffer solution, and the concentration of the exosome suspension is 50-80 μg / mL.

[0013] Preferably, the method for preparing the exosomes comprises the following steps:

[0014] 1) Culturing umbilical cord mesenchymal stem cells in a low-glucose DMEM medium containing 10-15% fetal bovine serum by volume, changing to a serum-free medium for culturing when the cell confluence reaches 70%-80%, and collecting the supernatant after 48-72 h;

[0015] 2) Performing differential centrifugation extraction on the supernatant to obtain the exosomes.

[0016] Preferably, the method for differential centrifugation extraction in step 2) is as follows:

[0017] ① Centrifuging at 300-400×g for 15-20 min, and collecting the supernatant;

[0018] ② Centrifuging the supernatant collected in step ① at 2100-2200×g for 15-20 min, and collecting the supernatant;

[0019] ③ Centrifuging the supernatant collected in step ② at 11000-12000×g for 20-25 min, and collecting the supernatant;

[0020] ④ Centrifuging the supernatant collected in step ③ at 11000-12000×g for 50-60 min, and collecting the precipitate.

[0021] The present invention also provides the application of the said drug in the preparation of anti-tumor drugs.

[0022] Preferably, the anti-tumor drug includes an anti-hepatocellular carcinoma drug.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses exosomes derived from umbilical cord mesenchymal stem cells as an efficient cell delivery carrier for tengmatrine, greatly improving its cell delivery efficiency and the exertion of its anti-tumor effect. Exosomes derived from umbilical cord mesenchymal stem cells, as an important medium for intercellular communication, contain a large number of active components, including proteins, lipids and nucleic acids, have a wide range of drug effects, and also play a positive role in anti-tumor. The drug containing exosomes prepared by the present invention can effectively improve the cell delivery efficiency and exert the synergistic effect of exosomes and tengmatrine.

[0025] In the present invention, febrifugine is loaded on exosomes and released into cells through molecular mediation on the cell membrane and stimulation of the internal environment, and directional treatment of tumors can be achieved by regulating the drug loading amount and release rate, thereby improving the bioavailability of febrifugine and greatly reducing the side effects of the drug. Detailed implementation mode

[0026] The present invention provides a drug containing exosomes, including exosomes and febrifugine; the mass ratio of the exosomes to febrifugine is preferably 1:4 to 6, and more preferably 1:4.5 to 5.5. In the present invention, the exosomes are preferably umbilical cord mesenchymal stem cell exosomes.

[0027] The present invention also provides a preparation method of the drug, including the following steps:

[0028] Mix the exosomes and buffer solution to obtain an exosome suspension; mix the exosome suspension and febrifugine, incubate at a temperature of 25 to 35 °C for 15 to 25 min, then freeze at -90 to -70 °C for 3 to 5 min, and thaw at room temperature. After repeating the freeze-thaw cycle three times, perform ultrafiltration centrifugation, and collect the precipitate to obtain the drug containing exosomes.

[0029] In the present invention, the preparation method of the exosomes includes the following steps:

[0030] 1) Culture umbilical cord mesenchymal stem cells with low-glucose DMEM medium containing 10 to 15% fetal bovine serum by volume fraction. When the cells reach 70% to 80% confluence, change to serum-free medium for culture, and collect the supernatant after 48 to 72 h;

[0031] 2) Perform differential centrifugation extraction on the supernatant to obtain the exosomes.

[0032] In the present invention, the differential centrifugation extraction method in step 2) is preferably:

[0033] ① Centrifuge at 300 to 400 × g for 15 to 20 min, and collect the supernatant;

[0034] ② Centrifuge the supernatant collected in step ① at 2100 to 2200 × g for 15 to 20 min, and collect the supernatant;

[0035] ③ Centrifuge the supernatant collected in step ② at 11000 to 12000 × g for 20 to 25 min, and collect the supernatant;

[0036] ④ Centrifuge the supernatant collected in step ③ at 11000 to 12000 × g for 50 to 60 min, and collect the precipitate;

[0037] More preferably:

[0038] ① Centrifuge at 320 - 380×g for 16 - 19 min and collect the supernatant;

[0039] ② Centrifuge the supernatant collected in step ① at 2120 - 2180×g for 16 - 19 min and collect the supernatant;

[0040] ③ Centrifuge the supernatant collected in step ② at 11200 - 11800×g for 21 - 24 min and collect the supernatant;

[0041] ④ Centrifuge the supernatant collected in step ③ at 11200 - 11800×g for 52 - 58 min and collect the precipitate.

[0042] In the present invention, the buffer is preferably PBS buffer, the concentration of the exosome suspension is preferably 50 - 80 μg / mL, more preferably 55 - 75 μg / mL; the tengmatrine is dissolved in ethanol as a solvent before mixing, and the concentration of the tengmatrine is preferably 20 - 50 μg / mL, more preferably 30 - 40 μg / mL.

[0043] In the present invention, the incubation temperature is preferably 28 - 32 °C; the incubation time is preferably 18 - 22 min; the freezing temperature is preferably -85 - -75 °C; the freezing time is preferably 3.5 - 4.5 min; the ultrafiltration centrifugation conditions are preferably 5000 - 6000 rpm, 5 - 10 min; more preferably 5200 - 5800 rpm, 6 - 9 min.

[0044] The present invention also provides the application of the described drug in the preparation of anti-tumor drugs. In the present invention, the anti-tumor drugs include anti-hepatocellular carcinoma drugs.

[0045] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0046] Example 1

[0047] A drug containing exosomes, composed of exosomes and tengmatrine; the mass ratio of the exosomes to tengmatrine is 1:5.

[0048] The preparation method of the drug is as follows:

[0049] The umbilical cord mesenchymal stem cells were cultured in low-glucose DMEM medium containing 12% fetal bovine serum by volume. When the cell confluence reached 75%, the cells were switched to serum-free medium for culture. After 60 h, the supernatant was collected; the supernatant was centrifuged at 350×g for 18 min, and the supernatant A was collected; the collected supernatant A was centrifuged at 2150×g for 18 min, and the supernatant B was collected; the collected supernatant B was centrifuged at 11500×g for 22 min, and the supernatant C was collected; the collected supernatant C was centrifuged at 11500×g for 55 min to collect the precipitate to obtain the exosomes.

[0050] 12 μg of exosomes and 200 μL of PBS solution were mixed to obtain an exosome suspension; 60 μg of tetrandrine was dissolved in 2000 μL of ethanol solvent to obtain a tetrandrine solution; the exosome suspension and the tetrandrine solution were mixed and incubated at 30 °C for 20 min, then frozen at -80 °C for 4 min and thawed at room temperature. After repeating the freeze-thaw cycle three times, ultrafiltration centrifugation (5500 rpm, 8 min) was performed, and the precipitate was collected to obtain the drug containing exosomes.

[0051] Example 2

[0052] A drug containing exosomes, which consists of exosomes and tetrandrine; the mass ratio of the exosomes to tetrandrine is 1:4.

[0053] The preparation method of the drug is as follows:

[0054] The umbilical cord mesenchymal stem cells were cultured in low-glucose DMEM medium containing 10% fetal bovine serum by volume. When the cell confluence reached 70%, the cells were switched to serum-free medium for culture. After 48 h, the supernatant was collected; the supernatant was centrifuged at 300×g for 15 min, and the supernatant A was collected; the collected supernatant A was centrifuged at 2100×g for 15 min, and the supernatant B was collected; the collected supernatant B was centrifuged at 11000×g for 20 min, and the supernatant C was collected; the collected supernatant C was centrifuged at 11000×g for 50 min to collect the precipitate to obtain the exosomes.

[0055] 12 μg of exosomes and 240 μL of PBS buffer were mixed to obtain an exosome suspension; 48 μg of tetrandrine was dissolved in 2400 μL of ethanol solvent to obtain a tetrandrine solution; the exosome suspension and the tetrandrine solution were mixed and incubated at 25 °C for 15 min, then frozen at -90 °C for 3 min and thawed at room temperature. After repeating the freeze-thaw cycle three times, ultrafiltration centrifugation (5000 rpm, 5 min) was performed, and the precipitate was collected to obtain the drug containing exosomes.

[0056] Example 3

[0057] A drug containing exosomes, which consists of exosomes and tetrandrine; the mass ratio of the exosomes to tetrandrine is 1:6.

[0058] The preparation method of the drug is as follows:

[0059] Cultivate umbilical cord mesenchymal stem cells with low-glucose DMEM medium containing 15% fetal bovine serum by volume. When the cells reach 80% confluence, change to serum-free medium for cultivation. After 72 hours, collect the supernatant; centrifuge the supernatant at 400×g for 20 minutes, and collect supernatant A; centrifuge the collected supernatant A at 2200×g for 20 minutes, and collect supernatant B; centrifuge the collected supernatant B at 12000×g for 25 minutes, and collect supernatant C; centrifuge the collected supernatant C at 12000×g for 60 minutes to collect the precipitate to obtain the exosomes.

[0060] Mix 12 μg of exosomes with 150 μL of PBS buffer to obtain an exosome suspension; dissolve 72 μg of tetrandrine in 1440 μL of ethanol solvent to obtain a tetrandrine solution; mix the exosome suspension and the tetrandrine solution, incubate at 35 °C for 25 minutes, then freeze at -70 °C for 5 minutes and thaw at room temperature. Repeat the freeze-thaw cycle three times and then perform ultrafiltration centrifugation (6000 rpm, 10 minutes) to collect the precipitate to obtain the drug containing exosomes.

[0061] Experimental Example 1

[0062] Respectively take hepatoma SMMC-7721 cells in the logarithmic growth phase (purchased from Shanghai Yansheng Industrial Co., Ltd.) and inoculate them into 96-well plates at a density of 5×10 7 cells / L / well, 100 μL per well, that is, about 5000 cells per well. Place the culture plate in an incubator at 37 °C and 5% CO 2 After 12 hours, add drugs according to the following grouping method. After adding the drug to each well, the final volume is 150 μL (make up the insufficient volume with serum-free RPMI 1640 culture medium). Set 3 replicates for each group and place them in an incubator at 37 °C and 5% CO 2 After culturing for 48 hours, add 10 μL of CCK-8 reagent to each well and continue culturing for 1 hour. Detect the absorbance value A with an enzyme-labeled instrument (measurement wavelength 450 nm, reference wavelength 655 nm), calculate the proliferation inhibition rate of each group, and repeat this experiment 3 times. Proliferation inhibition rate (%) = (1 - A experimental group / A control group) × 100%.

[0063] The grouping method is as follows, where: the pure umbilical cord mesenchymal stem cell exosome group, the tetrandrine group, and the drug containing exosomes prepared in Example 1; the control group is serum-free RPMI 1640 culture medium;

[0064] Pure umbilical cord mesenchymal stem cell exosome group: low concentration 5 μg / mL; high concentration 20 μg / mL;

[0065] Ferdinandus trichotomus group: low concentration 5 μg / mL; high concentration 20 μg / mL;

[0066] The drug containing exosomes prepared in Example 1: low concentration 5 μg / mL; high concentration 20 μg / mL.

[0067] Experimental results: As shown in Table 1.

[0068] Table 1 Proliferation inhibition rate of different treatment groups on hepatocellular carcinoma SMMC-7721 cells

[0069]

[0070] As can be seen from Table 1, the pure umbilical cord mesenchymal stem cell exosome group at low and high concentrations has a certain inhibitory effect on the proliferation of SMMC-7721 cells, but the inhibition rate is low; compared with the pure exosome group, the Ferdinandus trichotomus group at low and high concentrations is relatively higher, but both are lower than the drug containing exosomes prepared in Example 1 at low and high concentrations. The drug containing exosomes prepared in the present invention has a significant inhibitory effect on the proliferation of SMMC-7721 cells.

[0071] Experimental Example 2

[0072] BALB / c mice (purchased from Beijing Speifo Biotechnology Co., Ltd.) were acclimated for 1 week under SPF conditions in an independent ventilated cage box. After 1 week, 0.1 mL of SMMC-7721 cell suspension (containing 5×10 6 cells in the logarithmic growth phase) was subcutaneously inoculated into the axilla of BALB / c mice. When the tumor volume grew to about 100 mm 3 (about 12 days), grouping and intraperitoneal administration were started. The grouping is as follows, with 8 mice in each group, all administered by intraperitoneal injection for 14 consecutive days.

[0073] Control group: normal saline + 1% DMSO;

[0074] Drug group containing exosomes prepared in Example 1: 10 mg / (kg·d).

[0075] The nude mice were sacrificed on the second day after the end of administration, and the transplanted tumors were removed to measure the final volume and tumor inhibition rate.

[0076] Tumor inhibition rate (%) = (1 - tumor volume of the drug group containing exosomes prepared in Example 1 / tumor volume of the control group) × 100%.

[0077] Experimental results: As shown in Table 2.

[0078] Table 2 Terminal transplanted tumor volume and tumor inhibition rate of different treatment groups

[0079]

[0080] As can be seen from Table 2, compared with the control group, the tumor growth rate of the drug group containing exosomes prepared in Example 1 was significantly reduced, and the tumor volume was significantly decreased.

[0081] From the above examples and experimental examples, it can be seen that the drug containing exosomes prepared by the present invention has a significantly improved inhibitory effect on the proliferation of liver cancer cells in vivo and in vitro, excellent drug efficacy, and obvious anti-tumor effect.

[0082] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A drug containing exosomes, characterized in that: including exosomes and fusanolide; The mass ratio of the exosomes to fusinocampus solani is 1:4-6.

2. The drug according to claim 1, characterized in that The exosomes are umbilical cord mesenchymal stem cell exosomes.

3. The method for preparing the drug according to claim 1 or 2, characterized in that: The following steps are involved: The exosomes and buffer solution are mixed to obtain an exosome suspension; the exosome suspension is mixed with fusanolide, incubated at a temperature of 25 to 35°C for 15 to 25 minutes, and then frozen at -90 to -70°C for 3 to 5 minutes, and thawed at room temperature. The freeze-thaw cycle is repeated three times, and then ultrafiltration centrifugation is performed to collect the precipitate to obtain the drug containing exosomes.

4. The preparation method according to claim 3, characterized in that: The fusanolide is dissolved in ethanol as a solvent before mixing; the concentration of the fusanolide is 20-50 μg / mL.

5. The preparation method according to claim 3, characterized in that: The buffer solution is PBS buffer solution, and the concentration of the exosome suspension is 50-80 μg / mL.

6. The preparation method according to claim 3, characterized in that: The method for preparing exosomes comprises the following steps: 1) culturing the umbilical cord mesenchymal stem cells in a low-glucose DMEM medium containing 10-15% fetal bovine serum by volume, and switching to a serum-free medium when the cells are 70%-80% confluent, and collecting the supernatant after 48-72 hours; 2) The supernatant is subjected to differential centrifugation extraction to obtain the exosomes.

7. The preparation method according to claim 6, characterized in that: The method of differential centrifugation extraction in step 2) is: ① Centrifuge at 300-400×g for 15-20 min and collect the supernatant; ② Centrifuge the supernatant collected in step ① at 2100-2200×g for 15-20 min and collect the supernatant; ③ Centrifuge the supernatant collected in step ② at 11000-12000×g for 20-25 min and collect the supernatant; ④ Centrifuge the supernatant collected in step ③ at 11000-12000×g for 50-60 min and collect the precipitate.

8. Use of the drug according to claim 1 or 2 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The anti-tumor drugs include anti-liver cancer drugs.

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