A kind of atomizer containing stem cell exosomes and its preparation method and application

Abstract

The present invention proposes an atomizer containing stem cell exosomes, and a preparation method and application thereof, which belong to the field of exosome technology. Magnetic ferroferric oxide particles are prepared, the surface of which is modified with tannic acid, loaded with berberine and doxorubicin, and then mixed and incubated with exosomes secreted by adipose mesenchymal stem cells, centrifuged, added to physiological saline, and ultrasonically dispersed uniformly to obtain an atomizer containing stem cell exosomes. The atomizer containing stem cell exosomes prepared by the present invention not only has a simple preparation method and a wide source of raw materials, but the prepared atomizer is administered by spraying and directly enters the lungs. It has the unique performance of magnetic targeted drug delivery, releases drugs in a targeted and fixed-point manner, improves the efficacy of the drug, and effectively promotes tumor cell apoptosis, and plays an anti-lung cancer and anti-pneumonia role.

Description

Technical Field

[0001] The present invention relates to the technical field of exosomes, and in particular to an atomizer containing stem cell exosomes, and a preparation method and application thereof. Background Art

[0002] The lung is an important respiratory organ in the human body. Lung damage is mostly caused by factors such as lung trauma and infection, which not only leads to a decline in people's quality of life, but also seriously threatens human health and life span. Stem cells can promote the repair of damaged lung tissue, reduce proinflammatory cytokines, and reduce excessive inflammatory responses. They have a good preventive and therapeutic effect on anti-pulmonary fibrosis and lung inflammation. However, the effect of directly injecting stem cells into the body is limited.

[0003] The mesenchymal stem cell culture system contains a large number of active ingredients, such as stem cell growth factor (SCF), fibroblast growth factor (FGF), endothelial growth factor (VEGF), epidermal growth factor (EGF), colony stimulating factor, interleukin (IL), collagen, hyaluronic acid and more than 80 other active substances. Studies have shown that most of the active substances secreted by stem cells exist in the form of exosomes, which can fuse well with the cell membrane, thereby transporting the internal active substances to the inside of the targeted cells to exert their effects. Stem cell exosomes are tiny vesicles secreted by stem cells. The main active ingredients include mRNA, microRNA and protein. Many studies have shown that stem cell exosomes can be inhaled in the form of atomization, which can play a targeted role in repairing lung damage. Its intervention effect on lung diseases is better than directly using stem cells. Summary of the invention

[0004] The purpose of the present invention is to propose an aerosol containing stem cell exosomes and its preparation method and application. Not only is the preparation method simple and the raw material source is wide, the prepared aerosol is administered by spraying and directly enters the lungs. It has the unique performance of magnetic targeted drug delivery, releases drugs in a targeted and fixed point, improves the efficacy, and effectively promotes tumor cell apoptosis, playing an anti-lung cancer and anti-pneumonia role.

[0005] The technical solution of the present invention is achieved in this way:

[0006] The present invention provides a method for preparing an atomizer containing stem cell exosomes. The method comprises preparing ferroferric oxide magnetic particles, modifying the surface of the particles with tannic acid, loading berberine and doxorubicin, and then mixing and incubating the particles with exosomes secreted by adipose mesenchymal stem cells, centrifuging the particles, adding the particles to physiological saline, and uniformly dispersing the particles by ultrasonication to obtain an atomizer containing stem cell exosomes.

[0007] As a further improvement of the present invention, the following steps are included:

[0008] S1. Preparation of magnetic particles: dissolving ferric chloride and ferrous chloride in water, adding ammonia water dropwise under inert gas protection, heating and stirring to react, filtering, ball milling, washing, and drying to obtain magnetic particles;

[0009] S2. Surface modification: adding magnetic particles to water, adding tannic acid and a catalyst, heating and stirring to react, separating with a magnet, washing, and drying to obtain modified magnetic particles;

[0010] S3. Drug loading: dissolving berberine and doxorubicin in water, adding modified magnetic particles, heating and stirring to react, evaporating the solvent, washing, and drying to obtain drug-loaded magnetic particles;

[0011] S4. Extraction of exosomes: The collected abdominal subcutaneous fat tissue was rinsed with sterile PBS solution, cut into pieces and soaked in PBS solution containing double antibodies, washed and digested with type I collagenase, and DMEM medium containing 10-12wt% fetal bovine serum was added to terminate the digestion, centrifuged and resuspended, cultured, and subcultured when the cell confluence reached 80-90%, and the 3rd to 6th generation cells in the logarithmic growth phase were taken and the culture medium was replaced when the confluence reached 80-90%, and the culture supernatant was collected, and the exosomes were extracted by ultra-high-speed centrifugation combined with ultrafiltration tube method;

[0012] S5. Preparation of a nebulizer containing stem cell exosomes: Mix the drug-loaded magnetic particles and the exosome solution and add them to a DMEM culture medium containing 10-12wt% fetal bovine serum, incubate, centrifuge, collect the precipitate, add it to physiological saline, and disperse it evenly by ultrasonication to obtain a nebulizer containing stem cell exosomes.

[0013] As a further improvement of the present invention, the mass ratio of ferric chloride to ferrous chloride in step S1 is 3.24:1.26, the temperature of the heating and stirring reaction is 70-80°C, and the time is 3-5h.

[0014] As a further improvement of the present invention, the mass ratio of the magnetic particles, tannic acid and catalyst in step S2 is 10:3-5:0.5-1, the catalyst has a pH of 8.5-9.5 Tris-HCl solution, the temperature of the heating and stirring reaction is 35-45°C, and the time is 2-3h.

[0015] As a further improvement of the present invention, in step S3, the mass ratio of berberine, doxorubicin and modified magnetic particles is 1-2:0.8-1.2:10, and the temperature of the heating and stirring reaction is 30-40° C. and the time is 1-2 h.

[0016] As a further improvement of the present invention, the PBS solution containing dual antibodies in step S4 is a PBS solution with a pH of 7.2-7.4 containing 90-110 U / mL of penicillin and 100-120 μg / mL of streptomycin, and the culture conditions are 36-38°C, 4-6 v / v% CO2, and the culture is carried out for 2-3 weeks.

[0017] As a further improvement of the present invention, the mass volume ratio of the drug-loaded magnetic particles and the exosome solution in step S5 is 3-5 g:1-2 mL, the incubation conditions are 36-38° C., the time is 30-50 min, and the solid-liquid ratio of the precipitate and physiological saline is 1:5-7 g / mL.

[0018] The present invention further protects an aerosol containing stem cell exosomes obtained by the above-mentioned preparation method.

[0019] The present invention further protects the use of the above-mentioned atomizer containing stem cell exosomes in the preparation of anti-lung cancer drugs.

[0020] The present invention has the following beneficial effects:

[0021] The present invention prepares a magnetic particle with the unique performance of magnetic targeted drug delivery. At the same time, the loaded drugs berberine and doxorubicin are loaded with exosomes after incubation with exosomes. Under the action of the magnetic field, the composite magnetic particles generate heat energy and the magnetic drug-loaded particles are enriched in the patient's lesion site in large quantities and released in a controlled manner, thereby performing local heat therapy and targeted drug treatment on the lesion site.

[0022] Surface modification with tannic acid not only greatly improves the loading capacity of drugs such as berberine and doxorubicin, as well as the loading capacity for exosomes, but also the magnetic particles modified with tannic acid can find the target more quickly, thereby playing a role in targeted drug delivery and efficient release.

[0023] Adipose-derived mesenchymal stem cells have the characteristics of wide sources and simple preparation methods. The exosomes isolated and extracted can not only promote inflammatory response by inducing inflammatory cells, but also participate in inducing bronchial epithelial cells to differentiate into fibroblasts, and involve abnormal communication between bronchial epithelial cells and fibroblasts, and can participate in a variety of lung cancer pathological processes, including lung cancer cell proliferation and migration, angiogenesis, epithelial-mesenchymal transition, etc., thereby effectively promoting tumor cell apoptosis and playing an anti-lung cancer and anti-pneumonia role. In the present invention, there is also a synergistic effect between exosomes and the drugs berberine and doxorubicin. Exosomes regulate lung cancer cell proliferation and migration, angiogenesis, and epithelial-mesenchymal transition, and drugs inhibit tumor cell growth. Under the synergistic effect of the two, they can significantly play an anti-tumor role.

[0024] The aerosol containing stem cell exosomes prepared by the present invention has not only a simple preparation method and a wide source of raw materials, but also the prepared aerosol is administered by spraying and directly enters the lungs, has the unique performance of magnetic targeted drug delivery, releases drugs in a targeted and fixed point, improves the efficacy, and effectively promotes tumor cell apoptosis, playing an anti-lung cancer and anti-pneumonia role. DETAILED DESCRIPTION

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] This embodiment provides a method for preparing an aerosol containing stem cell exosomes, comprising the following steps:

[0028] S1. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 500 mL of water, 30 mL of ammonia water was added dropwise under nitrogen protection, heated to 70 ° C, stirred for 3 h, filtered, ball milled for 1 h, washed, and dried to obtain magnetic particles;

[0029] S2. Surface modification: 10 g of magnetic particles were added to 500 mL of water, 3 g of tannic acid and 0.5 g of catalyst were added, heated to 35 ° C, stirred for 2 h, separated by magnet, washed, and dried to obtain modified magnetic particles;

[0030] Tris-HCl solution of the catalyst with a pH of 8.5;

[0031] S3. Drug loading: 1 g of berberine and 0.8 g of doxorubicin were dissolved in 200 mL of water, 10 g of modified magnetic particles were added, heated to 30 ° C, stirred for 1 h, the solvent was evaporated, washed, and dried to obtain drug-loaded magnetic particles;

[0032] S4. Extraction of exosomes: The collected abdominal subcutaneous fat tissue was rinsed with sterile PBS solution, cut into pieces and soaked in PBS solution containing double antibodies, then digested with an equal volume of 0.25wt% type I collagenase, and 5 times the volume of DMEM medium containing 10wt% fetal bovine serum was added to terminate the digestion, and the cells were resuspended after centrifugation and cultured at 36°C, 4v / v% CO2 for 2 weeks. When the cell confluence reached 80-90%, the cells of the 3rd to 6th generation in the logarithmic growth phase were taken and the culture medium was replaced when the confluence reached 80-90%, and the culture supernatant was collected, and the exosomes were extracted by ultra-high-speed centrifugation combined with ultrafiltration tube method;

[0033] The PBS solution containing dual antibodies is a PBS solution with a pH of 7.2 and containing 90 U / mL of penicillin and 100 μg / mL of streptomycin;

[0034] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 30 min to remove large vesicles and proteins, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.9, and centrifuged again at 4°C and 32000 r / min for 60 min. The precipitate obtained is exosomes, which are resuspended in PBS with pH=6.9, and protein quantification is performed according to the steps of the BCA kit. By calculation, an exosome solution with a concentration of 80 μg / mL is finally obtained, which is frozen in a -80°C refrigerator;

[0035] S5. Preparation of a nebulizer containing stem cell exosomes: 3 g of drug-loaded magnetic particles and 1 mL of exosome solution were mixed and added to a DMEM culture medium containing 10 wt% fetal bovine serum, incubated at 36°C for 30 min, centrifuged, and the precipitate was collected. The precipitate was added to physiological saline with a solid-liquid ratio of 1:5 g / mL. Ultrasonic dispersion was performed at 1000 W for 20 min to obtain a nebulizer containing stem cell exosomes.

[0036] Example 2

[0037] This embodiment provides a method for preparing an aerosol containing stem cell exosomes, comprising the following steps:

[0038] S1. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 500 mL of water, 30 mL of ammonia water was added dropwise under nitrogen protection, heated to 80 ° C, stirred for 5 h, filtered, ball milled for 1 h, washed, and dried to obtain magnetic particles;

[0039] S2. Surface modification: 10 g of magnetic particles were added to 500 mL of water, 5 g of tannic acid and 1 g of catalyst were added, heated to 45 ° C, stirred for 3 h, separated by magnet, washed, and dried to obtain modified magnetic particles;

[0040] The catalyst has a pH of 9.5 in a Tris-HCl solution;

[0041] S3. Drug loading: 2 g of berberine and 1.2 g of doxorubicin were dissolved in 200 mL of water, 10 g of modified magnetic particles were added, heated to 40 ° C, stirred for 2 h, the solvent was evaporated, washed, and dried to obtain drug-loaded magnetic particles;

[0042] S4. Extraction of exosomes: The collected abdominal subcutaneous fat tissue was rinsed with sterile PBS solution, cut into pieces and soaked in PBS solution containing double antibodies, then digested with an equal volume of 0.25wt% type I collagenase, and 5 times the volume of DMEM medium containing 12wt% fetal bovine serum was added to terminate the digestion, and the cells were resuspended after centrifugation and cultured at 38°C, 6v / v% CO2 for 3 weeks. When the cell confluence reached 80-90%, the cells of the 3rd to 6th generations in the logarithmic growth phase were taken and the culture medium was replaced when the confluence reached 80-90%, and the culture supernatant was collected, and the exosomes were extracted by ultra-high-speed centrifugation combined with ultrafiltration tube method;

[0043] The PBS solution containing dual antibodies is a PBS solution with a pH of 7.4 and containing 110 U / mL of penicillin and 120 μg / mL of streptomycin;

[0044] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 30 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.9, and centrifuged again at 4°C and 32000 r / min for 60 min. The precipitate obtained is exosomes, which are resuspended in PBS with pH=6.9, and protein quantification is performed according to the steps of the BCA kit. By calculation, an exosome solution with a concentration of 120 μg / mL is finally obtained, which is frozen in a -80°C refrigerator;

[0045] S5. Preparation of a nebulizer containing stem cell exosomes: 5 g of drug-loaded magnetic particles and 2 mL of exosome solution were mixed and added to a DMEM culture medium containing 12 wt% fetal bovine serum, incubated at 38°C for 50 min, centrifuged, and the precipitate was collected. The precipitate was added to physiological saline, the solid-liquid ratio of the precipitate to the physiological saline was 1:7 g / mL, and ultrasonic dispersion was performed at 1000 W for 20 min to obtain a nebulizer containing stem cell exosomes.

[0046] Example 3

[0047] This embodiment provides a method for preparing an aerosol containing stem cell exosomes, comprising the following steps:

[0048] S1. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 500 mL of water, 30 mL of ammonia water was added dropwise under nitrogen protection, heated to 75 ° C, stirred for 4 h, filtered, ball milled for 1 h, washed, and dried to obtain magnetic particles;

[0049] S2. Surface modification: 10 g of magnetic particles were added to 500 mL of water, 4 g of tannic acid and 0.7 g of catalyst were added, heated to 40 ° C, stirred for 2.5 h, separated by magnet, washed, and dried to obtain modified magnetic particles;

[0050] A Tris-HCl solution of the catalyst with a pH of 9;

[0051] S3. Drug loading: 1.5 g of berberine and 1 g of doxorubicin were dissolved in 200 mL of water, 10 g of modified magnetic particles were added, heated to 35 ° C, stirred for 1.5 h, the solvent was evaporated, washed, and dried to obtain drug-loaded magnetic particles;

[0052] S4. Extraction of exosomes: The collected abdominal subcutaneous fat tissue was rinsed with sterile PBS solution, cut into pieces and soaked in PBS solution containing double antibodies, and then digested with an equal volume of 0.25wt% type I collagenase, and 5 volumes of DMEM medium containing 10wt% fetal bovine serum were added to terminate the digestion, and the cells were resuspended after centrifugation and cultured at 37°C, 5v / v% CO2 for 3 weeks. When the cell confluence reached 80-90%, the cells of the 3rd to 6th generations in the logarithmic growth phase were taken and the culture medium was replaced when the confluence reached 80-90%, and the culture supernatant was collected, and the exosomes were extracted by ultra-high-speed centrifugation combined with ultrafiltration tube method;

[0053] The PBS solution containing dual antibodies is a PBS solution with a pH of 7.3 and containing 100 U / mL of penicillin and 110 μg / mL of streptomycin;

[0054] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 30 min to remove large vesicles and proteins, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.9, and centrifuged again at 4°C and 32000 r / min for 60 min. The obtained precipitate is exosomes, which are resuspended in PBS with pH=6.9, and protein quantification is performed according to the steps of the BCA kit. By calculation, an exosome solution with a concentration of 100 μg / mL is finally obtained, which is frozen in a -80°C refrigerator;

[0055] S5. Preparation of a nebulizer containing stem cell exosomes: 4 g of drug-loaded magnetic particles and 1.5 mL of exosome solution were mixed and added to a DMEM culture medium containing 10 wt% fetal bovine serum, incubated at 37°C for 40 min, centrifuged, and the precipitate was collected. The precipitate was added to physiological saline, the solid-liquid ratio of the precipitate to the physiological saline was 1:6 g / mL, and ultrasonic dispersion was performed at 1000 W for 20 min to obtain a nebulizer containing stem cell exosomes.

[0056] Comparative Example 1

[0057] Compared with Example 3, the difference is that berberine is not added in step S3.

[0058] The details are as follows:

[0059] S3. Drug loading: Dissolve 2.5 g doxorubicin in 200 mL water, add 10 g modified magnetic particles, heat to 35°C, stir and react for 1.5 h, evaporate the solvent, wash, and dry to obtain drug-loaded magnetic particles.

[0060] Comparative Example 2

[0061] Compared with Example 3, the difference is that doxorubicin is not added in step S3.

[0062] The details are as follows:

[0063] S3. Drug loading: Dissolve 2.5 g of berberine in 200 mL of water, add 10 g of modified magnetic particles, heat to 35°C, stir and react for 1.5 h, evaporate the solvent, wash, and dry to obtain drug-loaded magnetic particles.

[0064] Comparative Example 3

[0065] Compared with Example 3, the difference is that step S3 is not performed.

[0066] The details are as follows:

[0067] S1. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 500 mL of water, 30 mL of ammonia water was added dropwise under nitrogen protection, heated to 75 ° C, stirred for 4 h, filtered, ball milled for 1 h, washed, and dried to obtain magnetic particles;

[0068] S2. Surface modification: 10 g of magnetic particles were added to 500 mL of water, 4 g of tannic acid and 0.7 g of catalyst were added, heated to 40 ° C, stirred for 2.5 h, separated by magnet, washed, and dried to obtain modified magnetic particles;

[0069] A Tris-HCl solution of the catalyst with a pH of 9;

[0070] S3. Extraction of exosomes: The collected abdominal subcutaneous fat tissue was rinsed with sterile PBS solution, cut into pieces and soaked in PBS solution containing double antibodies, and then digested with an equal volume of 0.25wt% type I collagenase, and 5 times the volume of DMEM medium containing 10wt% fetal bovine serum was added to terminate the digestion, and the cells were resuspended after centrifugation and cultured at 37°C, 5v / v% CO2 for 3 weeks. When the cell confluence reached 80-90%, the cells of the 3rd to 6th generations in the logarithmic growth phase were taken and the culture medium was replaced when the confluence reached 80-90%, and the culture supernatant was collected, and the exosomes were extracted by ultra-high-speed centrifugation combined with ultrafiltration tube method;

[0071] The PBS solution containing dual antibodies is a PBS solution with a pH of 7.3 and containing 100 U / mL of penicillin and 110 μg / mL of streptomycin;

[0072] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 700 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13000 r / min for 30 min to remove large vesicles and proteins, and the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, and the precipitate is resuspended in PBS with pH=6.9, and centrifuged again at 4°C and 32000 r / min for 60 min. The obtained precipitate is exosomes, which are resuspended in PBS with pH=6.9, and protein quantification is performed according to the steps of the BCA kit. By calculation, an exosome solution with a concentration of 100 μg / mL is finally obtained, which is frozen in a -80°C refrigerator;

[0073] S4. Preparation of a nebulizer containing stem cell exosomes: 4 g of modified magnetic particles and 1.5 mL of exosome solution were mixed and added to DMEM culture medium containing 10 wt% fetal bovine serum, incubated at 37°C for 40 min, centrifuged, and the precipitate was collected. The precipitate was added to physiological saline, the solid-liquid ratio of the precipitate to the physiological saline was 1:6 g / mL, and ultrasonic dispersion was performed at 1000 W for 20 min to obtain a nebulizer containing stem cell exosomes.

[0074] Comparative Example 4

[0075] Compared with Example 3, the difference is that steps S4 and S5 are not performed.

[0076] The details are as follows:

[0077] S1. Preparation of magnetic particles: 3.24 g of ferric chloride and 1.26 g of ferrous chloride were dissolved in 500 mL of water, 30 mL of ammonia water was added dropwise under nitrogen protection, heated to 75 ° C, stirred for 4 h, filtered, ball milled for 1 h, washed, and dried to obtain magnetic particles;

[0078] S2. Surface modification: 10 g of magnetic particles were added to 500 mL of water, 4 g of tannic acid and 0.7 g of catalyst were added, heated to 40 ° C, stirred for 2.5 h, separated by magnet, washed, and dried to obtain modified magnetic particles;

[0079] A Tris-HCl solution of the catalyst with a pH of 9;

[0080] S3. Drug loading: Dissolve 1.5g of berberine and 1g of doxorubicin in 200mL of water, add 10g of modified magnetic particles, heat to 35°C, stir and react for 1.5h, evaporate the solvent, wash, and dry to obtain drug-loaded magnetic particles, add to physiological saline, the solid-liquid ratio of the drug-loaded magnetic particles and physiological saline is 1:6g / mL, and ultrasonically disperse at 1000W for 20min to obtain an atomizer containing stem cell exosomes.

[0081] Test Example 1 MTT assay to detect cell growth inhibition

[0082] Human lung cancer A549 cells in the logarithmic growth phase were digested with 0.25% trypsin and resuspended in fresh growth medium. The cells were inoculated into 96-well culture plates and cultured overnight in a 37°C, 5% CO2 incubator. The cell density per well was adjusted to 4×10 4 / mL, add the atomizer solution containing stem cell exosomes prepared in Example 1-3 or Comparative Example 1-4 (diluted 100 times) to each well, the negative control group is normal saline, and the blank group is not added with drugs. Treat the cells in the 96-well plate, set up 6 duplicate wells in each group, and continue to culture in a 37°C, 5% CO2 incubator. After drug treatment for 24 hours, add (50 mg / L) MTT solution directly to each well, incubate at 37°C for 3 hours, stand at room temperature until the precipitate is completely dissolved (10 minutes), and use an enzyme marker (detection wavelength 570nm) to detect the absorbance (A) value of each well. Take the average value of the duplicate wells to calculate the proliferation inhibition rate.

[0083] Proliferation inhibition rate (%) = [(A value of negative control group - A value of blank group) - (A value of experimental group - A value of blank group)] / (A value of negative control group - A value of blank group) × 100%

[0084] The results are shown in Table 1.

[0085] Table 1

[0086] Group Proliferation inhibition rate (%) Example 1 43.3 Example 2 42.1 Example 3 44.0 Comparative Example 1 32.1 Comparative Example 2 29.6 Comparative Example 3 21.5 Comparative Example 4 17.4

[0087] It can be seen from the above table that the atomizer solution containing stem cell exosomes prepared in Examples 1-3 of the present invention has a good in vitro inhibitory effect on lung cancer cells.

[0088] Test Example 2

[0089] LLC cells (nude mouse lung cancer cells) in the logarithmic growth phase were discarded from the culture medium, gently rinsed with PBS buffer, digested with trypsin, slowly blown off with PBS buffer, collected in a centrifuge tube, centrifuged and resuspended, and stained with a cell counter to detect the cell number and survival rate (the survival rate must be greater than 90%). The resulting cells were then prepared into a concentration of 2×10 7 / mL of cell suspension. After preparing the skin of the right armpit of nude mice, 0.1mL / mouse was injected subcutaneously. After the injection, the right armpit was observed daily for tumor formation. Ten days after the injection, a soybean-sized mass visible or palpable to the naked eye formed in the right armpit of the nude mouse, indicating a successful modeling. The successfully modeled nude mice were randomly divided into a model group, Example 1-3 and Comparative Example 1-4 groups, with 10 mice in each group. The prepared atomizer solution containing stem cell exosomes was diluted 10 times and given 2mL / mouse, twice a week. The model group was given an equal amount of normal saline, both administered through the tail vein. The cisplatin group (2 mg / kg), twice a week, was used as the positive drug group. The experimental period was 2 weeks. After the experiment, the tumor was isolated and the tumor weight inhibition rate (%) was calculated.

[0090] Tumor weight inhibition rate (%) = (tumor weight of model group - tumor weight of test group) / tumor weight of model group × 100%

[0091] The results are shown in Table 2.

[0092] Table 2

[0093] Group Tumor weight inhibition rate (%) Model Group / Positive drug group 44.1 Example 1 65.3 Example 2 64.8 Example 3 65.9 Comparative Example 1 56.2 Comparative Example 2 53.4 Comparative Example 3 42.7 Comparative Example 4 39.8

[0094] It can be seen from the above table that the atomizer solution containing stem cell exosomes prepared in Examples 1-3 of the present invention has a good effect of inhibiting lung cancer in vivo.

[0095] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing an aerosol containing stem cell exosomes, characterized in that: The method comprises preparing ferroferric oxide magnetic particles, modifying the surface with tannic acid, loading berberine and doxorubicin, and then mixing and incubating with exosomes secreted by adipose mesenchymal stem cells, centrifuging, adding to physiological saline, and ultrasonically dispersing the particles uniformly to obtain an atomizer containing stem cell exosomes; the method comprises the following steps: S1. Preparation of magnetic particles: dissolving ferric chloride and ferrous chloride in water, adding ammonia water dropwise under inert gas protection, heating and stirring to react, filtering, ball milling, washing, and drying to obtain magnetic particles; S2. Surface modification: adding magnetic particles to water, adding tannic acid and a catalyst, heating and stirring to react, separating with a magnet, washing, and drying to obtain modified magnetic particles; S3. Drug loading: dissolving berberine and doxorubicin in water, adding modified magnetic particles, heating and stirring to react, evaporating the solvent, washing, and drying to obtain drug-loaded magnetic particles; S4. Extraction of exosomes: The collected abdominal subcutaneous fat tissue was rinsed with sterile PBS solution, cut into pieces and soaked in a PBS solution containing double antibodies, washed and digested with type I collagenase, and DMEM medium containing 10-12wt% fetal bovine serum was added to terminate the digestion, centrifuged and resuspended, cultured, and subcultured when the cell confluence was 80-90%, and the 3rd to 6th generation cells in the logarithmic growth phase were taken and the culture medium was replaced when the confluence was 80-90%, and the culture supernatant was collected, and the exosomes were extracted by ultra-high speed centrifugation combined with ultrafiltration tube method; the PBS solution containing double antibodies is a PBS solution with a pH of 7.2-7.4 containing penicillin 90-110U / mL and streptomycin 100-120μg / mL; S5. Preparation of a nebulizer containing stem cell exosomes: Mix the drug-loaded magnetic particles and the exosome solution and add them to a DMEM culture medium containing 10-12wt% fetal bovine serum, incubate, centrifuge, collect the precipitate, add it to physiological saline, and disperse it evenly by ultrasonication to obtain a nebulizer containing stem cell exosomes.

2. The preparation method according to claim 1, characterized in that: The mass ratio of ferric chloride to ferrous chloride in step S1 is 3.24:1.26, the temperature of the heating and stirring reaction is 70-80° C., and the time is 3-5 hours.

3. The preparation method according to claim 1, characterized in that: In step S2, the mass ratio of the magnetic particles, tannic acid and catalyst is 10:3-5:0.5-1, the catalyst has a pH of 8.5-9.5 in Tris-HCl solution, the temperature of the heating and stirring reaction is 35-45° C., and the time is 2-3 hours.

4. The preparation method according to claim 1, characterized in that: In step S3, the mass ratio of berberine, doxorubicin and modified magnetic particles is 1-2:0.8-1.2:10, and the temperature of the heating and stirring reaction is 30-40° C. and the time is 1-2 h.

5. The preparation method according to claim 1, characterized in that: The culture conditions in step S4 are 36-38° C., 4-6 v / v % CO 2 , and the culture period is 2-3 weeks.

6. The preparation method according to claim 1, characterized in that: In step S5, the mass volume ratio of the drug-loaded magnetic particles and the exosome solution is 3-5 g:1-2 mL, the incubation condition is 36-38° C., the time is 30-50 min, and the solid-liquid ratio of the precipitate and the physiological saline is 1:5-7 g / mL.

7. An aerosol containing stem cell exosomes obtained by the preparation method according to any one of claims 1 to 6.

8. Use of the aerosol containing stem cell exosomes as claimed in claim 7 in the preparation of anti-lung cancer drugs.