Extracellular vesicles derived from stephania epigaea and preparation method and application of extracellular vesicles

Abstract

The invention relates to a stephania epigaea-derived extracellular vesicle as well as a preparation method and application thereof. The extracellular vesicles are obtained by extracting and purifying radix stephaniae tetrandrae, the hydrodynamic particle size is 161.8 + / -2.68 nm, the average particle size is 124.9 + / -2 nm, the extracellular vesicles have negative surface charges of-33.82 + / -1.675 mV, and the extracellular vesicles contain lipids, proteins and active small molecule compounds. The preparation method comprises the following steps: pretreatment and extraction of radix stephaniae tetrandrae, gradient centrifugation, ultracentrifugation, filter membrane filtration and the like. The extracellular vesicles can be used for preparing anti-breast cancer drugs, have good biocompatibility, can inhibit growth and metastasis of breast cancer and promote apoptosis of breast cancer cells, can target tumor sites and stay for a long time, and can also be used as drug carriers for breast cancer targeted drug delivery systems.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of extracellular vesicles, in particular to an extracellular vesicle from Stephania delavayi and a preparation method and application thereof. BACKGROUND

[0002] Extracellular vesicles (EVs) are nanostructures (100-1000nm) produced by direct budding of the plasma membrane, a membrane structure group wrapped by a lipid bilayer, containing micro-particles secreted by cells, and widely exist in animals, plants, fungi and other organisms. The components contained in EVs are very diverse, the specific types depend on the plants or animals they come from, but most of them are composed of proteins, lipids and nucleic acids, and play a role in physiological and pathological processes. In recent years, studies have shown that plant-derived extracellular vesicles have good biocompatibility, low immunogenicity and natural targeting characteristics, and show great application potential in drug delivery systems.

[0003] Breast cancer is a highly lethal disease, which has become one of the most destructive cancers in the past few years, and the occurrence of breast cancer involves genetics and environment. Millions of new cases occur every year, posing a serious threat to women's health, and breast cancer is a type of cancer with high malignancy and temporary lack of targeted therapy, with a high metastasis rate. Although traditional chemical drug therapy can to some extent avoid the surgical risk accompanied by mainstream surgical resection and reduce premature death of patients, the chemotherapy drug treatment is often accompanied by serious side effects and drug resistance. Therefore, finding new anti-breast cancer drugs has become one of the important directions of current breast cancer research.

[0004] Stephania delavayi Diels Stephania epigaea H. S. Lo, SE Stephania delavayi Diels belongs to the genus of Stephania in Menispermaceae, mainly distributed in the southwest and southeast of Yunnan, and its main components are alkaloids such as sinomontanine and fibraureline, which have the effects of anti-inflammatory, antibacterial, immune regulation and the like. In recent years, with the in-depth study of SE, it is found that many active substances in SE have anti-tumor, antipyretic analgesic, antibacterial anti-inflammatory and anti-arrhythmic effects, but no one has extracted and separated the extracellular vesicles from Stephania delavayi for biological and pharmacological research in the current search. SUMMARY

[0005] In view of the problems of the prior art, the technical purpose of the present application is to provide an extracellular vesicle from Stephania delavayi and a preparation method and application thereof.

[0006] The present application is realized by the following technical scheme: Firstly, the present application provides an extracellular vesicle (SE-EVs) from Radix Aconiti, which is purified from Radix Aconiti, has a hydrodynamic particle size of 161.8±2.68 nm, an average particle size of 124.9±2 nm, a negative surface charge of-33.82±1.675 mV, and contains lipids, proteins and active small molecule compounds.

[0007] Preferably, the lipids include phosphatidyl ethanol, diacylglycerol, phosphatidyl choline and phosphatidyl serine, monogalactosyldiacylglycerol and digalactosyldiacylglycerol, which constitute the basic membrane structure of the vesicles, enabling the vesicles to maintain a stable form and carry various bioactive substances; the proteins include ribosome-related cell translation pathway proteins, autophagy-related cell transport and catabolism pathway proteins, signal transduction-related MAPK pathway proteins, and amino acid synthesis-related pathway proteins, which enable the SE-EVs to have functions related to cell processes such as translation, autophagy and apoptosis; and the active small molecule compounds include sinactine, sinomine, feruloyl tyramine, 5-hydroxyferulic acid and doxorubicin alcohol. These active small molecule compounds endow the extracellular vesicles with various biological activity functions.

[0008] Further, the present application also provides a preparation method of the above-mentioned extracellular vesicles, comprising the following steps: (1) raw material treatment: after washing and peeling the tuberous roots of Radix Aconiti, immerse them in pre-cooled phosphate buffer at 0-4℃, mechanically crush for 8-12 min, and filter to obtain juice; (2) gradient centrifugation purification: centrifuge the juice at 3000-4000×g for 20-40 min to remove plant debris; centrifuge the supernatant at 30000-50000×g for 35-45 min to remove macromolecular impurities; collect the supernatant and ultracentrifuge at 100000-150000×g for 100-140 min to obtain crude extracellular vesicle precipitate; (3) refining: resuspend the precipitate with PBS, centrifuge at 100000-150000×g for 50-70 min, and filter the obtained precipitate through a filter membrane to obtain high-purity extracellular vesicles (SE-EVs).

[0009] Preferably, in step (1), the mass-volume ratio of the tuberous roots of Radix Aconiti to the phosphate buffer is 1:1-2, and the pH value is 7.2-7.4.

[0010] Preferably, in step (3), the filter membrane has a pore size of 0.22μm.

[0011] Secondly, the present application also provides the use of the extracellular vesicles prepared by the preparation method in the preparation of an anti-breast cancer drug. Preferably, the drug inhibits the growth and metastasis of breast cancer. Preferably, the drug promotes apoptosis of breast cancer cells. Preferably, the drug can be targeted to the tumor site and retained for a long time.

[0012] Preferably, the drug further comprises a pharmaceutically acceptable excipient, which comprises any one of a diluent, a flavoring agent, a binder or a filler, or a combination of at least two of them, such as a combination of a binder and a diluent, a combination of a binder and a flavoring agent, a combination of a binder and a filler, and the like, and any other combination can be selected, which will not be described here one by one.

[0013] Preferably, the drug dosage form is any one of a tablet, a granule, a capsule, an oral liquid preparation, an injection, a microcapsule, a suppository, an ointment or a film.

[0014] Finally, the application provides the use of the above-mentioned extracellular vesicles in a breast cancer targeted drug delivery system.

[0015] Preferably, the above-mentioned extracellular vesicles act as drug carriers to deliver anti-breast cancer drugs to the target site.

[0016] Advantages of the present application: 1. The present application first extracts and purifies extracellular vesicles from the roots of Radix Araliae Contortae with high purity and high activity. Compared with traditional extraction methods, the method provided by the present application can more effectively maintain the biological activity and structural integrity of extracellular vesicles.

[0017] 2. The extracellular vesicles extracted from Radix Araliae Contortae have the effects of immune regulation, tumor self-targeting and anti-tumor, and have good inhibitory effect on the growth and metastasis of breast tumors. Experiments have shown that the IC 50 value of SE-EVs for 4T1 breast cancer cells is 3.125ug / ml, which is nearly 100 times lower than the IC 50 value of 304.5ug / ml for L02 normal liver cells, showing good selective anti-tumor activity. This high selectivity provides a new way for breast cancer treatment.

[0018] 3. Experimental results show that SE-EVs can effectively inhibit the migration ability of 4T1 cells and effectively penetrate 3D tumor spheres, which is of great significance for inhibiting tumor metastasis. In vivo experiments further confirmed that SE-EVs can spontaneously target tumor sites and be retained for a long time, significantly inhibit the growth of breast tumors in mice, and have no significant effect on the body weight of mice, showing good biocompatibility.

[0019] 4. The extracellular vesicles from the source of S. moellinum can not only be used as anti-breast cancer drugs, but also can be used as drug delivery systems to deliver other anti-tumor drugs to target sites, improve the targeting and therapeutic effect of drugs, and reduce the toxic side effects on normal tissues. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure is the characterization diagram of extracellular vesicles from S. moellinum, A is the transmission electron microscope diagram, B is the dynamic light scattering diagram, C is the Zeta potential diagram, and D is the average particle size diagram; Figure 2 Figure is the lipid composition diagram of extracellular vesicles from S. moellinum; Figure 3 Figure is the Masson's trichrome staining result (A) and protein subcellular localization analysis (CC) diagram (B) of the protein component of extracellular vesicles from S. moellinum; Figure 4 Figure is the biological function analysis diagram of the protein component of extracellular vesicles from S. moellinum; Figure 5 Figure is the biological pathway mainly participated by the protein in the extracellular vesicles from S. moellinum; Figure 6 Figure is the small molecule compound analysis diagram of extracellular vesicles from S. moellinum; Figure 7 Figure is the cell uptake experiment result of extracellular vesicles from S. moellinum; Figure 8 Figure is the activity diagram of extracellular vesicles from S. moellinum on 4T1 cells (A) and L02 cells (B) at different concentrations; Figure 9 Figure is the anti-4T1 cell migration ability experiment result of extracellular vesicles from S. moellinum, L, M, and H in the figure respectively represent low, medium, and high concentrations, and A is the wound healing rate, and B is the healing width after 24h; Figure 10 Figure is the Western blot analysis result, L, M, and H in the figure respectively represent low, medium, and high concentrations, and A is the protein quantification result, and B is the protein electrophoresis diagram; Figure 11 Figure is the in vitro 3D tumor sphere uptake experiment result of extracellular vesicles from S. moellinum; Figure 12 Figure is the in vivo biodistribution experiment result of extracellular vesicles from S. moellinum, A is the distribution of SE-EVs in mice, and B is the accumulation of SE-EVs in tumor tissues of mice with time; Figure 13 Figure is the in vivo anti-tumor effect experiment result of extracellular vesicles from S. moellinum, A is the change of tumor volume of mice, B is the tumor photo of mice, C is the change of tumor weight of mice, and D is the change of body weight of mice; Figure 14 H&E and TUNEL staining results of tumor tissues of mice after treatment with extracellular vesicles from Radix Stephaniae Tetrandrae. DETAILED DESCRIPTION

[0021] The application will be further described in conjunction with the accompanying drawings and examples, but in no way limit the application, any transformation or improvement based on the teaching of the application, falls within the protection scope of the application.

[0022] The process, conditions, reagents, experimental methods, etc. for implementing the application, except for the following specifically mentioned contents, are the general knowledge and common sense in the art, and the application has no special limitations. The experimental methods not specified in the specific conditions in each example are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer.

[0023] Radix Stephaniae Tetrandrae was collected from Kaiyuan, Honghe, Yunnan, and identified by Mr. Li Guodong of Yunnan University of Chinese Medicine as Radix Stephaniae Tetrandrae Stephania epigaea H. S. Lo, SE ).

[0024] Example 1 Isolation and characterization of extracellular vesicles from Radix Stephaniae Tetrandrae 1. Isolation of extracellular vesicles (SE-EVs) (1) Raw material treatment: After washing and peeling the tuber of Radix Stephaniae Tetrandrae, immerse it in 1 times volume of 4℃ pre-cooled phosphate buffer solution (PBS, pH 7.4) for 10 min, and mechanically crush it to obtain juice; (2) Gradient centrifugation purification: centrifuge the juice at 3000×g for 40 min to remove plant debris; centrifuge the supernatant at 40000×g for 40 min to remove macromolecular impurities; collect the supernatant and centrifuge at 110000×g for 120 min to obtain the crude extracellular vesicle precipitate; (3) Refining: resuspend the precipitate with PBS, centrifuge at 120000×g for 60 min, and filter the obtained precipitate through a 0.22μm filter membrane to obtain high-purity extracellular vesicles (SE-EVs).

[0025] Quantify the protein concentration using the BCA detection kit (B6169, UElandy, China), and store the obtained extracellular vesicles at -80℃ for further application. The particle size and Zeta potential of SE-EVs were determined by zeta method (sizer Nano ZS90). The particle concentration of SE-EVs was determined by nanoparticle tracking analyzer (Zetaview-PMX120-Z, Particle Metrix, Germany). Transmission electron microscope (JEM1400, JEOL, Japan) was used to characterize the morphology of SE-EVs.

[0026] The results are as follows Figure 1 As shown, SE-EVs present as exosome-like spherical particles ( Figure 1 A); Dynamic light scattering (DLS) results showed ( Figure 1 B), the hydrodynamic size of SE-EVs was 161.8±2.68 nm, with uniform size distribution (PDI = 0.068±0.046); Zeta potential showed ( Figure 1 C), SE-EVs have a negative surface charge (-33.82±1.675 mV); according to the results of nanoparticle tracking analysis (NTA), ( Figure 1 D), the average particle size of SE-EVs is 124.9 nm.

[0027] 2. SE-EVs lipid analysis An appropriate amount of sample was centrifuged to remove PBS and lyophilized. 200 μL of pre-chilled methanol / water (3 / 1, v / v), 1 mL of pre-chilled MTBE, and 100 μL of water were added sequentially. After each addition, the mixture was thoroughly vortexed, sonicated in an ice bath, and allowed to stand at room temperature for 10 min. Finally, the mixture was centrifuged at 14,000 g for 15 min at 4°C. The supernatant was evaporated to dryness in a high-speed vacuum centrifuge and reconstituted with 30 μL of pre-chilled isopropanol / methanol (1 / 1, v / v). The mixture was centrifuged at 20,000 g for 20 min at 4°C, vortexed, and transferred to an injection vial. An appropriate amount of the supernatant was injected and analyzed. Samples were analyzed using a SHIMADZU-LC30 ultra-high performance liquid chromatography (UHPLC) system using a Hypersil GOLD™ C18 (2.1 × 100 mm, 3 µm) column (injection volume 3 μL, column temperature 40°C, flow rate 0.3 mL / min). The mobile phase consisted of A: 10 mM ammonium formate in acetonitrile and water (acetonitrile:water = 4:6, v / v); B: a mixture of acetonitrile and isopropanol (acetonitrile:isopropanol = 1:9, v / v). The gradient elution program was as follows: linear gradient from 0% to 10% at 0-0.1 min; linear gradient from 10% to 40% at 0.1-4 min; linear gradient from 40% to 44% at 4-12 min; linear gradient from 44% to 55% at 12-5 min; linear gradient from 55% to 66% at 15-18.5 min; linear gradient from 66% to 71% at 18.5-20 min; B maintained at 71% at 20-26 min; linear gradient from 71% to 10% at 26-26.1 min; and B maintained at 10% at 26.1-30 min. Samples were maintained at 4°C throughout the autosampler analysis. The samples were separated by UHPLC and analyzed by mass spectrometry using a Q Exactive plus mass spectrometer (ThermoScientific™).

[0028] The results are as followsFigure 2 As shown, lipidomic analysis revealed that SE-EVs are primarily composed of phosphatidylethanol (PE, 14.55%), diacylglycerol (DG, 12.27%), phosphatidylcholine (PC, 11.36%), and phosphatidylserine (PS, 6.36%). These molecules are amphiphilic and provide a structural foundation for the stability of SE-EVs. PE is a membrane fusion promoter, enhancing the efficiency of vesicle-cell fusion and facilitating cellular uptake, while PC maintains vesicle stability, protects the contents, and is non-toxic. DG, by competing with DAG kinase, blocks the conversion of DAG to PA, thereby inhibiting the mTORC1 pathway, blocking cellular glycolysis and protein synthesis, and disrupting signal transduction and energy metabolism. It is worth noting the presence of plant-specific lipids (digalactosyldiglyceride) DGDG (4.55%) and (monogalactosyldiglyceride) MGDG (2.27%). Since these components are not found in animal cells, they can avoid autoimmune attacks. In addition, plant galactolipids themselves are TLR4 agonists, which can promote dendritic cell maturation and T cell activation, and are beneficial to immune activation.

[0029] 3. SE-EVs Protein Analysis After freeze-drying of SE-EVs, an appropriate amount of SDT lysis solution was added, transferred to an EP tube, then boiled in a water bath for 3 min, and ultrasonically broken for 2 min, centrifuged at 16000 g for 20 min at 4°C, and the supernatant was taken. Protein quantification was performed using the BCA method, and 15 μg of protein sample was taken and mixed with 5x loading buffer at a ratio of 5:1 (v / v), boiled in a water bath for 5 min, and subjected to 10% SDS-PAGE electrophoresis (constant voltage 100 V, 45 min), and the protein bands were stained with Coomassie brilliant blue to show the distribution of protein molecular weight. An appropriate amount of protein was taken from each sample for FASP enzymolysis, and the dried peptides were reconstituted with 0.1% FA, and the peptide concentration was determined for LC-MS analysis. A Vanquish Neo UHPLC system was used for chromatographic separation. Buffer: A liquid was 0.1% formic acid aqueous solution, B liquid was 0.1% formic acid acetonitrile aqueous solution (acetonitrile was 80%). The chromatographic column was equilibrated with 96% A liquid. After the sample was injected into the Trap Column (PepMap Neo 5 μm C18 300 μm X 5 mm, Thermo Scientific), it was subjected to gradient separation by the chromatographic analysis column (μPAC Neo High Throughput column, Thermo Scientific). The liquid phase gradient is set as follows: 0 min-0.1 min, B liquid linear gradient from 4%-6%; 0.1 min-1.1 min, B liquid linear gradient from 6%-12%; 1.1 min-4.3 min, B liquid linear gradient from 12%-22.5%; 4.3 min-6.1 min, B liquid linear gradient from 22.5%-45%; 6.1 min-8 min, B liquid maintained at 99%. After the separation of the peptides, Orbitrap Astral mass spectrometer (Thermo Scientific) was used for DIA (data-independent acquisition) mass spectrometry analysis. The analysis time was 8 min, the electrospray voltage was 2.2 kV, the detection mode was positive ion, the parent ion scan range was 380-980 m / z, the primary mass resolution was 240000, the AGC target was 500%, and the primary Maximum IT was 3 ms. The secondary mass resolution was 80000, the AGC target was 500%, the secondary Maximum IT was 3 ms, the RF-lens was 40%, the MS2 Activation Type was HCD, the Isolation window was 2 Th, the Normalized collision energy was 25%, the cycle time was 0.6.All mass spectrometry data were merged by software DIA-NN, and database search and further analysis were completed.

[0030] Results as shown in Figures 3-5 , according to the results of Coomassie blue showed that SE-EVs in the presence of a rich protein, and the molecular weight is mainly concentrated in 20-55KDa (see Figure 3 A), using liquid chromatography-mass spectrometry, protein analysis of SE-EVs, found that SE-EVs in the presence of 4985 protein. Using gene ontology (GO) database analysis of the biological function of these proteins, we found that these proteins and biological processes (Biological Process, BP), molecular function (Molecular Function, MF) and cellular components (Cellular Component, CC) of multiple processes or composition related. Among them, 2744 related to cell processes, 2299 related to catalytic activity, 2762 related to cellular anatomy entity (see Figure 4 ). According to the subcellular localization analysis (CC), the results show (see Figure 3 B) SE-EVs in the protein mainly from the cytoplasm (53.91%), with exosome source definition consistent with, according to the genome annotation path (KEGG) analysis shows that SE-EVs in the protein mainly with the following pathways related (see Figure 5 ), including ribosome associated with cellular translation pathway, with autophagy related cellular transport and catabolism pathway, with signal transduction related MAPK pathway, and amino acid synthesis related pathway, the above results suggest that SE-EVs may be associated with translation, autophagy, apoptosis and other cellular processes.

[0031] 4、SE-EVs small molecule analysis The SE-EVs were pre-cooled at 4°C, and the interference of buffer salts in the solvent was removed after passing through the column. The sample to be tested was eluted into a 5 mL EP tube in the collection container with 3 mL of methanol, and then blown to dryness with nitrogen. 300 μL of methanol-water (V:V=4:1, containing mixed internal standard, 4 μg / mL) was used for re-dissolution, vortexed for 1 min, ultrasonicated for 3 min, and then placed at -40°C overnight. After centrifugation for 10 min (12000 rpm, 4°C), 150 μL of supernatant was taken and loaded into an LC-MS sample vial with a foot inner liner for analysis. The analytical instrument used in this experiment was a liquid chromatograph-mass spectrometer (Waters ACQUITY UPLC I-Class plus / Thermo QE). The chromatographic column was ACQUITY UPLC HSS T3 (100 mm x 2.1 mm, 1.8 um). The mobile phase was A-water (containing 0.1% formic acid) and B-acetonitrile. The flow rate was 0.35 mL / min. The injection volume was 2 μL. The ion source was HESI. The sample mass signal was collected in positive and negative ion scanning modes. The data collection mode was DDA, and the scanning mode was Full MS / dd-MS2 (TOP 10).

[0032] The results show that SE-EVs contain a large number of alkaloids, phenylpropanoids and other compounds that have been well proven to have anti-tumor effects (see Figure 6 ), including sinomenine, sinomenine hydrochloride, feruloyl tyramine, 5-hydroxyferulic acid, doxorubicin alcohol, etc. These components combined with SE-EVs can provide a material basis for the application of SE-EVs in cancer treatment.

[0033] Example 2 Separation of extracellular vesicles from the roots of Sinopodophyllum emodi Wall. 1. Separation of extracellular vesicles (SE-EVs) (1) Raw material treatment: After washing and peeling the roots of Sinopodophyllum emodi Wall., immerse them in 2 times the volume of 4°C pre-cooled phosphate buffer solution (PBS, pH 7.2) and mechanically crush for 12 min to obtain juice by filtration; (2) Gradient centrifugation purification: centrifuge the juice at 4000 x g for 20 min to remove plant debris; centrifuge the supernatant at 30000 x g for 45 min to remove macromolecular impurities; collect the supernatant and centrifuge at 150000 x g for 100 min to obtain the crude extracellular vesicle precipitate; (3) Refining: resuspend the precipitate in PBS and centrifuge at 150000 x g for 50 min. The obtained precipitate is filtered through a 0.22 μm filter membrane to obtain high-purity extracellular vesicles (SE-EVs).

[0034] After detection, the obtained extracellular vesicles are consistent with Example 1.

[0035] Example 3 Isolation of extracellular vesicles from D. oppositifolia (1) Raw material processing: After washing and peeling the D. oppositifolia tuber, immerse it in 1 times volume of 4°C pre-cooled phosphate buffer solution (PBS, pH 7.2), mechanically crush for 8 min, and filter to obtain juice; (2) Gradient centrifugation purification: centrifuge the juice at 3000 x g for 30 min to remove plant debris; centrifuge the supernatant at 50000 x g for 45 min to remove macromolecular impurities; collect the supernatant and ultracentrifuge at 100000 x g for 140 min to obtain the crude extracellular vesicle precipitate; (3) Refining: resuspend the precipitate with PBS, centrifuge at 100000 x g for 70 min, filter the obtained precipitate through a 0.22 μm filter membrane to obtain high-purity extracellular vesicles.

[0036] It was detected that the obtained extracellular vesicles were consistent with Example 1.

[0037] Example 4 In vitro efficacy experiment of D. oppositifolia-derived extracellular vesicles (SE-EVs) 1. Cell culture The mouse breast cancer cell line (4T1) and the normal liver cell line (L02) were provided by the China Academy of Sciences Cell Bank (Shanghai, China). These cells were cultured in Dulbecco's modified eagle medium (DMEM) containing fetal bovine serum (10%, v / v) and penicillin / streptomycin (1%, w / v) at 37°C in a CO2 incubator.

[0038] 2. Cell uptake of D. oppositifolia-derived extracellular vesicles (SE-EVs) Cell uptake experiment to evaluate the uptake ability of 4T1 cells to FITC-SE-EVs. Fluorescent dye FITC (F8070, Solarbio, China) was conjugated with SE-EVs. Cells were seeded in a 24-well plate at 1 x 10 5 6.25 μg / ml) for 24 h, and then incubated with DAPI (KGE2505-10, KeyGEN, China) for nuclear labeling. The laser confocal microscope (LAM900, ZEISS, Germany) was used to take pictures at 0 h, 3 h, 6 h, and 12 h, respectively.

[0039] The results are shown in Figure 2. Figure 7As shown, the fluorescence images showed that blank cells had no green fluorescence signal, and green fluorescence began to appear after 3 h of incubation with FITC-labeled SE-EVs, and more than 60% of the cells showed green fluorescence and the fluorescence intensity was enhanced at 12 h. These observations indicated that SE-EVs could be effectively taken up by 4T1 cells in a time-dependent manner.

[0040] 3. Anti-tumor activity in vitro The anti-tumor activity of SE-EVs in vitro was determined by CCK-8 method. 4T1 cells and L02 cells were cultured in 96-well plates at a density of 1 x 104 cells / well overnight. They were incubated with different amounts of SE-EVs (protein concentration: 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 pg / mL) in serum-free medium for 24 and 48 hours, respectively. Then the medium containing SE-EVs was removed, and the cells were washed with PBS for 3 times. The cells were incubated with 10 pL CCK-8 (0.5 mg / mL) at 37°C for 2 hours, and the OD value was determined at 540 nm wavelength by an enzyme-labeled instrument.

[0041] Results are shown in Figure 8 , which showed that the survival rate of 4T1 cells gradually decreased with the increase of the concentration of SE-EVs after co-incubation with SE-EVs (see Figure 8 A). The IC 50 value of SE-EVs for 4T1 cells was 3.322 pg / ml, which was nearly 100 times lower than the IC 50 value of 308.8 pg / ml for L02 normal liver cells. These results clearly indicated that SE-EVs had the ability to inhibit the proliferation of breast tumor cells and only very slight cytotoxicity was found in normal cell lines (L02 normal liver cells) after SE-EVs treatment, revealing that SE-EVs had very good biocompatibility (see Figure 8 B).

[0042] 4. Cell migration analysis The anti-migration ability of SE-EVs on 4T1 cells was evaluated by cell scratch assay. Cells were seeded in 6-well plates at a density of 1 x 10 5 cells / well, and after 24 hours of culture, the confluent cell layer was scratched with a sterile 200 pL pipette tip. Then the cells were gently washed, and the wound was imaged before / after the addition of different concentrations of SE-EVs (protein concentration was 0, 3.125, 6.25, 12.5 pg / ml, respectively). The relative wound coverage area was analyzed using image j.

[0043] The results of cell scratch are shown in Figure 9, results showed that the treatment group of SE-EVs significantly inhibited the wound healing rate of 4T1 cells after 24h (see Figure 9 A), and the healing width also had no obvious change after 24h (see Figure 9 B).

[0044] 5. Western blot analysis The total protein of 4T1 cells treated with different concentrations of SE-EVs (treatment same as cell migration analysis) was extracted using high-efficiency RIPA lysis buffer (R0010, Solarbio) containing protease inhibitors (FJP0100, Solarbio) and phosphatase inhibitor cocktail (G2007, Servicebio), and quantified by BCA quantitative kit.

[0045] Protein electrophoresis, prepare 10% and 7.5% separation gel (PG212, Yamei), load 30μg total protein per well, place in electrophoresis liquid, compress gel at 80V for 30min, separation gel at 100V for 90min. Protein transfer, immerse PVDF membrane (IPVH00010, Millipore, Ireland) in methanol, then transfer with SDS-PAGE gel in transfer buffer, constant voltage at 100V under cold conditions for 1.5h. After transmembrane, block the membrane with skimmed milk (containing 5% BSA) at room temperature for 1h, then wash with TBST for 3 times, 5min each time, finally incubate with antibodies p-Akt (28731-1-AP, Proteintech, dilution 1:2000), Akt (10176-2-AP, Proteintech, dilution 1:2000), p-mTOR (AF3308, Affinity dilution 1:1000), mTOR (BD10315611, Bioss, dilution 1:1000), β-actin (20536-1-AP, Proteintech, dilution 1:2000) at 4℃ overnight. Protein membrane is washed with TBST for 3 times, 5min each time, then incubated with HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (SA00001-2, Proteintech, dilution 1:5000) at room temperature for 1h, and then washed with TBST for 3 times, 5min each time. Finally, use the developing solution Chemiluminescent HRP Substrate (WBKLS0500 Millipore) to incubate the membrane at room temperature for 1min, remove the excess developing solution, and expose in the developing instrument (5200 Multi, Tanon, China) for 5-10min to form an image.

[0046] In vivo Western blot analysis confirmed that SE-EVs treatment of breast cancer in mice depends on the PI3K / Akt / mTOR pathway, with low expression of p-Akt and p-mTOR (see Figure 10 ), achieving the function of inhibiting the growth and metastasis of triple-negative breast cancer.

[0047] 6. SE-EVs Uptake in 3D Tumor Spheroids in Vitro 3D tumor spheres are a medium-complexity model between monolayer cultured cancer cells and in vivo tumors. Compared with traditional monolayer cell experiments, they can significantly improve the reliability of research data. The penetration ability of FITC-SE-EVs into 4T1 cell tumor spheres was evaluated by 3D tumor sphere uptake experiments of SE-EVs. The cells were cultured at a rate of 1×10 4 Cells were seeded per well in a 96-well microplate (FULA962, BeyoGold) with minimal attachment and incubated with FITC-SE-EVs (protein concentration 6.25 μg / ml) after 24 hours of culture. Laser scanning and imaging were performed using the copolymer gel at 0, 1, 6, and 12 hours.

[0048] The penetration of FITC-SE-EVs into 3D tumor spheres is shown in Figure 2. Figure 11 As shown in the figure, the penetration layer of FITC-SE-EVs on 4T1 cell 3D tumor spheres increased with the increase of co-culture time, and the penetration layer reached 50 μm at 12 h. These observations indicate that 4T1 cell 3D tumor spheres can be effectively penetrated by SE-EVs, and the penetration degree is time-dependent.

[0049] Example 5 In vivo efficacy experiment 1. Biodistribution of SE-EVs in vivo Female BALB / c mice weighing 17–19 g were obtained from SPIEF Biotechnology Co., Ltd. (Beijing, China). Animal studies were approved by the Animal Care and Use Committee of Yunnan University of Traditional Chinese Medicine. To track the biodistribution of SE-EVs in vivo, SE-EVs were labeled with a DiR fluorescent probe (D4006, UElandy). 4T1 cells (1 × 10 6 ) were injected into the mammary gland of BALB / c mice. When the tumor volume reached 100 mm 3At 120 h, mice were administered DiR-SE-EVs (BCA: 3 mg / kg / mouse) with an equal DiR concentration via intravenous or intraperitoneal injection. Fluorescence images were taken using an IVIS Spectral Imaging System (IVIS Lumina III, PE, PerkinElmer, USA) at predetermined time points (0, 12, 24, 36, 48, 60, 72, 84, 96, 108, and 120 h). After 120 h of imaging, mice were euthanized, and major organs (liver, heart, spleen, lung, and kidney) and tumors were isolated, and ex vivo organ fluorescence images were captured.

[0050] The results are as follows Figure 12 As shown, SE-EVs were observed to distribute spontaneously to the liver and spleen of mice (see Figure 12 A) The maximum fluorescence accumulation in the tumor tissue occurs at 24 hours and then stabilizes (see Figure 12 B), and obvious fluorescence signals were observed at 24 h and 12 h after SE-EVs tail vein administration and intraperitoneal administration, respectively, and the fluorescence at the tumor site was still obvious after 120 h. This result shows that SE-EVs can target the tumor site and retain it for a long time, further illustrating that SE-EVs have a natural structural advantage of tumor self-targeting, and can be used not only as a drug, but also as a drug carrier to deliver anti-tumor drugs to the target site.

[0051] 2 Anti-tumor effects of SE-EVs in vivo Female BALB / c mice weighing 17–19 g were obtained from Spectrum Biotechnology Co., Ltd. (Beijing, China). All animal experimental procedures were performed in accordance with the guidelines and protocols of the Animal Experimentation Ethics Committee of Yunnan University of Traditional Chinese Medicine. Subcutaneous mammary tumors were used to evaluate the anti-breast cancer potential of SE-EVs in vivo. 4T1 cells suspended in PBS (100 μl) were inoculated into the mammary glands of BALB / c mice (1 million 4T1 cells per mouse). When the average tumor volume reached 100 mm, the cells were inoculated with 4T1 cells. 3 The mice were divided into the following groups: control group (NC), model control group (MC PBS iv), intraperitoneal injection group (SE-EVs ip 7 mg / kg), intravenous injection group (SE-EVs iv 7 mg / kg), and positive drug group (CISPLATIN iv 2 mg / kg). The tumor volume and body weight of the mice were recorded every 2 days (the formula for calculating the tumor volume is: width 2 × Length 2 ×1 / 2). Tumor tissues were removed on day 14 after administration, fixed in paraformaldehyde solution (4%, v / v), embedded in paraffin, and stained with hematoxylin and eosin (H&E) and a TUNEL kit.

[0052] The experimental results are shown in Figure 13 , and the results show that the tumor volume of the experimental mice develops rapidly within 14 days (see Figure 13 A), while SE-EVs, whether intraperitoneally injected or intravenously injected, significantly delay the growth trend of the tumor (see Figure 13 B). The average tumor weight of the intravenous and intraperitoneal SE-EVs treatment groups was 594.30 mg and 558.39 mg, respectively, while the average tumor weight of the PBS group was 983.44 mg (see Figure 13 C). At the same time, there was no significant change in the body weight of the mice during the treatment period (see Figure 13 D). The experimental results show that SE-EVs can effectively inhibit the growth of breast tumors in mice.

[0053] The results of H&E and TUNEL staining are shown in Figure 14 . The H&E staining images show that a high density of cancer cells is observed in the tumor tissue of the control group, and almost no necrotic cancer cells are detected. In contrast, the SE-EVs treatment group shows much fewer tumor cells than the control group, with a large area of tumor cell necrosis in the center and obvious cavities can be observed. In summary, the H&E staining results show that SE-EVs have the ability to inhibit breast tumors in vivo. The TUNEL staining images show that only a small amount of apoptosis fluorescence is observed in the tumor tissue of the control group, while the apoptosis fluorescence is clearly visible in the SE-EVs intravenous and intraperitoneal administration groups, further indicating that SE-EVs can inhibit tumors by promoting apoptosis.

[0054] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. However, if there is a conflict between the definitions of terms in the specification and the definitions of terms in the included definitions, the definitions in the specification shall prevail. Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent replacements to some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An extracellular vesicle derived from dapoxetine, characterized in that The extracellular vesicles are extracted and purified from the root of Euphorbia cerifera, have a hydrodynamic particle size of 161.8±2.68 nm, an average particle size of 124.9±2 nm, a negative surface charge of -33.82±1.675 mV, and contain lipids, proteins, and active small molecule compounds.

2. The extracellular vesicle according to claim 1, characterized in that The lipids include phosphatidylethanol, diacylglycerol, phosphatidylcholine and phosphatidylserine, monogalactosyl diglyceride, and digalactosyl diglyceride; the proteins include cellular translation pathway proteins related to ribosomes, cellular transport and catabolism pathway proteins related to autophagy, MAPK pathway proteins related to signal transduction, and pathway proteins related to amino acid synthesis; the active small molecule compounds include chelidonine, sinomenine hydrochloride, feruloyltyramine, 5-hydroxyferulic acid, and doxorubicin.

3. A method for preparing the extracellular vesicles according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) Raw material processing: Wash and peel the root tubers of the Chinese yam, immerse them in 0-4℃ pre-cooled phosphate buffer, mechanically crush them for 8-12 minutes, and filter to obtain the juice; (2) Gradient centrifugation purification: The juice was centrifuged at 3000-4000 × g for 20-40 min to remove plant debris; The supernatant was centrifuged at 30,000-50,000 × g for 35-45 min to remove macromolecular impurities; The supernatant was collected and ultracentrifuged at 100,000-150,000 × g for 100-140 min to obtain a crude extracellular vesicle pellet; (3) Purification: The precipitate was resuspended in PBS and purified by centrifugation at 100,000-150,000 × g for 50-70 min. The resulting precipitate was filtered through a filter membrane to obtain high-purity extracellular vesicles.

4. The preparation method according to claim 3, characterized in that In step (1), the mass volume ratio of the root tuber of Dibuconazole to the phosphate buffer solution is 1:1-2.

5. Use of the extracellular vesicles according to any one of claims 1 to 2 or the extracellular vesicles prepared by the preparation method according to claim 3 in the preparation of anti-breast cancer drugs.

6. The application according to claim 5, characterized in that The drug promotes apoptosis of breast cancer cells and inhibits growth and metastasis of breast cancer.

7. The use according to claim 5, characterized in that The drug can be targeted to the tumor site and retained for a long time.

8. The application according to claim 5, characterized in that: The pharmaceutical dosage form is any one of tablets, granules, capsules, oral liquid preparations, injections, microcapsules, suppositories, ointments or films.

9. Use of the extracellular vesicle according to any one of claims 1 to 2 or the extracellular vesicle prepared by the preparation method according to claim 3 in a breast cancer targeted drug delivery system.

10. The use according to claim 9, characterized in that: The extracellular vesicles serve as drug carriers to deliver anti-breast cancer drugs to target sites.

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