Application of lily extracellular vesicles in preparation of tumor treatment drugs

Abstract

The invention belongs to the technical field of biological agents, and particularly discloses application of lily extracellular vesicles in preparation of tumor treatment drugs. The preparation method of the lily extracellular vesicles comprises the following steps: S1, juicing lily bulb tissues, and filtering to obtain a lily bulb stock solution; s2, treating the lily bulb stock solution obtained in the step S1 to obtain a lily extracellular vesicle extracting solution; s3, concentrating and purifying the lily extracellular vesicle extracting solution obtained in the step S2 to obtain lily extracellular vesicles; wherein in the step S2, the treatment comprises centrifugation and filter membrane filtration. The lily extracellular vesicle provided by the invention can effectively promote polarization of M2-type macrophages to M1-type macrophages, shows remarkable immunoregulation ability, is good in biocompatibility, and has the potential of preparing anti-cancer drugs.

Description

Technical Field

[0001] The invention relates to the technical field of biological preparations, and relates to application of lily extracellular vesicles in preparing tumor therapeutic drugs. Background Art

[0002] Lilium is widely cultivated worldwide for its excellent ornamental, edible and medicinal value. Lilies are rich in amino acids, polysaccharides, and bioactive components such as phenols, flavonoids and saponins, which have been shown to have a variety of medicinal values. Although some studies have explored the potential of lily extracts in cancer treatment, the specific components that exert anti-tumor effects have not yet been fully elucidated.

[0003] Extracellular vesicles are a type of vesicles with a lipid membrane that encapsulates active macromolecules such as proteins, lipids, and nucleic acids. They are secreted from cells into the apoplast and are essential for intercellular communication. In 1967, plant extracellular vesicles were first discovered in carrots. Plant extracellular vesicles are rich in biologically active substances such as nucleic acids and proteins. Compared with animal-derived extracellular vesicles, plant extracellular vesicles are derived from edible plants, so they are not only non-toxic, but also have high yields, good biocompatibility, and are non-immunogenic. Extensive studies have shown that extracellular vesicles extracted from various edible plants have multiple therapeutic effects such as anti-inflammatory, anti-cancer, anti-viral, and antioxidant, and have the potential to be developed into pharmaceutical preparations and effectively treat a series of human diseases; and biologically active plant extracellular vesicles can also be used as carriers to deliver biological macromolecules such as nucleic acids, polysaccharides, and proteins, such as encapsulating drugs and synergistically enhancing their therapeutic effects. In recent years, plant-derived extracellular vesicles have become a new research hotspot due to their rich biological properties and application potential.

[0004] Macrophages are an important part of the monocyte-macrophage system and are responsible for immune defense and tissue repair in the body. Since macrophages can recognize and ingest foreign nanovesicles, they become ideal targets in the development of nanomedicines. Studies have shown that natural or modified extracellular vesicles derived from mammalian cells can induce anti-tumor responses in macrophages and inhibit tumor growth. In the tumor microenvironment, tumor-associated macrophages (TAMs) are an important part of the tumor, and TAMs are known to play an important role in promoting tumor growth, angiogenesis, and metastasis. Therefore, TAMs are considered to be a potential target for cancer immunotherapy. Changing the phenotype of TAMs from the tumor-promoting M2 type to the tumor-suppressing M1 type is currently an important anti-cancer strategy.

[0005] Chinese invention patent CN116726152A provides a tumor vaccine chimeric with Chinese herbal medicine-derived vesicle-like particles, and its preparation method and application. The tumor vaccine is prepared by fusing Chinese herbal medicine vesicle-like particles with tumor cell membranes. The fusion ratio of Chinese herbal medicine vesicle-like particles and tumor cell membranes is 1:1-1:10 (w / w). The preparation method includes separating and preparing Chinese herbal medicine vesicle-like particles, extracting autologous tumor cell membranes, fusing the Chinese herbal medicine vesicle-like particles with tumor cell membranes, and obtaining a personalized tumor vaccine chimeric with Chinese herbal medicine vesicle-like particles. The personalized tumor vaccine of this technology can induce dendritic cell maturation while enhancing dendritic cell phagocytosis, produce efficient anti-tumor immune response and anti-tumor immune memory, and ultimately inhibit tumor recurrence and metastasis. However, the technology does not provide lily extracellular vesicles that can regulate the transformation of macrophages from M2 to M1 and their preparation methods.

[0006] Chinese invention patent CN117210384A provides an extracellular vesicle derived from Paris polyphylla and its separation and extraction method and application. The method is to first grind Paris polyphylla tubers to obtain Paris polyphylla tuber juice, remove large particles of Paris polyphylla tissue by multiple low-speed centrifugation, and at the same time pass 8% PEG8000 to precipitate the Paris polyphylla extracellular vesicles after low-speed centrifugation; after precipitation, purify the extracellular vesicles by sucrose gradient ultracentrifugation. This separation and extraction method has a wide range of material sources, low cost, easy reagents, simple operation, and can efficiently separate and extract Paris polyphylla extracellular vesicles, which is conducive to the development of plant cell-derived extracellular vesicle extraction technology and the later development of new nano drug-carrying materials. However, the technology does not provide lily extracellular vesicles that can regulate the transformation of macrophages from M2 to M1 and their preparation methods.

[0007] In summary, the prior art has failed to provide a lily extracellular vesicle that can regulate the transformation of macrophages from M2 to M1 and a preparation method thereof. Summary of the invention

[0008] In view of the problem that the prior art fails to provide a lily extracellular vesicle that can regulate the transformation of macrophages from M2 to M1 and a preparation method thereof, the present invention provides an application of lily extracellular vesicles in the preparation of tumor therapeutic drugs. In the present invention, extracellular vesicles (Lilium derived exosomes, LDEs) were successfully isolated and purified from lily bulbs for the first time. Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA) showed that these extracellular vesicles had a typical saucer-like structure, were complete and stable, and had a particle size distribution of about 235 nm. Its good biocompatibility was verified by cell death and live staining and CCK-8 tests. Subsequently, M2 macrophages were treated with lily-derived extracellular vesicles, and it was found that M2 macrophages were successfully polarized to M1. Immunofluorescence staining and flow cytometry further supported this conclusion, confirming the significant role of lily-derived extracellular vesicles in regulating macrophage polarization. Overall, this invention reveals for the first time the potential of lily-derived extracellular vesicles in macrophage immunoregulation, demonstrates its application prospects in tumor suppression, and provides new ideas for the development of cancer immunotherapy based on plant-derived extracellular vesicles.

[0009] To achieve the above-mentioned object of the invention, on the one hand, the present invention provides a use of lily extracellular vesicles in preparing tumor treatment drugs, and the preparation method of the lily extracellular vesicles comprises the following steps: S1, squeezing the lily bulb tissue and filtering to obtain the lily bulb stock solution; S2, treating the lily bulb stock solution obtained in step S1 to obtain a lily extracellular vesicle extract; S3, concentrating and purifying the lily extracellular vesicle extract obtained in step S2 to obtain lily extracellular vesicles; Wherein, in step S2, the processing includes centrifugation and membrane filtration.

[0010] The drug can be prepared into dosage forms such as pills, capsules, granules, oral liquids, powders, tablets, lozenges and injections, and suitable drug carriers in the art can be selected for different dosage forms.

[0011] The pharmaceutical carrier used can be solid, liquid or gas. Examples of solid carriers include lactose, kaolin, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0012] When preparing drugs in oral dosage forms, any convenient pharmaceutical medium can be used. For example, water, ethanol, oil, alcohol, flavoring agents, preservatives, colorants, etc. can be used to form oral liquid preparations, such as suspensions, elixirs and solutions; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulating agents, emulsifiers, lubricants, binders, disintegrants can be used to form oral solid preparations, such as powders, capsules and tablets. Tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers due to their ease of administration. Tablets can be coated using standard aqueous or non-aqueous techniques.

[0013] Tablet drugs containing the lily extracellular vesicles prepared by the present invention can be prepared by tableting or molding, and one or more auxiliary ingredients or adjuvants can be used optionally. The active ingredient can be tableted in a free-flowing form (such as powder or granules) in a suitable machine, and can be optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant to prepare a tablet. Molded tablets can be molded in a suitable machine, that is, a powdered compound mixture moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of active ingredient, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of active ingredient. For example, a preparation intended for oral administration to humans may contain about 0.5 mg to about 5 g of active drug, mixed with an appropriate and convenient carrier material, which may account for about 5% to 95% of the total composition. Dosage unit forms will generally contain from about 1 mg to about 2 g of active ingredient, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

[0014] The medicine suitable for parenteral administration in the present invention can be prepared as an aqueous solution or suspension. Suitable surfactants, such as hydroxypropylcellulose, can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and oil mixtures thereof. In addition, preservatives can be added to prevent the harmful growth of microorganisms.

[0015] The drugs suitable for injection use in the present invention include sterile aqueous solutions or dispersions. In addition, the drug can be in the form of sterile powders for the extemporaneous preparation of such sterile injections or dispersions. In all cases, the final injection form must be sterile and must be an effective liquid so that the injectable drug component must remain stable under production and storage conditions; therefore, it is best to preserve it to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils and suitable mixtures thereof.

[0016] The medicine of the present invention can be in a form suitable for topical use, such as an aerosol, cream, ointment, lotion, powder or the like. In addition, the medicine can be in a suitable form for transdermal administration devices. These prescriptions can be prepared using the medicine of the present invention by conventional processing methods. For example, a cream or ointment with a desired consistency can be prepared by mixing a hydrophilic material and water, and about 5wt% to about 10wt% of the compound.

[0017] The medicine of the present invention can be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture is made into a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be prepared by first forming a composition containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0018] In addition to the above-mentioned carrier components, the above-mentioned drugs may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. In addition, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants and flavoring agents, etc., may be added. The preparation is made isotonic with the blood of the intended recipient. The components containing the lily extracellular vesicles of the present invention can also be prepared in the form of powder or concentrate.

[0019] Preferably, the active ingredient of the tumor therapeutic drug includes the lily extracellular vesicles.

[0020] More preferably, the active ingredient of the tumor therapeutic drug is the lily extracellular vesicles.

[0021] Preferably, the tumor therapeutic drug is a tumor immunotherapy drug.

[0022] More preferably, the tumor immunotherapy drug is a tumor region macrophage activation immunotherapy drug.

[0023] More preferably, the tumor region macrophage activation immunotherapy drug is a tumor immunotherapy drug that regulates the transformation of tumor region macrophages from M2 type to M1 type.

[0024] Preferably, in step S1, the lily bulb tissue is selected from at least one of Lanzhou lily dry bulb tissue and fresh Lanzhou lily bulb tissue.

[0025] More preferably, in step S1, the juicing comprises the following steps: washing the lily bulb tissue, mixing it with a PBS solution, and juicing it using a juicer.

[0026] More preferably, in step S1, the mixing is specifically: Each gram of fresh bulb tissue of Lilium lanzhouense was mixed with 1 mL of PBS solution, or each gram of dried bulb tissue of Lilium lanzhouense was mixed with 1 mL of PBS solution.

[0027] More preferably, the time for extracting juice using a juicer is 2-5 minutes.

[0028] More preferably, the temperature of the PBS solution is 3-5°C, and even more preferably 4°C.

[0029] More preferably, in step S1, the filtration is performed using sterile gauze.

[0030] Preferably, in step S2, the treatment further comprises enzymolysis.

[0031] Preferably, in step S3, the concentration and purification comprises at least one of concentration and purification using an ultrafiltration tube and concentration and purification using a chromatographic column.

[0032] On the other hand, the present invention provides a tumor treatment drug prepared in the above application.

[0033] Compared with the prior art, the beneficial effects of the present invention include: The invention provides a lily extracellular vesicle and a preparation method thereof. The extracellular vesicle prepared by the method has good biocompatibility, can significantly induce macrophage polarization to M1 type, activate macrophage immune effect, and thus play an anti-tumor role. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a transmission electron micrograph of the lily extracellular vesicles prepared in Example 1.

[0035] Figure 2 This is a transmission electron micrograph of the lily extracellular vesicles prepared in Example 2.

[0036] Figure 3 These are the particle size analysis results of the lily extracellular vesicles prepared in Example 1.

[0037] Figure 4 These are the particle size analysis results of the lily extracellular vesicles prepared in Example 2.

[0038] Figure 5 These are confocal photographs of the lily extracellular vesicles prepared in Example 1 being taken up by macrophages; wherein (a) is a confocal photograph of the cytoskeleton stained with the phalloidin fluorescent probe; (b) is a confocal photograph of the distribution of lily extracellular vesicles stained with the DiD red fluorescent probe in macrophages; (c) is a channel fusion confocal photograph of (a) and (b); and (d) is a locally enlarged confocal photograph of (c).

[0039] Figure 6 The cytotoxicity test results of the lily extracellular vesicles prepared in Example 1 and Example 2 are histograms; wherein, (a) represents the cytotoxicity test results of the lily extracellular vesicles prepared in Example 1 on RAW264.7 cells; (b) represents the cytotoxicity test results of the lily extracellular vesicles prepared in Example 2 on RAW264.7 cells; (c) represents the cytotoxicity test results of the lily extracellular vesicles prepared in Example 1 on bone marrow mesenchymal stem cells (BMSCs); (d) represents the cytotoxicity test results of the lily extracellular vesicles prepared in Example 2 on BMSCs cells; wherein, "" indicates a significant difference and p < 0.01, and "ns" indicates no significant difference.

[0040] Figure 7 The test results of the lily extracellular vesicles provided in Example 1 on the expression of TNF-α and CCL17 in macrophages; wherein, (a) represents the test results of the lily extracellular vesicles prepared in Example 1 on the expression of CCL17 in RAW264.7 cells; (b) represents the test results of the lily extracellular vesicles prepared in Example 1 on the expression of TNF-α in RAW264.7 cells; wherein, Indicates that there is a significant difference and p < 0.01.

[0041] Figure 8 The test results of the lily extracellular vesicles provided in Example 2 on the expression of TNF-α and CCL17 in macrophages; wherein, (a) represents the test results of the lily extracellular vesicles prepared in Example 2 on the expression of CCL17 in RAW264.7 cells; (b) represents the test results of the lily extracellular vesicles prepared in Example 2 on the expression of TNF-α in RAW264.7 cells; wherein, Indicates significant difference and p < 0.0001.

[0042] Fig. 9 The flow cytometry results of the effect of lily extracellular vesicles provided in Example 1 on the expression of CD206 on the surface of macrophages; wherein (a) represents the blank control group, and (b) represents the Example 1 group. DETAILED DESCRIPTION

[0043] Terms and Claims of the Present Invention: 1. The articles “a”, “an” and “the” include plural referents unless expressly limited to one or more referents otherwise.

[0044] 2. Numerical range: Unless otherwise expressly stated, all ranges or ratios disclosed herein will be understood to include any and all sub-ranges or sub-ratios contained therein. For example, a range or ratio of 1 to 30 stated should be considered to be included between a minimum value of 1 and a maximum value of 30, and any sub-range or sub-ratio, integer, decimal, or sub-range or sub-ratio consisting of integers or decimals including the end points.

[0045] The following non-limiting examples can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is merely an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0046] The present invention is further described below by way of specific examples. The various chemical reagents used in the examples of the present invention are obtained through conventional commercial channels unless otherwise specified. Unless otherwise specified, the contents described below are all weight contents. Unless otherwise specified, it is understood that the process is carried out at room temperature.

[0047] The PBS solution used in the present invention is a mixed aqueous solution of 2.0 mM KH2PO4, 137 mM NaCl, 10.0 mM Na2HPO4, and 2.7 mM KCl, and the pH of the solution is 7.4.

[0048] The ultrapure chromatographic column model used in the present invention is: IZON qEV ultrapure chromatographic column, and the column bed volume is 8.5 mL.

[0049] Example 1 Preparation of extracellular vesicles from lily.

[0050] S1. Take 200 g of fresh Lanzhou lily bulb tissue in a clean bench, cut it into pieces, and wash it with distilled water and PBS solution with a pH of 7.2-7.4 in turn; pour 200 mL of PBS solution at 4°C and pH 7.2-7.4 into a juicer, and squeeze juice with 200 g of fresh lily for 2-5 minutes to obtain juice; filter the juice with sterile gauze to obtain lily bulb stock solution.

[0051] S2, centrifuge the lily bulb stock solution obtained in step S1 at 4°C and 1000g for 10 minutes, take the supernatant; centrifuge at 4°C and 3000g for 20 minutes, take the supernatant; centrifuge at 4°C and 5000g for 2h, take the supernatant. Then filter the supernatant using 0.8μM and 0.45μM filter membranes in turn to obtain a filtrate. Then centrifuge the filtrate at 4°C and 20000g for 1.5h, take the supernatant, filter it using a 0.22μm filter membrane, centrifuge it at 120000g for 90min at 4°C, remove the supernatant and retain the precipitate. Add 1mL of PBS solution to blow and resuspend the precipitate, then add 1mL of PBS solution to blow and mix, then add 1mL of PBS solution to blow and mix to obtain a mixed solution. Filter the mixed solution using a 0.22μm filter membrane to obtain a lily extracellular vesicle extract.

[0052] S3. The lily extracellular vesicle extract obtained in step S2 was concentrated to 0.5 mL by centrifugation at 4 ° C and 3000g using a 100kDa ultrafiltration tube, and then 0.5 mL of lily extracellular vesicle concentrate was purified using an IZON qEV ultrapure column with a column bed volume of 8.5 mL. Take 0.5 mL of extracellular vesicle concentrate and add it to the ultrapure column. After all samples enter the column, add 2 mL of PBS immediately and collect 2.5 mL of void volume. After the liquid flows out, continue to add 2.8 mL of PBS, collect the liquid of the 8th to 14th fractions (i.e., the eluent), a total of 2.8 mL of the target fraction volume, and combine. Concentrate the combined liquid with a 100kDa ultrafiltration tube, and obtain lily extracellular vesicles based on particle size and morphology analysis.

[0053] Example 2 Preparation of extracellular vesicles from lily.

[0054] S1. Take 50 g of Lanzhou lily dry bulb tissue and place it in a clean bench, and wash it with distilled water and PBS solution with a pH of 7.4 in sequence; pour 200 mL of PBS solution at 4°C and pH 7.4 into a juicer, and squeeze juice with 50 g of air-dried lily for 4 minutes to obtain juice; filter the juice with sterile gauze to obtain lily bulb stock solution.

[0055] S2. Centrifuge the lily bulb stock solution obtained in step S1 at 4°C and 1000g for 10 minutes, and take the supernatant; centrifuge at 4°C and 3000g for 20 minutes, and take the supernatant. Mix the supernatant with hemicellulase, pectinase and cellulase to obtain an enzyme mixture. The hemicellulase accounts for 0.2% of the mass fraction of the enzyme mixture, the pectinase accounts for 0.1% of the mass fraction of the enzyme mixture, and the cellulase accounts for 0.1% of the mass fraction of the enzyme mixture. Digest at room temperature for 2-3 hours, centrifuge at 4°C and 5000g for 2h, take the supernatant, filter using 0.8μm and 0.45μm filter membranes in turn, centrifuge at 4°C and 120000g for 90min, remove the supernatant and obtain a precipitate. Mix the precipitate with 1mL PBS solution, resuspend the precipitate by blowing, add 1mL PBS solution, mix by blowing, add 1mL PBS solution, mix by blowing, and then add 1mL PBS solution, mix by blowing to obtain a mixed solution. The mixed solution was centrifuged at 120000g for 90 min at 4°C, and the supernatant was removed to obtain a precipitate. The precipitate was mixed with 1 mL of PBS solution, and the precipitate was resuspended by pipetting, and then 1 mL of PBS solution was added, and pipetting was mixed, and then 1 mL of PBS solution was added, and pipetting was mixed to obtain a secondary mixed solution. The secondary mixed solution was filtered using a 0.22 μm filter membrane to obtain a lily extracellular vesicle extract.

[0056] S3. The lily extracellular vesicle extract provided in step S2 is concentrated to 0.5 mL by centrifugation at 4° C. and 3000 g using a 100 kDa ultrafiltration tube, and lily extracellular vesicles are obtained based on particle size and morphology analysis.

[0057] Example 3 Characterization and analysis of the morphology and composition of lily extracellular vesicles.

[0058] (1) Morphological observation using transmission electron microscopy: 10 μL of the extracellular vesicles of lily prepared in Example 1 and Example 2 were respectively taken with a pipette gun and dropped on a copper mesh for 1 min, and the floating liquid was removed by filter paper. 10 μL of uranyl acetate was dropped on a copper mesh for 1 min, and the floating liquid was removed by filter paper. After drying at room temperature for 5 minutes, electron microscopy was performed at 80 kV to obtain transmission electron microscopy imaging results, which are as follows: Figure 1 , Figure 2 As shown in the figure, the obtained lily extracellular vesicles appear as cup-shaped vesicle-like structures under electron microscope.

[0059] (2) Particle size analyzer to measure particle size and concentration: 1 μL of lily extracellular vesicles from Example 1 and Example 2 were respectively taken and diluted with 999 μL of PBS. The diluted lily extracellular vesicle samples were taken with a new needle using the PARTICLE METRIX PMX120 instrument for NTA detection. The results were as follows: Figure 3 , Figure 4The size of the extracellular vesicles extracted from fresh lily in Example 1 is about 234.1 nm, and the concentration is 2.4×10 10 Particles / mL; The size of the extracellular vesicles extracted from lily stem in Example 2 was about 180.7 nm, and the concentration was 5.5×10 11 Particles / mL, which are consistent with the general size of plant-derived extracellular vesicles.

[0060] (3) Protein concentration determination: The protein was extracted by conventional total cell protein extraction method, and the protein concentration of the extract was determined by BCA method. The total protein concentration of the extracellular vesicles of fresh lily prepared in Example 1 was 2.208×10 -12 mg / Particles. The total protein concentration of the extracellular vesicles of lily stem cells prepared in Example 2 was 0.826×10 -12 Combined with the concentration of lily extracellular vesicle nanoparticles, it can be found that the lily extracellular vesicles obtained by this preparation method have a high purity.

[0061] The morphology, particle size distribution and protein concentration of the lily extracellular vesicles obtained in Example 1 and Example 2 were tested and the results are summarized in Table 1.

[0062] Table 1

[0063] Example 4 Experiment on uptake of lily extracellular vesicles by macrophages.

[0064] In order to detect the uptake of lily-derived extracellular vesicles, 1×10 11 The extracellular vesicles prepared in Example 1 were labeled in PBS and stained for 20 minutes at room temperature in the dark. After labeling, unbound free dye was removed by ultracentrifugation (120,000 g, 90 minutes). The labeled extracellular vesicles were resuspended in cell culture medium and treated with macrophages at a concentration of 10 μg of extracellular vesicles per well. Macrophages were cultured at 5×10 4 The cells were seeded at a density of 10 cells / well in a 48-well plate and incubated at 37°C and 5% CO2 until the cells adhered to the wall. After adding fluorescently labeled extracellular vesicles, the incubation continued for 8 hours. At the end of the incubation, the cells were washed three times with PBS to remove the uningested extracellular vesicles. Next, the cells were fixed with 4% paraformaldehyde and the cytoskeleton was stained with phalloidin. Finally, the distribution of intracellular fluorescence signals was observed and recorded using a confocal microscope to analyze the uptake of extracellular vesicles in detail. The results are shown in Figure 2. Figure 5 As shown, it can be seen that the lily extracellular vesicles prepared in Example 1 can be phagocytosed by macrophages.

[0065] Example 5 Evaluation of cytotoxicity of extracellular vesicles from Lilium spp.

[0066] Using mouse mononuclear macrophage RAW264.7 cells and bone marrow mesenchymal stem cells BMSCs as models, the cytotoxicity of the lily extracellular vesicles prepared in Example 1 and Example 2 was evaluated. Mouse mononuclear macrophage RAW264.7 cells and bone marrow mesenchymal stem cells BMSCs were prepared into 5×10 5 / mL and 5×10 4 / mL of cell suspension. Take a 96-well plate, plant 100 μL of cell suspension in each well and place it in a carbon dioxide incubator, and culture it overnight at 5% CO2 concentration and 37°C temperature. After removing the culture medium the next day, add 100 mL of lily extracellular vesicle solution with LEDs concentration gradients of 0 μg / mL, 0.1 μg / mL, 1 μg / mL, and 10 μg / mL prepared with DMEM culture medium. Refer to the instructions of the CCK-8 kit (Cell Proliferation and Toxicity Detection Kit, Beyotime, C0037) to detect cell activity, add 10 μL of CCK-8 reagent to each well, incubate at room temperature away from light for 1 hour, and measure the absorbance of each well in the two groups at a wavelength of 450 nm. The results are as follows Figure 6 As shown, it can be seen that 0-10 μg / mL lily exosomes prepared in Example 1 and Example 2 have no inhibitory effect on the activity of RAW264.7 cells and BMSCs cells.

[0067] Example 6 Characterization of lily extracellular vesicles promoting polarization of M2 macrophages to M1 macrophages.

[0068] (1) Immunofluorescence: First, RAW264.7 macrophages were cultured at 5 × 10 4 / cm 2 The cells were seeded at a density of 100 μg / mL in a well plate, and after 6 hours of attachment, they were treated with 20 ng / mL IL-4 for 24 hours to polarize the macrophages to M2 type. Subsequently, 10 μg / mL of lily extracellular vesicles prepared in Example 1 and Example 2 were added to the experimental groups, respectively, and incubated for 36 hours to identify the effect of lily extracellular vesicles on the phenotype of macrophages.

[0069] (2) Enzyme-linked immunosorbent assay (ELISA) TNF-a and CCL17 represent two different phenotypes of macrophages, M1 and M2, respectively. In the tumor microenvironment, M2 macrophages are believed to have a tumor-promoting effect, while M1 macrophages have a tumor-suppressing effect. Therefore, changing the phenotype of TAMs from M2 to M1 is currently an important anti-cancer strategy.

[0070] The expression levels of TNF-α and CCL17 in co-incubated macrophages in (1) were detected according to the instructions of the Mouse TNF-α ELISA kit (Invitrogen, 88-7324-88Y529-05) and the Mouse CCL17 ELISA kit (R&D, DY529-05). Each group of experiments was tested four times in parallel, and the average value and standard deviation were calculated.

[0071] The above ELISA method was used to detect the effects of the lily extracellular vesicles obtained in Example 1 and Example 2 on the expression of TNF-α and CCL17 in macrophages. The results are summarized in Figure 7 , Figure 8 and Table 2.

[0072] Table 2

[0073] From the data in Table 2, it can be seen that after adding the extracellular vesicles provided in Example 1, the expression of macrophage TNF-α can be significantly increased while the expression of CCL17 can be reduced, indicating that the lily extracellular vesicles provided in Example 1 can promote the polarization of macrophages to M1 type, thereby inhibiting tumors. After adding the lily extracellular vesicles provided in Example 2, although the expression of macrophage TNF-α and CCL17 was increased at the same time, the increase in TNF-α reached more than 130 times, while the increase in CCL17 was only 4.94 times, and the increase in TNF-α was significantly higher than that of CCL17, which indicates that the effect of macrophage polarization to M2 type is significantly lower than that to M1 type. It can be seen that the extracellular vesicles provided in Example 2 can also inhibit tumors.

[0074] (3) Flow cytometry First, macrophages treated with lily extracellular vesicles provided in Example 1 were collected using a cell scraper and fixed with 4% paraformaldehyde at room temperature for 15 minutes. After fixation, the cells were washed with PBS and filtered through a 100 μM cell sieve. Subsequently, the cells were dispensed into flow cytometer tubes, and 5 μL of CD206 fluorescently labeled antibody (Elabscience, E-AB-F1135H) was added to each tube according to the dosage recommended in the instructions, and incubated at 4 ° C in the dark for 30 minutes. Finally, the cells were washed with PBS, and the fluorescence signal was then analyzed by flow cytometry to evaluate the effect of extracellular vesicles on the macrophage surface marker CD206.

[0075] The results are as follows Fig. 9 As shown. The CD206 expression level of M2 macrophages not treated with the lily extracellular vesicles provided in Example 1 was 93.27%; while the CD206 expression level of M2 macrophages treated with the lily extracellular vesicles provided in Example 1 was significantly reduced to 80.43%. These results show that lily-derived extracellular vesicles can effectively induce M2 macrophages to polarize to M1, demonstrating its application value in tumor treatment.

[0076] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A use of lily extracellular vesicles in the preparation of tumor therapeutic drugs, characterized in that: The preparation method of lily extracellular vesicles comprises the following steps: S1, squeezing the lily bulb tissue and filtering to obtain the lily bulb stock solution; S2, treating the lily bulb stock solution obtained in step S1 to obtain a lily extracellular vesicle extract; S3, concentrating and purifying the lily extracellular vesicle extract obtained in step S2 to obtain lily extracellular vesicles; Wherein, in step S2, the processing includes centrifugation and membrane filtration.

2. The use according to claim 1, characterized in that: The effective ingredients of the tumor treatment drug include the lily extracellular vesicles.

3. The use according to claim 2, characterized in that: The active ingredient of the tumor treatment drug is the lily extracellular vesicles.

4. The use according to claim 1, characterized in that: The tumor therapeutic drug is a tumor immunotherapy drug.

5. The use according to claim 4, characterized in that: The tumor immunotherapy drug is a tumor region macrophage activation immunotherapy drug.

6. The use according to claim 5, characterized in that: The tumor region macrophage activation immunotherapy drug is a tumor immunotherapy drug that regulates the transformation of tumor region macrophages from M2 type to M1 type.

7. The use according to claim 1, characterized in that: In step S1, the lily bulb tissue is selected from at least one of Lanzhou lily dry bulb tissue and fresh Lanzhou lily bulb tissue.

8. The use according to claim 1, characterized in that: In step S2, the treatment further includes enzymatic hydrolysis.

9. The use according to claim 1, characterized in that: In step S3, the concentration and purification includes at least one of concentration and purification using an ultrafiltration tube and concentration and purification using a chromatographic column.

10. The tumor therapeutic drug prepared by the use according to any one of claims 1 to 9.

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