Application of camellia nitidissima exosome-like nano-vesicles in preparation of antitumor drugs

Abstract

The invention discloses application of camellia nitidissima exosome-like nano-vesicles in preparation of antitumor drugs, and belongs to the technical field of biological medicines. The exosome-like nano-vesicles are extracted from golden camellia for the first time, and the exosome-like nano-vesicles are proved to have remarkable anti-tumor activity.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and particularly to the application of exosome-like nanovesicles of Camellia nitidissima in the preparation of anti-tumor drugs. Background Art

[0002] Tumor is one of the major diseases seriously threatening human health, with high incidence and mortality rates. Currently, the main treatment methods for tumors include surgery, radiotherapy, chemotherapy, etc., but these methods generally have problems such as large side effects, easy recurrence, and drug resistance, and it is difficult to meet the needs of clinical treatment. Therefore, the development of new anti-tumor strategies with high efficiency and low toxicity has become the focus of current research.

[0003] Camellia nitidissima C.W.Chi is a plant of the genus Camellia in the family Theaceae. Because it is rich in various bioactive components (such as polyphenols, flavonoids, saponins, etc.), it has been proven to have significant pharmacological effects such as anti-tumor, antioxidant, anti-inflammatory, and immunomodulatory effects. However, current research on Camellia nitidissima mainly focuses on the chemical composition and biological activities of its extracts, and there is no research on its exosome-like nanovesicles. Summary of the Invention

[0004] The main purpose of this application is to provide the application of exosome-like nanovesicles of Camellia nitidissima in the preparation of anti-tumor drugs, extract exosome-like nanovesicles from Camellia nitidissima for the first time, and verify their anti-tumor activity.

[0005] To achieve the above purpose, this application provides the application of exosome-like nanovesicles of Camellia nitidissima in the preparation of anti-tumor drugs.

[0006] In some embodiments, the method for preparing the exosome-like nanovesicles of Camellia nitidissima includes the following steps:

[0007] Wash and crush fresh Camellia nitidissima leaves, then homogenize and perform ultra-high speed centrifugation to extract exosome-like nanovesicle precipitate;

[0008] Perform sucrose density gradient ultracentrifugation on the exosome-like nanovesicle precipitate to obtain purified exosome-like nanovesicles of Camellia nitidissima.

[0009] In some embodiments, the extraction of exosome-like nanovesicle precipitate by ultra-high speed centrifugation includes:

[0010] Take the homogenate obtained by homogenization, centrifuge at 2000g - 4000g for 10 minutes - 30 minutes, and take the upper first supernatant;

[0011] Centrifuge the first supernatant at 10000g - 15000g for 30 minutes - 60 minutes, and take the upper second supernatant;

[0012] Centrifuge the second supernatant at a condition of not less than 100,000 g for 60 minutes to 120 minutes, discard the supernatant, and obtain the exosome-like nanovesicle precipitate.

[0013] In some embodiments, the sucrose density gradient ultracentrifugation of the exosome-like nanovesicle precipitate includes:

[0014] Resuspend the exosome-like nanovesicle precipitate with PBS buffer, use 30%, 45%, 60% sucrose density gradient, centrifuge at a condition of not less than 100,000 g for 60 minutes to 120 minutes, and collect the substance at the 30%-45% sucrose interface, which is the purified exosome-like nanovesicles of Camellia nitidissima.

[0015] In some embodiments, the tumor includes lung cancer cells, breast cancer cells, colorectal cancer cells or gastric cancer cells.

[0016] In some embodiments, the application of the exosome-like nanovesicles of Camellia nitidissima as a tumor cell proliferation inhibitor in an anti-tumor drug.

[0017] In some embodiments, the application of the exosome-like nanovesicles of Camellia nitidissima as a tumor cell migration inhibitor and / or a tumor cell invasion inhibitor in an anti-tumor drug.

[0018] In some embodiments, the application of the exosome-like nanovesicles of Camellia nitidissima as a tumor cell apoptosis inducer in an anti-tumor drug.

[0019] In some embodiments, the application of the exosome-like nanovesicles of Camellia nitidissima as a tumor cell cycle blocker in an anti-tumor drug.

[0020] In some embodiments, the exosome-like nanovesicles of Camellia nitidissima are in the shape of a saucer, have a bilayer membrane structure, and the particle size is 30 nm - 200 nm.

[0021] In the embodiments of the present application, exosome-like nanovesicles are extracted from Camellia nitidissima for the first time, and it is confirmed that they have significant anti-tumor activity, can inhibit the proliferation of various tumor cells, induce cell apoptosis, block the cell cycle, increase the intracellular ROS level, and inhibit the migration and invasion of tumor cells, providing new ideas and methods for tumor treatment and having important application prospects. Description of the Drawings

[0022] To more clearly illustrate the technical solutions in the embodiments of the present drawings or the related art, the following will briefly introduce the drawings required for use in the description of the embodiments or the related art. Obviously, the drawings in the following description are only some embodiments of the present drawings. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0023] Figure 1 Schematic diagram of the extraction process of a kind of camellia nitidissima exosome-like nanovesicles provided by the embodiment of the present application;

[0024] Figure 2 Transmission electron microscope photographs and particle size distribution diagrams of camellia nitidissima exosome-like nanovesicles before and after purification provided by the embodiment of the present application;

[0025] Figure 3 Test result diagram of the inhibitory effect of camellia nitidissima exosome-like nanovesicles on tumor cell proliferation detected by the CCK-8 experiment provided by the embodiment of the present application;

[0026] Figure 4 Test result diagram of observing the endocytosis of camellia nitidissima exosome-like nanovesicles by tumor cells in the DiO exosome tracing experiment provided by the embodiment of the present application;

[0027] Figure 5 Test result diagram of the apoptosis effect of camellia nitidissima exosome-like nanovesicles on tumor cells detected by flow cytometry provided by the embodiment of the present application;

[0028] Figure 6 Test result diagram of the effect of camellia nitidissima exosome-like nanovesicles on the cell cycle of tumor cells detected by flow cytometry provided by the embodiment of the present application;

[0029] Figure 7 Test result diagram of the effect of camellia nitidissima exosome-like nanovesicles on the ROS level of tumor cells detected by flow cytometry provided by the embodiment of the present application;

[0030] Figure 8 Test result diagram of the effect of camellia nitidissima exosome-like nanovesicles on the migration ability of tumor cells detected by the scratch experiment provided by the embodiment of the present application;

[0031] Figure 9 Test result diagram of the effect of camellia nitidissima exosome-like nanovesicles on the migration ability of tumor cells detected by the Transwell experiment provided by the embodiment of the present application;

[0032] Figure 10 Test result diagram of the effect of camellia nitidissima exosome-like nanovesicles on the invasion ability of tumor cells detected by the Transwell experiment provided by the embodiment of the present application.

[0033] The realization, functional features, and advantages of the present application will be further described in conjunction with embodiments with reference to the accompanying drawings. Detailed Embodiments

[0034] Hereinafter, the embodiments of the application of the camellia nitidissima exosome-like nanovesicles disclosed in the present application in the preparation of anti-tumor drugs will be specifically and detailedly described with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary details are omitted. For example, there are cases where the detailed description of well-known matters and the repeated description of actually identical structures are omitted. This is to prevent the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0035] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundary of a specific range. Unless otherwise specified, the range defined in this way can include the end values or not include the end values, and can be combined arbitrarily, that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 - 120 and 80 - 110 are listed for a specific parameter, ranges of 60 - 110 and 80 - 120 are also contemplated. In addition, if the minimum range values 1 and 2 are listed, and if the maximum range values 3, 4, and 5 are listed, then the following ranges are all contemplated: 1 - 3, 1 - 4, 1 - 5, 2 - 3, 2 - 4, and 2 - 5. In the present application, unless otherwise stated, the numerical range "a - b" represents an abbreviated representation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0 - 5" means that all real numbers between "0 - 5" have been fully listed herein, and "0 - 5" is only an abbreviated representation of these numerical combinations. Additionally, when stating that a certain parameter is an integer ≥2, it is equivalent to disclosing that the parameter is, for example, the integer 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0036] If there is no special instruction, all embodiments and alternative embodiments of the present application can be combined with each other to form new technical solutions.

[0037] To make the above objects, features, and advantages of the present application more apparent and understandable, the technical solutions of the present application will be further described below in conjunction with the accompanying drawings and embodiments. However, the present application is not limited to the listed embodiments and should also include any other well-known changes within the scope of the rights required by the present application.

[0038] In recent years, exosomes, as a novel intercellular communication carrier, have shown great potential in the field of tumor diagnosis and treatment. Exosomes are nanoscale vesicles secreted by cells, which are rich in various bioactive substances such as proteins, nucleic acids, and lipids. They can participate in the regulation of processes such as the proliferation, apoptosis, migration, invasion, and immune escape of tumor cells by mediating intercellular signal transduction. In particular, exosome-like nanovesicles (ELNs) derived from plants have gradually become a research hotspot in anti-tumor research due to their advantages such as natural origin, high biocompatibility, low toxicity, and easy large-scale preparation. Research has shown that plant exosome-like nanovesicles can not only directly inhibit the growth of tumor cells but also play a synergistic anti-tumor role by regulating the tumor microenvironment and enhancing the body's immune response, providing new ideas for reducing the side effects of traditional treatments and developing safer and more effective tumor treatment strategies.

[0039] Camellia nitidissima is a plant of the genus Camellia in the family Theaceae. It has been proven to have significant pharmacological effects such as anti-tumor, antioxidant, anti-inflammatory, and immunomodulatory effects due to its rich bioactive components (such as polyphenols, flavonoids, saponins, etc.). However, current research on Camellia nitidissima mainly focuses on the chemical composition and biological activities of its extracts, and there is no report on the extraction, identification, and anti-tumor effects of its exosome-like nanovesicles. Therefore, exploring the anti-tumor activity and mechanism of action of exosome-like nanovesicles from Camellia nitidissima not only helps to expand the application of plant exosome-like nanovesicles in the field of tumor treatment but also provides important theoretical basis and practical value for the development of new anti-tumor drugs based on natural products.

[0040] The embodiments of this application provide the application of exosome-like nanovesicles from Camellia nitidissima in the preparation of anti-tumor drugs. Exosome-like nanovesicles were first extracted from Camellia nitidissima and proven to have significant anti-tumor activity. They can inhibit the proliferation of various tumor cells, induce apoptosis, arrest the cell cycle, increase the intracellular ROS level, and inhibit the migration and invasion of tumor cells, providing new ideas and methods for tumor treatment and having important application prospects.

[0041] In some embodiments, the preparation method of exosome-like nanovesicles from Camellia nitidissima includes the following steps S10 to S20:

[0042] Step S10, after washing and crushing fresh Camellia nitidissima leaves, homogenize them and perform ultra-high-speed centrifugation to extract exosome-like nanovesicle precipitate;

[0043] The flowers and leaves of Camellia nitidissima contain various substances beneficial to human health. In the embodiments of this application, fresh leaves of Camellia nitidissima are used as raw materials. The fresh leaves of Camellia nitidissima are washed with sterile water, crushed, and then added to PBS buffer for homogenization in an ice bath to obtain a homogenate. The homogenate is ultracentrifuged to extract the precipitate of exosome-like nanovesicles. Ultracentrifugation has a high rotation speed and a centrifugal force that can reach hundreds of thousands of times the acceleration of gravity, and can be used for the extraction of exosome-like nanovesicles in the embodiments of this application.

[0044] In some embodiments, the extraction of exosome-like nanovesicles by ultracentrifugation includes the following steps S11 to S13:

[0045] Step S11: Take the homogenate obtained from homogenization, centrifuge it at 2000g - 4000g for 10 minutes - 30 minutes, and take the upper first supernatant.

[0046] Step S12: Centrifuge the first supernatant at 10000g - 15000g for 30 minutes - 60 minutes, and take the upper second supernatant.

[0047] Step S13: Centrifuge the second supernatant at no less than 100000g for 60 minutes - 120 minutes, discard the supernatant, and obtain the precipitate of exosome-like nanovesicles.

[0048] In the embodiments of this application, staged centrifugation is carried out with different centrifugal forces to gradually separate substances with different sedimentation coefficients, and finally obtain the precipitate of exosome-like nanovesicles. First, centrifuge at a relatively small centrifugal force of 2000g - 4000g, and take the first supernatant after centrifugation. Centrifuge the first supernatant at a medium centrifugal force of 10000g - 15000g, and take the second supernatant after centrifugation again. Finally, centrifuge at a large centrifugal force of no less than 100000g, discard the upper supernatant, and obtain the precipitate of exosome-like nanovesicles at the bottom.

[0049] Step S20: Perform sucrose gradient ultracentrifugation on the precipitate of exosome-like nanovesicles to obtain purified exosome-like nanovesicles of Camellia nitidissima.

[0050] After obtaining the precipitate of exosome-like nanovesicles, it can also be purified to improve its homogeneity and purity. In the embodiments of this application, the method of sucrose gradient ultracentrifugation is used for purification, with sucrose as the density gradient solvent. The density gradient centrifugation method can enable each substance particle to balance in the corresponding solvent density according to its respective specific gravity to form a zone after centrifugation.

[0051] In some embodiments, the sucrose gradient ultracentrifugation of exosome-like nanovesicle precipitate includes the following steps: resuspend the exosome-like nanovesicle precipitate with PBS buffer, use 30%, 45%, 60% sucrose density gradient, centrifuge at no less than 100,000g for 60 minutes to 120 minutes, and collect the substance at the 30%-45% sucrose interface, which is the purified exosome-like nanovesicles of Camellia nitidissima.

[0052] In some embodiments, referring to Figure 1 , Figure 1 is a schematic diagram of the extraction process of exosome-like nanovesicles of Camellia nitidissima provided in the embodiments of the present application. Figure 1 shows the extraction and purification process of exosome-like nanovesicles of Camellia nitidissima. First, wash, crush and homogenize fresh Camellia nitidissima leaves, centrifuge at 3000g for 10 minutes to 30 minutes to remove residual tissues or cells, and take the first supernatant; the first supernatant is then centrifuged at 10,000g for 30 minutes to 60 minutes to remove large particle impurities such as cell debris, and take the second supernatant; then the second supernatant is ultracentrifuged at no less than 100,000g for 60 minutes to 120 minutes to obtain the exosome-like nanovesicle precipitate. After the obtained exosome-like nanovesicle precipitate is resuspended with PBS buffer, sucrose gradient ultracentrifugation (using 30%, 45%, 60% sucrose density gradient) is carried out, and it is centrifuged again at no less than 100,000g for 60 minutes to 120 minutes, and the purified exosome-like nanovesicles of Camellia nitidissima at the 30%-45% sucrose interface are collected.

[0053] In some embodiments, the preparation raw materials of the anti-tumor drug further include a pharmaceutical carrier.

[0054] In some embodiments, the pharmaceutical carrier includes at least one of a diluent, an excipient, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetening agent and a flavoring agent.

[0055] In some embodiments, the excipient includes at least one of lactose, polyethylene glycol, sodium citrate, dicalcium phosphate and water.

[0056] In some embodiments, the filler includes at least one of starch, lactose, sucrose, glucose, mannitol and silicic acid.

[0057] In some embodiments, the binder includes at least one of cellulose derivatives, alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic.

[0058] In some embodiments, the cellulose derivative includes carboxymethyl cellulose.

[0059] In some embodiments, the alginate includes alginate.

[0060] In some embodiments, the humectant comprises glycerol.

[0061] In some embodiments, the disintegrant comprises at least one of agar, calcium carbonate, sodium bicarbonate, potato starch, tapioca starch, alginic acid, silicate, and sodium carbonate.

[0062] In some embodiments, the absorption enhancer comprises a quaternary ammonium compound.

[0063] In some embodiments, the surfactant comprises cetyl alcohol.

[0064] In some embodiments, the adsorption carrier comprises at least one of kaolin, bentonite, and saponite.

[0065] In some embodiments, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate, sodium lauryl sulfate, and polyethylene glycol.

[0066] In some embodiments, the dosage form of the drug is various conventional dosage forms in the art.

[0067] In some embodiments, the tumor includes lung cancer cells, breast cancer cells, colorectal cancer cells, or gastric cancer cells.

[0068] In some embodiments, the application of the camellia nitidissima exosome-like nanovesicles as a tumor cell proliferation inhibitor in an anti-tumor drug.

[0069] In some embodiments, the application of the camellia nitidissima exosome-like nanovesicles as a tumor cell migration inhibitor and / or a tumor cell invasion inhibitor in an anti-tumor drug.

[0070] In some embodiments, the application of the camellia nitidissima exosome-like nanovesicles as a tumor cell apoptosis inducer in an anti-tumor drug.

[0071] In some embodiments, the application of the camellia nitidissima exosome-like nanovesicles as a tumor cell cycle blocker in an anti-tumor drug.

[0072] Example 1: Extraction and identification of camellia nitidissima exosome-like nanovesicles.

[0073] 1. Extraction of camellia nitidissima exosome-like nanovesicles:

[0074] 1.1 Preliminary centrifugation: Take 500 g of fresh camellia nitidissima leaves, wash them clean with sterile water, cut them into pieces, add 300 mL of PBS buffer, homogenize in an ice bath to obtain a homogenate. Centrifuge the homogenate at 3000 g for 10 minutes to 30 minutes, take the first supernatant, and remove the remaining tissues or cells.

[0075] 1.2 Further centrifugation: Centrifuge the first supernatant at 10,000 g for 30 to 60 minutes to remove large particulate impurities such as cell debris, and take the second supernatant.

[0076] 1.3 Ultra-high speed centrifugation: Centrifuge the second supernatant at no less than 100,000 g for 60 to 120 minutes, discard the supernatant, and obtain the exosome-like nanovesicle precipitate.

[0077] 1.4 Purification by sucrose density gradient ultracentrifugation: Resuspend the exosome-like nanovesicle precipitate with PBS buffer and perform sucrose density gradient ultracentrifugation. Use 30%, 45%, and 60% sucrose density (W / V) gradients and centrifuge again at no less than 100,000 g for 60 to 120 minutes, and collect the purified camellia nitidissima exosome-like nanovesicles at the 30% - 45% sucrose interface.

[0078] 2. Identification of camellia nitidissima exosome-like nanovesicles:

[0079] 2.1 Transmission electron microscopy observation: Take 10 μL of camellia nitidissima exosome-like nanovesicles, drop it on a copper grid and let it precipitate for 1 min, then use filter paper to absorb the floating liquid; Pipette 15 μL of 2% staining agent uranyl acetate and drop it on the copper grid to precipitate for 1 min, use filter paper to absorb the floating liquid, drop deionized water, and immediately dry it to remove the excess staining agent; Dry it at room temperature for several minutes and perform electron microscopy detection and imaging at 100 kV to obtain the transmission electron microscopy imaging results and confirm that it is a typical nanoscale vesicle structure.

[0080] 2.2 Particle size distribution detection: Take 100 μL of camellia nitidissima exosome-like nanovesicles and dilute them to the optimal detection concentration range of the instrument, and use a nanoparticle tracking analyzer (ZetaView) to detect the particle size distribution and particle size range of the camellia nitidissima exosome-like nanovesicles.

[0081] Figure 2 For the transmission electron microscopy photos and particle size distribution diagrams of camellia nitidissima exosome-like nanovesicles before and after purification, transmission electron microscopy (scale bar is 200 nm) was used to observe the camellia nitidissima exosome-like nanovesicles before and after purification by sucrose density gradient ultracentrifugation. Among them, A is the morphology before purification and B is the morphology after purification. The results show that its morphology is in the shape of a tea tray and has a complete bilayer membrane structure. The nanoparticle tracking analyzer was used to detect the particle size of the nanovesicles. The particle size distribution before purification was (151.7 ± 56.4) nm, and after purification it was (87.5 ± 55.9) nm, meeting the condition of a particle size between 30 nm and 200 nm. Compared with before purification, the particle size distribution of the camellia nitidissima exosome-like nanovesicles after purification by sucrose density gradient ultracentrifugation decreased significantly, indicating that this purification method can effectively improve the uniformity and purity of the nanovesicles and shows obvious advantages in the separation and purification process.

[0082] Example 2: Detection of the anti-tumor activity of Camellia nitidissima exosome-like nanovesicles.

[0083] 1. CCK-8 cytotoxicity assay:

[0084] Seed 5000 cells of four types of tumor cells, A549, MDA-MB-231, SW620, and HGC-27, into each well of a 96-well plate. The culture medium is DMEM / F12 (containing 10% fetal bovine serum and 1% penicillin-streptomycin), and incubate for 24 hours. Add Camellia nitidissima exosome-like nanovesicles at different concentrations (0, 50, 100, 150, 200 μg / mL) respectively, and set 3 replicates for each concentration. After continuing to incubate for 12, 24, 36, and 48 hours, add 10 μL of CCK-8 solution to each well and incubate at 37 °C in the dark for 3 hours. Measure the absorbance value at 450 nm using a microplate reader, calculate the cell proliferation inhibition rate, and evaluate the inhibitory effect of Camellia nitidissima exosome-like nanovesicles on tumor cell proliferation.

[0085] 2. DiO exosome tracing experiment:

[0086] 2.1 Label exosome-like nanovesicles with DiO: Prepare a staining working solution according to the DiO dye instruction manual. Mix an equal volume of the DiO staining working solution with Camellia nitidissima exosome-like nanovesicles and incubate at 37 °C in the dark for 30 minutes. Centrifuge to remove unbound dye and resuspend with PBS buffer to obtain DiO-labeled Camellia nitidissima exosome-like nanovesicles.

[0087] 2.2 Co-culture with tumor cells: Seed 20000 cells of four types of tumor cells, A549, MDA-MB-231, SW620, and HGC-27, into each well of a 24-well plate. The culture medium is DMEM / F12 (containing 10% fetal bovine serum and 1% penicillin-streptomycin), and incubate for 24 hours. Add different concentrations of DiO-labeled Camellia nitidissima exosome-like nanovesicles respectively, and set 3 replicates for each concentration. Continue to incubate for 24 and 48 hours.

[0088] 2.3 Staining and photographing: Wash the cells 3 times with PBS, fix the cells with 4% paraformaldehyde for 15 minutes, and stain the cell nuclei with DAPI. Observe the internalization of DiO-labeled exosome-like nanovesicles by tumor cells using a fluorescence microscope and take pictures for recording.

[0089] 3. Flow cytometry detection of cell apoptosis, cell cycle, and ROS level:

[0090] 3.1 Co-culture with tumor cells: Seed 1*10 cells of four types of tumor cells, A549, MDA-MB-231, SW620, and HGC-27, into each well of a 6-well plate. 5Cells were cultured in DMEM / F12 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) for 24 hours. Different concentrations of Camellia nitidissima exosome-like nanovesicles were added, with 3 replicates for each concentration. After continued culture for 24 hours, the cells were collected.

[0091] 3.2 Detection of cell apoptosis: The Annexin V-FITC / PI double staining method was used to detect the apoptosis rate of cells. The cells were resuspended in binding buffer, Annexin V-FITC and PI staining solutions were added, and after incubation in the dark for 15 minutes, the apoptosis of cells was detected using a flow cytometer.

[0092] 3.3 Detection of cell cycle: The PI single staining method was used to detect the cell cycle distribution. After the cells were fixed and treated with 70% ethanol, they were incubated with PI staining solution in the dark for 30 minutes, and the distribution of each phase (G0 / G1, S, G2 / M) of the cell cycle was analyzed using a flow cytometer.

[0093] 3.4 Detection of ROS level: The DCFH-DA fluorescent probe was used to detect the intracellular ROS level. The cells were co-incubated with the DCFH-DA probe for 20 minutes, washed with serum-free medium, and the fluorescence intensity was detected using a flow cytometer to evaluate the change in intracellular ROS level.

[0094] 4. Scratch, migration, and invasion experiments of tumor cells:

[0095] 4.1 Scratch experiment: Four types of tumor cells, A549, MDA-MB-231, SW620, and HGC-27, were seeded in 24-well plates, with 3 replicates for each concentration. After the cells grew to confluence, a straight line was scratched in the cell layer using a 10 μL pipette tip. The scratched cells were removed by washing with PBS, and the medium containing different concentrations of Camellia nitidissima exosome-like nanovesicles was added. After culturing for 24 hours, the healing of the scratch was observed, and data analysis was performed using Image J software.

[0096] 4.2 Transwell migration experiment: Four types of tumor cells, A549, MDA-MB-231, SW620, and HGC-27, were seeded in 24-well plates with Transwell chambers, with 3 replicates for each concentration. The tumor cells were seeded in the upper chamber of the Transwell, and the medium containing different concentrations of Camellia nitidissima exosome-like nanovesicles was added to the lower chamber. After culturing for 24 and 48 hours, the non-migrated cells in the upper chamber were wiped off with a cotton swab, the cells in the lower chamber were fixed with 4% paraformaldehyde, stained with crystal violet, and the migration of tumor cells was observed under a microscope.

[0097] Transwell invasion experiment: Matrigel matrix gel was coated on the upper chamber of the Transwell, and the remaining steps were the same as those in the migration experiment.

[0098] Figure 3 This is a test result graph for detecting the inhibitory effect of Camellia nitidissima exosome-like nanovesicles on tumor cell proliferation by CCK-8 assay. Using four tumor cell lines, A549, MDA-MB-231, SW620, and HGC-27, and based on the protein concentration of Camellia nitidissima exosome-like nanovesicles, different concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, and 200 μg / mL were set for treatment. At multiple time points of 12, 24, 36, and 48 hours, the CCK-8 method was used to detect the inhibition rate of nanovesicles on tumor cells. Figure 3 In it, P represents the degree of significant difference. The results show that at different action concentrations and action times, the inhibitory ability of Camellia nitidissima exosome-like nanovesicles on tumor cells is significantly different and shows time- and dose-dependence. After 24 hours of treatment, the inhibition rates of three tumor cells, A549, MDA-MB-231, and SW620, exceeded 90% at a concentration of 150 μg / mL; the inhibition rate of HGC-27 exceeded 90% at a concentration of 100 μg / mL.

[0099] Figure 4 This is a test result graph for observing the endocytosis of Camellia nitidissima exosome-like nanovesicles by tumor cells through DiO exosome tracing experiment. Using four tumor cell lines, A549, MDA-MB-231, SW620, and HGC-27, a control group and a treatment group with Camellia nitidissima exosome-like nanovesicles were set. According to the protein concentration of nanovesicles, 0, 100, and 150 μg / mL of DiO-labeled nanovesicles were added to the A549, MDA-MB-231, and SW620 groups respectively, and 0, 50, and 100 μg / mL of DiO-labeled nanovesicles were added to the HGC-27 group. Photos were taken under a fluorescence microscope at time points of 24 hours and 48 hours. Figure 4 The results in it show that with the prolongation of time and the increase of concentration, the absorption amount of nanovesicles by tumor cells increases, indicating that each tumor cell line can endocytose Camellia nitidissima exosome-like nanovesicles, thus producing an effect of inhibiting tumor cells.

[0100] Figure 5 This is a test result graph for detecting the apoptotic effect of Camellia nitidissima exosome-like nanovesicles on tumor cells by flow cytometry. Figure 5 It presents the apoptotic conditions of each cell line detected by flow cytometry after treatment with the control group and treatment groups with different concentrations of Camellia nitidissima exosome-like nanovesicles (the concentrations of the A549, MDA-MB-231, and SW620 groups are 0, 100, and 150 μg / mL, and the concentrations of the HGC-27 group are 0, 50, and 100 μg / mL). The results show that with the increase of treatment concentration, the apoptosis ratio shows a significant upward trend, and the apoptosis ratio of each tumor cell reaches more than 50% at high concentrations.

[0101] Figure 6This is a test result graph for detecting the effect of Camellia nitidissima exosome-like nanovesicles on the cell cycle of tumor cells by flow cytometry. Figure 6 It shows that in each cell line, under the control group and different concentrations of Camellia nitidissima exosome-like nanovesicles treatment groups (concentrations of 0, 100, 150 μg / mL for A549, MDA-MB-231, SW620 groups, and concentrations of 0, 50, 100 μg / mL for HGC-27 group), the cell proportions in each cell cycle phase (G0 / G1 phase, S phase, G2 / M phase) have changed, reflecting that Camellia nitidissima exosome-like nanovesicles have a regulatory effect on the cell cycle process.

[0102] Figure 7 This is a test result graph for detecting the effect of Camellia nitidissima exosome-like nanovesicles on the ROS level of tumor cells by flow cytometry. Figure 7 It shows that in each cell line, under the control group and different concentrations of Camellia nitidissima exosome-like nanovesicles treatment groups (concentrations of 0, 100, 150 μg / mL for A549, MDA-MB-231, SW620 groups, and concentrations of 0, 50, 100 μg / mL for HGC-27 group), the ROS levels in each cell line have increased, indicating that Camellia nitidissima exosome-like nanovesicles can induce the production of ROS in tumor cells, thereby playing an inhibitory role on tumor cells.

[0103] Figures 5 to 7 These results comprehensively show that Camellia nitidissima exosome-like nanovesicles can affect different tumor cell lines through multiple pathways such as inducing apoptosis, regulating the cell cycle, and increasing the ROS level.

[0104] Figure 8 This is a test result graph for detecting the effect of Camellia nitidissima exosome-like nanovesicles on the migration ability of tumor cells by scratch assay. Figure 8 It shows the cell scratch assay images of the control group and different concentrations of Camellia nitidissima exosome-like nanovesicles treatment groups (concentrations of 0, 100, 150 μg / mL for A549, MDA-MB-231, SW620 groups, and concentrations of 0, 50, 100 μg / mL for HGC-27 group) at different time points (0 h and 24 h). By comparing the results at 0 h and 24 h, it can be seen that compared with the control group, the cells treated with Camellia nitidissima exosome-like nanovesicles showed a significant inhibitory effect on the healing of scratched cells at low doses. After quantitative analysis, the data show that Camellia nitidissima exosome-like nanovesicles have a significant inhibitory effect on the migration ability of different tumor cell lines.

[0105] Figure 9 This is a test result graph for detecting the effect of Camellia nitidissima exosome-like nanovesicles on the migration ability of tumor cells by Transwell assay. Figure 9The stained images of tumor cell migration experiments at 24 h and 48 h time points of four tumor cell lines in the control group and the treatment groups with different concentrations of Camellia nitidissima exosome-like nanovesicles (concentrations of 0, 100, 150 μg / mL for A549, MDA-MB-231, SW620 groups, and concentrations of 0, 50, 100 μg / mL for HGC-27 group) are respectively shown. Compared with the control group, the number of cells migrating to the back of the Transwell chamber within the same time is significantly reduced in the cells treated with Camellia nitidissima exosome-like nanovesicles. This indicates that Camellia nitidissima exosome-like nanovesicles can significantly inhibit the migration ability of each tumor cell line, and the inhibitory effect is more obvious with the extension of the treatment time and the increase of the concentration.

[0106] Figure 10 The figure shows the test results of the effect of Camellia nitidissima exosome-like nanovesicles on the invasion ability of tumor cells detected by Transwell experiment. Figure 10 The invasion of tumor cells at 24 h and 48 h time points of each cell line in the control group and the treatment groups with different concentrations of Camellia nitidissima exosome-like nanovesicles (concentrations of 0, 100, 150 μg / mL for A549, MDA-MB-231, SW620 groups, and concentrations of 0, 50, 100 μg / mL for HGC-27 group) is shown. Compared with the control group, the number of cells passing through the Matrigel is significantly reduced in the cells treated with Camellia nitidissima exosome-like nanovesicles. This indicates that Camellia nitidissima exosome-like nanovesicles have an obvious inhibitory effect on the invasion of tumor cells.

[0107] It should be noted that this application is not limited to the above embodiments. The above embodiments are only examples, and the embodiments with the same structure and the same effect as the technical idea within the technical solution scope of this application are all included in the technical scope of this application. In addition, within the scope not departing from the main idea of this application, various deformations that those skilled in the art can think of imposed on the embodiments, and other ways constructed by combining some constituent elements of the embodiments are also included in the scope of this application.

Claims

1. Application of Camellia nitidissima exosome-like nanovesicles in the preparation of anti-tumor drugs.

2. The application according to claim 1, characterized in that The preparation method of the Camellia nitidissima exosome-like nanovesicles comprises the following steps: After washing and crushing fresh Camellia nitidissima leaves, homogenize them and perform ultra-high speed centrifugation to extract exosome-like nanovesicle precipitate; Perform sucrose density gradient ultra-high speed centrifugation on the exosome-like nanovesicle precipitate to obtain purified Camellia nitidissima exosome-like nanovesicles.

3. The application according to claim 2, wherein The extraction of exosome-like nanovesicle precipitate by ultra-high speed centrifugation includes: Take the homogenate obtained from homogenization, centrifuge it at 2000g - 4000g for 10 minutes - 30 minutes, and take the upper first supernatant; Centrifuge the first supernatant at 10000g - 15000g for 30 minutes - 60 minutes, and take the upper second supernatant; Centrifuge the second supernatant at no less than 100000g for 60 minutes - 120 minutes, discard the supernatant, and obtain exosome-like nanovesicle precipitate.

4. The application according to claim 2, characterized in that The sucrose density gradient ultra-high speed centrifugation on the exosome-like nanovesicle precipitate includes: Resuspend the exosome-like nanovesicle precipitate with PBS buffer, use 30%, 45%, 60% sucrose density gradient, centrifuge it at no less than 100000g for 60 minutes - 120 minutes, and collect the substance at the 30% - 45% sucrose interface, which is the purified Camellia nitidissima exosome-like nanovesicles.

5. The application according to claim 1, wherein The tumors include lung cancer cells, breast cancer cells, colorectal cancer cells or gastric cancer cells.

6. The application according to claim 1, characterized in that, The application of the Camellia nitidissima exosome-like nanovesicles as a tumor cell proliferation inhibitor in anti-tumor drugs.

7. The application according to claim 1, characterized in that, The application of the Camellia nitidissima exosome-like nanovesicles as a tumor cell migration inhibitor and / or a tumor cell invasion inhibitor in anti-tumor drugs.

8. The application according to claim 1, characterized in that, The application of the Camellia nitidissima exosome-like nanovesicles as a tumor cell apoptosis inducer in anti-tumor drugs.

9. The application according to claim 1, wherein The application of the Camellia nitidissima exosome-like nanovesicles as a tumor cell cycle blocker in anti-tumor drugs.

10. The application according to claim 1, wherein The Camellia nitidissima exosome-like nanovesicles are in the shape of a saucer, have a double-membrane structure, and the particle size is 30nm - 200nm.

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