Chinese pulsatilla root and stem exosome-like vesicle as well as preparation method and application thereof

Abstract

The invention relates to Chinese pulsatilla root and stem exosome-like vesicles as well as a preparation method and application thereof. The preparation method comprises the following steps: juicing Chinese pulsatilla roots, filtering, and collecting filtrate; and centrifuging the filtrate, collecting the precipitate, re-suspending, and filtering to obtain a solution containing the exosome-like vesicles of the Chinese pulsatilla roots. Compared with the prior art, the Chinese pulsatilla root and stem exosome-like vesicles extracted by the method can inhibit malignant progression, including proliferation and migration, of lung cancer, and cell uptake and animal living imaging prove that the Chinese pulsatilla root and stem exosome-like vesicles can be taken by cells and can be enriched in tumor tissues at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of Chinese herbal medicines, and in particular to exosome-like vesicles of Pulsatilla striata rhizomes and a preparation method and application thereof. Background Art

[0002] According to the latest data report from the International Agency for Research on Cancer, lung cancer has become the number one killer of cancer-related deaths. Chemotherapy resistance is an important reason for poor prognosis and even death in lung cancer patients. Metabolic changes in lung cancer tissue trigger aerobic glycolysis, which is the key to tumor chemotherapy resistance and malignant progression. Therefore, recoding tumor metabolism has important clinical significance for improving the chemotherapy resistance niche of lung cancer and thus inhibiting the malignant progression of lung cancer.

[0003] Pulsatilla is a plant of the Ranunculaceae family, also known as wild father-in-law, naihe grass, white-headed man, etc. The main medicinal part is the root, which is bitter and cold in nature and enters the stomach and large intestine meridians. The effective ingredients of Pulsatilla are triterpenoid saponins, triterpenoid acids, rhamnose, pulsatillae, 2,3-hydroxybetulinic acid, carrot glycosides and other chemical components. From the perspective of modern pharmacology, Pulsatilla has the effects of clearing away heat and detoxification, cooling blood and stopping dysentery, treating vaginal itching, and clearing dampness and heat in Yangming. It has biological activities such as antibacterial, anti-inflammatory, insecticidal, anti-tumor, sedative, and analgesic, and is widely used in clinical practice. The active ingredients of traditional Chinese medicine often have the problem of easy loss of activity in practical applications, and traditional drugs may cause damage to the body and have safety issues. The existing technology lacks effective Chinese herbal medicines with strong therapeutic effects, which can target and inhibit tumors. There is no report on the relevant research on extracting exosomes from Pulsatilla and using them for anti-tumor treatment. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the above-mentioned prior art and provide exosome-like vesicles from Pulsatilla striata rhizomes and a preparation method and application thereof.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The technical solution of the present invention provides the use of exosome-like vesicles from Pulsatilla striata rhizomes in the preparation of anti-tumor drugs.

[0007] Furthermore, a method for preparing the exosome-like vesicles from the rhizome of Pulsatilla chinensis is provided, comprising the following steps:

[0008] S1. Squeeze juice from the rhizome of Pulsatilla chinensis, filter and collect the filtrate;

[0009] S2. Centrifuge the filtrate obtained in step S1, collect the precipitate, resuspend it, and filter it to obtain a solution containing exosome-like vesicles from the rhizomes of Pulsatilla striata.

[0010] Furthermore, in step S1, the Pulsatilla chinensis rhizome needs to be washed with sterile water before being squeezed and crushed, and mixed with a pre-cooled PBS buffer solution. Preferably, the PBS solution is pre-cooled at 4°C.

[0011] Furthermore, in step S1, the ratio of adding Pulsatilla striata rhizomes to PBS solution is (1-1.2) g: (1-1.2) mL; in some specific embodiments, the ratio of adding Pulsatilla striata rhizomes to PBS solution is preferably 1 g: 1 mL.

[0012] Furthermore, in step S1, the filtration is performed using a 0.45 μm filter membrane.

[0013] Furthermore, in step S2, the filtrate is centrifuged several times at 4°C and 2000-120000 g, with each centrifugation time being 15-135 min.

[0014] Further, in step S2, the filtrate is centrifuged at 4°C and 2000-3000g for 15-20 min to collect supernatant I; supernatant I is centrifuged at 4°C and 10000-20000g for 30-45 min to collect supernatant II; supernatant II is centrifuged at 4°C and 100000-120000g for 90-135 min to collect the precipitate.

[0015] In some specific embodiments, in step S2, preferably: the filtrate is centrifuged at 4°C and 2000g for 15 min to collect supernatant I; supernatant I is centrifuged at 4°C and 10000g for 30 min to collect supernatant II; supernatant II is centrifuged at 4°C and 120000g for 90 min to collect the precipitate.

[0016] In some specific embodiments, in step S2, the precipitate is resuspended in a PBS solution and then filtered using a 0.22 μm filter membrane.

[0017] Furthermore, exosome-like vesicles from Pulsatilla dasyphylla rhizomes are provided, which are obtained based on the preparation method described in the above technical scheme.

[0018] In some specific embodiments, the particle size of the exosome vesicles is 180-220 nm.

[0019] Furthermore, the tumor in the application refers to non-small cell lung cancer cells;

[0020] Furthermore, in the application, Bge-Exo is dispersed and dissolved with physiological saline;

[0021] Furthermore, the Bge-Exo has a targeting effect on tumor cells, can be taken up by cells, and can be enriched in tumor cell tissues.

[0022] Furthermore, the application includes: the application of Bge-Exo as a tumor cell growth and proliferation inhibitor, a tumor cell migration inhibitor and a tumor cell apoptosis promoter in the preparation of anti-tumor drugs.

[0023] Furthermore, when Bge-Exo was used as a growth and proliferation inhibitor / tumor cell migration inhibitor / tumor cell apoptosis promoter to inhibit HCC-1833 cells / A549 cells, the concentration was 100-500 μg / mL; when Bge-Exo was used in a nude mouse model, the injected dose was 135 mg / kg.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The extraction process of exosome-like vesicles Bge-Exo from the rhizomes of Pulsatilla chinensis is simple, safe, environmentally friendly and pollution-free. Moreover, the raw material Pulsatilla chinensis is low-cost and easy to obtain on a large scale, providing sufficient and economical material support for subsequent research and application.

[0026] (2) The Bge-Exo treatment is an innovative extension of traditional Chinese medicine treatment. It has many excellent properties, such as high safety, stability of effective drug ingredients, extremely low toxicity, good solubility and precise targeting. These properties make Bge-Exo show extremely promising application prospects in related treatment fields.

[0027] (3) The preliminary research results of this application show that Bge-Exo can be highly enriched in tumor tissues. At the same time, it has a significant inhibitory effect on the malignant progression of lung cancer, specifically by inhibiting the growth, proliferation and migration ability of tumor cells, blocking the cell cycle process and promoting tumor cell apoptosis.

[0028] (4) The exosome-like vesicles prepared from the rhizome of Pulsatilla chinensis of the present invention can be well integrated into the field of traditional Chinese medicine. It has high safety, stability of effective drug components, low toxicity and good solubility, and these advantages together give it a wide range of application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 To isolate and characterize exosome-like vesicles (Bge-Exo) from the rhizomes of Pulsatilla chinensis;

[0030] Reference numerals:

[0031] (A) Transmission electron microscopy to detect the morphology of Bge-Exo; (B) Bge-Exo particle size analysis results; (C) HCC-1833 and A549 cells uptake of PKH67-labeled Bge-Exo; (D) Schematic diagram of the Bge-Exo preparation process;

[0032] Figure 2The results of the experiment of Bge-Exo inhibiting lung cancer cells and tumor proliferation in vivo in Example 2 are shown in FIG.

[0033] Reference numerals:

[0034] (A) Images of A549 cells and HCC-1833 cells under a microscope in the EdU detection experiment; (B) Statistics of the percentage of EdU-positive labeled cells in A549 cells; (C) Statistics of the percentage of EdU-positive labeled cells in HCC-1833 cells; (D) HCC-1833 cell tumor growth graph; (E) Subcutaneous tumor volume statistics; (F) Tumor volume statistics; (G) Ki67 immunohistochemical staining; (H) Statistical analysis in the Ki67 immunohistochemical staining experiment;

[0035] Figure 3 The results of the experiment of Bge-Exo inhibiting lung cancer cell migration and in vivo transplanted tumor enrichment in Example 3 are shown in FIG.

[0036] Reference numerals:

[0037] (A) Fluorescence inverted microscope image of A549 cell Transwell detection; (B) Statistical graph of migration of A549 cells before and after Bge-Exo treatment; (C) Fluorescence inverted microscope image of HCC-1833 cell Transwell detection; (D) Statistical graph of migration of HCC-1833 cells before and after Bge-Exo treatment; (E) Tail vein injection of HCC-1833 cells, in vivo imaging showing tumor enrichment in vivo; (F) Tumor enrichment and statistical graphs in different groups; (G) HE staining showing changes in tumor number after drug administration; (H) Numerical statistics of tumors in lung tissue;

[0038] Figure 4 This is the result diagram of Bge-Exo inhibiting tumor growth and enriching in tumor tissue in Example 4; PKH67 marked the distribution of drugs in subcutaneous tumors at different time points. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0040] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0041] The concentration of PBS buffer used in all examples of the present application is 0.0067 M, and the pH is 7.4.

[0042] The complete culture medium was DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0043] The paraformaldehyde fixative was 4% (w / v) and purchased from biosharp (BL539A).

[0044] The crystal violet staining solution was 0.1% (w / v) and was purchased from Beyotime (Cat No. C0121-500ml).

[0045] Example 1: Preparation and morphological characterization of exosome-like vesicles from Pulsatilla striata rhizomes (Bge-Exo)

[0046] (I) Preparation of exosome-like vesicles from Pulsatilla chinensis rhizomes (Bge-Exo), specific procedures (such as Figure 1 D) steps are as follows:

[0047] Take 500 g of fresh rhizomes of Pulsatilla chinensis, wash them thoroughly with sterile water, chop them into small pieces, add 500 mL of PBS solution (precooled at 4°C), and use a screw juicer to extract the juice (voltage / frequency / power: 220 V, 50 Hz, 1.2 KW). Collect the juice and filter it with a 0.45 μm filter membrane, then collect the filtrate.

[0048] The filtrate was placed in a 50 mL centrifuge tube and centrifuged at 2000 g for 15 min at 4 °C to collect the supernatant I;

[0049] Place supernatant I in a 50 mL centrifuge tube, centrifuge at 10,000 g for 30 min at 4 °C, and collect supernatant II;

[0050] Place supernatant II in an ultracentrifuge tube, centrifuge at 120,000 g for 90 min at 4°C, and collect the precipitate;

[0051] The precipitate was resuspended in 1 mL of pre-cooled PBS in each tube, and the reaction was repeated three times. The filtrate was filtered through a 0.22 μm filter membrane. The filtrate was the solution containing exosome-like vesicles from the rhizomes of Pulsatilla striata (Bge-Exo), which was stored at 4°C for future use.

[0052] (ii) Morphological characterization of Bge-Exo under transmission electron microscopy, such as Figure 1 As shown in A, Bge-Exo exhibits a double-layer membrane structure.

[0053] (III) Particle size determination results of Bge-Exo, such as Figure 1 As shown in B, the results show that the size of Bge-Exo is around 180-220nm.

[0054] (IV) PKH26 experiment, such as Figure 1 C, the specific steps are as follows:

[0055] ①PKH26 staining experiment:

[0056] PKH26 is a lipophilic fluorescent dye.

[0057] Take 60μL of the exosome solution diluted with phosphate buffered saline (PBS) and gently add it to 0.5mL of diluent C (purchased from Sigma-Aldrich). At the same time, add 4μL of PKH26 dye to 0.5mL of diluent C, then mix it thoroughly with the exosome solution and incubate for 4 minutes. To bind excess dye, add 1mL of 0.5% BSA / PBS. Place the labeled exosomes in a centrifuge and ultracentrifuge at 100,000g for 1 hour. After centrifugation, carefully discard the supernatant and collect the exosome pellet. Subsequently, resuspend the pellet in 100μL of phosphate buffered saline (PBS), gently pipette to mix, and the resulting solution can be used for subsequent uptake experiments.

[0058] ②Intake experiment:

[0059] There are 2 groups:

[0060] Control group: only HCC-1833 cells / A549 cells without exosome treatment.

[0061] Experimental group: HCC-1833 cells / A549 cells + 1 μL PKH26 staining medium (after adding PKH26-stained exosomes to complete medium, filter them with a 0.22 μm filter and then add them to the 6-well plate cells), staining for 24 h.

[0062] The experimental group and the control group were stained with DAPI: 500 μL of DAPI staining solution was added (just enough to cover the bottom of the dish), and incubated at room temperature for 15 minutes to allow the stain to fully combine with the cells; after mixing, the cells were washed 2-3 times with PBS, each time for 3-5 minutes. Finally, the cells were placed under a microscope for observation and photography.

[0063] The results of the photo taken under the mirror are as follows Figure 1 As shown in C, the exosomes stained with PKH26 appear red ( Figure 1 C column 1), cells stained with DAPI appear blue ( Figure 1 C column 2), the blue fluorescence and red fluorescence parts of the cells after uptake are almost overlapped, so the position and color of the fluorescence in the figure can be used to judge whether the exosomes Bge-Exo can be taken up by HCC-1833 cells / A549 cells.

[0064] Example 2: Experiment on inhibition of non-small cell lung cancer cells and tumor proliferation in vivo by Bge-Exo

[0065] The HCC-1833 cell line was derived from tumor tissue from a patient with lung adenocarcinoma. Lung adenocarcinoma is a type of non-small cell lung cancer (NSCLC) and one of the most common subtypes of lung cancer. It is preserved and distributed by the American Type Culture Collection (ATCC). The A549 cell line was derived from lung adenocarcinoma tissue from a 58-year-old white male. It was obtained and established by DJ Giard et al. through resection surgery in 1972 and is also preserved and promoted by ATCC.

[0066] (I) EDU experiment, such as Figure 2 As shown in AC, the experimental groups are: Bge-L (low concentration Bge-Exo treatment group) and Bge-H (high concentration Bge-Exo treatment group); among them, the Bge-L group of A549 cells is 100μg / mL, and the Bge-H group is 200μg / mL; the Bge-L group of HCC-1833 cells is 250μg / mL, and the Bge-H group is 500μg / mL. The control group (NC group) was treated with an equal amount of PBS buffer. The treatment steps refer to the uptake experimental steps provided in Example 1.

[0067] The specific operation steps of the cancer cell proliferation inhibition experiment are as follows:

[0068] ① Take cells in the logarithmic growth phase (including two types of cells: HCC-1833 cells and A549 cells) and add 4×10 5 Cells were seeded in 6-well plates, with 100 μL in each well; 100 μL of culture medium containing 50 μM EdU was added to each well; incubated in a cell culture incubator at 37°C and 5% CO2 for 2 h, the culture medium was discarded, and the cells were washed twice with PBS, each time for 5 min;

[0069] ② Add 1 mL of 4% paraformaldehyde fixative (PBS buffer containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min, discard the fixative, add 1 mL of 2 mg / mL glycine to each well, incubate on a decolorizing shaker (the speed of the decolorizing shaker is set at 40 rpm, the same below) for 5 min, and discard the glycine solution;

[0070] ③ Add 1 mL PBS, wash on a decolorizing shaker for 5 minutes, and discard PBS; add 1 mL 0.5% TritonX-100, incubate on a decolorizing shaker for 10 minutes, and wash with PBS once, each time for 5 minutes;

[0071] ④ Add 500 μL of 1X Apollo staining reaction solution to each well (the Apollo staining solution contains a fluorescently labeled azide, which forms a stable covalent bond through a click chemical reaction with the acetylene group of EdU, thereby labeling proliferating cells). Incubate in the dark at room temperature on a shaker for 30 min, and discard the staining reaction solution. Add 1 mL of permeabilizer and wash 3 times on a shaker, 10 min each time. Discard the permeabilizer, wash 2 times with PBS, 5 min each time. Add 500 μL of 1X Hoechst 33342 reaction solution (1X Hoechst 33342 reaction solution is a commonly used nuclear fluorescent dye that binds to the minor groove of the DNA double helix (AT-rich region) and is used to label the nuclei of live or fixed cells). Incubate in the dark at room temperature on a shaker for 30 min, discard the staining reaction solution, and add 1 mL of PBS to wash 3 times; take pictures and count with a high-content instrument.

[0072] Count and analyze the cells showing blue or green in the field of view respectively. Calculate according to the formula EDU positive (% of cells) = number of green EDU / number of blue DAPI × 100%, and obtain the EDU cell positive ratio and plot it in Figure 2 A - C.

[0073] Figure 2 A - C are as follows: The effects of Bge-L and Bge-H on the proliferation of HCC-1833 and A549 cells were detected by the EDU experiment. *** in the figure indicates 0.0001 < p < 0.001. The results show that the area occupied by the cells with green fluorescence in the control group is significantly larger and the number is more than that in the drug-administered group in the field of view. It can be seen that Bge-L and Bge-H have a significant inhibitory effect on the proliferation of A549 / HCC-1833 cells.

[0074] (II) Subcutaneous tumor implantation

[0075] Select 4-week-old female BALB / c nude mice weighing 18 - 22 g, purchased from Shanghai Jihui Laboratory Animal Breeding Co., Ltd., and adaptively raise them in an SPF environment for 1 week before the experiment.

[0076] After HCC-1833 cells are digested with 0.25% trypsin and centrifuged, they are resuspended with PBS and counted. According to 5 × 10 per nude mouse 5Tumor-bearing was carried out with a quantity and volume of 100 μL. The tumor-bearing site was selected on the lower dorsal skin; the tumor cell suspension was pipetted and mixed well to ensure uniform cell distribution. A 1 mL syringe was used to aspirate the cell tumor suspension and the air in it was expelled, preparing for tumor-bearing; the skin of the nude mouse was gently pinched with the thumb and index finger of the left hand, and the needle tip was slowly and gently inserted about 2 / 3 from the skin clamped by the left thumb with the other hand, and the tumor cell suspension was injected at a constant speed. At this time, a small bulge could be felt between the index finger and thumb of the left hand. After the injection, pause for a few seconds to ensure that the cell suspension fully enters the subcutaneous tissue, and then slowly withdraw the needle. After withdrawal, the left hand continued to pinch the skin for a few seconds to prevent leakage of the cell suspension; The growth of the tumor was observed starting from the 7th day after subcutaneous tumor-bearing. The tumor volume was measured with a vernier caliper every 2 days. The nude mice were sacrificed on the 21st day after tumor-bearing. The tumor volume calculation formula = long side × short side² / 2. The entire experimental process was carried out in a laminar flow hood, strictly following aseptic operating procedures. Pipette tips, syringes, etc. used were all subjected to high-pressure sterilization treatment.

[0077] Figure 2 It is the result graph of the experiment on the inhibition of lung cancer cells and in vivo tumor proliferation by Bge-Exo in Example 2; (D) Tumor growth graph of HCC-1833 cells; (E) Statistical graph of subcutaneous tumor volume; (F) Statistical graph of tumor volume size. *** in the figure indicates 0.0001 < p < 0.001. The results show that Bge-L and Bge-H have a significant inhibitory effect on the proliferation of A549 / HCC-1833 cells in nude mice.

[0078] (III) Ki67 immunohistochemistry

[0079] The tissue obtained in (II) was placed in 4% paraformaldehyde and fixed for 4 hours, dehydrated with 75% alcohol for 1.5 h, 95% alcohol for 1.5 h, 95% alcohol for 1 h, absolute ethanol for 1.5 h, absolute ethanol for 1 h, xylene No. 1 for 0.5 h, and xylene No. 2 for 0.5 h, and then the tissue was embedded. Sections were made and dewaxed and hydrated, an antigen repair solution was prepared for antigen repair. Endogenous peroxidase and biotin were inactivated, serum was blocked, the Ki67 primary antibody was incubated overnight at 4 °C, incubated at room temperature for 30 minutes, washed 2 times with PBS, incubated with the secondary antibody for 10 minutes, developed with DAB for 5 minutes, counterstained with hematoxylin for 1 minute, dehydrated and cleared, and then mounted. Photographs were taken with a fluorescence microscope and counted.

[0080] Figure 2 It is the result graph of the inhibition of the degree of malignant progression of tumors in vivo by Bge-Exo in Example 2; (G) Ki67 immunohistochemical staining; (H) Statistical analysis in the Ki67 immunohistochemical staining experiment. ** in the figure indicates 0.001 < p < 0.01, and *** in the figure indicates 0.0001 < p < 0.001. It can be seen that Bge-L and Bge-H can inhibit the expression of the tumor growth marker protein Ki67 and show a dose-dependent manner.

[0081] Example 3: Experiment on the inhibition of lung cancer cell migration and in vivo xenograft enrichment by Bge-Exo

[0082] (1) Transwell assay

[0083] The experimental groups were: Bge-L (low-concentration Bge-Exo treatment group), Bge-H (high-concentration Bge-Exo treatment group); among them, the Bge-L group of A549 cells was 100 μg / mL, and the Bge-H group was 200 μg / mL; the Bge-L group of HCC-1833 cells was 250 μg / mL, and the Bge-H group was 500 μg / mL. The control group (NC group) was treated with an equal volume of PBS buffer. The treatment steps refer to the uptake experiment steps provided in Example 1.

[0084] Cells (including two types of cells: HCC-1833 cells, A549 cells) were cultured with normal administration until the logarithmic growth phase, digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, washed twice with PBS, suspended with serum-free medium, and seeded at 1×10 5 / mL on the upper chamber of the chamber, 300 μL of 20% complete medium was added to the lower chamber. After 24 h, the medium was discarded, washed twice with PBS, fixed with paraformaldehyde fixative for 30 minutes, washed twice with PBS, stained with crystal violet for 15 minutes, washed thoroughly with PBS, photographed under a microscope and counted.

[0085] Figure 3 A-D are: the effects of Bge-L and Bge-H on the Transwell migration of A549 and HCC-1833 cells (20×) and the quantitative analysis histogram; it can be seen that Bge-L and Bge-H both have inhibitory effects on the migration of A549 and HCC-1833 cells, and show a dose-dependent relationship. In the figure, ## indicates 0.001 < p < 0.01; in the figure, ***, indicate 0.0001 < p < 0.001.

[0086] (2) HE staining

[0087] Place the tissue slices in an in situ hybridization apparatus at 70°C and bake for 30 minutes. After baking, quickly soak the slices in xylene twice, each for 5 minutes. Place in 100%, 90%, 80%, and 70% ethanol for 5 minutes each, and place in distilled water for 5 minutes. Drain the water, add hematoxylin staining solution, stain for 5 minutes, rinse off the hematoxylin staining solution with running water, and observe the degree of staining under a microscope. Differentiate with 1% hydrochloric acid alcohol for 3 seconds, wash with water, and soak in tap water for 7 minutes to turn blue; add 1 drop of 0.5% eosin staining solution, stain for 10 seconds, and observe under a microscope after washing with distilled water. Dehydrate with 80%, 90%, 95%, and 100% ethanol. Soak in xylene twice, each for 5 minutes, and dry at room temperature. Add a drop of neutral gum on the tissue and cover the slide with a coverslip. Observe and take pictures under a microscope after 24 hours.

[0088] Figure 3 E~H are (EF) tumor enrichment and statistical graphs of different groups; (G) HE staining shows the changes in the number of tumors after administration; (H) numerical statistics of tumors in lung tissue. The results showed that Bge-Exo (L) and Bge-Exo (H) could inhibit the occurrence of tumor lesions and promote the apoptosis of cancer cells in a dose-dependent manner.

[0089] Example 4: Enrichment of Bge-Exo in tumor tissue

[0090] (I) Construction of tumor nude mouse model, the steps are the same as Example 2.

[0091] (II) In vivo imaging to detect exosome enrichment

[0092] 1. PKH26 staining of LE-Exo (the staining steps are the same as in Example 1), ultra-high speed centrifugation followed by dissolution in PBS;

[0093] 2. Tail vein injection, taking photos and collecting samples at different time periods: Tail vein injection was given to nude mice with subcutaneous tumors in the experimental group. The injection dose for each nude mouse was 135 mg / kg (calculated and injected according to the body weight of the nude mice). The control group was injected with an equal volume of PBS buffer solution. Photos were taken and samples were collected at different time periods (1h, 3h, 6h, 12h, 24h, 48h);

[0094] 3. Take photos of each organ and tissue at different time periods and count them.

[0095] Figure 4 The figure is the result of enrichment of Bge-Exo in tumor tissue in Example 4; drug distribution at different time points of subcutaneous tumor labeled by PKH26. It can be seen that Bge-Exo is mainly enriched in liver, lung, kidney and tumor tissue, and can be maintained for at least 24 hours.

[0096] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. Application of exosome-like vesicles from Pulsatilla chinensis rhizomes in the preparation of anti-tumor drugs, characterized in that: The method for preparing the exosome-like vesicles from the rhizomes of Pulsatilla chinensis comprises the following steps: S1. Squeeze juice from the rhizome of Pulsatilla chinensis, filter and collect the filtrate; S2. Centrifuge the filtrate obtained in step S1, collect the precipitate, resuspend it, and filter it to obtain a solution containing exosome-like vesicles from the rhizomes of Pulsatilla striata.

2. According to claim 1, the use of exosome-like vesicles from Pulsatilla chinensis rhizomes in the preparation of anti-tumor drugs, in step S1, the Pulsatilla chinensis rhizomes are washed with sterile water before being squeezed and crushed, and mixed with pre-cooled PBS buffer.

3. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 2 in the preparation of anti-tumor drugs, characterized in that: In step S1, the ratio of adding Pulsatilla striata rhizome to PBS solution is (1-1.2) g: (1-1.2) mL.

4. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 1 in the preparation of anti-tumor drugs, characterized in that: In step S1, the filtration is: filtering using a 0.45 μm filter membrane.

5. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 1 in the preparation of anti-tumor drugs, characterized in that: In step S2, the filtrate is centrifuged several times at 4°C and 2000-120000 g, with each centrifugation time being 15-135 min.

6. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 5 in the preparation of anti-tumor drugs, characterized in that: In step S2, the filtrate is centrifuged at 4°C and 2000-3000g for 15-20 minutes to collect supernatant I; supernatant I is centrifuged at 4°C and 10000-20000g for 30-45 minutes to collect supernatant II; supernatant II is centrifuged at 4°C and 100000-120000g for 90-135 minutes to collect precipitate; In step S2, the precipitate was resuspended in PBS solution and filtered through a 0.22 μm filter membrane.

7. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The particle size of the exosome-like vesicles from the rhizome of Pulsatilla dasyphylla is 180-220 nm.

8. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor in the application refers to non-small cell lung cancer cells.

9. The use of the exosome-like vesicles from the rhizome of Pulsatilla chinensis according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The application includes: the application of the exosome-like vesicles of Pulsatilla chinensis rhizomes as tumor cell growth and proliferation inhibitors, tumor cell migration inhibitors and tumor cell apoptosis promoters in the preparation of anti-tumor drugs.

10. The use of the exosome-like vesicles from Pulsatilla chinensis rhizome according to claim 1 in the preparation of anti-tumor drugs, characterized in that: When the exosome-like vesicles from the rhizomes of Pulsatilla chinensis are used as growth and proliferation inhibitors / tumor cell migration inhibitors / tumor cell apoptosis promoters, the concentration is 100 to 500 μg / mL.

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