Extraction method of prunella vulgaris exosome and application of prunella vulgaris exosome in antitumor drugs

Abstract

The invention belongs to the field of Chinese herbal medicine application, and particularly relates to an extraction method of prunella vulgaris exosomes and application of the prunella vulgaris exosomes in antitumor drugs. According to the invention, a Chinese herbal medicine plant selfheal is taken as an object, an exosome vesicle structure is successfully separated and prepared by crushing the selfheal and an ultra-high-speed centrifugation method, and the exosome is taken as a prodrug and is applied to anti-tumor treatment. The prunella vulgaris exosome can target tumor cells, is taken by the tumor cells, is enriched in the tumor cells, inhibits growth and proliferation and cell migration of the tumor cells, inhibits the cell cycle and promotes cell apoptosis. Compared with the prior art, the plant exosome vesicles provided by the invention can well avoid activity loss of effective components of traditional Chinese medicines, improve the treatment effect and tumor targeting property, weaken the body injury and safety problems caused by traditional medicines, and have wide application prospects in tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese herbal medicines, and specifically relates to a method for extracting exosomes of Prunella vulgaris and an application thereof in anti-tumor drugs. Background Art

[0002] According to the latest data report from the International Agency for Research on Cancer, lung cancer has become the number one cause of cancer-related deaths. Malignant progression such as invasion and metastasis is an important cause of poor prognosis and even death in lung cancer patients.

[0003] With the development of traditional Chinese medicine, Chinese herbal medicine plays an important role in improving the tumor microenvironment, including Prunella vulgaris. Prunella vulgaris is bitter, pungent, cold in nature, enters the liver meridian, has the effect of clearing liver fire and dispersing depression, and is often used clinically for liver heat red eyes, pain or redness and swelling. For liver heat red eyes, swelling and pain, it is often used in combination with drugs such as chrysanthemum and dandelion. For liver heat and stagnation eye pain, it can be used in combination with Cyperus rotundus, which can soothe the liver. For liver deficiency and heat eye pain, it is often used in combination with drugs such as Angelica sinensis and White Peony Root. Prunella vulgaris is also used for scrofula and goiter caused by phlegm and fire stagnation, and can be used in combination with drugs such as kelp and seaweed to soften and disperse nodules. In addition, Prunella vulgaris is also used for hypertension, which is more suitable for those with liver heat. Prunella vulgaris is also often used for pulmonary tuberculosis and cancers such as lymphosarcoma and mediastinal tumors. How to improve the efficacy, solubility and safety of the active ingredients in Chinese medicine based on the traditional processing has always been a hot topic of concern.

[0004] 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 safety issues. The existing technology lacks effective Chinese herbal medicines with strong therapeutic effects, which can target and inhibit tumors. There are no reports on extracting exosomes from Prunella vulgaris and using them in anti-tumor treatment. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting Prunella Spike-Exosomes and their application in anti-tumor drugs in view of the lack of effective Chinese herbal medicines with strong therapeutic effects, which can target and inhibit tumors in the above-mentioned prior art. The present application isolates Prunella Spike-Exosomes (SP-Exo) from Prunella vulgaris, which can carry bioactive substances, directly target lung cancer cell tissues and significantly inhibit the malignant progression of lung cancer.

[0006] Based on the above objectives, this application proposes the following technical solutions:

[0007] The technical solution of the present invention provides the use of Prunella vulgaris exosomes (SP-Exo) in the preparation of anti-tumor drugs.

[0008] Furthermore, a method for preparing the above-mentioned Prunella vulgaris exosomes SP-Exo is provided, comprising the following steps:

[0009] (1) crushing fresh Prunella vulgaris to obtain crushed liquid;

[0010] (2) After filtering the above-mentioned crushed liquid, ultrahigh-speed centrifugation was used to separate and collect Prunella vulgaris exosomes SP-Exo.

[0011] Furthermore, the fresh Prunella vulgaris in step (1) needs to be washed with sterile water before being crushed, and mixed with pre-cooled PBS buffer;

[0012] Furthermore, the mixing ratio is Prunella vulgaris: PBS buffer = 1 g: (1-1.1) mL; the concentration of PBS buffer is 5-15 mM; pH = 7.4-7.6;

[0013] Furthermore, the crushing in step (1) is preferably performed by using a juicer to extract juice;

[0014] In some specific embodiments of the present application, a spiral juicer is used for juice extraction, and the parameters of the juicer are set as voltage / frequency / power: 220V, 50HZ, 1.2KW;

[0015] Furthermore, the filtration in step (2) refers to filtering using a 0.45 μm filter membrane to obtain a filtrate;

[0016] Furthermore, the ultra-high speed centrifugation method in step (2) comprises the following steps:

[0017] a) centrifuging the filtrate at 4°C, 2000-3000 g for 15-25 min, and collecting the supernatant I;

[0018] b) centrifuging the supernatant I at 4°C, 10,000-20,000 g for 30-45 min, and collecting the supernatant II;

[0019] c) centrifuging the supernatant II at 4°C, 100,000-120,000 g for 90-120 min, and collecting the precipitate;

[0020] d) Resuspend the precipitate in each tube with pre-cooled PBS, repeat 3 to 5 times, and then filter with a 0.22 μm filter membrane. The obtained filtrate contains SP-Exo and is stored at 4°C for later use.

[0021] In some specific embodiments of the present invention, the filtrate, supernatant I and supernatant II are preferably placed in a 50 mL centrifuge tube for centrifugation; however, in actual applications, other types of centrifuge containers and equipment may be selected.

[0022] Furthermore, the SP-Exo is soluble in physiological saline and can exist at 4°C for 1 month.

[0023] Furthermore, the SP-Exo obtained by the above preparation method has a size of about 160 to 200 nm, is spherical under an electron microscope, and has a double-layer membrane structure.

[0024] Furthermore, the tumor in the application refers to lung cancer cells;

[0025] Furthermore, in the application, SP-Exo is dispersed and dissolved with physiological saline;

[0026] Furthermore, the SP-Exo has a targeting effect on tumor cells, can be taken up by cells, and can be enriched in tumor cell tissues.

[0027] Furthermore, the application includes: the use of SP-Exo as a tumor cell growth and proliferation inhibitor, a tumor cell migration inhibitor, a tumor cell cycle inhibitor and a tumor cell apoptosis promoter in the preparation of anti-tumor drugs.

[0028] Furthermore, in the application, the concentration of SP-Exo is preferably 50 to 400 μg / mL.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] (1) The SP-Exo extraction process is simple, safe and pollution-free; the raw material, Prunella vulgaris, is cheap and easy to obtain in large quantities.

[0031] (2) SP-Exo therapy is an innovation based on traditional Chinese medicine treatment. It has sufficient safety, stability of effective drug ingredients, low toxicity, solubility, and targeting, and has good application prospects.

[0032] (3) The preliminary study of this application confirmed that SP-Exo can be enriched in tumor tissues and inhibit the malignant progression of lung cancer, including inhibiting growth proliferation and cell migration, inhibiting the cell cycle, and promoting cell apoptosis, providing new support for the future treatment of cancer with exosomes.

[0033] (4) The Prunella vulgaris exosomes provided by the present invention contain multiple active ingredients, which are different from the research on traditional single-component Prunella vulgaris extracts, and provide new basis for the potential therapeutic effects and mechanisms of traditional Chinese herbal medicines in the treatment of diseases such as tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The isolation and characterization of Prunella vulgaris exosomes (SP-Exo) in Examples 1 and 2; the numbers in the figure indicate: (A) Schematic diagram of the separation process of SP-Exo in fresh Prunella vulgaris; (B) Transmission electron microscopy detection of SP-Exo morphology; (C) SP-Exo particle size analysis; (D) Fluorescence microscopy shows the uptake of PKH26-labeled SP-Exo by HCC1833 cells;

[0035] Figure 2 The results of the experiment of SP-Exo inhibiting lung cancer cell proliferation in Example 3 are shown in the figure; the numbers in the figure indicate: (A) the response of A549 cells to the SP-Exo concentration in the CCK8 detection experiment; (B) the response of HCC1833 cells to the SP-Exo concentration in the CCK8 detection experiment; (C) the response of Besa2b cells to the SP-Exo concentration in the CCK8 detection experiment; (D) the images of clone spots of A549 cells and HCC1833 cells under a microscope; (E) the amount of clone spot formation of A549 cells; (F) the amount of clone spot formation of HCC1833 cells; (G) the images of A549 cells and HCC1833 cells under a microscope in the EdU detection experiment; (H) the statistical percentage of EdU-positive labeled cells in A549 cells; (I) the statistical percentage of EdU-positive labeled cells in HCC1833 cells;

[0036] Figure 3 The results of the SP-Exo inhibition of lung cancer cell migration experiment in Example 4 are shown in Figure 4; (A) Fluorescence inverted microscope images of A549 and HCC1833 cell Transwell assays; (B) Migration of A549 cells before and after SP-Exo treatment; (C) Migration of HCC1833 cells before and after SP-Exo treatment; (D) Electron microscope images of A549 and HCC1833 cells in wound healing experiments; (E) Statistical analysis of A549 cell migration ability; (F) Statistical analysis of HCC1833 cell migration ability;

[0037] Figure 4 The results of the effects of SP-Exo on cell cycle and cell apoptosis in Example 5 are shown in Figure 5. (A) A549 cell cycle detection; (B) HCC1833 cell cycle detection; (C) A549 cell cycle detection statistical analysis; (D) HCC1833 cell cycle detection statistical analysis; (E) A549 cell flow cytometer analysis results; (F) A549 cell apoptosis rate statistics; (G) HCC1833 cell flow cytometer analysis results; (H) HCC1833 cell apoptosis rate statistics;

[0038] Figure 5 The results of SP-Exo inhibiting tumor growth and enriching in tumor tissue in Example 6; (A) subcutaneous tumor experiment flow chart; (B) HCC1833 cell tumor growth chart; (C) nude mouse body weight; (D) tumor volume; (E) electron microscopy image in Ki67 immunohistochemical staining experiment; (F) statistical analysis in Ki67 immunohistochemical staining experiment; (G) in vivo imaging at different time points after tail vein injection of PKH67-labeled SP-Exo. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0040] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

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

[0042] All instruments used need to be sterilized before use.

[0043] Example 1: Preparation and morphological characterization of SP-Exo

[0044] (I) The preparation of SP-Exo mainly includes crushing and ultra-high-speed centrifugation collection ( Figure 1 A), specifically including the following steps:

[0045] Take 500 g of fresh Prunella vulgaris, wash it thoroughly with sterile water, chop it, add 500 mL PBS (precooled at 4°C), use a screw juicer to squeeze the juice (voltage / frequency / power: 220 V, 50 Hz, 1.2 KW), collect the juice, filter it with a 0.45 μm filter membrane, and collect the filtrate;

[0046] 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;

[0047] Place supernatant I in a 50 mL centrifuge tube, centrifuge at 10,000 g for 30 min at 4 °C, and collect supernatant II;

[0048] Place supernatant II in an ultracentrifuge tube, centrifuge at 120,000 g for 90 min at 4°C, and collect the precipitate;

[0049] Resuspend each tube of precipitate with 1 mL of pre-cooled PBS, repeat 3 times, and then filter with a 0.22 μm filter membrane. The filtrate contains SP-Exo and is stored at 4°C for later use.

[0050] (ii) Morphological characterization of SP-Exo under transmission electron microscopy, such as Figure 1 As shown in B, it shows that SP-Exo presents a double-layer membrane structure.

[0051] (III) Particle size determination results of SP-Exo, such as Figure 1 As shown in C, the results show that the size of SP-Exo is around 160-200nm.

[0052] Example 2: Uptake of SP-Exo by HCC1833 cells

[0053] HCC1833 cells are a type of human lung cancer adenocarcinoma cells. To demonstrate the uptake of SP-Exo by HCC1833 cells, PKH26 staining solution was used for labeling and observation under a fluorescence microscope ( Figure 1 D), the specific operation steps of the experimental group are as follows:

[0054] ①Staining of SP-Exo with PKH26 staining solution

[0055] Add 60 μL SP-Exo (diluted in PBS) to 0.5 mL diluent C (Sigma-Aldrich); add 4 μL PKH26 dye to the above diluent C with SP-Exo added to obtain a mixed solution, and incubate the mixed solution at 25°C for 4 minutes to allow the dye and SP-Exo to fully combine to obtain SP-Exo labeled with PKH26 dye.

[0056] In order to bind excess dye, 1 mL of 0.5% BSA / PBS was added to the mixed solution after the above incubation; the labeled exosomes were then washed at 100,000 g for 1 h, and the exosome precipitate was collected, diluted in 100 μL PBS and used for uptake experiments.

[0057] ②Uptake of SP-Exo by HCC1833 cells

[0058] Treatment system of experimental group: 1 μL PKH26-labeled SP-Exo was added to HCC1833 cells;

[0059] The blank control group consisted of HCC1833 cells only without exosome treatment.

[0060] The experimental steps are as follows:

[0061] The cells in the control group and experimental group were cultured in 4 mL DMEM complete medium for 24 hours, fixed with paraformaldehyde for 15-20 minutes, and washed 3 times with PBS. 400 μL DAPI staining solution (covering the bottom of the dish) was added and cultured at room temperature for 3-5 minutes to allow the stain to fully bind to the cells. After the culture was completed, the cells were washed 2-3 times with PBS to remove the unbound dye, each time for 3-5 minutes, and finally observed under a microscope.

[0062] The results of fluorescence microscopy were Figure 1 As shown in D, the exosomes stained with PKH26 appear red, and the cells stained with DAPI appear green. According to the position and color of the fluorescence in the figure, it can be judged that SP-Exo can be taken up and absorbed by HCC1833 cells.

[0063] Example 3: SP-Exo inhibits lung cancer cell proliferation

[0064] This example verifies the inhibitory effect of SP-Exo on lung cancer cells by various methods, as follows:

[0065] (I) CCK8 detection experiment

[0066] Experimental group: cells that took up SP-Exo, the uptake operation was the same as in Example 2; the experimental group was divided into three groups, using A549 cells (human non-small cell lung cancer cells), HCC1833 cells (human lung cancer adenocarcinoma cells), and Besa2b cells (human normal lung bronchial epithelial cells); in this example, the concentration gradient of SP-Exo added was set to 50, 100, 200, 300, and 400 μg / mL. NC control group: The SP-Exo of the above three experimental groups was replaced with an equal volume of PBS, that is, the concentration of SP-Exo added was 0. The specific operation is as follows:

[0067] ①Preparation of single cell suspension: When the confluence of cells that have taken up SP-Exo is about 80%-90%, discard the medium in the bottle, wash with PBS, add trypsin for digestion, then add DMEM medium to terminate digestion, centrifuge at 1500rpm for 4min, collect the cell pellet, and adjust the cell density to 2×10 with serum-free DMEM medium. 5 / mL, that is, the cell suspension; in the subsequent experiments involving the preparation of the cell suspension, refer to this example unless otherwise specified;

[0068] ②Take out the pre-cooled matrix gel (make sure to keep it on ice), the pipette tip and the 24-well plate, and drop 200 μL of matrix gel in the middle of each well of the 24-well plate (avoid bubbles); make three replicate wells for each of the experimental and control groups; place the 24-well plate with matrix gel in a 37°C incubator for 1 hour to solidify and set aside; steps ① and ② can be performed simultaneously;

[0069] ③ The cell suspension obtained in step ① was plated on a 24-well plate containing matrix gel at a density of 10,000 cells per well, and then placed in a 37°C incubator for culture; after 4 to 6 hours, the 24-well plate was taken out, 10 μL of CCK8 reagent was added to each well, and the cells were observed and photographed under a microscope. The cell viability at different concentrations was calculated based on the photographic results. The results of the three groups of cells were as follows: Figure 2As shown in A - C, the analysis results. The principle of the CCK8 reagent is that the secondary products metabolized by mitochondria can produce visible color changes to indicate the overall cell condition. Simply put, the more cells there are, the more mitochondria there are, the more metabolic products there are, the darker the color, and the higher the absorbance value. In the figure, *** indicates 0.0001 < p < 0.001, ** indicates 0.001 < p < 0.01, and * indicates 0.01 < p < 0.05. In the figure, *** indicates 0.0001 < p < 0.001, and ns indicates non - significant difference.

[0070] The results showed that the IC50 concentrations of SP - Exo - treated A549 and HCC1833 cells were 150 and 200 μg / mL respectively, while the IC50 concentration of Besa2b cells was greater than 800 μg / mL, indicating that tumor cells were more sensitive to SP - Exo than normal lung epithelial cells. At the same time, CCK8 detection confirmed that the inhibitory effect of SP - Exo on the proliferation ability of A549, HCC1833, and Besa2b cells was dose - dependent, and SP - Exo had less toxicity to Besa2b and greater toxicity to tumor cells, suggesting that SP - Exo had good anti - tumor effects.

[0071] (II) Colony - forming assay

[0072] Experimental group: A549 cells that had taken up SP - Exo, with concentration gradients set at 150 and 200 μg / mL, and HCC1833 cells that had taken up SP - Exo, with concentration gradients set at 200 and 400 μg / mL; Control group: Replace the SP - Exo in the experimental group with an equal volume of PBS, i.e., the concentration is 0; The uptake operation is the same as in Example 2. The operation steps of the experimental group are as follows:

[0073] ① Take A549 / HCC1833 cells that have taken up SP - Exo in the logarithmic growth phase, digest them with 0.25% trypsin, and make a single - cell suspension for counting.

[0074] ② Inoculate each group of cell suspensions into a 6 - well plate at 300 cells / dish, place them in a 5% CO2, 37 °C constant - temperature incubator for incubation, and terminate the incubation when colonies visible to the naked eye grow.

[0075] ③ Wash twice with PBS buffer, fix with methanol for 15 min, discard, stain with crystal violet for 10 min, rinse with running water, and dry in air at room temperature.

[0076] ④ Count under a microscope, and count the number of colonies with ≥ 50 cells. Calculate the colony - forming rate according to the following formula:

[0077] Colony - forming rate = number of colonies / number of inoculated cells × 100%.

[0078] A549, HCC1833, and NC control groups, and the results are shown in Figure 2 D - F, Figure 2 In D, the upper three microscopic examination images are of A549 cells, and the lower three microscopic examination images are of HCC1833 cells. It can be seen that more and larger colonies are formed by the two types of cells without adding SP-Exo, that is, the growth and proliferation are faster, while fewer colonies with a smaller area are formed when SP-Exo is added, and it is dose-related; the microscopic examination results are counted and analyzed. Figures E and F respectively correspond to the relationship diagrams of the clone numbers of the two types of cells and the added amount of SP-Exo. *** in the figures indicates 0.0001 < p < 0.001; it can be seen that SP-Exo has a significant inhibitory effect on the proliferation of A549 / HCC1833 cells.

[0079] (III) EdU experiment

[0080] Experimental group: A549 or HCC1833 cells that have taken up SP-Exo, with an SP-Exo concentration of 50 μg / mL; Control group: Replace the SP-Exo in the experimental group with an equal volume of PBS, that is, the concentration is 0; The uptake operation is the same as in Example 2. The operation steps of the experimental group are as follows:

[0081] ① Preparation of cell suspension: Refer to the preparation method in (I);

[0082] ② Inoculate 100,000 A549 / HCC1833 cells that have taken up SP-Exo (final concentration of 50 μg / mL) per well into a 96-well plate, and add EdU working solution ( 488 Click-iT EdU Universal Cell Proliferation Detection Kit, #C6043L, Bioscience Co., Ltd., showing green), and perform cell labeling at room temperature according to the instructions of the kit;

[0083] ③ Add 0.1 mL of 0.5% Triton X-100 (prepared with PBS) to each well, and incubate at room temperature for 20 min to fix the cells and promote osmosis;

[0084] ④ Add Hoechst 33342 solution (showing blue) and stain for 5 min, then rinse 3 times with PBS to counterstain the cell nuclei;

[0085] ⑤ Perform fluorescence microscopy detection and photographing on the above fluorescently labeled cells ( Figure 2 G), and perform statistical analysis on the results ( Figure 2 H - I).

[0086] As Figure 2In group G, the cells containing green fluorescence in the control group occupied a relatively large area and had a relatively large number in the field of view, while the cells containing green fluorescence in the experimental group were very few or almost invisible. The cells showing blue or green in the field of view were counted and analyzed respectively. According to the formula EDU positive (% of cells) = the number of EdU-stained green cells / the number of DAPI-stained blue cells × 100%, the EdU cell positive ratio was calculated and plotted in Figure 2 H and I, where *** indicates 0.0001 < p < 0.001; according to the results of A549, HCC1833 and the NC control group, it can be seen that SP-Exo has a significant inhibitory effect on the proliferation of A549 / HCC1833 cells.

[0087] Example 4: SP-Exo inhibits the migration of lung cancer cells

[0088] (I) Transwell assay

[0089] Experimental group (SP-Exo group): A549 cells / HCC1833 cells that have taken up SP-Exo (final concentration of 150 μg / mL); NC control group: Replace the SP-Exo in the experimental group with an equal volume of PBS. The specific detection steps of the experimental group are as follows, and the blank control is adjusted adaptively:

[0090] ① Prepare a single-cell suspension: When the confluence of A549 cells / HCC1833 cells that have taken up SP-Exo reaches about 80% - 90%, discard the medium in the flask, wash it with PBS, add trypsin for digestion, then add DMEM medium to terminate the digestion, centrifuge at 1500 rpm for 4 min, collect the cell pellet, and adjust the cell density to 2×10 5 / mL with serum-free DMEM medium to obtain the cell suspension;

[0091] ② Add 50 μL of the cell suspension to the Transwell chamber, incubate in a 5% CO2 incubator at 37℃ for 30 min, then add 500 μL of complete DMEM medium to the lower chamber of each chamber, and finally carefully transfer the chamber to the lower chamber, making sure not to generate bubbles (or inject from the side), and place it in a 5% CO2 incubator and culture at 37℃ for 12 h;

[0092] ③ Fixation: Take out the chamber and place it in an adjacent blank well, rinse the upper and lower chambers with 600 μL of PBS (the lower chamber is placed in a 24-well plate and just add PBS to rinse), add 600 μL of 4% paraformaldehyde pre-cooled at 4℃ to each well, fix for 20 min, then invert the chamber, air-dry it, and carefully wipe off the cells inside the chamber with a cotton swab;

[0093] ④ Staining: Drop 0.1% crystal violet solution on the upper end of the inverted chamber to evenly cover the bottom of the chamber. A few drops of crystal violet staining are sufficient. Stain for 15 min, rinse 3 times with deionized water, then wipe the inner side of the membrane of the chamber with a cotton swab and thoroughly air dry.

[0094] ⑤ Photographing: Observe and take pictures with a fluorescence inverted microscope ( Figure 3 A), and randomly count the number of cells in 6 fields of view to calculate the migration ability ( Figure 3 B–C).

[0095] Figure 3 As shown in A, the cells in the left control group are very dense, while the cells in the right experimental group are relatively sparse, indicating that the migration ability of cancer cells in the group with added exosomes is significantly inhibited.

[0096] Figure 3 In the statistical data graphs of B and 3C, *** indicates 0.0001 < p < 0.001, showing that SP-Exo has a significant inhibitory effect on the migration of HCC1833 cells and A549 cells.

[0097] (II) Scratch assay

[0098] Experimental group (SP-Exo group): A549 cells / HCC1833 cells that have taken up SP-Exo (final concentration of 150 μg / mL); NC control group: Replace the SP-Exo in the experimental group with an equal volume of PBS. The specific detection steps for the experimental group are as follows:

[0099] ① Preparation of cell suspension, refer to Example 3.

[0100] ② The ruler is irradiated with ultraviolet light for 30 min before operation. First, draw two horizontal lines on the back of the 12-well plate. According to the experimental design groups, add about 1.5×10 5 cells after different treatments into the wells, with the criterion that the cells reach a confluence of more than 90% the next day;

[0101] ③ Replace with DMEM medium containing 1% fetal bovine serum the next day, and make a scratch perpendicular to the horizontal line in the well with a 10 μL pipette tip; Gently rinse 2–3 times with PBS, add DMEM medium containing 1% fetal bovine serum, and culture in an incubator at 37 °C and 5% CO2. Observe and take pictures under a fluorescence microscope at 0 h, 24 h, and 48 h time points ( Figure 3 D), with the central shaded area of the 12-well plate as the reference, and the scratch in the center of the picture.

[0102] ④ According to the pictures after scratching, and using the formula cell migration rate (%) = (initial scratch width - scratch width after 24 h) / initial scratch width × 100%, calculate the cell migration rate of each group ( Figure 3 E–F).

[0103] Figure 3 D shows that the cells on both sides of the scratch in the control group grew faster towards the scratch, while in the experimental group, the cells on both sides hardly migrated towards the middle; Figure 3 E–F After statistical analysis, it was found that SP-Exo could significantly inhibit the migration of A549 and HCC1833 cells. ** in the figure indicates 0.001 < p < 0.01, and *** indicates 0.0001 < p < 0.001.

[0104] Example 5: Effects of SP-Exo on cell cycle and apoptosis

[0105] (I) Effects of SP-Exo on cell cycle

[0106] Experimental group (SP-Exo group): A549 cells / HCC1833 cells that took up SP-Exo (final concentration of 150 μg / mL); NC control group: Replace SP-Exo in the experimental group with an equal volume of PBS. EdU staining can label the cells in the S phase of the cell cycle. The specific detection steps of the experimental group are as follows:

[0107] ① Prepare a cell suspension and seed 1×10 5 HCC1833 cells / A549 cells that took up SP-Exo per well in a 96-well plate, and add EdU working solution ( 594Click-iT EdU Universal Cell Proliferation Detection Kit, #C6045L, Bioscience Co., Ltd.) for cell labeling;

[0108] ② Add 0.1 mL of 0.5% Triton X-100 (prepared with PBS) to each well and incubate at room temperature for 20 min to fix the cells and promote osmosis;

[0109] ③ Add Hoechst 33342 solution (blue) for staining for 5 min, and then wash 3 times with PBS to counterstain the cell nuclei;

[0110] ④ Use a fluorescence microscope to detect, take pictures, and perform statistical analysis on the results ( Figure 4 A–D).

[0111] Figure 4 In A and Figure 4 B, the green shadow represents the cells in the G1 phase; the yellow shadow represents the cells in the S phase; the blue shadow represents the cells in the G2 phase; the text in the figure indicates (taking Figure 4Taking A as an example): RMS (Root Mean Square) is the root mean square value, indicating the goodness of curve fitting; Freq G1 represents the proportion of cells in the G1 phase, Mean G1 is the average number of cells in the G1 phase, G2 / G1 = 1.96, indicating that the number of cells in the G2 phase is 1.96 times that of the cells in the G1 phase; the CV value represents the coefficient of variation, which is mainly affected by the instrument sensitivity, sample preparation and staining process (such as parameter setting and fixation time). The smaller the CV value, the narrower and sharper the peak shape, and generally about 5% is more appropriate. Figure 4 In C and 4D, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001. The results show that through cell cycle detection, it is confirmed that SP-Exo can arrest A549 cells in the S phase and HCC1833 cells in the G2 / M phase.

[0112] (II) Flow cytometry apoptosis detection

[0113] Experimental group (SP-Exo group): A549 cells / HCC1833 cells that uptake SP-Exo (final concentration is 150 μg / mL); NC control group: Replace SP-Exo in the experimental group with an equal volume of PBS.

[0114] AnnexinⅤ is a Ca2+-dependent phospholipid-binding protein with a molecular weight of 35 - 36 kDa, which can bind to PS with high affinity. Fluorescein FITC is used to label Annexin V, and flow cytometry or fluorescence microscopy can be used to detect the occurrence of cell apoptosis. Propidium Iodide (PI) is a dye that can bind to DNA. It cannot penetrate the intact cell membrane of normal cells or early apoptotic cells, but in apoptotic late-stage cells and dead cells, PI can penetrate the cell membrane and stain the nucleus red. The specific detection steps of the experimental group are as follows:

[0115] ① Pretreatment before staining: Resuspend the collected cells once with pre-cooled PBS, centrifuge at 300g for 5 min, discard the supernatant, and collect the cells; Add 300 μL of 1×Binding Buffer to resuspend the cells and adjust the cell concentration to 1×10 6 / mL.

[0116] ② Annexin V / PI staining: Take flow tubes, add 100 μL of cell suspension filtered through a 200-mesh cell filter membrane to different groups respectively, and add 5 μL of Annexin V-Alexa Fluor TM 488 and mix gently.

[0117] ③Add 10 μL PI propidium iodide and mix gently, then incubate at room temperature in the dark for 10 to 20 minutes.

[0118] ④ Add 400 μL 1× Binding Buffer, then place in an ice bath and test on the instrument. The selected channels are as follows: Alexa Fluor TM 488: 488nm excitation, acquisition band 530 / 30; PI: 561nm excitation, acquisition band 610 / 20.

[0119] The collection results of A549 cells and HCC1833 cells are as follows Figure 4 E and G show that the apoptosis rates were statistically Figure 4 As shown in F and H, *** in the figure means 0.0001 <p<0.001。

[0120] The above experiments confirmed that SP-Exo can significantly promote apoptosis of A549 and HCC1833 cells.

[0121] Example 6: SP-Exo inhibits tumor growth and is enriched in tumor cells

[0122] (I) Subcutaneous tumor experiment

[0123] ① 100 μL containing 2×10 6 PBS containing HCC1833 cells was subcutaneously injected into 4-6 week-old female C57 nude mice to obtain mice bearing subcutaneous tumors;

[0124] ②Starting from the fifth day, 5×10 8 particles / kg SP-Exo was used as the SP-Exo treatment group, and the same amount of PBS was injected as the NC group control (refer to the flow chart of subcutaneous tumor experiment Figure 5 A).

[0125] Tumor size in living mice was measured every 5 days using a vernier caliper ( Figure 5 D), and found that the tumor size continued to grow during the experiment, and the tumor size of the experimental group grew more slowly, and the volume was always smaller than that of the control group; one month later, the mice were killed by dissection of the neck and the tumor volume was measured ( Figure 5 B), it can be seen that the tumor size of the experimental group was smaller than that of the control group; the weight of the mice was recorded every 2 days ( Figure 5 C), indicating that injection of HCC1833 cells to create subcutaneous tumors had little effect on the physical signs of mice.

[0126] Figure 5B-D show that the tumor volume and weight of the SP-Exo treatment group were significantly lower than those of the NC group, indicating that SP-Exo can effectively reduce the volume and weight of the tumor, further indicating that SP-Exo inhibits tumor growth.

[0127] (II) Immunohistochemical detection of Ki67 expression and apoptosis in tumor tissues

[0128] ① Tumor tissue sections: The tumor tissues collected in the above subcutaneous tumor experiment were embedded in paraffin, and the embedded paraffin sections were dewaxed to water: the sections were sequentially placed in xylene I for 15 min-xylene II for 15 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-85% alcohol for 5 min-75% alcohol for 5 min-distilled water for washing;

[0129] ② Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH 8.0) and perform antigen repair in a microwave oven. Set the microwave oven parameters as follows: medium heat for 8 minutes, stop the fire for 8 minutes, and switch to medium-low heat for 7 minutes. During this process, excessive evaporation of the buffer should be prevented and the sections should not be dried. After natural cooling, place the tissue sections in PBS (pH 7.4) and shake on a decolorizing shaker to wash 3 times, 5 minutes each time.

[0130] ③ BSA blocking: After the tissue sections are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), add 3% BSA in the circle, evenly cover the tissue, and block at room temperature for 30 minutes;

[0131] ④Add primary antibody (red): Gently shake off the blocking solution, add Ki67 primary antibody (PBS) on the slices, and incubate the slices flat in a light-proof humid box at 4°C overnight;

[0132] ⑤ DAPI counterstaining of cell nuclei (blue): Place tissue sections in PBS (pH 7.4) and wash on a decolorizing shaker for 3 times, 5 min each time; After the tissue sections are slightly dried, add DAPI staining solution in the circle and incubate at room temperature away from light for 10 min;

[0133] ⑥ Sealing: Place the tissue sections in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time; after the sections are slightly dried, seal them with anti-fluorescence quenching sealing media;

[0134] ⑦ Microscopic examination and photography: The sections were observed and images were collected under a Nikon inverted fluorescence microscope ( Figure 5 E), and calculated the expression of Ki67 protein ( Figure 5 F).

[0135] Figure 5In F, *** indicates 0.0001 < p < 0.001. The results showed that the inhibition of the expression of the tumor growth marker protein Ki67 by SP-Exo was confirmed by immunohistochemistry.

[0136] In addition, in vivo imaging was performed at different time points after the tail vein injection of PKH67-labeled SP-Exo. The results showed that SP-Exo could exist in the body for more than 24 h, and SP-Exo could be enriched in tumor tissues ( Figure 5 G).

[0137] In summary, the Prunella vulgaris exosome SP-Exo provided by the present invention can be enriched in cancer cells and tumor tissues, can inhibit the malignant progression of tumor cells such as the proliferation and migration of tumor cells, can arrest tumor cells in the S phase or G2 phase, and significantly promote the apoptosis of tumor cells. The SP-Exo treatment is an innovation based on traditional Chinese medicine treatment, and has sufficient safety, stability of effective drug components, low toxicity, and solubility.

[0138] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. 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 efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Application of Prunella Vulgaris exosomes in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The method for preparing Prunella vulgaris exosomes comprises the following steps: (1) crushing fresh Prunella vulgaris to obtain crushed liquid; (2) After filtering the above-mentioned disrupted liquid, the SP-Exo was separated and collected by ultrahigh-speed centrifugation.

3. The use according to claim 2, characterized in that: In step (1) of the preparation method, the fresh Prunella vulgaris needs to be washed with sterile water before being crushed and mixed with pre-cooled PBS buffer.

4. The use according to claim 3, characterized in that: In step (1) of the preparation method, the mixing ratio is Prunella vulgaris: PBS buffer = 1 g: (1-1.1) mL; the concentration of the PBS buffer is 5-15 mM; and the pH is 7.4-7.

6.

5. The use according to claim 2, characterized in that: The filtration in step (2) of the preparation method refers to filtering using a 0.45 μm filter membrane to obtain a filtrate.

6. The use according to claim 2, characterized in that: The ultracentrifugation method of step (2) of the preparation method comprises the following steps: a) centrifuging the filtrate at 4°C, 2000-3000 g for 15-25 min, and collecting the supernatant I; b) centrifuging the supernatant I at 4°C, 10,000-20,000 g for 30-45 min, and collecting the supernatant II; c) centrifuging the supernatant II at 4°C, 100,000-120,000 g for 90-120 min, and collecting the precipitate; d) resuspending the precipitate with pre-cooled PBS, repeating 3 to 5 times, and then filtering with a 0.22 μm filter membrane, and the obtained filtrate contains Prunella vulgaris exosomes; The SP-Exo is soluble in physiological saline and can exist at 4°C for 1 month.

7. The use according to claim 1, characterized in that: The Prunella vulgaris exosomes have a size of 160 to 200 nm, are spherical under an electron microscope, and have a double-layer membrane structure.

8. The use according to claim 1, characterized in that: The tumor is lung cancer cells.

9. The use according to claim 1, characterized in that: The Prunella vulgaris exosomes are enriched in tumors and are used as tumor cell growth and proliferation inhibitors, tumor cell migration inhibitors, tumor cell cycle inhibitors and tumor cell apoptosis promoters in the preparation of anti-tumor drugs.

10. The use according to claim 1, characterized in that: In the application, the concentration of SP-Exo is preferably 50 to 400 μg / mL.

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