Application of rabdosia rubescens exosome in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of Chinese herbal medicines, and relates to a nano-drug preparation technology, in particular to application of a rabdosia rubescens exosome (DLC-Exo) in preparation of antitumor drugs. According to the invention, a Chinese herbal medicine plant rabdosia rubescens is taken as an object, an exosome vesicle structure is successfully separated and prepared by breaking the rabdosia rubescens and adopting an ultra-high-speed centrifugation method, and the exosome is taken as a prodrug and is applied to anti-tumor treatment. The DLC-Exo can target tumor cells, is taken by the tumor cells and is enriched in tumor tissues, tumor cell proliferation and cell migration are inhibited, and tumor growth and invasion are inhibited. Compared with the prior art, the rabdosia rubescens exosome provided by the invention contains a plurality of active components which are different from a traditional single-component rabdosia rubescens extract, the development of the plant exosome drug improves the anti-tumor effect of traditional Chinese herbal medicines, and meanwhile, the problems of toxicity, poor solubility and the like of a monomer drug are solved; the safety and stability of the Chinese herbal medicine are improved, and the application effect and value of the Chinese herbal medicine can be improved to the greatest extent.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese herbal medicines, relates to a nano drug preparation technology, and specifically relates to the application of Rubescens rubescens exosomes (DLC-Exo) in the preparation of 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 killer of cancer deaths. Malignant progression such as invasion and metastasis is an important reason for poor prognosis and even death of lung cancer patients. With the development of traditional Chinese medicine, Chinese herbal medicine plays an important role in improving the tumor microenvironment, including wintergrass. Wintergrass tastes bitter and sweet, is slightly cold in nature, and enters the lung, stomach, and liver meridians. It has the effects of clearing away heat and detoxifying, promoting blood circulation and relieving pain. It is clinically used for sore throat, lumps and masses, and snake and insect bites.

[0003] As a traditional Chinese medicinal material, Rubescens rubescens has attracted much attention in recent years for its anti-tumor activity. It contains a variety of active ingredients, such as Rubescensine A, which play an important role in the anti-tumor process.

[0004] For example, patent CN105919984A (porous particle inhalation of Rubescensine A and its application in treating primary lung cancer) discloses a porous particle inhalation of Rubescensine A and the application of the porous particle inhalation of Rubescensine A in treating primary lung cancer, wherein the porous particle inhalation of Rubescensine A has good anticancer effect, especially good anticancer effect on non-small cell lung cancer; CN118416050A (a pharmaceutical composition, a pharmaceutical preparation and its application in preparing antitumor drugs) provides a pharmaceutical composition, a pharmaceutical preparation and its application in preparing antitumor drugs, wherein the pharmaceutical composition comprises Rubescensine A and Rubescensine B, and the invention discloses a pharmaceutical composition, a pharmaceutical preparation and its application in preparing antitumor drugs. Studies have found that the main components of oridonin and oridonin B in oridonium rubescens have a strong proliferation inhibitory activity on cancer cells. Within a specific ratio range, the pharmaceutical composition obtained by the combined use of oridonin and oridonin B can have a synergistic inhibitory effect on cancer treatment and can be used to prepare anti-tumor drugs. CN110693873A (Preparation and application of oridonium rubescens active ingredient composition) provides a preparation and application of oridonium rubescens active ingredient composition, which is composed of oridonin A, oridonin B, rosmarinic acid, ferulic acid and quercetin. The active ingredients in the composition can produce a synergistic effect, thereby effectively treating NAFLD.

[0005] The above existing patents all prove the application of Rubescensine A, Rubescensine B and other active ingredients of Rubescens in anti-tumor drugs. It can be seen that the important active ingredients in Rubescens have high bioavailability and stable properties, and have clinical use value.

[0006] However, at present, the research on Rubescens rubescens is basically limited to certain active ingredients (such as Rubescens rubescens A, Rubescens rubescens B, diterpenes, and triterpenes), and rarely involves the research on extracts containing complex active ingredients such as exosomes, and even less involves the application of Rubescens rubescens exosomes in anti-tumor and other aspects. Summary of the invention

[0007] The prior art lacks research on extracts containing complex active ingredients such as Rubescens exosomes, and there are few studies involving the application of Rubescens exosomes in anti-tumor and other aspects. In view of the above problems, the purpose of the present invention is to provide the application of Rubescens exosomes (DLC-Exo) in the preparation of anti-tumor drugs.

[0008] The present application separates Prunella DLCike-Exosomes (DLC-Exo) from Prunella rubescens. The DLC-Exo can carry bioactive substances, directly target lung cancer cell tissues and significantly inhibit the malignant progression of lung cancer.

[0009] Based on the above objectives, this application proposes the following technical solutions:

[0010] The technical solution of the present invention provides the use of Rubescens rubescens exosomes (DLC-Exo) in the preparation of anti-tumor drugs.

[0011] Furthermore, a method for preparing the above-mentioned Rubescens rubescens exosomes DLC-Exo is provided, comprising the following steps:

[0012] (1) crushing fresh Rubescens rubescens to obtain crushed liquid;

[0013] (2) After filtering the above-mentioned crushed liquid, ultra-high speed centrifugation was used to separate and collect the exosomes DLC-Exo of Rubescens rubescens.

[0014] Furthermore, the fresh Rubescens rubescens in step (1) needs to be washed with sterile water before being crushed, and mixed with pre-cooled PBS buffer;

[0015] The mixing ratio is Rubescens rubescens: PBS buffer = 1 g: (1-1.1) mL; the concentration of PBS buffer is 5-15 mM; pH = 7.4-7.6;

[0016] Furthermore, the crushing in step (1) is preferably performed by using a juicer to extract juice;

[0017] 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;

[0018] Furthermore, the filtration in step (2) refers to filtering using a 0.45 μm filter membrane to obtain a filtrate;

[0019] Furthermore, the ultra-high speed centrifugation method in step (2) comprises the following steps:

[0020] a) centrifuging the filtrate at 4°C, 2000-3000 g for 15-25 min, and collecting the supernatant I;

[0021] b) centrifuging the supernatant I at 4°C, 10,000-20,000 g for 30-45 min, and collecting the supernatant II;

[0022] c) centrifuging the supernatant II at 4°C, 100,000-120,000 g for 90-120 min, and collecting the precipitate;

[0023] d) Resuspend each tube of precipitate with pre-cooled PBS, repeat 3 to 5 times, and then filter with a 0.22 μm filter membrane. The obtained filtrate is the solution containing DLC-Exo, and the filtrate is stored at 4°C for later use.

[0024] 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.

[0025] Furthermore, the DLC-Exo is soluble in physiological saline and can exist at 4°C for 1 month.

[0026] Furthermore, the DLC-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.

[0027] Furthermore, the tumor in the application refers to lung cancer cells.

[0028] Furthermore, in the application, DLC-Exo is dispersed and dissolved in physiological saline.

[0029] Furthermore, the DLC-Exo has a targeting effect on tumor cells and can be enriched in tumor cell tissues after being taken up by tumor cells.

[0030] Furthermore, the application includes: use of DLC-Exo as a tumor cell growth and proliferation inhibitor and a tumor cell migration inhibitor in the preparation of anti-tumor drugs.

[0031] Furthermore, the concentration of DLC-Exo added in the application is 0.107-4.3 mg / mL.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] (1) The DLC-Exo extraction process is simple, safe and pollution-free; the raw material, Rubescens rubescens, is cheap and easy to obtain in large quantities.

[0034] (2) DLC-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.

[0035] (3) The preliminary study of this application confirmed that DLC-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.

[0036] (4) The exosomes of Rubescens rubescens provided by the present invention contain multiple active ingredients, which are different from the research on traditional single-component Rubescens rubescens extracts, and provide new basis for the potential therapeutic effects and mechanisms of traditional Chinese herbal medicines in treating diseases such as tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The separation and characterization of Rubescens rubescens exosomes (DLC-Exo) in Examples 1 and 2;

[0038] The numbers in the figure indicate: (A) Schematic diagram of the separation process of DLC-Exo from fresh Rubescens; (B) DLC-Exo particle size analysis; (C) Fluorescence microscopy showing the uptake of PKH26-labeled DLC-Exo by HCC1833 cells;

[0039] Figure 2 This is a graph showing the results of the DLC-Exo inhibition of lung cancer cell proliferation experiment in Example 3;

[0040] The numbers in the figure indicate: (A) the response of HCC1833 cells to DLC-Exo concentration in CCK8 detection experiment; (B) the response of A549 cells to DLC-Exo concentration in CCK8 detection experiment; (C) the image of HCC1833 cells under the microscope in EdU detection experiment; (D) the percentage of HCC1833 cells EdU-positive labeled cells; (E) the image of A549 cells under the microscope in EdU detection experiment; (F) the percentage of A549 cells EdU-positive labeled cells;

[0041] Figure 3 This is a graph showing the results of the DLC-Exo inhibition of lung cancer cell migration experiment in Example 4;

[0042] The numbers in the figure indicate: (A) microscopic image of HCC1833 cells in Transwell assay; (B) statistical analysis of HCC1833 cell migration ability; (C) microscopic image of A549 cells in Transwell assay; (D) statistical analysis of A549 cell migration ability; (E) microscopic image of HCC1833 cells in wound healing experiment; (F) statistical analysis of HCC1833 cell healing ability; (G) microscopic image of A549 cells in wound healing experiment; (H) statistical analysis of A549 cell healing ability;

[0043] Figure 4 This is a result diagram of the effect of DLC-Exo on the lung metastasis model of nude mice injected with tail vein in Example 5;

[0044] Numbers in the figures indicate: (A) lung metastasis results of HCC1833 tail vein injection; (B) HE staining lung lesion number results; (C) electron microscopy image in Ki67 immunohistochemical staining experiment; (D) statistical analysis of HE staining lung lesion number; (E) statistical analysis in Ki67 immunohistochemical staining experiment;

[0045] Figure 5 These are in vivo images of PKH67-labeled DLC-Exo at different time points after tail vein injection in Example 5. DETAILED DESCRIPTION

[0046] 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.

[0047] 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.

[0048] The PBS buffer concentration used in all examples of the present application is 0.0067 M, and the pH is 7.4.

[0049] All instruments used need to be sterilized before use and kept ready for use; all operations should be performed in a clean environment to avoid contamination.

[0050] Example 1: Preparation and morphological characterization of DLC-Exo

[0051] (I) The preparation of DLC-Exo mainly includes crushing and ultra-high-speed centrifugation collection ( Figure 1A), specifically including the following steps:

[0052] 500 g of fresh Rubescens rubescens was taken, washed thoroughly with sterile water, chopped, added with 500 mL of PBS (precooled at 4° C.), and juiced using a spiral juicer (voltage / frequency / power: 220 V, 50 Hz, 1.2 KW);

[0053] The collected juice was filtered through a 0.45 μm filter membrane and the filtrate was collected;

[0054] 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;

[0055] Place supernatant I in a 50 mL centrifuge tube, centrifuge at 10,000 g for 30 min at 4 °C, and collect supernatant II;

[0056] Place supernatant II in an ultracentrifuge tube, centrifuge at 120,000 g for 90 min at 4°C, and collect the precipitate;

[0057] 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 obtained filtrate contains DLC-Exo, and the filtrate is stored at 4°C for future use.

[0058] (ii) Particle size determination results of DLC-Exo, such as Figure 1 As shown in B, the results show that the size of DLC-Exo is around 160-200nm.

[0059] Example 2: Uptake of DLC-Exo by HCC1833 cells

[0060] HCC1833 cells are a type of human lung cancer adenocarcinoma cells. To demonstrate the uptake of DLC-Exo by HCC1833 cells, PKH26 staining solution was used for labeling and fluorescence microscopy was used for observation ( Figure 1 C), the specific operation steps of the experimental group are as follows:

[0061] (1) Staining of DLC-Exo with PKH26 staining solution

[0062] The experimental steps are as follows:

[0063] a. Add 60 μL DLC-Exo (diluted in PBS) to 0.5 mL diluent C (Sigma-Aldrich);

[0064] b. Add 4 μL of PKH26 dye to the diluent C to which DLC-Exo was added to obtain a mixed solution, and incubate the mixed solution at 25° C. for 4 min to allow the dye and DLC-Exo to fully combine to obtain DLC-Exo labeled with PKH26 dye.

[0065] 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.

[0066] (2) Uptake of DLC-Exo by HCC1833 cells

[0067] Experimental group treatment system: 1 μL PKH26-labeled DLC-Exo was added to HCC1833 cells;

[0068] The blank control group consisted of HCC1833 cells only without exosome treatment.

[0069] The experimental steps are as follows:

[0070] a. The cells in the control group and experimental group were cultured in 4 mL of DMEM complete medium for 24 hours, fixed with paraformaldehyde for 15-20 minutes, and washed with PBS three times;

[0071] b. Add 400 μL DAPI staining solution (just enough to cover the bottom of the dish) and incubate at room temperature for 3 to 5 minutes to allow the stain to fully bind to the cells;

[0072] c. After the culture is completed, wash with PBS 2 to 3 times to remove unbound dye, each time for 3 to 5 minutes, and finally observe under a microscope.

[0073] The results of fluorescence microscopy were Figure 1 As shown in Figure C, the exosomes stained with PKH26 appear red (left picture), the cells stained with DAPI appear blue (middle picture), and the positions of the red and blue fluorescence in the right picture basically overlap and appear purple. Therefore, based on the position and color of the fluorescence in the figure, it can be judged that DLC-Exo is successfully taken up and absorbed by HCC1833 cells.

[0074] Example 3: DLC-Exo inhibits lung cancer cell proliferation

[0075] This example uses a variety of methods (CCK8 detection experiment, EdU experiment, immunohistochemistry experiment) to verify the inhibitory effect of DLC-Exo on lung cancer cells, as follows:

[0076] (I) CCK8 detection experiment

[0077] Experimental group: Cells that ingested DLC-Exo, and the ingestion operation was the same as in Example 2; The experimental group was divided into two different groups of cells, namely A549 cells (human non-small cell lung cancer cells) and HCC1833 cells (human lung adenocarcinoma cells); In this example, the concentration gradients of DLC-Exo added were set to 4.3, 0.43, 0.215, 0.107, 0.071, 0.053, 0.043, 0 mg / mL, and the solvent of DLC-Exo was PBS buffer; Among them, a concentration of 0 indicated that only PBS buffer was added and no DLC-Exo was contained, which could be used as a control group.

[0078] The specific operation of the CCK8 detection experiment is as follows:

[0079] (1) Preparation of single-cell suspension:

[0080] First, let A549 cells and HCC1833 cells ingest DLC-Exo at the above-set concentrations (refer to Example 2 for the ingestion experimental method) and culture them in DEME medium (37 °C); When the confluence of the cells that ingested DLC-Exo reached 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 precipitate, and adjust the cell density to 2×10 5 / mL with serum-free DMEM medium to obtain the cell suspension;

[0081] (2) CCK8 reagent staining:

[0082] Seed the cell suspension obtained in the above step into a 96-well plate at a density of 10,000 cells per well and place it back in the 37 °C incubator for culture; After 4-6 h, take out the 96-well plate, add 10 μL of CCK8 reagent to each well, and detect the absorbance at 450 nm in an enzyme-linked immunosorbent assay reader. Three wells of cells for each concentration were detected as duplicates.

[0083] After taking the average value of the above absorbance results for statistical analysis, plot them in Figure 2 A (HCC1833 cells) and Figure 2 B (A549 cells) for result analysis. The principle of action 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 metabolites 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.

[0084] The results in the figure show that for HCC1833 cells, when the concentration of DLC-Exo taken is 0.215-4.3 mg / mL, it has a significant inhibitory effect on the proliferation of cancer cells; when the concentration of DLC-Exo taken is 0.043-0.107 mg / mL, it has basically no effect on the proliferation of cancer cells or even promotes it. For A549 cells, when the concentration of DLC-Exo taken is 0.107-4.3 mg / mL, it has an inhibitory effect on the proliferation of cancer cells; when the concentration of DLC-Exo taken is 0.043-0.071 mg / mL, it has basically no effect on the proliferation of cancer cells or even promotes it.

[0085] The IC50 concentrations (half inhibitory concentration) of DLC-Exo at OD450 for HCC1833 and A549 cells were 0.22 and 0.43 mg / mL, respectively. This indicates that the inhibitory effect of DLC-Exo on the proliferation of HCC1833 and A549 cells is dose-dependent, suggesting that DLC-Exo has a good anti-tumor effect.

[0086] (II) EdU experiment

[0087] EdU is a thymine analog that is embedded in chromatin during cell replication and presents itself as fluorescence in the final result. EdU can directly indicate the number of new cells after replication and locate the markers, which is an absolute quantitative detection method.

[0088] Experimental group: A549 or HCC1833 cells that took up DLC-Exo, with DLC-Exo concentrations of 0.43 and 0.22 mg / mL, respectively;

[0089] NC (negative control) control group: The DLC-Exo in the experimental group was replaced with an equal volume of PBS, that is, the concentration of DLC-Exo added was 0.

[0090] The operating steps of the experimental group are as follows:

[0091] ① Preparation of cell suspension:

[0092] First, A549 cells and HCC1833 cells were allowed to absorb (the absorption experimental method refers to Example 2) the DLC-Exo of the above-set concentration and cultured in DEME medium (37°C); when the confluence of the cells that absorbed DLC-Exo was about 80%-90%, the medium in the bottle was discarded, and the cells were washed with PBS and then digested with trypsin. Then, an appropriate amount of DMEM medium was added to terminate the digestion, and the cells were centrifuged at 1500 rpm for 4 minutes to collect the cell precipitate. The cell density was adjusted to 2×10 5 / mL, which is the cell suspension;

[0093] ② Seed 100,000 A549 or HCC1833 cells that had taken up DLC-Exo into each well of a 96-well plate, and add the EdU working solution ( 488Click-iT EdU Universal Cell Proliferation Detection Kit, #C6043L, Bioscience Co., Ltd., showing green), and perform cell labeling at room temperature according to the kit's instructions;

[0094] 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;

[0095] ④ Add Hoechst 33342 solution (showing blue) for staining for 5 min, then rinse 3 times with PBS to counterstain the cell nuclei;

[0096] ⑤ Perform fluorescence microscopy detection and photography on the above fluorescence-labeled cells ( Figure 2 C and Figure 2 E), and perform statistical analysis on the results ( Figure 2 D and Figure 2 F).

[0097] As Figure 2 C and Figure 2 E show, in the control group, the area occupied by cells with green fluorescence in the field of view is larger and the number is more, indicating that when the control group is not treated, the cancer cells proliferate faster. In the experimental group, the cells with green fluorescence are very few or almost invisible, indicating that the cancer cells hardly proliferate after the treatment in the experimental group. Count the cells showing blue or green in the field of view respectively. According to the formula EDU positive (% of cells) = number of EdU-stained green cells / number of DAPI-stained blue cells × 100%, calculate the EdU cell positive ratio and plot it in Figure 2 D and Figure 2 F. Among them, *** indicates 0.0001 < p < 0.001; it is found that the positive ratio of the exosome treatment group is significantly lower than that of the blank control group. According to the results of the exosome treatment experimental group and the NC control group of A549 cells and HCC1833 cells, it can be seen that DLC-Exo has a significant inhibitory effect on the proliferation of A549 or HCC1833 cells.

[0098] (III) Immunohistochemistry experiment

[0099] To further verify the effect of DLC-Exo on tumor growth, a nude mouse subcutaneous tumor model was constructed in this application, divided into the NC group and the DLC-Exo treatment group.

[0100] (1) The construction method of the nude mouse subcutaneous tumor model is as follows:

[0101] 100 μL of HCC1833 cells (1×10 7 The DLC-Exo treatment group was injected subcutaneously into nude mice, and the DLC-Exo treatment group was injected intraperitoneally with DLC-Exo (5 mice in each group). The tumor size was measured with a vernier caliper every 5 days, and the tumor was removed one month later for photography and Ki67 immunohistochemistry.

[0102] (2) The steps of immunohistochemistry experiment are as follows:

[0103] ①Tumor tissue sections:

[0104] The tumor tissue collected in the above subcutaneous tumor experiment was 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;

[0105] ②Antigen retrieval:

[0106] 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 heat for 8 minutes, and switch to medium-low heat for 7 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the sections. After natural cooling, place the tissue sections in PBS (pH 7.4) and wash them on a decolorizing shaker for 3 times, 5 minutes each time.

[0107] ③BSA blocking:

[0108] After the tissue sections were slightly dried, a circle was drawn around the tissue with a histochemical pen (to prevent the antibody from flowing away), and 3% BSA was added dropwise in the circle to evenly cover the tissue, and the tissue was blocked at room temperature for 30 minutes.

[0109] ④Add primary antibody (red):

[0110] Gently shake off the blocking solution, add Ki67 primary antibody (PBS) on the slices, and incubate the slices flat in a light-proof humidified box at 4°C overnight; place the tissue slices in PBS (pH 7.4) and wash them 3 times on a decolorizing shaker, 5 minutes each time, and then add the secondary antibody and incubate for 30 minutes;

[0111] ⑥DAPI counterstaining of cell nuclei (shown in blue):

[0112] The tissue sections were placed in PBS (pH 7.4) and washed three times on a decolorizing shaker, each time for 5 min. After the tissue sections were slightly dried, DAPI staining solution was added dropwise into the circle and incubated at room temperature in the dark for 10 min.

[0113] ⑥Seal the film:

[0114] Place the tissue sections in PBS buffer and shake and wash them 3 times on a脱色shaker for 5 min each time; after the sections are slightly dried by shaking, seal them with an anti-fluorescence quenching mounting medium;

[0115] ⑦Microscopic examination and photography:

[0116] Observe and collect images of the sections under a Nikon inverted fluorescence microscope( Figure 2 I), and calculate the expression level of Ki67 protein( Figure 2 J).

[0117] Experimental results:

[0118] The tumor tissues obtained from the subcutaneous tumor experiment are as Figure 2 shown in G. It can be seen that the cancer cells in the exosome treatment group have smaller diameters and smaller volumes; the tumor size in live mice was measured with a vernier caliper every 5 days( Figure 2 H, *** indicates 0.0001 < p < 0.001). It was found that the tumor size increased continuously during the experiment, and the tumor size in the experimental group increased more slowly, and the volume was always significantly smaller than that of the control group;

[0119] The results of photography under the microscope are as Figure 2 shown in I. It can be seen that the positive part is brownish-yellow, the cell nuclei are light blue-violet, with a distinct contrast and no background staining; Figure 2 J is the relative expression level of the tumor growth marker protein Ki67. It can be seen that the Ki67 expression level in the experimental group is significantly lower than that in the control group.

[0120] The above experiments show that DLC-Exo can significantly inhibit the proliferation of cancer cells.

[0121] Example 4: DLC-Exo inhibits the migration of lung cancer cells

[0122] In this example, multiple methods (Transwell assay, scratch assay) were used to verify the inhibitory effect of DLC-Exo on the migration of lung cancer cells, as follows:

[0123] (I) Transwell assay

[0124] The basic principle of the Transwell experiment is to place a small chamber in a culture plate. The inside of the small chamber is called the upper chamber, and the inside of the culture plate is called the lower chamber. The upper and lower culture media are separated by a polycarbonate membrane. The upper chamber is filled with the upper culture medium, and the lower chamber is filled with the lower culture medium. The cells to be studied are seeded in the upper chamber. Since the polycarbonate membrane is permeable, the components in the lower culture medium can affect the growth, movement, etc. of the cells in the upper chamber.

[0125] It should be noted that there is an unclear expression "脱色摇床" in the original text, which is directly translated as "脱色shaker" here. You may need to correct it according to the actual situation.Experimental group (DLC-Exo group): A549 cells or HCC1833 cells that took up DLC-Exo; the concentrations of DLC-Exo were 0.43 mg / mL and 0.22 mg / mL, respectively;

[0126] NC control group: DLC-Exo in the experimental group was replaced with an equal volume of PBS buffer.

[0127] The specific detection steps of the experimental group are as follows, and the blank control is adjusted adaptively:

[0128] ①Preparation of single cell suspension:

[0129] First, A549 cells and HCC1833 cells were allowed to absorb (the method of the absorption experiment refers to Example 2) the DLC-Exo of the above-set concentration, and cultured in DEME medium (37°C); when the confluence of the cells that absorbed DLC-Exo was about 80%-90%, the medium in the bottle was discarded, and the cells were washed with PBS and then digested with trypsin, and then an appropriate amount of DMEM medium was added to terminate the digestion, and the cells were centrifuged at 1500 rpm for 4 minutes, and the cell pellets were collected, and the cell density was adjusted to 2×10 5 / mL, which is the cell suspension;

[0130] ② Add 50 μL of cell suspension to the Transwell chamber and incubate at 37°C in a 5% CO2 incubator for 30 min; then add 500 μL of DMEM complete medium to the lower chamber of each chamber, and finally carefully transfer the chamber to the lower chamber without creating bubbles (you can also push them in from the side), place in a 5% CO2 incubator, and culture at 37°C for 12 h;

[0131] ③Fixed:

[0132] Take out the chamber and place it in the adjacent blank well, rinse the upper and lower chambers with 600 μL PBS (the lower chamber is placed in a 24-well plate and can be rinsed with PBS), add 600 μL of 4% paraformaldehyde precooled at 4°C to each well, fix for 20 minutes, turn the chamber upside down, air dry, and carefully wipe off the cells inside the chamber with a cotton swab;

[0133] ④Dyeing:

[0134] Drop 0.1% crystal violet solution on the top of the inverted chamber to evenly cover the bottom of the chamber. A few drops of crystal violet staining will do. Stain for 15 minutes. Rinse with deionized water three times, then wipe the inside of the membrane of the chamber with a cotton swab and air dry thoroughly.

[0135] ⑤ Take photos:

[0136] Observe and take pictures with an inverted fluorescence microscope ( Figure 3 A and Figure 3C), and randomly count the number of cells in 6 fields of view to calculate the migration ability ( Figure 3 B and Figure 3 D).

[0137] Figure 3 A and Figure 3 C shows that in the left control group, a large area of blue appears in the field of view, that is, the number of cells is very large, indicating that the migration ability of cancer cells treated in the blank group is very strong, while the migration amounts of the two kinds of cancer cells treated with exosomes in the experimental group are significantly reduced;

[0138] Figure 3 In the statistical data graphs of B and 3C, *** indicates 0.0001 < p < 0.001. The cell migration amount in the exosome treatment group is significantly lower than that in the control group. It can be seen that DLC-Exo has a significant inhibitory effect on the migration of HCC1833 cells and A549 cells.

[0139] (II) Scratch assay

[0140] The cell scratch assay, because it is similar to the in vitro wound healing process, is also known as the wound healing assay.

[0141] Experimental group (DLC-Exo group): A549 cells or HCC1833 cells that uptake DLC-Exo, and the DLC-Exo concentrations are 0.43 mg / mL and 0.22 mg / mL respectively;

[0142] NC control group: Replace the DLC-Exo in the experimental group with an equal volume of PBS buffer.

[0143] The specific detection steps of the experimental group are as follows:

[0144] ① Preparation of cell suspension:

[0145] First, let A549 cells and HCC1833 cells uptake (the uptake experimental method refers to Example (2) the above-set concentration of DLC-Exo, and culture them in DEME medium (37 °C); when the confluence of the cells that uptake DLC-Exo is about 80%-90%, discard the medium in the flask, wash it with PBS, add trypsin for digestion, then add an appropriate amount of DMEM medium to terminate the digestion, centrifuge at 1500 rpm for 4 min, collect the cell precipitate, and adjust the cell density to 2×10 5 / mL to obtain the cell suspension;

[0146] ② Labeling:

[0147] Irradiate the ruler 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 5Different treated cells, with the confluence of cells reaching over 90% the next day as the standard;

[0148] ③ Scratch:

[0149] On the second day, replace the DMEM medium containing 1% fetal bovine serum, 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 the DMEM medium containing 1% fetal bovine serum, and place it in an incubator at 37 °C and 5% CO₂ for culture. Observe and take pictures under a fluorescence microscope at the time points of 0 h, 24 h, and 48 h ( Figure 3 F and Figure 3 G), with the central shaded area of the 12 - well as the reference, and the scratch in the center of the picture.

[0150] According to the pictures observed under the microscope after scratching ( Figure 3 E and Figure 3 G), and 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 F and Figure 3 H).

[0151] Figure 3 E and Figure 3 G show that in the control group, the cells on both sides of the scratch grew faster towards the scratch, while in the experimental group, the cells on both sides hardly migrated towards the middle; Figure 3 F and Figure 3 H is the statistical analysis. In the figure, ** indicates 0.001 < p < 0.01, *** indicates 0.0001 < p < 0.001, and it is found that DLC - Exo can significantly inhibit the migration of A549 and HCC1833 cells.

[0152] Example 5: DLC - Exo inhibits tumor growth and enriches in tumor cells

[0153] (I) Subcutaneous tumor experiment

[0154] ① Construction of subcutaneous tumor mouse model:

[0155] Inject 100 μL of PBS containing 2 × 10 6 HCC1833 cells subcutaneously into 4 - 6 - week - old female C57 nude mice to obtain mice with subcutaneous tumors;

[0156] ② Starting from the fifth day, perform intraperitoneal injection every 3 days with 5 × 10 8 particles / kg DLC - Exo (that is, determine the injection dose according to the mouse body weight) as the DLC - Exo treatment group (confirm the concentration and volume of DLC - Exo according to cell counting), and inject the same volume of PBS as the NC group control.

[0157] ③ In vivo imaging was performed at different time points after tail vein injection of PKH67-labeled DLC-Exo. The results are as follows Figure 4 A shows that DLC-Exo can exist in the body for more than 24 hours, and DLC-Exo can be enriched in tumor tissues, indicating that DLC-Exo has a good aggregation effect on tumors.

[0158] (II) HE staining experiment

[0159] HE staining lung histological evaluation can directly reflect the degree of lung injury. After HE staining, the inflammatory cell infiltration, edema and damage of the entire left lung tissue can be observed under a low-power microscope to evaluate the distribution of lung damage; while under a high-power microscope, the alveolar structure can be identified and the degree of lung injury can be evaluated and scored.

[0160] Experimental group: mouse lung tissue obtained by dissection after subcutaneous tumor experiment;

[0161] Control group: DLC-Exo in the experimental group was replaced with an equal volume of PBS buffer;

[0162] The experimental group was operated as follows, and the control group was adjusted adaptively:

[0163] ① Material collection and fixation:

[0164] The lung tissue was taken and placed in a pre-prepared fixative (10% formalin, Bouin's fixative) to denature and coagulate the proteins in the tissues and cells to prevent autolysis of the cells after death or decomposition by bacteria, thereby maintaining the original morphological structure of the cells.

[0165] ②Dehydration and transparency:

[0166] Use alcohol from low concentration to high concentration as dehydrating agent (the concentration is 70%, 80%, 90%, 95% and 100% respectively) to gradually remove the water in the tissue block; then place the tissue block in xylene, a transparent agent that is soluble in both alcohol and paraffin, to make it transparent. Xylene is used to replace the alcohol in the tissue block before wax immersion and embedding.

[0167] ③Wax embedding:

[0168] Place the transparent tissue block in the melted paraffin and put it in a wax melting box to keep it warm. After the paraffin is completely immersed in the tissue block, embed it: first prepare a container (such as a folded small paper box), pour the melted paraffin, and quickly clamp the tissue block soaked in paraffin and put it in. Cool and solidify into a block. The embedded tissue block becomes hard before it can be cut into very thin slices on the slicer.

[0169] ④ Slicing and patching:

[0170] Fix the embedded wax block on the microtome and cut into 4-μm thin sections. The cut sections are often wrinkled and need to be flattened in heated water, then pasted onto glass slides and dried in an incubator at 45°C.

[0171] ⑤ HE staining:

[0172] Place the sections that have been put into distilled water into hematoxylin aqueous solution for staining for several minutes; differentiate in acid water and ammonia water for several seconds each; rinse with running water for 1 hour and then place in distilled water for a moment; dehydrate in 70% and 90% alcohol for 10 min each; stain with alcoholic eosin staining solution for 2-3 min.

[0173] HE staining is commonly used for dewaxing to increase the color difference of various parts of the tissue cell structure, facilitating observation.

[0174] Hematoxylin (H) is a basic dye that can stain cell nuclei and ribosomes in cells blue-purple. Structures stained by basic dyes are basophilic.

[0175] Eosin (E) is an acidic dye that can stain cytoplasm red or light red. Structures stained by acidic dyes are eosinophilic. Before staining, the paraffin in the sections must be removed with xylene, then through alcohol from high concentration to low concentration, and finally into distilled water for staining.

[0176] ⑥ Dehydration and clearing:

[0177] The stained sections are dehydrated with absolute alcohol and then cleared with xylene.

[0178] ⑦ Sealing:

[0179] Drop Canada balsam on the cleared sections and cover with cover slips for sealing. After the balsam is slightly dry, attach labels and the section specimens can be used.

[0180] The results detected under the microscope are as Figure 4 shown in B. The darker-colored parts are the tumor lesions. The statistical results of the number of lesions are as Figure 4 shown in D. In the figure, *** indicates 0.0001 < p < 0.001. It can be seen that the number of tumor lesions in the tissue without exosome treatment is significantly more than that in the experimental treatment group.

[0181] (3) Immunohistochemistry to detect the expression and apoptosis of Ki67 in tumor tissues

[0182] The steps of preparing lung tissue sections and immunohistochemical staining for subcutaneous tumor-bearing mice are the same as those in Example 3. The results are shown in Figure 4 C and E.

[0183] Figure 4 The brown-stained parts in C are the positive parts, indicating that the lesions in the lung tissue after exosome treatment are significantly reduced; Figure 4 In E, *** indicates 0.0001 < p < 0.001. The results showed that the inhibition of the expression of the tumor growth marker protein Ki67 by DLC-Exo was confirmed by immunohistochemistry.

[0184] (IV) In vivo imaging to detect the enrichment of DLC-Exo in a mouse model

[0185] As described in (I), a subcutaneous tumor model was constructed. PKH26-labeled DLC-Exo was injected via the tail vein for treatment. In vivo imaging was performed at different time points, and then the nude mice were sacrificed to remove the organs for in vivo imaging detection.

[0186] The results showed that DLC-Exo could be enriched in the liver, kidney, spleen, intestine, and tumor tissues, indicating that DLC-Exo could stably exist in tumor tissues to exert its anti-tumor effect ( Figure 5 ).

[0187] In summary, the Rabdosia rubescens exosome DLC-Exo provided by the present invention can be enriched in cancer cells and tumor tissues, and can inhibit the malignant progression of tumor cells such as the proliferation and migration of tumor cells. The treatment with DLC-Exo is an innovation based on traditional Chinese medicine treatment, and has sufficient safety, stability of effective drug components, low toxicity, and solubility.

[0188] The above description of the embodiments is intended 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 Rubescens rubescens exosomes in the preparation of anti-tumor drugs.

2. The use according to claim 1, characterized in that: The method for preparing Rubescens rubescens exosomes comprises the following steps: (1) crushing fresh Rubescens rubescens to obtain crushed liquid; (2) After filtering the above-mentioned crushed liquid, ultrahigh-speed centrifugation was used to separate and collect the exosomes of Rubescens rubescens.

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

4. The use according to claim 3, characterized in that: In step (1) of the preparation method, the mixing ratio is Rubescens rubescens: 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 this process 3 to 5 times, and then filtering the precipitate with a 0.22 μm filter membrane. The obtained filtrate is a solution containing exosomes of Rubescens rubescens, and the filtrate is stored at 4° C. for later use.

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

10. The use according to claim 1, characterized in that: The concentration of Rubescens rubescens exosomes added in the application is 0.107-4.3 mg / mL, and they are dispersed and dissolved in physiological saline.

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