Lithospermum erythrorhizon rhizome exosome vesicles, preparation method thereof and application of exosome vesicles in preparation of antitumor drugs

Abstract

The invention relates to lithospermum erythrorhizon rhizome exosome vesicles as well as a preparation method and application thereof in preparation of antitumor drugs. The preparation method comprises the following steps: juicing lithospermum erythrorhizon, filtering, and collecting filtrate; and centrifuging the filtrate, collecting the precipitate, re-suspending, and filtering to obtain a solution containing the lithospermum erythrorhizon exosome vesicles. Compared with the prior art, the lithospermum erythrorhizon rhizome exosome vesicles extracted by the method can inhibit malignant progression, including proliferation and migration, of lung cancer, and cell and animal living imaging proves that the lithospermum erythrorhizon rhizome exosome 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 vesicles of Lithospermum erythrorhizon roots and their preparation methods and applications in the preparation of anti-tumor drugs. Background Art

[0002] Lung cancer (LC), as a malignant tumor of the primary bronchopulmonary, is one of the cancers with the highest incidence worldwide and is also a major cause of cancer death. Exploring the causes of lung cancer, revealing its regulatory mechanisms and seeking new treatment methods have become an urgent clinical need.

[0003] Lithospermum erythrorhizon has a long history as a medicine in China. It was first recorded in "Shennong Ben Cao Jing", and later recorded in all subsequent materia medica. "Shennong Ben Cao Jing" expounded on the medicinal value of Lithospermum erythrorhizon: "Bitter in taste, cold in nature. It is mainly used for treating pathogenic factors in the heart and abdomen, five types of jaundice, tonifying the middle qi, benefiting the nine orifices, and dredging the water channels." Lithospermum erythrorhizon is sweet, salty, and cold in nature, and belongs to the heart and liver meridians. Its main functions are clearing heat and cooling blood, promoting blood circulation and detoxifying, and promoting eruption and eliminating macules. It can be used for excessive heat and toxin, purple and black macules, unsmooth measles, sores, eczema, and scalds caused by fire and water. The chemical components of Lithospermum erythrorhizon are mainly naphthoquinone components, and at the same time contain alkaloids, phenols, polysaccharides, esters and other components. People have studied the main component shikonin of Lithospermum erythrorhizon and confirmed that shikonin has various pharmacological activities such as anti-tumor, antioxidant, anti-inflammatory, antiviral, anti-thrombotic, immunomodulatory, and promoting wound healing.

[0004] Patent CN117645727A discloses a supramolecular complex based on shikonin and divalent copper ions and its preparation method. In this invention, shikonin and divalent copper salt are respectively dissolved in ethanol, mixed to obtain a mixed solution; after stirring the mixed solution, it is added to water for purification treatment to obtain the supramolecular complex, and its molecular structural formula is: where n takes an integer greater than 100. The shikonin-copper (II) supramolecular complex of this invention can solve the problem of poor solubility of shikonin, and has stronger anti-tumor activity, does not affect normal organs, and has good safety. Patent CN109223737A discloses the application of shikonin compounds in the preparation of drugs to promote the anti-tumor activity of TRAIL. This invention provides the application of shikonin compounds in the preparation of drugs to enhance the anti-tumor activity of A, where A is TRAIL recombinant protein or TRAIL modifier. Experiments have proved that after pretreatment with shikonin (SKN) and then co-stimulation with a low dose of TRAIL, it can significantly increase the killing effect of TRAIL on A549, HeLa, PANC1 and HCT116, and has no cytotoxic effect on the normal cell line HEK 293. It shows that shikonin compounds can be used to enhance the anti-tumor activity of TRAIL, TRAIL recombinant protein or TRAIL modifier.

[0005] However, at present, there are certain limitations in the exploration of Lithospermum erythrorhizon. Currently, most research focuses almost entirely on the active ingredient shikonin in Lithospermum erythrorhizon, and a large number of studies have been carried out on its pharmacological effects, extraction processes, etc. However, extracts containing complex active ingredients such as exosomes are rarely involved in the research of Lithospermum erythrorhizon. Exosomes contain a variety of unique bioactive substances. Moreover, in key application fields such as anti-tumor, research on Lithospermum erythrorhizon exosomes is extremely rare. This research status limits our comprehensive and in-depth understanding of Lithospermum erythrorhizon and also restricts the development of its potential applications in a wider medical field. Summary of the Invention

[0006] The object of the present invention is to overcome the defects of the above-mentioned existing technologies and provide Lithospermum erythrorhizon rhizome exosome vesicles, a preparation method thereof, and an application thereof in the preparation of anti-tumor drugs. In this application, Lithospermum erythrorhizon rhizome exosome vesicles (Lithospermum erythrorhizon Siebold-Exosome, LE-Exo) are isolated from Lithospermum erythrorhizon. The Lithospermum erythrorhizon rhizome exosome vesicles can carry bioactive substances and can directly target lung cancer cell tissues and significantly inhibit the malignant progression of lung cancer.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] One of the technical solutions of the present invention provides an application of Lithospermum erythrorhizon rhizome exosome vesicles in the preparation of anti-tumor drugs;

[0009] Furthermore, a preparation method of the Lithospermum erythrorhizon rhizome exosome vesicles is provided, including the following steps:

[0010] S1. Juice the Lithospermum erythrorhizon rhizome, filter, and collect the filtrate;

[0011] S2. Centrifuge the filtrate obtained in step S1, collect the precipitate, resuspend it, and filter to obtain a solution containing Lithospermum erythrorhizon rhizome exosome vesicles, and the Lithospermum erythrorhizon rhizome exosome vesicles contain shikonin.

[0012] Furthermore, in step S1, before juicing and crushing the Lithospermum erythrorhizon rhizome, it needs to be washed with sterile water and mixed with pre-cooled PBS buffer. Preferably, the PBS solution is pre-cooled at 4°C.

[0013] Furthermore, in step S1, the addition ratio of Lithospermum erythrorhizon rhizome to PBS solution is (1 - 1.2) g : (1 - 1.2) mL;

[0014] In some specific embodiments, the ratio of Lithospermum erythrorhizon rhizome to PBS solution is preferably 1 g : 1 mL.

[0015] Furthermore, in step S1, the filtration is performed using a 0.45 μm filter membrane.

[0016] Further, in step S2, the filtrate is centrifuged several times at 4°C and a rotation speed of 2000 - 120000 g, and the centrifugation time for each time is 15 - 135 min.

[0017] Further, in step S2, the filtrate is centrifuged at 4°C and a rotation speed of 2000 - 3000 g for 15 - 20 min to collect supernatant I; supernatant I is centrifuged at 4°C and 10000 - 20000 g for 30 - 45 min to collect supernatant II; supernatant II is centrifuged at 4°C and 100000 - 120000 g for 90 - 135 min to collect the precipitate.

[0018] In some specific embodiments, in step S2, it is preferably:

[0019] The filtrate is centrifuged at 4°C and a rotation speed of 2000 g for 15 min to collect supernatant I;

[0020] Supernatant I is centrifuged at 4°C and 10000 g for 30 min to collect supernatant II;

[0021] Supernatant II is centrifuged at 4°C and 120000 g for 90 min to collect the precipitate.

[0022] In some specific embodiments, in step S2, after the precipitate is resuspended with PBS solution, it is filtered through a 0.22 μm filter membrane.

[0023] Further, purple perilla rhizome exosome vesicles are provided, obtained based on the preparation method described in the above technical solution.

[0024] In some specific embodiments, the particle size of the exosome vesicles is about 220 - 260 nm.

[0025] Further, the tumor in the application refers to non - small cell lung cancer (NSCLC cells).

[0026] Further, the application includes: the application of LE - Exo as an inhibitor of tumor cell growth and proliferation, an inhibitor of tumor cell migration, and a promoter of tumor cell apoptosis in the preparation of anti - tumor drugs.

[0027] Further, the application also includes: LE - Exo as a carrier of active ingredients to transport the active ingredients to the pathological site.

[0028] In some specific embodiments, the tumor is caused by subcutaneous injection of non - small cell lung cancer Luc - 1833 cells (HCC - 1833 cells). Meanwhile, this technical solution may also be applicable to other types of tumors.

[0029] In some specific embodiments, the anti-tumor drug inhibits tumor cells and / or delivers shikonin in tumor cells.

[0030] In some specific embodiments, the anti-tumor drug is dissolved in normal saline.

[0031] In some specific embodiments, the addition concentration of LE-Exo in the anti-tumor drug is 100 - 500 μg / mL.

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

[0033] (1) The extraction process is simple and easy for large-scale preparation.

[0034] Traditional extraction methods are complex, requiring professional equipment and technology, with high costs and low yields. However, by optimizing the process and adopting mild treatment means, the present invention has a simple operation process, low requirements for equipment, can significantly reduce costs, and achieve a large amount of acquisition of exosome vesicles from purple perilla roots, laying a foundation for its wide application.

[0035] (2) It is soluble and easy to be taken up, inhibiting tumor growth.

[0036] The drug solubility and cell uptake ability are related to the curative effect. The exosome vesicles from purple perilla roots of the present invention can be well dissolved in normal saline, facilitating the development of preparations and clinical applications. Specific membrane proteins and ligands on its surface can bind to the receptors of non-small cell lung cancer cells to achieve efficient uptake. After entering the cells, it can regulate cytokines and signaling pathways in the microenvironment, improve the hypoxic and acidic environment that is originally conducive to tumor growth. At the same time, it can also activate immune cells, enhance the body's immune function, and inhibit the growth and proliferation of tumor cells through multiple means.

[0037] (3) Delivering shikonin to target and block tumor growth.

[0038] Shikonin has anti-tumor activity, and the exosome vesicles from purple perilla roots of the present invention are its ideal carriers. During extraction, shikonin is encapsulated therein to avoid degradation and inactivation. The substances on the surface of the exosomes guide them to target and bind to non-small cell lung cancer cells, cut off the tumor energy supply, and effectively inhibit the growth and metastasis of tumor cells.

[0039] (4) Combining with traditional Chinese medicine, it has broad application prospects.

[0040] Traditional Chinese medicine has a long history, and the combination of the exosome vesicles from purple perilla roots of the present invention with it is of great significance. It has high safety, is derived from purple perilla roots, is a natural active substance, is not easy to cause immune reactions, and is suitable for clinical applications. The effective drug components have good stability, which can ensure the continuous exertion of the drug effect. In addition, it also has low toxicity and good solubility. These advantages make it have broad application prospects in the field of traditional Chinese medicine and can promote the modernization development of traditional Chinese medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 For the isolation and characterization of exosome vesicles (LE-Exo) from Lithospermum erythrorhizon roots

[0042] Reference numerals:

[0043] (A) Schematic diagram of the isolation process of LE-Exo from fresh Lithospermum erythrorhizon roots; (B) Particle size analysis and separation of LE-Exo; (C) Transmission electron microscopy detection of the morphology of LE-Exo; (D) Uptake of PKH67-labeled LE-Exo by HCC-1833 and A549 cells

[0044] Figure 2 For LE-Exo to deliver its shikonin (SKN) to inhibit the proliferation of NSCLC cells

[0045] Reference numerals:

[0046] (A) Proliferation of HCC-1833 cells treated with LE-Exo at different time intervals; (B) Proliferation of A549 cells treated with LE-Exo at different time intervals; (C) Proliferation of Beas-2B cells treated with LE-Exo at different time intervals; (D) Proliferation of HCC-1833 cells treated with SKN at different time intervals; (E) Proliferation of A549 cells treated with SKN at different time intervals; (F) Proliferation of Beas-2B cells treated with SKN at different time intervals

[0047] Figure 3 For the inhibitory effect of LE-Exo delivering SKN on the proliferation of non-small cell lung cancer cell lines

[0048] Reference numerals:

[0049] (A-C) Detection of the effects of LE-Exo and SKN on the proliferation of HCC-1833 and A549 cells using the EDU assay; (D-F) Cell colony formation assay indicating that LE-Exo and SKN inhibit the colony formation of HCC-1833 and A549 cells

[0050] Figure 4 For in vitro experiments to verify that LE-Exo delivers SKN to inhibit the invasion and migration of NSCLC cells

[0051] Reference numerals:

[0052] (A) Effects of LE-Exo and SKN on Transwell migration of A549 and HCC-1833 cells (20×) and the bar graph of quantitative analysis; (B) Scratch areas of A549 cells treated with LE-Exo and SKN at 0 h, 24 h, and 48 h time points (10×) and the bar graph of their quantitative analysis; (C) Scratch areas of HCC-1833 cells treated with LE-Exo and SKN at 0 h, 24 h, and 48 h time points (10×) and the bar graph of their quantitative analysis;

[0053] Figure 5 For in vivo experiments to verify the inhibition of subcutaneous xenograft tumor growth by LE-Exo and SKN in nude mice;

[0054] Reference numerals:

[0055] (A) Fluorescence images of treating nude mice with subcutaneous tumors of HCC-1833 with LE-Exo extracted from Lithospermum erythrorhizon; (B-D) Effects of LE-Exo and SKN on volume and weight; (E-F) Ki67 immunohistochemistry to detect the effects of LE-Exo and SKN on the expression of tumor growth marker protein Ki67; (G-H) Fluorescence signal results of Tunel staining of LE-Exo and SKN;

[0056] Figure 6 For in vivo experiments to verify the inhibition of lung metastasis in nude mice by LE-Exo and SKN;

[0057] Reference numerals:

[0058] (A) After tail vein injection of HCC-1833 cells, the in vivo enrichment of tumors after drug administration; (B-C) After treatment with LE-Exo and SKN, the tumors in the lungs of nude mice were significantly reduced, and HE staining showed a significant decrease in the number of tumors after drug administration. Detailed implementation manners

[0059] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and gives detailed implementation manners and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0060] In the following examples and comparative examples, unless otherwise specified for raw materials or processing techniques, it means that they are all conventional commercially available raw material products or conventional processing techniques in the art.

[0061] The concentration of the PBS buffer solution used in all examples of this application is: 0.0067 M, and the pH is 7.4.

[0062] The complete culture medium is DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody.

[0063] The paraformaldehyde fixative was 4% (w / v) and was purchased from Biosharp (BL539A).

[0064] The crystal violet staining solution was 0.1% (w / v) and was purchased from Beyotime (Cat No. C0121-500ml).

[0065] Example 1: Preparation and morphological characterization of SP-Exo

[0066] (I) Preparation of exosome vesicles from the roots and rhizomes of Lithospermum erythrorhizon (as Figure 1 shown in A), and the specific operation steps are as follows:

[0067] ① Take 500 g of fresh roots and rhizomes of Lithospermum erythrorhizon, wash them thoroughly with sterile water, chop them up, add 500 mL of PBS solution (pre-cooled at 4°C), and use a spiral juicer to extract the juice (voltage / frequency / power: 220 V, 50 Hz, 1.2 KW). Collect the juice and filter it through a 0.45 μm filter membrane, and then collect the filtrate;

[0068] ② Place the filtrate in a 50 mL centrifuge tube, centrifuge at 2000 g for 15 min at 4°C, and collect the supernatant I;

[0069] ③ Place the supernatant I in a 50 mL centrifuge tube, centrifuge at 10000 g for 30 min at 4°C, and collect the supernatant II;

[0070] ④ Place the supernatant II in an ultracentrifuge tube, centrifuge at 120000 g for 90 min at 4°C, and collect the precipitate;

[0071] ⑤ Resuspend the precipitate in 1 mL of pre-cooled PBS per tube, repeat 3 times, and filter through a 0.22 μm filter membrane. The filtrate is the solution containing exosome vesicles from the roots and rhizomes of Lithospermum erythrorhizon (LE-Exo), and it is stored at 4°C for later use.

[0072] (II) Morphological characterization of LE-Exo under a transmission electron microscope, as Figure 1 shown in B, LE-Exo presents a bilayer membrane structure.

[0073] (III) Results of the particle size measurement of LE-Exo, as Figure 1 shown in C, the results show that: the size of LE-Exo is about 220 - 260 nm.

[0074] (IV) PKH26 experiment, as Figure 1 shown in D, and the specific steps are as follows:

[0075] ① PKH26 staining experiment:

[0076] PKH26 is a lipophilic fluorescent dye.

[0077] 60 μL of exosomes diluted in PBS were added to 0.5 mL of diluent C (Sigma-Aldrich). Meanwhile, 4 μL of PKH26 dye was added to 0.5 mL of diluent C and incubated with the exosome solution for 4 min. To bind the excess dye, 1 mL of 0.5% BSA / PBS was added. The labeled exosomes were centrifuged at 100,000 g for 1 h, and the exosome pellet was diluted in 100 μL of PBS and used for the uptake experiment.

[0078] ② Uptake experiment:

[0079] The uptake experiment refers to allowing the experimental cells to uptake PKH26-stained exosomes LE-Exo, which will also be used in the subsequent examples. This experiment is divided into a control group and an experimental group:

[0080] Control group: Only HCC-1833 cells / A549 cells, without exosome treatment.

[0081] Experimental group: HCC-1833 cells / A549 cells + 1 μL of PKH26-stained LE-Exo (after adding PKH26-stained exosomes to the complete DMEM medium, first filter through a 0.22 μm filter, and then add to the cells in a 6-well plate), and stain for 24 h.

[0082] ① After culturing the above control group and experimental group cells in 4 mL of complete DMEM medium for 24 h, they were fixed with paraformaldehyde for 15 - 20 min (a conventional fixation method in the art), and washed 3 times with PBS;

[0083] ② DAPI staining: Add 400 μL of DAPI staining solution (just covering the bottom of the dish), and incubate at room temperature for 15 min to allow the staining agent to fully bind to the cells;

[0084] ③ After mixing, wash 2 - 3 times with PBS, 3 - 5 min each time. Finally, place under the microscope for photography and observation.

[0085] The results of photography under the microscope are as Figure 1 shown in D. In the figure, the exosomes after PKH26 staining appear red ( Figure 1 the second column of D), the cells after DAPI staining appear blue ( Figure 1 the third column of D). The blue fluorescence and red fluorescence parts in the cells after uptake are almost coincident. Therefore, according to the position and color of the fluorescence in the figure, it can be judged that exosome LE-Exo can be taken up and absorbed by human non-small cell lung cancer adenocarcinoma cells.

[0086] Example 2: LE-Exo delivers shikonin (SKN) to inhibit the proliferation of NSCLC cells

[0087] NSCLC cells, namely Non-Small Cell Lung Cancer cells, are the most common type of lung cancer, accounting for approximately 80%-85% of all lung cancer cases. NSCLC cells mainly originate from the tumor tissues of NSCLC patients. These tumor tissues can be obtained through surgical resection, percutaneous biopsy, etc., and then, after a series of treatments and cultures, a cell line that can continuously grow in vitro is established. Common NSCLC cell lines include: A549 cells, HCC-1833 cells.

[0088] Shikonin (SKN) is a natural naphthoquinone compound extracted from plants of the Boraginaceae family, with various biological activities and broad application prospects in the fields of medicine, cosmetics, etc. Shikonin mainly exists in the roots of plants of the Boraginaceae family, such as Arnebia euchroma (Royle) Johnst., Arnebia guttata Bunge, Lithospermum erythrorhizon Sieb. et Zucc., etc.

[0089] (1) The CCK-8 experiment aims to quantitatively evaluate cell proliferation, toxicity, and viability by detecting the dehydrogenase activity in cells. The experimental results are as Figure 2 shown, and the specific operation steps are as follows:

[0090] 1. Seeding cells on the plate: Digest the cells in the logarithmic growth phase with trypsin to prepare a cell suspension, and inoculate 5000 cells per well into a 96-well plate, adding 100 μL of cell suspension to each well; Place the 96-well plate in a CO 2 (5%) incubator and culture overnight at 37°C for cell attachment, and fill the edge wells with sterile PBS.

[0091] The cells in the logarithmic growth phase include 3 types of cells: HCC-1833 cells (human lung adenocarcinoma cells HCC-1833, cancer cells), A549 cells (human lung alveolar basal epithelial cells of lung cancer, cancer cells), and Beas-2B cells (human normal lung epithelial cells, normal cells). The HCC-1833 cell line is derived from the tumor tissue of a patient with lung adenocarcinoma. Lung adenocarcinoma is a type of non-small cell lung cancer (NSCLC) and is one of the most common subtypes of lung cancer, which is preserved and distributed by the American Type Culture Collection (ATCC). A549 cells are derived from the lung adenocarcinoma tissue of a 58-year-old white male. In 1972, D.J. Giard et al. obtained and established the cell line through surgical resection, and it is also preserved and promoted by ATCC. Beas-2B cells are an immortalized cell line established by infecting normal human bronchial epithelial cells with an adenovirus 12-SV40 virus hybrid. The above cells can be obtained by applying to ATCC to purchase the cell line, or can be obtained from domestic qualified cell banks or research institutions.

[0092] 2. Perform drug treatment according to the specific experimental protocol, with 3 replicates set for each sample concentration.

[0093] The specific experimental protocol is as follows:

[0094] Experimental groups:

[0095] A: 0 - 200 μg / mL LE-Exo + 100 μL HCC-1833 cell suspension;

[0096] B: 0 - 500 μg / mL LE-Exo + 100 μL A549 cell suspension;

[0097] C: 0 - 400 μg / mL LE-Exo + 100 μL Beas-2B cell suspension;

[0098] D: 0 - 7 μM SKN + 100 μL HCC-1833 cell suspension;

[0099] E: 0 - 25 μM SKN + 100 μL A549 cell suspension;

[0100] F: 0 - 25 μM SKN + 100 μL Beas-2B cell suspension;

[0101] Negative control group: Each group is 100 μL cell suspension of the corresponding cells;

[0102] Blank control group: Each group is 100 μL high-glucose DMEM medium.

[0103] 3. CCK-8 reaction: Add 10 μL CCK-8 solution and 90 μL high-glucose DMEM medium to all wells respectively, mix evenly, and incubate in the incubator for 1 hour.

[0104] 4. Measure the absorbance value: Use a microplate reader to measure the light absorbance value at 450 nm, and calculate the inhibition rate of the drug on cells according to the formula.

[0105] Experimental groups: Absorbance value of the experimental group cells after adding CCK-8 solution

[0106] Negative control: Absorbance value of the control cells after adding CCK-8 solution

[0107] Blank control: Absorbance value of the medium after adding CCK-8 solution

[0108] Proliferation rate = (experimental group - blank control) / (negative control - blank control) × 100%

[0109] Inhibition rate = 1 - (experimental group - blank control) / (negative control - blank control) × 100%

[0110] Figure 2 To deliver shikonin (SKN) contained therein by LE-Exo and inhibit the proliferation of non-small cell lung cancer (NSCLC) cells. In the figure, * indicates 0.01 < p < 0.05, ** indicates 0.001 < p < 0.01, and *** indicates 0.0001 < p < 0.001.

[0111] (A) The proliferation of HCC-1833 cells treated with LE-Exo at different time periods, confirming that the median lethal dose of LE-Exo for the proliferation ability of HCC-1833 cells is 200 μg / mL; (B) The proliferation of A549 cells treated with LE-Exo at different time periods, confirming that the median lethal dose of LE-Exo for the proliferation ability of A549 cells is 500 μg / mL; (C) The proliferation of Beas-2B cells treated with LE-Exo at different time periods, showing no obvious inhibitory effect;

[0112] (D) The proliferation of HCC-1833 cells treated with SKN at different time periods, reaching the IC50 at 4 μM; (E) The proliferation of A549 cells treated with SKN at different time periods, reaching the IC50 at 5 μM; (F) The proliferation of Beas-2B cells treated with SKN at different time periods, showing an inhibitory effect.

[0113] Thus, it can be seen that the LE-Exo, like SKN, has an inhibitory effect on tumor cells HCC-1833 cells and A549 cells. However, LE-Exo has no obvious inhibitory effect on the proliferation of normal cells Beas-2B cells, and it is a safe and highly selective active substance.

[0114] Example 3: Inhibition of the proliferation of NSCLC cells by LE-Exo delivering shikonin (SKN) (I) The EDU experiment aims to study the inhibitory effects of LE-Exo and SKN on the proliferation of HCC-1833 cells and A549 cells. As shown in Figure 3 A-C, three groups are set for each type of cell. The experimental groups are: LE-Exo (L) (low-concentration LE-Exo treatment group), LE-Exo (H) (high-concentration LE-Exo treatment group), and SKN group;

[0115] Among them, the concentration of the LE-Exo (L) group for A549 cells is 250 μg / mL, the concentration of the LE-Exo (H) group is 500 μg / mL, and the added concentration of SKN in the SKN group is 5 μM;

[0116] The concentration of the LE-Exo(L) group of HCC-1833 cells was 100 μg / mL, and the concentration of the LE-Exo(H) group was 200 μg / mL; the concentration of LE-Exo in the SKN group was 4 μM.

[0117] 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.

[0118] The specific operation steps for the experiment of inhibiting cancer cell proliferation are as follows:

[0119] ① Take cells in the logarithmic growth phase (including two types of cells: HCC-1833 cells, A549 cells), and seed 5 the cells at a density of 4×10

[0120] cells per well in a 6-well plate, 100 μL per well; add 100 μL of medium containing 50 μM EdU to each well; incubate for 2 h, discard the medium, wash twice with PBS for 5 min each time;

[0121] 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 shaker for 5 min, and discard the glycine solution;

[0122] ③ Add 1 mL of PBS, wash on a shaker for 5 min, and discard the PBS; add 1 mL of 0.5% TritonX-100, incubate on a shaker for 10 min, wash once with PBS for 5 min each time;

[0123] ④ Add 500 μL of 1X Apollo staining reaction solution to each well, 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 on a shaker three times for 10 min each time, discard the permeabilizer, wash twice with PBS for 5 min each time, add 500 μL of 1X Hoechst 33342 reaction solution, 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 three times; Take pictures and count with a high-content instrument. Figure 3 B - C.

[0124] Figure 3A-C were: Detecting the effects of LE-Exo and SKN on the proliferation of HCC-1833 and A549 cells using EDU experiments. # in the figure indicates 0.01 < p < 0.05; ## in the figure indicates 0.001 < p < 0.01; ***, in the figure indicate 0.0001 < p < 0.001. The results showed that: The area and number of cells containing green fluorescence in the control group were larger in the field of view, while the number of cells containing green fluorescence in the experimental group was smaller. It can be seen that LE-Exo has a significant inhibitory effect on the proliferation of A549 / HCC-1833 cells.

[0125] (II) Cell colony formation assay

[0126] Take cells in the logarithmic growth phase (including two types of cells: HCC-1833 cells, A549 cells), suspend them in 1 mL of complete medium and count (using a cell counting plate for cell counting).

[0127] Disperse the cells evenly into a 6-well plate at a density of 1000 cells / well, and place them in a cell incubator (5% CO 2 , 37 °C) for 2 weeks. Change the medium every 2 days and observe the cell status. Carefully aspirate the original medium, and slowly add 1 mL of fresh complete medium to each well, avoiding flushing away the cells, and observe the cell status.

[0128] When visible clones appear in the culture dish, terminate the culture. Wash twice with PBS, add 1 mL of 4% paraformaldehyde to each well for fixation for 30 min, wash twice with PBS, add 1 mL of crystal violet staining solution to each well, and stain for 20 min; Wash the cells several times with PBS, air dry, take pictures and count. The number of clones = the number of colonies in each well.

[0129] Figure 3 D-F were: Detecting the effects of LE-Exo and SKN on the proliferation of HCC-1833 and A549 cells using cell colony formation experiments. # in the figure indicates 0.01 < p < 0.05; ## in the figure indicates 0.001 < p < 0.01; ***, in the figure indicate 0.0001 < p < 0.001. The results showed that: The cell colony formation experiment indicated that the number of cell colony clones in the control group was larger, and the number of cell colony clones in the LE-Exo and SKN groups was significantly reduced. It can be seen that LE-Exo and SKN have a significant inhibitory effect on the colony formation of HCC-1833 and A549 cells.

[0130] Example 4: In vitro experiments to verify the inhibition of NSCLC cell invasion and migration by LE-Exo delivering SKN

[0131] (I) Transwell experiment

[0132] Cells (including two types of cells: HCC-1833 cells and A549 cells) were cultured with normal administration until the logarithmic growth phase. The cells were digested with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, washed twice with PBS, suspended in serum-free medium, and plated at a density of 1×10 5 / mL in the upper chamber of the Transwell insert. 300 μL of 20% complete medium was added to the lower chamber. After 24 h, the medium was discarded, the cells were washed twice with PBS, fixed with paraformaldehyde fixative for 30 minutes, washed twice with PBS, stained with crystal violet for 15 minutes, thoroughly washed with PBS, and photographed and counted under a microscope.

[0133] Figure 4 A shows the effects of LE-Exo and SKN on the Transwell migration of A549 and HCC-1833 cells (20×) and the quantitative analysis histogram. The results showed that the cell migration of LE-Exo (L), LE-Exo (H), and SKN was significantly lower than that of the control group. LE-Exo (L), LE-Exo (H), and SKN all inhibited the migration of A549 and HCC-1833 cells, and the inhibitory strength was LE-Exo (H) > SKN > LE-Exo (L). ## in the figure indicates 0.001 < p < 0.01; *** and in the figure indicate 0.0001 < p < 0.001.

[0134] (2) Cell scratch assay

[0135] Cells in the logarithmic growth phase were digested into single-cell suspensions with 0.25% trypsin and seeded in 6-well culture plates at a density of 5×10 5 cells per well. The cells were cultured in a cell incubator at 37°C and 5% CO 2 with a relative humidity of about 95%. After 24 h, drugs were administered according to different groups. When the confluence rate reached 100% at 48 h, a 200 μL pipette tip was used to scratch vertically along the horizontal line on the back of the plate; on the back of the 6-well plate, horizontal lines were evenly drawn every 1 cm with a marker pen, and 3 horizontal lines were drawn in each well. The cells were washed 3 times with PBS to remove the scratched cells, and serum-free medium (serum-free medium is DMEM high-glucose medium supplemented with HEPES [-], phenol red [+], 4.5 g / L D-glucose [+], 4 mM L-glutamine [+], and 0.11 g / L sodium pyruvate [+]) was added. Photos were taken at 0 h, 24 h, and 48 h respectively. Analyzed and counted with Image J (Open the taken photos in Image J software, use the straight line tool to measure the scratch width, select 3 points at different positions of each scratch for measurement, and take the average value as the width of the scratch). The formula for cell migration rate (%) = (initial scratch width - scratch width after 24 h) / initial scratch width × 100%, and the cell migration rates of each group were calculated.

[0136] Figure 4 B is the bar graph of the scratch area (10×) of LE-Exo and SKN acting on A549 cells at the time points of 0 h, 24 h, and 48 h and its quantitative analysis (24 h, 48 h); Figure 4 C is the bar graph of the scratch area (10×) of LE-Exo and SKN acting on HCC-1833 cells at the time points of 0 h, 24 h, and 48 h and its quantitative analysis (24 h, 48 h). The results show that LE-Exo (L), LE-Exo (H), and SKN all have inhibitory effects on the migration of A549 and HCC-1833 cells, and can slow down the speed of scratch recovery. # in the figure indicates 0.01 < p < 0.05; ## in the figure indicates 0.001 < p < 0.01; ***, in the figure indicate 0.0001 < p < 0.001.

[0137] Example 5: In vivo experiment to verify the inhibition of the growth of subcutaneous xenografts in nude mice by LE-Exo and SKN

[0138] (I) Construction of a tumor mouse model

[0139] Female BALB / c nude mice at 4 weeks old and weighing 18 - 22 g were selected and purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd. They were adaptively fed in an SPF environment for 1 week before the experiment.

[0140] After HCC-1833 cells were digested with 0.25% trypsin and centrifuged, they were resuspended with PBS and counted. Tumor inoculation was carried out according to the quantity and volume of 5×10 5 / 100 μL per nude mouse. The tumor inoculation 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 discharged, and tumor inoculation was prepared; 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 into the skin clamped by the left thumb of 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 was completed, it was paused for a few seconds to ensure that the cell suspension fully entered the subcutaneous tissue, and the needle was slowly withdrawn. After withdrawal, the left hand continued to pinch the skin for a few seconds to prevent the leakage of the cell suspension; the growth of the tumor was observed starting from the 7th day after subcutaneous tumor inoculation. The tumor volume was measured once every 2 days using a vernier caliper. The mice were sacrificed on the 21st day after tumor inoculation. The tumor volume calculation formula = long side × short side2 / 2. The whole experimental process was carried out in a laminar flow hood, and strict aseptic operation procedures were followed. Pipette tips, syringes, etc. used needed to be autoclaved.

[0141] (II) In vivo imaging to detect the enrichment of exosomes in vivo

[0142] 1. PKH26 stain LE-Exo (the staining steps are the same as in Example 1), dissolve it in PBS after ultra-high speed centrifugation (centrifuge at 100,000 g for 1 h);

[0143] 2. Inject it into the nude mice with subcutaneous tumors in the experimental groups (LE-Exo(L) group, LE-Exo(H) group, SKN group) via the tail vein. The injection dose for each nude mouse is 135 mg / kg (calculated and injected according to the body weight of the mice). The control group is injected with an equal volume of PBS buffer solution. Take pictures and samples at different time intervals (1 h, 3 h, 6 h, 12 h, 24 h, 48 h);

[0144] 3. Take pictures and count the viscera tissues at different time intervals.

[0145] The experimental results are as Figure 5 shown in A - D:

[0146] Figure 5 In A: LE-Exo extracted from Lithospermum erythrorhizon was used to treat nude mice with subcutaneous tumors of HCC-1833, and the uptake time of LE-Exo in vivo was explored. It showed that the drug enrichment in vivo reached the best at 3 h, and LE-Exo could persist in vivo for 48 h.

[0147] Figure 5 B - D show that the therapeutic effects of LE-Exo and SKN are significant, including the inhibition of volume and weight. In the figure, ## indicates 0.001 < p < 0.01; in the figure, ***, indicate 0.0001 < p < 0.001.

[0148] (3) Ki67 immunohistochemistry

[0149] Ki67 immunohistochemistry is used to detect the degree of lung cancer differentiation. The specific experimental steps of Ki67 immunohistochemistry are as follows:

[0150] Put the tissues taken in step (2) into 4% paraformaldehyde for fixation for 4 hours, then dehydrate them successively 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 embed the tissues. Section and dewax, hydrate, prepare antigen repair solution, and perform antigen repair. Inactivate endogenous peroxidase and biotin, block with serum, incubate with Ki67 primary antibody at 4 °C overnight, incubate at room temperature for 30 minutes, wash 2 times with PBS, incubate with secondary antibody for 10 minutes, develop color with DAB for 5 minutes, counterstain with hematoxylin for 1 minute, dehydrate and clear, mount the slides, take pictures and count under a fluorescence microscope.

[0151] Figure 5In E-F, Ki67 immunohistochemical detection showed that LE-Exo and SKN could inhibit the expression of the tumor growth marker protein Ki67. In the figure, ** indicates 0.001 < p < 0.01; in the figure, *** indicates 0.0001 < p < 0.001.

[0152] (III) Tunel staining

[0153] Tunel staining aims to label the DNA breaks of apoptotic cells and visually observe the apoptosis of cells through color development, providing a basis for studying disease mechanisms, drug efficacy, and cell fate regulation. The specific experimental steps of Tunel staining are as follows:

[0154] After dewaxing and rehydrating the sections, incubate with 20 μg / mL proteinase K at 37 °C for 15 minutes, and incubate with 3% H 2 O 2 Incubate at room temperature for 20 minutes. According to the instructions of the colorimetric Tunel cell apoptosis detection kit, incubate the biotin-labeled solution at 37 °C for 60 minutes and the Streptavidin-HRP working solution at room temperature for 30 minutes. After DAB color development and hematoxylin staining for 20 seconds, mount the slides. Analyze the average optical density of the images with ImageJ. Apoptotic cell ratio = DAB absorbance / (DAB + eosin absorbance).

[0155] Figure 5 Tunel staining in G-H showed enhanced fluorescence signals of LE-Exo and SKN, suggesting that LE-Exo and SKN could promote the apoptosis of tumor tissues. In the figure, *** indicates 0.0001 < p < 0.001.

[0156] Example 6: In vivo experiment to verify the inhibition of lung metastasis in nude mice by LE-Exo and SKN

[0157] HE staining can clearly distinguish tumor tissues from normal tissues, helping to accurately identify the tumor morphology and count the number, providing an intuitive basis for the diagnosis and study of tumors. The specific experimental steps of HE staining are as follows:

[0158] Place the sections in a 70°C in situ hybridization instrument and bake the sections for 30 min. Quickly put the baked sections into xylene twice, for 5 min each time. Place them in 100%, 90%, 80%, and 70% ethanol for 5 min each, and in distilled water for 5 min. Blot dry the water, add hematoxylin staining solution dropwise, stain for 5 min, rinse off the hematoxylin staining solution with running water, and observe the staining degree under a microscope. Differentiate with 1% hydrochloric acid alcohol for 3 s, wash with water, and soak in tap water for 7 min for blueing; add 1 drop of 0.5% eosin staining solution, stain for 10 s, wash with distilled water, and observe under a microscope. Dehydrate with 80%, 90%, 95%, and 100% ethanol. Soak in xylene twice, for 5 min each, and air dry at room temperature. Add a drop of neutral gum on the tissue and cover with a coverslip. Observe and take pictures under a microscope after 24 h.

[0159] Figure 6 In vivo experiments were carried out to verify the inhibition of lung metastasis in nude mice by LE-Exo and SKN. Figure 6 A shows the in vivo enrichment of tumors after tail vein injection of HCC-1833 cells and administration of drugs; Figure 6 B-C show that the tumors in the lungs of nude mice were significantly reduced after treatment with LE-Exo and SKN, and HE staining showed a significant reduction in the number of tumors after drug administration.

[0160] 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 exosome vesicles from lithospermum rhizome in the preparation of anti-tumor drugs, characterized in that: The method for preparing exosome vesicles from Lithospermum officinale rhizomes comprises the following steps: S1, extracting juice from the rhizome of Lithospermum officinale, filtering and collecting the filtrate; S2. Centrifuge the filtrate obtained in step S1, collect the precipitate, resuspend it, and filter it to obtain a solution containing exosome vesicles from the rhizomes of Lithospermum officinale, wherein the exosome vesicles from the rhizomes of Lithospermum officinale contain shikonin.

2. According to claim 1, the use of exosome vesicles from Lithospermum officinale rhizomes in the preparation of anti-tumor drugs, in step S1, the Lithospermum officinale rhizomes need to be washed with sterile water before juicing and crushing, and mixed with pre-cooled PBS buffer.

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

4. The use of the exosome vesicles from Lithospermum rhizomes 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 vesicles from Lithospermum rhizomes 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 vesicles from Lithospermum rhizomes 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 vesicles from Lithospermum rhizomes according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The particle size of the exosome-like vesicles of the lithospermum rhizome is 220-260 nm.

8. The use of the exosome vesicles from Lithospermum rhizomes 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.

9. The use of the exosome vesicles from Lithospermum officinale rhizomes according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The applications include: using LE-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; and using LE-Exo as a carrier of active ingredients to transport the active ingredients to pathological sites.

10. The use of the exosome vesicles from Lithospermum officinale rhizomes according to claim 9 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug inhibits tumor cells and / or delivers shikonin in tumor cells.

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