Application of solanum nigrum exosome in preparation of antitumor drugs

Abstract

The invention belongs to the technical field of Chinese herbal medicines, and particularly relates to application of solanum nigrum exosomes in preparation of antitumor drugs. The invention creatively develops a drug delivery system based on the plant exosome, and exosome nanoparticles with a complete membrane structure are separated from solanum nigrum tissues by combining a plant crushing technology with a differential ultra-high-speed centrifugation method. A characteristic surface marker verifies that the obtained exosome can be used as a natural drug carrier targeting tumor tissue, is taken by tumor cells and is enriched in the tumor cells. The solanum nigrum exosome is used as a natural transmission medium of a bioactive component, and accurate delivery is realized through specific recognition of tumor cell surface receptors. The obtained exosome has dual functions of a therapeutic agent and a drug carrier, provides a creative solution for developing an efficient and low-toxicity anti-tumor preparation, and has an important application value in the field of tumor targeted therapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of Chinese herbal medicines, and particularly relates to the application of Solanum nigrum exosomes in the preparation of anti-tumor drugs. Background Art

[0002] Lung cancer (LC) is a global health problem. Approximately 75% of lung cancer cases are diagnosed with non-small cell lung cancer (NSCLC). Survival rates for patients with advanced or distant metastatic NSCLC are low, with a particularly low 5-year survival rate of <4%. An estimated 1.2 million people are diagnosed with lung cancer each year, making it the leading cause of cancer-related death. Drug resistance is the primary factor leading to treatment failure in NSCLC, contributing to the majority of cancer-related deaths, tumor recurrence, and disease progression. Therefore, more effective NSCLC treatment options are needed to improve both progression-stage and 5-year overall survival.

[0003] With the development of traditional Chinese medicine, Chinese herbal medicines, including Solanum nigrum, have played a significant role in improving the tumor microenvironment. The entire plant, Solanum nigrum, is used as a medicinal herb. It is cold in nature, bitter in taste, and enters the lung and bladder meridians. It is mildly toxic and possesses the effects of clearing heat and detoxifying, promoting blood circulation and reducing swelling, and promoting diuresis and relieving stranguria. It is commonly used clinically to treat tumors, furuncles, carbuncles, erysipelas, bruises, sprains, chronic tracheitis, and acute nephritis. Solanum nigrum, combined with wild chrysanthemum, honeysuckle, and dandelion, can treat thromboangiitis obliterans. Solanum nigrum, combined with herbs such as Lysimachia chinensis, Plantago asiatica, and Lycopodiella serrata, can treat damp-heat-type cholelithiasis and urinary tract stones. Solanum nigrum, combined with herbs such as Patrinia chinensis, Peach Kernel, and Gardenia Root, can treat chronic prostatitis and chronic urethritis. Solanum nigrum is also commonly used in cancers such as liver cancer, lung cancer, stomach cancer, and bladder cancer. Improving the efficacy, solubility, and safety of active ingredients within traditional Chinese medicine preparations has long been a hot topic.

[0004] Patent CN116898946A discloses a Chinese medicine composition for treating non-small cell lung cancer and its application. The Chinese medicine composition is composed of Solanum nigrum and more than ten other Chinese herbal medicines. The Chinese medicine composition can inhibit the proliferation of non-small cell lung cancer A549 cells, promote the release of ROS, and promote cell apoptosis, thereby effectively inhibiting the occurrence of non-small cell lung cancer. Patent CN115505022B discloses an alkaloid glycoside extracted from Solanum nigrum. The alkaloid glycoside has a significant inhibitory effect on the growth and metastasis of human non-small cell lung cancer cells and human colorectal cancer cells. It can be used to prepare drugs for treating or preventing non-small cell lung cancer and colorectal cancer, as well as treating or preventing the metastasis of non-small cell lung cancer and colorectal cancer, providing important guidance for the clinical treatment or prevention of non-small cell lung cancer and colorectal cancer. The above examples illustrate that Solanum nigrum contains multiple effective chemical components.

[0005] However, the active ingredients of traditional Chinese medicine often lose their activity easily during actual application, and traditional medicines may cause damage to the body and safety issues. Summary of the Invention

[0006] The purpose of the present invention is to address the problems existing in the above-mentioned prior art and provide the use of Solanum nigrum exosomes in the preparation of anti-tumor drugs. The prior art lacks effective Chinese herbal medicines with strong therapeutic effects that can target and inhibit tumors, and there are no reports on the extraction of exosomes from Solanum nigrum and their use in anti-tumor treatment. The present invention isolates Solanum nigrum exosomes (plant-derived exosome-like nanovesicles, PELNs) from Solanum nigrum. The PELNs can carry bioactive substances and can directly target lung cancer cell tissue and significantly inhibit the malignant progression of lung cancer.

[0007] Based on the above objectives, the present invention proposes the following technical solutions: The technical solution of the present invention provides the use of Solanum nigrum exosomes (PELNs) in the preparation of anti-tumor drugs.

[0008] Furthermore, a method for preparing the above-mentioned Solanum nigrum exosomes is provided, comprising the following steps: (1) Crush fresh Solanum nigrum to obtain crushed liquid; (2) After filtering the above-mentioned crushed liquid, the Solanum nigrum exosomes were separated and collected by ultrahigh-speed centrifugation.

[0009] Furthermore, the fresh Solanum nigrum in step (1) needs to be washed with sterile water before being crushed, and mixed with pre-cooled PBS buffer; Furthermore, the crushing in step (1) refers to squeezing juice with a spiral juicer; the parameters of the juicer are set as voltage / frequency / power: 220V, 50HZ, 1.2KW; Furthermore, the filtration in step (2) refers to filtering using a 0.45 μm filter membrane to obtain a filtrate; Furthermore, the ultracentrifugation method in step (2) comprises the following steps: a) Centrifuge the filtrate at (1500-2500) g for (10-20) min under low temperature conditions and collect the supernatant I; b) centrifuging the supernatant I at (8000-12000) g for (20-40) min under low temperature conditions to collect the supernatant II; c) centrifuging the supernatant II at (100,000-140,000) g for (30-120) min under low temperature conditions and collecting the precipitate; d) Resuspend the precipitate in pre-chilled PBS, repeat this process three times, and then filter through a 0.22 μm filter membrane to obtain a concentrate containing Solanum nigrum exosomes.

[0010] Furthermore, in some embodiments, the ultracentrifugation method in step (2) includes the following steps: a) Place the filtrate in a 50 mL centrifuge tube and centrifuge at 2000 g for 15 min at 4°C to collect the supernatant I. b) Place supernatant I in a 50 mL centrifuge tube and centrifuge at 10,000 g for 30 min at 4°C to collect supernatant II. c) Place supernatant II in an ultracentrifuge tube and centrifuge at 120,000 g for 30-120 min at 4°C to collect the precipitate. d) Resuspend each tube of precipitate with 1 mL of pre-chilled PBS, repeat three times, and then filter through a 0.22 μm filter membrane to obtain a concentrate containing Solanum nigrum exosomes.

[0011] Furthermore, the low temperature is 0-5°C, preferably 4°C.

[0012] Furthermore, the Solanum nigrum exosomes are soluble in physiological saline and can exist at 4° C. for 1 month.

[0013] Furthermore, the Solanum nigrum exosomes obtained by the above preparation method are about 160-200 nm in size, appear spherical under an electron microscope, have a double-layer membrane structure, carry bioactive substances and target lung cancer cell tissues; The active substances include solanine, but are not limited thereto. Active ingredients also include other alkaloids (such as solanine, solanine, etc.), flavonoid glycosides, and steroidal saponins.

[0014] Furthermore, the tumor in the application refers to lung cancer; Furthermore, in the application, the PELNs are dispersed and dissolved with physiological saline; Furthermore, the Solanum nigrum exosomes have a targeting effect on tumor cells, can be taken up by cells, and can be enriched in tumor cell tissues.

[0015] Furthermore, the application includes: use of Solanum nigrum exosomes as tumor cell growth and proliferation inhibitors, tumor cell migration inhibitors, tumor cell cycle inhibitors and tumor cell apoptosis promoters in the preparation of anti-tumor drugs.

[0016] Furthermore, when Solanum nigrum exosomes are used as a tumor cell growth and proliferation inhibitor, the concentration used is 0-200 μg / mL, and is not 0, and the IC50 value for lung cancer cells is 55 μg / mL; when Solanum nigrum exosomes are used as a tumor cell migration inhibitor, the concentration used is 0-200 μg / mL, and is not 0; when Solanum nigrum exosomes are used as a tumor cell apoptosis promoter, the dosage used in animals is 55 mg / kg, and is not 0.

[0017] Furthermore, the Solanum nigrum exosomes can arrest the cell cycle of lung cancer cells at the S phase.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) PELNs extracted by differential ultracentrifugation have a complete double-layer membrane structure, which can protect the internal active ingredients from damage by the external environment. They can be stably stored for one month at 4°C, solving the problem of easy inactivation of effective ingredients in traditional Chinese medicine. At the same time, they are derived from natural Solanum nigrum tissue, have good biocompatibility and low toxicity, and the extraction process is safe and pollution-free. The raw materials are inexpensive and easy to obtain in large quantities, making them suitable for large-scale production.

[0019] (2) Solanum nigrum exosome 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. Solanum nigrum exosomes are both natural carriers of bioactive ingredients and have their own anti-tumor activity. The active substances such as solanine (such as alkaloids and flavonoid glycosides) carried by them can be directly delivered to lung cancer cells through targeted delivery. At the same time, the membrane structure and components of the exosomes themselves can synergistically inhibit tumor proliferation and migration, achieving the dual efficacy of delivery system + therapeutic agent and enhancing the anti-tumor effect.

[0020] (3) Preliminary studies of the present invention have confirmed that Solanum nigrum exosomes can be enriched in tumor tissues and inhibit the malignant progression of lung cancer, including inhibiting growth and proliferation, cell migration, arresting the cell cycle, and promoting cell apoptosis. Solanum nigrum exosomes can be efficiently enriched in tumor tissues by specifically recognizing lung cancer cell surface receptors, reducing the impact on normal tissues and reducing toxic side effects. Compared with the injection of pure solanine monomer, the active ingredients delivered by PELNs are more absorbed in tumors, with obvious targeting advantages.

[0021] (4) Solanum nigrum exosomes can not only inhibit the growth and proliferation of lung cancer cells (A549, HCC1833) (IC50 is 55 μg / mL, dose- and time-dependent) and migration (both Transwell and scratch experiments confirmed the inhibitory effect), but also arrest the tumor cell cycle at the S phase and promote its apoptosis (increase in TUNEL-positive cells), inhibiting the malignant progression of lung cancer in multiple dimensions, providing a more comprehensive solution for clinical treatment.

[0022] (5) Soluble in physiological saline, easy to prepare into various dosage forms (such as injection, etc.), suitable for different administration modes. In addition to being directly used as an antitumor drug, it can also be used as a drug carrier to load other antitumor components, further expanding its application scenarios in tumor targeted therapy, especially in the field of lung cancer treatment, which has important transformation value. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 to separate and characterize Solanum nigrum exosomes (PELNs); FIG. 1 is a schematic diagram of the separation process of PELNs in fresh Solanum nigrum; FIG. 2 is a transmission electron microscope detection of the morphology of PELNs; FIG. 3 is a particle size analysis of PELNs; Figure 2 FIG. 4 is a chemical structure of (A) solasonine; (B) HPLC detection results of solasonine in PELNs; (C) HPLC detection results of solasonine standard; Figure 3 FIG. 5 is a flowchart of the subcutaneous tumor experiment; FIG. 6 is a tumor growth chart of HCC1833 cells; FIG. 7 is a tumor volume size; FIG. 8 is a tumor weight size; Figure 4 FIG. 9 is a Ki67 and TUNEL immunohistochemical staining to confirm that PELNs respectively inhibit and promote the expression of Ki67 and TUNEL in tumor tissues; FIG. 10 is a Ki67 staining result; FIG. 11 is a Ki67 staining result statistics; FIG. 12 is a TUNEL staining result; FIG. 13 is TUNEL staining result statistics; Figure 5 FIG. 14 is a live imaging result; FIG. 15 is a PKH26-labeled PELNs in nude mice with the passage of time; FIG. 16 is a PKH26-labeled PELNs in nude mice with the passage of time in various organs or tissues (heart, liver, spleen, lung, kidney, tumor, intestine); FIG. 17 is a PKH26-labeled PELNs in the tumor of nude mice with the passage of time; Figure 6 FIG. 18 is a specific uptake fluorescence imaging statistics of PKH26-labeled PELNs in nude mice and various organs and tumors of nude mice with the passage of time, PELNs can exist in vivo for more than 24 h, and PELNs can be enriched in tumor tissues; FIG. 19 is a PKH26-labeled PELNs in the tumor of nude mice with the passage of time; FIG. 20 is a PKH26-labeled PELNs in nude mice with the passage of time; FIG. 21 is a PKH26-labeled PELNs in nude mice with the passage of time in various organs or tissues (heart, liver, spleen, lung, kidney, intestine); Figure 7 This is the result of Maldi-tof detecting whether solanine is enriched in tumors after 24 hours; Figure 8 Fluorescence microscopy shows the uptake of PKH26-labeled PELNs by A549 (A) or HCC1833 cells (B); Figure 9 CCK8 assay confirmed that PELNs inhibited the proliferation of A549 (A, C) and HCC1833 (B, D) cells in a dose- and time-dependent manner, suggesting that PELNs have a good anti-tumor effect. Figure 10 Figure 2: (A) Schematic diagram of colony formation assay results, (B) Statistical analysis of A549 and HCC1833 cell proliferation results; PELNs can significantly inhibit the proliferation of A549 / HCC1833 cells. Figure 11 (A) EdU assay results, (B) A549 and HCC1833 cell proliferation results. The assay confirmed that PELNs significantly inhibited the proliferation of A549 / HCC1833 cells and arrested them in the S phase. Figure 12 (A) Fluorescence inverted microscopy images of Transwell assays, (B) statistical analysis of A549 and HCC1833 cell migration results; PELNs treatment significantly inhibited the migration of both lung cancer cell lines. Figure 13 (A) The results of T scratch assay, (B) the statistical results of A549 and HCC1833 cell scratch assays; it was confirmed that PELNs treatment can significantly inhibit the migration of A549 / HCC1833 cells. DETAILED DESCRIPTION

[0024] 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 with reference to specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but are not intended to 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 be made without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0025] All raw materials of the present invention are not particularly limited in their sources and can be purchased from the market or prepared according to conventional methods well known to those skilled in the art.

[0026] Example 1: Preparation and morphological characterization of PELNs (I) The preparation of PELNs mainly includes crushing and ultra-high-speed centrifugation collection (the separation process of PELNs in fresh Solanum nigrum is referenced Figure 1 A), specifically including the following steps: Take 500 g of fresh Solanum nigrum, wash it thoroughly with sterile water, chop it, add 500 mL of PBS (pre-cooled at 4°C), and use a screw juicer to extract the juice (voltage / frequency / power: 220 V, 50 Hz, 1.2 kW). Collect the juice and filter it with a 0.45 μm filter membrane to collect the filtrate. 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; Place supernatant I in a 50 mL centrifuge tube and centrifuge at 10,000 g for 30 min at 4°C to collect supernatant II; Place supernatant II in an ultracentrifuge tube and centrifuge at 120,000 g for 30 min at 4°C to collect the precipitate; Resuspend each tube of precipitate with 1 mL of pre-cooled PBS, repeat three times, and then filter with a 0.22 μm filter membrane. The concentrated solution obtained after filtration contains PELNs, and the concentrated solution is stored at 4°C for future use.

[0027] (2) The PELNs in the above concentrate were characterized by morphology under a transmission electron microscope, such as Figure 1 As shown in B, it shows that PELNs present a double-layer membrane structure.

[0028] (III) Particle size determination results of PELNs, such as Figure 1 As shown in C, the results show that the size of PELNs is around 160-200 nm.

[0029] (IV) One of the main active ingredients in PELNs is solanine (structural formula: Figure 2 A) test results, such as Figure 2 B (experimental group results) and Figure 2 As shown in C (standard product result), it can be seen that PELNs contain solanine.

[0030] Example 2: PELNs inhibit tumor growth and accumulate in tumor cells and nude mice (1) Subcutaneous tumor experiment ① Subcutaneously inject 100 μL of PBS containing 2×10^6 HCC1833 cells into 4-6 week old female BALB / c nude mice to obtain nude mice with subcutaneous tumors (refer to the experimental flow chart of subcutaneous tumors). Figure 3 A); ② Starting from the fifth day, 55 mg / kg PELNs were injected intraperitoneally every 2 days as the PELNs treatment group, and the same amount of PBS was injected as the NC group control.

[0031] ③ Use a vernier caliper to measure the tumor size every 2 days ( Figure 3 B) The weight of the nude mice was recorded every 2 days, and the tumor weight and volume were measured after the nude mice were euthanized one month later ( Figure 3 C- Figure 3 D).

[0032] The results showed that the tumor volume and weight of the PELNs-treated group were significantly lower than those of the NC group, indicating that PELNs can effectively reduce the volume and weight of tumors, further demonstrating that PELNs inhibited tumor growth.

[0033] (II) Immunohistochemical detection of Ki67 and TUNEL levels in tumor tissues ① Tumor tissue sections: The collected tumor tissues were embedded in paraffin, and the embedded paraffin sections were dewaxed to water: the sections were sequentially placed in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and then washed with distilled water; ② Antigen retrieval: Place the tissue sections in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0) and perform antigen retrieval in a microwave oven. Set the microwave oven parameters as follows: medium heat for 8 minutes, off heat for 8 minutes, and medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer and do not let the sections dry out. After natural cooling, place the tissue sections in PBS (pH 7.4) and wash them on a decolorizing shaker three times for 5 minutes each time. ③ BSA blocking: After the tissue sections are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), add 3% BSA in the circle, evenly cover the tissue, and block at room temperature for 30 minutes; ④Add primary antibody (red): Gently shake off the blocking solution, add Ki67 and TUNEL primary antibodies (PBS) on the slices, and incubate the slices flat in a light-proof humidified box at 4°C overnight; ⑤ DAPI counterstaining of cell nuclei (blue): Place tissue sections in PBS (pH 7.4) and wash three times on a decolorizing shaker for 5 minutes each time; after the tissue sections are slightly dried, add DAPI staining solution to the circle and incubate at room temperature in the dark for 10 minutes; ⑥ Mounting: Place the tissue sections in PBS (pH 7.4) and wash them three times on a decolorizing shaker, each time for 5 minutes. Shake the sections slightly dry and then mount them with anti-fluorescence quenching mounting medium. ⑦ Microscopic examination and photography: The sections were observed and images were collected under a Nikon inverted fluorescence microscope ( Figure 4 A and Figure 4 C), and the expression levels of Ki67 and TUNEL were calculated ( Figure 4 B and Figure 4 D).

[0034] The results showed that immunohistochemistry confirmed that PELNs inhibited and promoted the expression of tumor growth marker proteins Ki67 and TUNEL, respectively.

[0035] (III) In vivo uptake experiment in nude mice ① PKH26 was used to label Solanum nigrum PELNs. 55 mg / kg PELNs were injected into tumor-bearing nude mice (i.e., nude mice with subcutaneous tumors) via the tail vein. An in vivo imaging system was used to detect PKH26 uptake by various organs and tumors in the nude mice at designated times. Figure 5 , AE), and calculate ( Figure 6 , AH).

[0036] like Figure 5 The figure shows the uptake of PKH26-labeled PELNs by nude mice and their various organs and tumors over time. The experiment was performed over a time course (0-24 hours). Figures A and E show the uptake of PKH26-labeled PELNs in nude mice over a 24-hour period, confirming that PELNs can be taken up by nude mice. Figures B and D show the uptake of PKH26-labeled PELNs in various organs and tissues (heart, liver, spleen, lung, kidney, tumor, and intestine) in nude mice over a 24-hour period, demonstrating that PELNs can be taken up by these three organs. Figure C is a magnified image of the uptake of PKH26-labeled PELNs in a nude mouse tumor over a 24-hour period, demonstrating that PELNs can be taken up by tumors. These experimental results demonstrate that PELNs can be taken up by nude mice.

[0037] like Figure 6 As shown in the figure, the X-axis (Total Radiant Efficiency) represents the total radiant efficiency. Higher values ​​indicate stronger bioluminescent signals, which in turn indicate higher levels / activity of the target (e.g., labeled cells or molecules). The Y-axis (Time) represents the time point of the assay (e.g., 1 hour, 4 hours, 8 hours, ... 24 hours). Figure 6 In A (Tumor), high radiation efficiency is concentrated in 20-24 hours (yellow and orange columns), and the corresponding X-axis value (2-3×10^9 p / s) is significantly higher than that at other time points, indicating that the luminescence signal at the tumor site is strongest at 20-24 hours, suggesting that the activity / distribution of labeled tumor cells in the tumor-bearing nude mouse model is most significant during this time period. Figure 6 In B (Mouse), the signal intensity is significantly lower than that of the tumor (only 1-2.4×10^6 p / s on the X-axis), and the difference at each time point is small, indicating that the bioluminescence background of the mouse's own tissue is low and has weak interference with the tumor signal; or the marker is mainly enriched in the tumor and has little distribution throughout the body. Figure 6 C (Heart), Figure 6 D (Liver), Figure 6E (Spleen), Figure 6 F (Lung), Figure 6 G (Kidney), Figure 6 H (Colorectum) indicates that the signal intensity of the organ part is higher than that of the tumor site.

[0038] In summary, the radiation efficiency of tumor is much higher than that of other tissues, indicating that PELNs can exist in the body for more than 24 h and can be enriched in tumor tissue.

[0039] ② Maldi-tof was used to detect the enrichment of solanine (one of the most abundant components in Solanum nigrum PELNs) in tumors that had taken up PKH26 for 24 hours ( Figure 7 The redder the color and the more abundant the solanine in the tumor tissue, the greater its uptake. The results show: on the left, the uptake of solanine in nude mouse tumors 24 hours after injection with solanine; on the right, the uptake of solanine in nude mouse tumors 24 hours after injection with PELNs containing a nearly identical dose of solanine. This demonstrates that tumor uptake of solanine delivered by PELNs is superior to that of pure solanine alone.

[0040] Example 3: Uptake of PKH26-labeled PELNs by A549 and HCC1833 cells A549 and HCC1833 cells are both human lung cancer adenocarcinoma cells. To demonstrate the uptake of PELNs by A549 and HCC1833 cells, they were labeled with PKH26 staining solution and observed using a fluorescence microscope. The specific experimental steps are as follows: ①Staining of PELNs with PKH26 staining solution 60 μL of exosomes diluted in PBS were added to 0.5 mL of diluent C (Sigma-Aldrich); 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 excess dye, 1 mL of 0.5% BSA / PBS was added; the labeled exosomes were washed at 100,000 g for 1 h, and the exosome pellet was diluted in 100 μL of PBS and used for uptake experiments.

[0041] ②Uptake of PELNs by A549 and HCC1833 cells Control group: A549 and HCC1833 cells only; Experimental group: 1 μL PKH26-labeled PELNs+A549 cells; 1 μL PKH26-labeled PELNs+ HCC1833 cells; Cells in the control and experimental groups were cultured in DMEM complete medium for 24 hours, fixed with paraformaldehyde for 15-20 minutes, and washed three times with PBS. 400 μL of DAPI staining solution (just enough to cover the bottom of the dish) was added, incubated at room temperature for 3-5 minutes, and washed two to three times with PBS, each time for 3-5 minutes, before being observed under a microscope.

[0042] The results are as follows Figure 8 A- Figure 8 As shown in B, the three-channel detection of DAPI (blue, marking cell nuclei), PKH26 (red, marking PELNs) and the superposition of the two (Merge) shows that DAPI clearly stains the cell nucleus while PELNs show red fluorescence signals after being labeled with PKH26. After merging, the positional relationship between the nucleus and the labeled signal can be observed. It can be seen from the figure that after staining, the labeled PELNs can be taken up by both cells, indicating that PELNs can be taken up by A549 and HCC1833.

[0043] Example 4: PELNs inhibit lung cancer cell proliferation This example uses multiple methods to verify the inhibitory effect of PELNs on lung cancer cells.

[0044] (I) CCK8 detection experiment Experimental group: PELNs-treated group; control group: DMEM medium only group; three replicates per group. The experimental group procedures are as follows: ① Detect IC50 A 96-well plate with 5×10^3 A549 or HCC1833 cells was placed in a 37°C incubator for 6 h to allow them to adhere to the wall. The 96-well plate was removed, the PELNs-treated group was washed with PBS, and 0-200 μg / mL PELNs were added to the plate. After incubation for 24 h, 10% CCK8 detection reagent was added and the plate was placed back in a 37°C incubator. After incubation, the plate was detected with a microplate reader at 450 nm. The results are as follows ( Figure 9 A- Figure 9 B) As shown, the IC values ​​of A549 and HCC1833 cells treated with PELNs were obtained. 50 The concentration was 55 μg / ml and the effects were dose-dependent.

[0045] ② Detection time dependence A 96-well plate with 5×10^3 A549 or HCC1833 cells was placed in a 37°C incubator for 6 h to allow them to adhere to the wall. The 96-well plate was removed, the PELNs-treated group was washed with PBS, and 55 μg / mL PELNs were added to the plate. After incubation for 0 h, 24 h, 48 h, and 72 h, 10% CCK8 detection reagent was added and the plate was placed back in a 37°C incubator. After incubation, the plate was detected by a microplate reader at 450 nm. The results are as follows ( Figure 9 C- Figure 9 D) shows that the optimal inhibition time for PELNs treatment of A549 and HCC1833 cells is 24 h, and the inhibition is time-dependent.

[0046] (II) Colony formation assay A549 or HCC1833 cells in the logarithmic growth phase that had taken up PELNs (final concentration 55 μg / mL) were digested with 0.25% trypsin and made into single-cell suspensions for counting; The cell suspension of each group was seeded into a 6-well plate at 1×10^3 cells / dish and incubated in a 5% CO2, 37°C constant temperature incubator. The culture was terminated when colonies were visible to the naked eye. The sections were washed twice with PBS (pH 7.4), fixed with methanol for 15 min, discarded, stained with crystal violet for 10 min, rinsed with running water, and dried in air at room temperature; like Figure 10 A- Figure 10 As shown in B, A549 and HCC1833 cells were used as control groups, and the inhibitory effect of PELNs on the proliferation of A549 / HCC1833 cells was observed.

[0047] (3) EdU experiment EdU staining can mark cells in the S phase of the cell cycle. The specific steps are as follows: 5 × 10^4 A549 / HCC1833 cells that had taken up PELNs (final concentration 55 μg / mL) were seeded per well in a 24-well plate. EdU working solution (YF®596 Click-iT EdU Universal Cell Proliferation Detection Kit, Biosense Biotechnology Co., Ltd., red color) was added, and cell labeling was performed at room temperature according to the kit's instructions. Add 0.1 mL of 0.5% Triton X-100 (in PBS) to each well and incubate at room temperature for 20 min to fix and enhance cell permeability. After adding Hoechst 33342 solution (blue color) for staining for 5 min, the cells were rinsed three times with PBS and the cell nuclei were counterstained; EdU was detected by fluorescence microscopy, photographed, and the results were statistically analyzed.

[0048] like Figure 11 A- Figure 11 B, A549 / HCC1833 cells were used as the control group, and the inhibitory effect of PELNs on the proliferation of A549 / HCC1833 cells was observed.

[0049] Example 5: PELNs inhibit the migration of lung cancer cells (1) Transwell assay The Transwell assay was set up as follows: Experimental group (PELNs group): A549 / HCC1833 cells were pretreated with PELNs (final concentration of 55 μg / mL); Blank control (Control group): A549 / HCC1833 cells were pretreated with an equal volume of PBS; The specific testing steps for the intervention experimental group are as follows, and the blank control group is adaptively adjusted: ① Preparation of single-cell suspension: When the confluence of A549 or HCC1833 cells containing PELNs (final concentration of 55 μg / mL) is approximately 80%-90%, discard the culture medium in the flask, wash with PBS, and then add trypsin to digest. Digestion is terminated with DMEM medium. Centrifuge at 1000 rpm for 3 min, collect the cell pellet, and adjust the cell density to 5 × 10^5 cells / mL with serum-free DMEM medium to prepare the cell suspension; ② Add 100 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. Finally, carefully transfer the chamber to the lower chamber without creating bubbles (you can also push it in from the side). Place it in a 5% CO2 incubator and culture at 37°C for 24 h. Set up three replicates for each group. ③ Fixation: Remove the chamber and place it in an 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 pre-cooled at 4°C to each well. Fix for 20 minutes, then turn the chamber upside down. After air drying, carefully wipe off the cells inside the chamber with a cotton swab. ④ Staining: Drop 0.1% crystal violet solution on the top of the inverted chamber so that it evenly covers the bottom of the chamber. A few drops of crystal violet stain will suffice. 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. ⑤ Take photos: Use a fluorescent inverted microscope to observe and take photos, and count the number of cells in the field of view.

[0050] like Figure 12 A- Figure 12 B, Transwell assay showed that PELNs inhibited the migration of A549 and HCC1833 cells.

[0051] (2) Scratch test The scratch settings are grouped as follows: Experimental group (PELNs group): A549 / HCC1833 cells were pretreated with Solanum nigrum PELNs (final concentration of 55 μg / mL); Blank control (Control group): A549 / HCC1833 cells were pretreated with the same volume of PBS; Three replicate wells were set up in each group; The specific testing steps for the intervention experimental group are as follows, and the blank control group is adaptively adjusted: ① Sterilize all instruments and set aside for use.

[0052] ② Add approximately 5×10^5 cells of different treatments to each well of a 6-well plate, and consider the cells to reach a confluence of more than 90% the next day. On the second day, replace the DMEM medium containing 1% fetal bovine serum and scratch the well perpendicular to the horizontal line with a 10 μL pipette tip. Rinse gently with PBS 2-3 times, add DMEM medium containing 1% fetal bovine serum, and culture in a 37°C, 5% CO2 incubator. Observe and photograph under a fluorescence microscope at 0 h, 24 h, and 48 h, respectively ( Figure 13 A) The scratch is in the center of the image, with the shaded area in the center of the 6-well plate as a reference.

[0053] ③ According to the pictures after scratching, the cell migration rate of each group was detected by comparing the scratch width of each group ( Figure 13 B).

[0054] like Figure 13 A- Figure 13 B, Scratch test showed that Solanum nigrum PELNs could significantly inhibit the migration of A549 and HCC1833 cells.

[0055] In summary, the Solanum nigrum exosomes PELNs provided by the present invention can be enriched in cancer cells and tumors in animals, can inhibit the malignant progression of tumor cells such as tumor cell proliferation and migration, can arrest tumor cells in the S phase and significantly promote tumor cell apoptosis. Solanum nigrum PELNs treatment is an innovation based on traditional Chinese medicine treatment, with sufficient safety, stability of effective drug ingredients, low toxicity and solubility.

[0056] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. Application of Solanum nigrum exosomes in the preparation of anti-tumor drugs.

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

3. The use according to claim 2, characterized in that Before being crushed in step (1), the fresh Solanum nigrum is washed with sterile water and mixed with pre-cooled PBS buffer.

4. The use according to claim 2, characterized in that The filtration in step (2) refers to filtering using a 0.45 μm filter membrane to obtain a filtrate.

5. The use according to claim 2, characterized in that The ultracentrifugation method in step (2) comprises the following steps: a) Centrifuge the filtrate at (1500-2500) g for (10-20) min under low temperature conditions and collect the supernatant I; b) centrifuging the supernatant I at (8000-12000) g for (20-40) min under low temperature conditions to collect the supernatant II; c) centrifuging the supernatant II at (100,000-140,000) g for (30-120) min under low temperature conditions and collecting the precipitate; d) resuspending the precipitate in pre-chilled PBS, and filtering it through a 0.22 μm filter membrane to obtain a concentrate containing Solanum nigrum exosomes; The low temperature mentioned above is 0-5°C.

6. The use according to any one of claims 2 to 5, characterized in that: The preparation method of the Solanum nigrum exosomes is: a) Wash fresh Solanum nigrum with sterile water, mix with 4°C pre-cooled PBS at a 1:1 mass-to-volume ratio, crush using a juicer at 220V, 50Hz, and 1.2kW, and collect the crushed liquid. b) Filter the disrupted liquid through a 0.45 μm filter membrane, centrifuge at 2000 g for 15 min at 4°C, and collect the supernatant I; c) Centrifuge supernatant I at 10,000 g for 30 min at 4°C and collect supernatant II; d) Supernatant II was centrifuged at 120,000 g for 30-120 min at 4°C. The precipitate was collected, resuspended three times with pre-cooled PBS, and filtered through a 0.22 μm filter to obtain a concentrated solution containing Solanum nigrum exosomes.

7. The use according to claim 1, characterized in that The Solanum nigrum exosomes are 160-200 nm in size, appear spherical under an electron microscope, have a double-layer membrane structure, carry bioactive substances and target lung cancer cell tissues; the active substances include solanine.

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

9. The use according to claim 1, characterized in that The Solanum nigrum exosomes are used as tumor cell growth and proliferation inhibitors, tumor cell migration inhibitors and tumor cell apoptosis promoters in the preparation of anti-tumor drugs.

10. The use according to claim 1, characterized in that The concentration of Solanum nigrum exosomes in the tumor cell growth and proliferation inhibitor is 0-200 μg / mL, and is not 0; the concentration of Solanum nigrum exosomes in the tumor cell migration inhibitor is 0-200 μg / mL, and is not 0.

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