CN114432328A

CN114432328A - Pharmaceutical composition for treating hepatocellular carcinoma and application thereof

Info

Publication number: CN114432328A
Application number: CN202011221395.XA
Authority: CN
Inventors: 韩睿
Original assignee: First Affiliated Hospital of Guangzhou University of Chinese Medicine
Current assignee: First Affiliated Hospital of Guangzhou University of Chinese Medicine
Priority date: 2020-11-05
Filing date: 2020-11-05
Publication date: 2022-05-06

Abstract

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition for treating hepatocellular carcinoma and application thereof. The medicinal composition is a tribuloside liposome nanoparticle compound, and the tribuloside is coated in the liposome. The tribuloside has obvious CAXII inhibitor characteristics and high-efficiency and low-toxicity liver cancer cell proliferation resistance, and can further play a role in 'synergy and attenuation' on the tumor inhibition effect of the tribuloside with the help of a novel cationic nanoparticle LNP-DP1 targeted delivery system.

Description

Pharmaceutical composition for treating hepatocellular carcinoma and application thereof

Technical Field

The invention relates to the technical field of medicines, in particular to a pharmaceutical composition for treating hepatocellular carcinoma and application thereof.

Background

Primary liver cancer is the sixth most common malignant disease worldwide and also the second leading cause of cancer-related death. Hepatocellular Carcinoma (HCC) accounts for 85% -90% of primary liver cancer onset and is the fourth most common cause of cancer-related death worldwide. Early HCC can be treated by local ablation, surgical resection or liver transplantation, but the high recurrence rate remains a serious challenge for radical surgery, and the five-year survival rate is only about 35%; more than half of HCC patients are already advanced at diagnosis, and treatment options for advanced patients are more limited (e.g., chemotherapy, radiation therapy, targeted drugs, and immune checkpoint inhibitors). Although extensive clinical trials are conducted to explore more optimal dosing regimens, the clinical benefits are still not optimistic. Therefore, the global demand for the development of new effective drugs for liver cancer treatment is still urgent.

Carbonic Anhydrase (CA) XII is a transmembrane zinc metalloenzyme, and is involved in the regulation of tumor microenvironment, contributing to cancer cell survival, promoting cancer cell invasion and migration, and maintaining cancer cell pluripotency, etc. Recent studies found that CAXII is in an abnormally high expression state in hepatocellular carcinoma patients, and the expression level thereof has a significant negative correlation with the prognosis of HCC patients, and is an independent prognostic factor. Liver cancer forms a highly hypoxic tumor microenvironment due to its rapid growth rate and the surrounding fibrotic tissue produced by chronic inflammation, further promoting the overproduction of pyruvate, lactate and carbonate, aggravating the hypoxic/acidic microenvironment and leading to enhanced tumor infiltration capacity. Tumor immune surveillance escape and local inflammation are driven. CAXII is used as regulator of hypoxia stress and acidity in vivo, and can influence tumor microenvironment (such as pH value) by regulating proteins such as 14V-ATPase and 15V-ATPase, and promote liver cancer progression. Therefore, CAXII inhibitors are expected to be a strategy to control HCC progression and reduce immunosuppressive stress mediated by hypoxia/acidic metabolism, especially considering their potential combinations (e.g. combination therapy of immune checkpoint based immunotherapy). However, international research on the antitumor mechanism of CAXII inhibitors is still in the beginning and needs to be deeply revealed, so the development of antitumor CAXII inhibitors has become a new international research hotspot in recent years. Meanwhile, the existing antitumor CAXII inhibitor lacks the targeting property for treating tumor lesions, has high systemic drug concentration after administration, has certain side effects, and has better high-efficiency and low-toxicity antitumor CAXII inhibitor to be explored.

Disclosure of Invention

In order to solve the technical problems, the invention provides a pharmaceutical composition for treating hepatocellular carcinoma, which has good targeting property and strong anti-tumor effect.

The invention adopts the following technical scheme:

in one aspect, the invention provides the use of tribuloside in the preparation of a medicament for the treatment of hepatocellular carcinoma.

On the other hand, the invention also provides a pharmaceutical composition for treating hepatocellular carcinoma, which is a tribuloside liposome nanoparticle complex and comprises tribuloside and liposome, wherein the tribuloside is coated in the liposome.

Further, mixing oxalic acid and the liposome nanoparticles in equal volume, centrifuging, concentrating, and standing to obtain the liposome nanoparticles coated with the tribuloside.

Further, the raw material components of the liposome nanoparticle include 1, 2-dioleyl-3-dimethylamino-propane, L- α -phosphatidylcholine, cholesterol, and cholesterol-polyethylene glycol (polyoxyethylene cholesteryl sebacate).

Further, the molar ratio of 1, 2-dioleyl-3-dimethylamino-propane, L-alpha-phosphatidylcholine, cholesterol and cholesterol-polyethylene glycol (polyoxyethylene cholesteryl sebacate) is (9-10): (3-4): (7-8): (1-2). Preferably, the molar ratio is 9:3:7: 1.

Further, 1, 2-dioleyl-3-dimethylamino-propane, phosphatidylcholine, cholesterol and cholesterol-polyethylene glycol (polyoxyethylene cholesteryl sebacate) are respectively dissolved into alcohol solutions in a water bath environment at 60 ℃, and are mixed according to the molar ratio of (9-10) to (3-4) to (7-8) to (1-2), then the mixture is slowly dripped into a rapidly stirred 4-hydroxyethyl piperazine ethanesulfonic acid solution to obtain a mixed liquid with the alcohol concentration of 35%, and then alcohol in the mixed solution is dialyzed, removed and filtered to obtain a liposome nano material solution.

Further, 20mM HEPES was used as the dialysis solution, and the dialysis solution was changed every 2 hours during dialysis for 8-12 hours.

Further, the pharmaceutical composition is used for treating hepatocellular carcinoma cells of which liver cancer cells are Hep3B and SNU-449.

The invention also provides application of the pharmaceutical composition in preparing a medicament for treating hepatocellular carcinoma.

Tribulus terrestris L, which is a plant of Zygophylaceae (Zygophylaceae) tribulus, also known as caltrop, Zygophyllaceae, tribulus terrestris, ascending and pushing and the like, is used as a common medicinal plant of traditional Chinese medicine and Indian ayurvedic medicine system, has wide and long medicine use history, is considered to enter liver meridian by record of Chinese pharmacopoeia, and can 'calm liver, relieve depression, activate blood and dispel wind'; the book of the meeting appointment medical mirror is recorded in the book of the meeting appointment medical mirror: "purging lung qi to dispel liver wind"; shen nong Ben Cao Jing (Shen nong's herbal classic) Yun he Nei Tu: "major blood stasis, broken knot accumulation"; the book Ben Cao Hui Yan also considers that it can be used for the syndrome of blood stasis; the domestic literature also reports effective clinical experience of tumor treatment. Modern researches find that the tribulus terrestris extract has a remarkable inhibiting effect on various tumors, but the effective components and a specific tumor inhibiting mechanism are still poorly known. Tribulus terrestris (Tiliroside, TS) is one of the main active ingredients of Tribulus terrestris, belongs to the family of flavonoids, and the study on the Tribulus terrestris and the active ingredients thereof mostly takes steroid saponins as the main ingredient, and the study on the flavonoid ingredients, especially the Tribulus terrestris is less. Through a large number of researches, the tribuloside is found to have remarkable CAXII inhibitor characteristics and high-efficiency and low-toxicity anti-hepatoma cell proliferation capacity.

The liposome nanoparticle (LNP-DP1) prepared by the invention has the characteristics of higher encapsulation and delivery efficiency, better safety and economy, easier acquisition and the like. The liposome nanoparticles can provide focus targeting for the wrapped medicine, have a double-layer and spherical liposome structure, have good encapsulation, loading efficiency and stability on the medicine, have strong Enhanced Penetration and Retention (EPR) effect, can be preferentially retained at a liver tumor part and enter tumor cells after intravenous injection, so that the portable medicine is preferentially accumulated to the tumor tissue and released, simultaneously ensure the low concentration of the medicine in other tissues of the periphery of the tumor tissue, and have the functions of synergy and attenuation on the carried medicine. Liposomal nanoparticles (LNP-DP1) provide an ideal choice for a delivery system for Tribuloside (TS) liver cancer therapy.

The invention provides a pharmaceutical composition for treating hepatocellular carcinoma, which is a tribuloside liposome nanoparticle complex, and the tribuloside is coated in liposome. The tribuloside has obvious CAXII inhibitor characteristics and high-efficiency and low-toxicity anti-hepatoma cell proliferation capacity, and can further play a role in 'synergism and attenuation' on the tumor inhibition effect of the tribuloside with the help of a novel cationic nanoparticle LNP-DP1 targeted delivery system.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a schematic structural diagram of a tribuloside liposome nanoparticle complex in the invention;

FIG. 2 is a graph showing the results of the effect of tribuloside on the relative concentration of CAXII in HCC cells in the present invention;

FIG. 3 is a schematic diagram showing the results of the effect of tribuloside on the CAXII esterase activity of HCC cells in the present invention;

FIG. 4 is a schematic diagram showing the effect of tribuloside on the expression of CAXII protein in HCC cells in the present invention;

FIG. 5 shows the IC50 values of the tribuloside, LNP-DP1 tribuloside complex of the present invention at 72 hours for the cell lines tested;

FIG. 6 is a schematic diagram showing the results of the inhibition rate of different concentrations of tribuloside on HCC cell lines and normal liver cells in the present invention;

FIG. 7 is a schematic diagram showing the results of the inhibition rate of the present invention LNP-DP1 and MC3 encapsulating tribuloside on HCC cell line and hepatic normal cell line, respectively;

FIG. 8 is a graph showing the effect of LT on the migration ability of Hep3B cells in the present invention;

FIG. 9 is a graph showing the results of LT on the migratory capacity of SNU-449 cells in the present invention;

FIG. 10 is a graph showing the results of the inhibition of clonality of hepatocarcinoma cell colonies by LT in the present invention;

FIG. 11 is a graph showing the effect of LT on the invasion capacity of hepatoma cells in the present invention;

FIG. 12 is a graph showing the effect of LT on the three-dimensional organoid formation ability of hepatoma cells in the present invention;

FIG. 13 is a bioluminescent signal image of a mouse SNU-449 hepatoma transplantation tumor according to the present invention;

FIG. 14 is a schematic diagram showing the tumor growth rate of SNU-449 hepatoma transplanted tumor mice in the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: formulation of Liposomal nanoparticles (LNP-DP1)

The raw material components and the amounts of the liposome nanoparticles (LNP-DP1) used in this example are shown in Table 1.

TABLE 1 Liposomal nanoparticle (LNP-DP1) formulation raw materials

The preparation method of liposome nanoparticle (LNP-DP1) is as follows:

the first day: preheating 100% alcohol to 60 deg.C, weighing all liquid components in Table 1 according to molar ratio, adding into preheated alcohol according to requirement, and dissolving completely. The four liquid components are dissolved into an alcohol solution (DODMA/EggPC/Chol/PEG-liqid ═ 45:15:35:5) in proportion (the dissolution is carried out in a water bath environment at 60 ℃), and the mixed solution is slowly dropped into a rapidly stirred HEPES solution (20mM, pH7.4) by using a syringe until the solution is a new mixed liquid with the alcohol concentration of 35%. The alcohol in the solution was removed by dialysis using MWCO 10,000Dalton Float-A-Lyzer (Spectrum Laboratories, Canada) placed in a stirrer and rotated, and 20mM HEPES was used as a dialysis solution, and the dialysis solution was changed every 2 hours during dialysis for 8 to 12 hours.

The next day: filtering the mixed solution after dialysis with 0.22um standard syringe filter, and storing the filtered nano material solution at 4 deg.C to obtain LNP-DP 1.

Specifically, the sources of reagents used in this example are as follows:

DODMA：Dimethylamino-1,2-dioleyloxy-propane，C₄₁H₈₁NO₂purchased from Sigma-Aldrich, USA;

cholesterol: cholesterol, C₂₇H₄₆O, available from Sigma-Aldrich, USA;

egg PC: l- α -phosphatidylholine [ 95% ], available from Sigma-Aldrich, USA;

PEG-Chol: Cholesterol-PEG 600 available from Sigma-Aldrich, USA;

ETOH: ethanol solution, available from ATCC corporation, usa;

HEPES (high efficiency particulate air): HEPES buffer, purchased from Sigma-Aldrich, USA.

Example 2: preparation of tribuloside liposome nanoparticle compound

Adding LNP-DP1 and tribuloside (mixed at a volume ratio of 1: 1) in equal volume into a centrifuge tube, centrifuging at 2100g for 20min, centrifuging the nanoparticle tribuloside complex, concentrating to 200 μ l, centrifuging, and standing for 20 min. Diluting according to required concentration, adding medicine, and fresh preparing before each use. The Tribulus terrestris liposome nanoparticle complex (LNP-DP 1-Tribulus terrestris, LT for short) is shown in FIG. 1.

Example 3: study of pharmacology

Action target of tribuloside and research on influence of action target on expression and activity of hepatoma carcinoma cell CAXII

3.1 ELISA and CAXII esterase Activity assays

Quantitative determination of CAXII was performed by using the human carbonic anhydrase 12(CA-12) ELISA kit (CUSABIO, USA). Hep3B and SNU-449 cells were treated with Tiliroside or DMSO, respectively, and cultured for different times (24h, 48h, and 72h) in 2D and 3D systems, respectively. Cell lysates and ELISA assays were performed according to the manufacturer's protocol. The absorbance (OD) was measured at a wavelength of 450 nm. Three replicates per sample were analyzed. Relative concentrations were calculated by comparing the values of the treated and control groups. For the CAXII esterase activity inhibition assay, all reagents were prepared as described in the previous study. Cell lysates were then prepared for CAXII enzyme activity examination. The reaction was started by adding substrate and the enzyme activity was detected at 400nm wavelength of 4 minutes every 3 minutes by monitoring the production of the coloured product 4-nitrophenol (4-NP/pNP) (SIGMA-ALDRICH, USA) for about 2h at 37 ℃. In the absence and absence of levulose, the absorbance of substrate only and of spontaneous hydrolysis in the presence of levulose is subtracted from the esterase activity. In addition, a potent CAXII inhibitor (U-104) (EMD Millipore, USA) (215901) was added to DMSO as a positive control. The slope of the initial rate of enzyme activity was determined by plotting absorbance (Y-axis) and time (minutes) (X-axis) and then converted to a percentage of enzyme activity. Percent inhibition was calculated by setting the enzyme activity in the absence of levulose glycoside to 100%. IC50 values were obtained by nonlinear least squares using Prism 8 (version 8.3.0).

The effect of TS on the amount of CAXII in HCC cells was tested using the human carbonic anhydrase 12(CAXII) ELISA kit. As shown in FIGS. 2 and 3, in both 2D and 3D culture systems, Hep3B and SNU-449 significantly reduced the amount of CAXII at 24h, 48h and 72h compared to the NC group. For example, in the two-dimensional Hep3B experiment, the relative CAXII concentration in the Tiliroside group was significantly lower than that in the NC group (0.23. + -. 0.05, P <0.001) at 48 h. In addition, the activity of the CAXII esterase was determined 1 hour after TS intervention. The activity curves were dose-dependent, with IC50 for Hep3B and SNU-449 of 47.54. + -. 3.6. mu.M and 34.67. + -. 2.7. mu.M, respectively.

3.2 Western blot analysis

The invention uses western printing method to detect the expression of CAXII protein of two HCC cell lines. Hep3B and SNU-449 cells were divided into a TS-stem cell (treatment group) and an NC (control group), and after 48 hours of intervention, cell lysates were prepared, loaded onto 10% polyacrylamide gels, and then subjected to nitrocellulose membrane transfer according to standard Western blot analysis procedures. 5% skim milk was added to PBS for blocking and incubated in the mixture at 37 ℃ for 1 hour. The corresponding antibody (anti-CAXII antibody, obtained from Abcam, USA, 1/1000) to the specific protein was added, incubated at 37 ℃ for 2 hours, followed by detection with a suitable horseradish peroxidase-conjugated secondary antibody (Ab205718(1/2000, Abcam, USA) placed at 37 ℃ for 1 hour after the final PBS wash, signals were generated by the ECL detection system and the relative photographic density was quantified by a gel recording and analysis system (Alpha Imager 2000, ALPHA INNOTCH CORPORATION, USA.) the results are shown in FIG. 4, where the expression of the TS group CAXII protein was lower in Hep3B cells than in the NC group, and similarly, in SNU-449 cells, the expression of the NC group CAXII protein was higher than in the TS group, indicating that TS inhibits the expression of CAXII in Hep3B and SNU-449 cells.

Example 4: study of pharmacodynamics

4.1 methods for purchasing and culturing cell lines

4.1.1 cell lines

Human hepatocellular carcinoma cell line Hep3B (ATCC company, usa); human hepatocellular carcinoma cell line SNU-449 (ATCC corporation, USA); human hepatocellular carcinoma cell line HepG2 (American ATCC company); the human normal liver cell line THLE-3 (ATCC company, USA); human breast cancer cell line MCF-7 (American ATCC company); human triple-negative breast cancer cell line MDA-MB-231 (ATCC company, USA); human normal mammary epithelial cell line MCF-10A (ATCC company, USA); human brain glioblastoma cell line a-172 (ATCC company, usa); human brain glioblastoma cell line U-87 (ATCC company, USA); human lung cancer cell line a549 (ATCC corporation, usa); human cervical cancer cell line HeLa (ATCC company, usa); human prostate cancer cell line DU145 (ATCC company, usa); human non-hodgkin lymphoma cell line Farage (ATCC corporation, usa); human acute promyelocytic leukemia cell line HL-60 (ATCC, Inc., USA).

4.1.2 identification of cells

STR analysis was applied to all cell lines to identify cell line species.

4.1.3 methods of culturing cell lines

The human liver cancer cells Hep3B and HepG2 are cultured by mixing 10% fetal calf serum, 1% penicillin and streptomycin with EMEM culture medium; SNU-449 cells were cultured in RPMI-1640 medium + 10% fetal bovine serum and 1% penicillin, streptomycin; THLE-3 was cultured in BEGM medium (deprenomycin/amphotericin and epinephrine, plus 5ng/mL epidermal growth factor, 70ng/mL ethanolamine phosphate, 10% fetal bovine serum) prepared by BEGM Bullet Kit (ATCC, USA). MCF-7 and MDA-MB-231 cells were cultured in DMEM plus 10% fetal calf serum and 1% penicillin and streptomycin; MCF-10A cells were cultured in MEBM medium containing 100ng/mL cholera toxin; u-87 was cultured in EMEM medium containing 10% fetal bovine serum, 1% penicillin and streptomycin; culturing A-172 cells with DMEM, 10% fetal calf serum, 1% penicillin and streptomycin; a549 is cultured in F12K medium containing 10% fetal calf serum; 10% fetal calf serum + 1% penicillin, streptomycin RPMI-1640 culture medium for HeLa, Farage cells; the culture medium of the Du145 cells is cultured by mixing 10 percent fetal calf serum and 1 percent complete culture medium of penicillin and streptomycin with EMEM culture solution; HL-60 cells were cultured in IMDM medium containing 20% fetal bovine serum. All cells were cultured at 37 ℃ in 5% CO₂Changing the liquid for 2-3 times every week in a cell incubator with saturated humidity; adherent cells were digested with 0.25% trypsin containing 0.02% EDTA and non-tumor normal cells MCF-10A and THLE-3 were passaged using 0.05% trypsin containing 0.02% EDTA.

4.1.3.1 revival of cells

Preheating a water bath to 37 ℃ in advance, taking out the freezing tube with the cells from the liquid nitrogen tank, quickly placing the freezing tube into the preheated water bath, and slightly shaking the freezing tube in water to accelerate dissolution under the condition that the tube opening of the freezing tube is always above the liquid level, so that the dissolution process is not more than 2 minutes. Subsequently, the thawed cells were aspirated and added to 4-6mL of complete medium, 125g, centrifuged for 5-10 minutes, and the supernatant discarded. Resuspend cells in 4-6mL complete medium, gently blow-mixAfter homogenization, the cell suspension was transferred to a culture dish for culture under the following conditions: 37 ℃ and 5% CO₂An incubator. The liquid was changed 2 times per week after the next liquid change.

4.1.3.2 passage method of adherent cells

Taking out the culture bottle containing cultured adherent cells from the incubator (adherent area reaches about 90%), sucking out with a dropper, discarding the old culture solution, gently washing the cell surface with 2-3mL of 1 × PBS liquid, discarding the waste liquid, and returning to 75cm²2-3mL trypsin-EDTA was added to the cell culture flask and the flask was replaced in the 37 ℃ incubator (5-10 min), digestion was stopped by adding 4-6mL complete medium after the cells were resuspended off the wall of the flask as seen in the inverted microscope and the mixture was centrifuged in a centrifuge tube (125g, 5-10 min). Discarding the supernatant, adding 4-6mL of complete medium, repeatedly blowing and beating the cells to resuspend the cells, transferring the resuspension solution to a new culture flask, and continuing culturing at a passage ratio of 1:3-1: 5. The culture conditions are as follows: 37 ℃ and 5% CO₂。

4.1.3.3 method for passaging suspension cells

Taking out the culture bottle with cells from the incubator, digesting and transferring the cells into a centrifuge tube, placing the centrifuge tube into a centrifuge with 300g for centrifugation for 5min, carefully removing supernatant, adding complete culture medium, gently blowing the solution under the page by a pipette until the precipitated cells are uniformly resuspended to prepare single cell particle suspension, and after cell counting is finished, inoculating the cell suspension into a new cell suspension according to proper concentration

The number of passage cells in T-75 culture flask is controlled at 1X 10⁵cells/mL to 1X 10⁶Within cells/mL. Ensuring that the culture flask contains enough culture solution and is cultured at 37 ℃ and 5% CO₂The incubator of (2) is filled with the liquid 2-3 times per week.

4.2 Primary reagents and instruments

EMEM culture solution (American ATCC company)

DMEM culture solution (American ATCC company)

RPMI-1640 culture solution (American ATCC company)

BEGM Bullet Kit (American ATCC company)

BEGM culture medium (American ATCC company)

MEBM broth (GIBCO USA)

IMDM broth (American ATCC company)

F12K culture solution (GIBCO, USA)

1 XPBS buffer (GIBCO USA)

Fetal bovine serum (GIBCO corporation, USA)

Streptomycin, penicillin (life Technologies, USA)

MTS (Sigma USA)

Trypsin-ethylenediaminetetraacetic acid (Life Technologies, USA)

Soybean trypsin inhibitor (life Technologies, USA)

Tribulus terrestris glycoside (pharmaceutical purity standard) (Lot Number 44344) (Sigma Co., USA)

DODMA,2,3-Dioleyloxy-1- (dimethylimine) propane (Sigma, USA)

Cholesterol (Sigma USA)

Egg PC (Sigma company, USA)

PEG-Chol (Sigma company, USA)

HEPES solution (SigmaAldrich, USA)

Slide-A-Lyzer^TMDialysis box (Slide-A-Lyzer)^TMDialysis Cassettes (ThermoFisher company, USA)

Epoch microplate spectrophotometer (American Bio-Tek company)

Thermo Scientific Sorvall Legend T Benchtop centrifuge (Kendro, Germany)

Vistivion microscope inverted microscope

BB5060 type 5% CO₂Constant temperature cell culture box (Germany Heraeus company)

MINI vortex mixer (Fisher Scientific, USA)

A/B3 type super clean bench (American Spectra lab corporation)

E12130 electronic balance (Ohaus, USA)

General Purpose purpos Series cryogenic refrigerator (Fisher Scientific, USA)

Heating magnetic stirrer/electric stove (Fisher Scientific Co., USA)

Digital-Control water bath (Fisher Scientific company, USA)

4.3 methods

4.3.1 MTS method for detecting the proliferation inhibition of the complex of the tribuloside and the LNP-DP 1-tribuloside on various tumors and common cell lines respectively and calculating the median lethal dose

Hep3B, SNU-449, HepG2, THLE-3, MCF-7, MDA-MB-231, MCF-10A, U-87, A-172, A549, HeLa, DU145, Farage, HL-60 in logarithmic growth phase were collected, centrifuged, resuspended in each complete medium, adjusted to a cell density of 2-4 k/100. mu.L, seeded on 96-well plates, 100. mu.L per well of cell suspension, placed at 37 ℃ in 5% CO₂Culturing in an incubator, replacing the culture medium after 24h, adding intervention solution into each well according to groups (adding different concentrations of tribuloside into a treatment group, adding corresponding equal amount of double distilled water-DMSO mixed solution into a control group), setting 3 multiple wells for each test concentration and different observation time, and observing for 4 time points (24h, 48h, 72h and 96 h). At each time point, 20. mu.l of MTS solution was added to the wells to be assayed, and the mixture was incubated at 37 ℃ in 5% CO₂Keeping the culture box in the dark for 2h, measuring the corresponding absorbance OD value of each hole of each group by using an enzyme-labeling instrument under the condition of 490nm wavelength, and calculating the survival rate and the inhibition rate of the cells to calculate the parasitism lethal dose.

4.3.2 Experimental groups

Measuring the inhibition effect of tribuloside on Hep3B, SNU-449, HepG2, THLE-3, MCF-7, MDA-MB-231, MCF-10A, U-87, A-172, A549, HeLa, DU145, Farage and HL-60, and calculating corresponding IC50 to be used as a treatment intervention group; the growth conditions of Hep3B, SNU-449, HepG2, THLE-3, MCF-7, MDA-MB-231, MCF-10A, U-87, A-172, A549, HeLa, DU145, Farage and HL-60 under the double distilled water with the corresponding concentration are measured and used as a control group.

Simultaneously determining the inhibition effect of the LNP-DP 1-tribuloside complex or MC 3-tribuloside complex on Hep3B, SNU-449, HepG2, THLE-3, MCF-7, MDA-MB-231, MCF-10A, U-87, A-172, A549, HeLa, DU145, Farage and HL-60 as a treatment intervention group; measuring the growth conditions of Hep3B, SNU-449, HepG2, THLE-3, MCF-7, MDA-MB-231, MCF-10A, U-87, A-172, A549, HeLa, DU145, Farage and HL-60 under the same amount of LNP to obtain a Mock group; a blank control group was added.

3.3 calculation method

Calculating the inhibition rate of the drug to the growth of tumor cells according to the OD value measured by the MTS experiment, wherein the formula is as follows:

cell inhibition rate of 100% -cell survival rate

The half inhibitory concentration of drug on cells was calculated by Graphpad Prism 8 software (IC 50).

4.3.4 statistical method

Data were processed using SPSS16.0 software for analysis of variance. The measured data obtained by each group adopts the mean value plus or minus standard deviation

Mean comparisons between groups were tested by t-test. Test level α ═ 0.05, p<0.05 is statistically significant.

Tribulus terrestris was compared to the cell lines tested at 72h IC50, as shown in Table 2 and FIG. 5.

TABLE 2 IC50 values at 72 hours for tested cell lines with tribuloside

The results of IC50 values of TS (Tribulus terrestris), LNP-DP1-TS (LNP-DP1 Tribulus terrestris complex) for 72h of each cell tested are shown in Table 2 and FIG. 5. TS has low IC50 value on liver-derived cancer cells (Hep3B, SNU-449, HepG2), and is most sensitive to SNU-449 cells and weakest sensitive to the glioma cell line A172. And LNP-DP1 can reduce IC50 value of TS to Hep3B, SNU-449, HepG2 and THLE-3 cell line, and IC50 value of TS of other source tumor cells and common cells is reduced or maintained at original level after LNP encapsulation.

The inhibitory effect of different concentrations of tribuloside on HCC cell lines and hepatic normal cells at different times was tested, and the results are shown in table 3 and fig. 6.

TABLE 3 inhibition ratio (%) (of different concentrations of Tribulus terrestris glycoside) on HCC cell lines and hepatic normal cells

As shown in Table 3 and FIG. 6, the inhibition of TS was significantly higher in hepatocellular carcinoma cells Hep3B and SNU-449 than in normal cells THLE-3 (at 24h, 48h, 72h and 96h, respectively) (note: P < 0.05;. P < 0.01;. P < 0.001; NS: non-significan difference in statistical significance compared with the control group).

Tribulus terrestris was encapsulated with LNP-DP1 and MC3, respectively, and tested for inhibition of HCC cell lines and hepatic normal cell lines, with the results shown in Table 4 and FIG. 7.

TABLE 4 inhibition ratio of HBCs cell line and liver normal cell line by respectively encapsulating Tribulus terrestris glycoside with LNP-DP1 and MC3

As can be seen, the inhibition rate of 30 mu M LNP-DP1-TS on the hepatocellular carcinoma cell lines SNU-449 and Hep3B is respectively obviously higher than that on the normal cell THLE-3 (P <0.001), and is better than that of the MC3-TS group.

Example 5: study on influence of LT (LNP-DP 1-tribuloside complex) on migration ability, colony cloning ability, invasion ability and organoid formation ability of hepatoma carcinoma cells

5.1 reagents and cell lines

EMEM broth (ATCC corporation, usa);

DMEM medium (ATCC company, usa);

RPMI-1640 medium (ATCC company, USA);

1 XPBS buffer (GIBCO, USA);

fetal bovine serum (GIBCO, usa);

streptomycin, penicillin (life Technologies, usa);

HDAC Activity Colorimetric Assay kit (BioVision, USA);

human hepatoma cell line Hep3B (ATCC company, usa);

human liver cancer cell line SNU-449 (ATCC corporation, USA);

human hepatocellular carcinoma cell line HepG2 (American ATCC company);

tribulus terrestris glycoside (pharmaceutical purity standard) (Lot Number: 44344) (Sigma, USA).

5.2 instruments and reagents

An Epoch microplate spectrophotometer (BioTek, usa);

thermo Scientific Sorvall Legend T Benchtop centrifuge (Kendro, Germany);

vislavion microscope inverted microscope (VWR corporation, usa);

fluorescence microscope (Olympus, japan);

BB5060 type 5% CO2 thermostatted cell culture chamber (Heraeus, germany);

MINI vortex mixer (Fisher Scientific, USA);

model A/B3 clean bench (Spectra lab, USA);

e12130 electronic balance (Ohaus, usa);

6-well plates (Falcon corporation, usa);

4% paraformehyde (sigma, usa);

general Purpose plasma Series cryo-refrigerator (Fisher Scientific, USA);

a heated magnetic stirrer/electric furnace (Fisher Scientific, USA);

Digital-Control water bath (Fisher Scientific, USA);

adjustable pipettes (fennish baide corporation);

crystal violet stain (sigma corporation, usa);

3.0 μm pore size transwell cell system (Falcon corporation, usa);

matrigel (Corning corporation, usa);

4% paraformehyde (sigma, usa);

ImageJ 1.52a version (NHI public resources usa).

5.3 Experimental methods

5.3.1 cell culture

Culturing human hepatoma cell Hep3B by mixing 10% fetal calf serum, 1% penicillin and streptomycin in EMEM culture medium; SNU-449 cells were cultured in RPMI-1640 medium + 10% fetal bovine serum and 1% penicillin, streptomycin.

5.3.2 preparation of Experimental drugs

This is the same as in examples 1 and 2.

5.3.3 study of the Effect of LT on the migration ability of liver cancer cells

Hep3B, SNU-449 cells were used for scar healing experiments. Approximately 1 × 10⁶Cells were plated in 6-well plates and cultured at 37 ℃ with 5% CO₂An incubator. After 24h when the cells have grown to the bottom wall, the wells are gently streaked with a 100 μ l tip and the images are recorded under the mirror, at which time the treatment group intervenes with LT and the control group is supplemented with an equal amount of double distilled water, all cells being cultured in 2mL of medium containing 0.1% FBS. Incubation and observation were continued and images were recorded.

5.3.4 study of the Effect of LT on the clonality of hepatocarcinoma cell colonies

Hep3B, SNU-449 cells, 2000 cells each, were seeded in 6-well plates, 2ml of complete medium was added, and gently shaken to allow uniform plating of the cells. Treatment ofGroups received LT intervention and control groups received equal amounts of double distilled water. Each group of cells was cultured at 37 ℃ in 5% CO₂The incubator is used for 10 days, and fresh culture medium is changed every 2-3 days. After cell colonies were formed, they were gently rinsed with 1 × PBS water, fixed with 4% parafumaldehyde, stained with crystal violet, and colonies containing more than 30 cells were counted.

5.3.5 LT study on influence of invasion capacity of liver cancer cells

A Transwell cell system with a 3.0 μm pore size was used for this study. The digestion count of Hep3B and SNU-449 cells was 1X 10⁴Cells were seeded in the upper layer of the chamber, and 100. mu.l of medium containing 10% Matrigel and 0.1% FBS was added to the upper layer. The lower chamber layer was added with 600. mu.l of complete medium containing 10% FBS. Treatment group received LT intervention and control group received equal amount of double distilled water; hep3B, SNU-449 for 48 h. The membrane was removed, and the cells on the upper chamber side were gently wiped off with a cotton swab, fixing the cells that passed through the membrane to the lower chamber side. Fixed with 4% parafumaldehyde and stained with crystal violet. The amount of cells under the microscope in different fields was calculated for analysis.

5.3.6 LT study on the influence of the ability of three-dimensional formation (Cell line-derived 3D organoid) of hepatocellular carcinoma Cell organoid

And culturing the stable luciferase expression cell strain for the three-dimensional sphere forming experiment. 750 cells were suspended upside down in 30. mu.L of solution (phenyl-red free matrigel [ Corning, USA ] + EMEM or PRMI-1640 complete medium), divided into LNP-DP1-TS (50. mu.M) and NC groups, one set of experiments was cultured continuously to record images for 96 hours and the 3D model cross-sectional area was determined with the Imagej program (version 1.52 a; https:// Imagej. nih. gov/ij /). In another set of experiments, hanging drop cells were taken and added with D-luciferin (150. mu.L/mL) (PerkinElmer, USA), and bioluminescence signals (bioluminescence signal) were recorded and calculated using a GloMax 96Microplate Luminometer (Promega, USA) with 3 wells (24, 48, 72, 96 and 120 hours) at each time point.

5.4 results of the experiment

5.4.1 inhibition of migration of liver cancer cells by LT

The effect of LT on the migration ability of Hep3B cells is shown in Table 5 and FIG. 8, and the ratio of scar width to initial width gradually decreased at 24h and 48h after LT action on the cells. LT cells healed more slowly compared to NC group, 81.44 + -3.04% at 24h, greater than 69.77 + -2.28% in NC group (P < 0.001). Scar ratios in the 48h LT group were 70.45 + -2.77%, while the NC group was only 30.45 + -2.38% of the initial width (P < 0.001).

TABLE 5 influence of LT on the migration Capacity of Hep3B

The effect of LT on the migratory capacity of SNU-449 cells, as shown in table 6 and fig. 9, had a similar trend to that of Hep3B cells.

TABLE 6 influence of LT on migration Capacity of SNU-449

5.4.22. LT inhibition of clonality of hepatoma cell colony

The colony formation of Hep3B, SNU-449 cells after 10 days is shown in FIG. 10 (P < 0.001). As is clear from Table 7, the number of colonies of LT cells from Hep3B was 52.01. + -. 2.51(P <0.001) compared with NC, and the number of colonies of LT cells from SNU-449 cells was 73.44. + -. 2.17(P <0.001) compared with NC.

Relative clone formation efficiency of Table 72 cells

5.4.2.3 LT Effect on the invasive Capacity of liver cancer cells

As shown in FIG. 11 and Table 8, Hep3B cells were fixed after 48h, and the number of cells visible on the membrane per field was 105.67. + -. 12.04(LT) and 204. + -. 9.2(NC) (P < 0.001); SNU-449 cells were fixed at 48h, with the number of cells visible on the membrane in each field being 93.33. + -. 10.27(LT) and 145.33. + -. 9.81(NC) (P < 0.001).

TABLE 8 cell counts per field of view of transwell chamber

	NC	VA
			Hep3B field cell mass	204±9.2	105.67±12.04
SNU-449 visual field cell number	145.33±9.81	93.33±10.27

5.4.2.4 LT influence on three-dimensional organoid forming ability of liver cancer cell

As shown in FIG. 12, in SNU-449 and Hep3B cells, LNP-DP1-TS showed smaller cross-sectional area and lower relative cross-sectional area formation rate (P <0.001), and LNP-DP1-TS showed more than 20% inhibition of 3D spheroid formation after 48h in both SNU-449 and Hep3B cells (note: P < 0.05;: P < 0.01;: P < 0.001; NS: non-significan difference was not statistically significant compared to the control group).

Example 6: animal experiments

Research on cationic liposome nanoparticle-coated tribuloside for resisting nude mouse liver cancer orthotopic transplantation tumor

6.1 Experimental animals: SPF grade, Athymic nude mice, male, 4 weeks old, 18-20 g, total 12.

Instruments and consumables:

epoch microplate spectrophotometer (American Bio-Tek company)

Thermo Scientific Sorvall Legend T Benchtop centrifuge (Kendro, Germany)

vislavsion microscope inverted microscope (VWR corporation, USA)

BB5060 type 5% CO₂Constant temperature cell culture box (German Heraeus company)

MINI vortex mixer (Fisher Scientific, USA)

A/B3 type super clean bench (American Spectralab company)

E12130 electronic balance (Ohaus, USA)

General Purpose purpos Series cryogenic refrigerator (Fisher Scientific, USA)

CMV-Firefly luciferase-IRES-Puro lentivirus and transfection kit (Cellomics technology, USA)

IVIS Lumina LT Small animal Living body optical imaging System (Xenogen Corp./Caliper life Science Co., USA)

Living

4.3.1Software (Caliper Life Science, USA)

MWCO 10,000Dalton Float-A-Lyzer (Spectrum Laboratories, Inc. Canada)

0.22um system filter (Falcon company, USA)

Isofluorane (Sigma-Aldrich, USA)

Glass jar for mouse anesthesia (Falcon company, USA)

Mouse fixer (Falcon company, USA)

Procreate software IOS version (Corel corporation, USA)

96Microplate Luminometer (Promega corporation, USA)

6.2 Experimental methods

6.2.1 cell culture

6.2.1.1 transfection of cell lentiviruses

SNU-449 and Hep3B cells grown in log phase were used for construction of transfected cell lines for fluorescent lentiviruses. Before transfection, 1X 10 cells were transfected⁵Cells/2 ml of medium were cultured in 6-well plates for 12 to 18 h. Polybrene was added at 6. mu.g/ml to the complete medium to continue culturing the cells. The frozen lentiviral samples were thawed slowly on ice, pipetted gently to dislodge the lentiviral samples into transfection medium (RPMI-1640 or EMEM medium + 10% heat-inactivated FBS) at a MOI (multiplex of infection) of 5. After 8 hours, the transfection medium was replaced by fresh complete medium and the temperature was continued at 37 ℃ with 5% CO₂The incubator cultures the cells.

6.2.1.2 Stable cell line selection

Transfected cells were selected with puromycin (1. mu.l/ml once every three days) and cultured for at least 14 days. After 14 days, D-luciferin (150. mu.l/ml) was mixed with the cells and the luminescence of the cells was measured using a 96Microplate Luminometer, and a high luminescence value indicated that the transfected cell lines were stable.

6.2.2 establishment of animal models

After purchasing the mice, the mice were kept in a yarrow house for 1 week to acclimatize.

The Matrigel was first placed in a 4 ℃ freezer from-20 ℃ to be dissolved. And (3) when the SNU-449 cell line of the lentivirus successfully transfected with the luciferase gene is stable, taking cells in the logarithmic growth phase, centrifuging for 5min at 500g, discarding supernatant, and counting 5 multiplied by 105 cells to obtain the injection amount of a single mouse. 5 x 10 of the counted number⁵The individual cells were mixed in 50ul of medium and then mixed with 50ul of Matrigel, taking care to avoid beating during mixing, the cell solution was mixed with a vortex mixer to avoid air bubbles, and the cell solution was aspirated out and placed in a 4 ℃ environment with a 1.5ml needle tube in combination with a 30g needle (the whole preparation was carried out in a 4 ℃ room).

After the injection of the cell solution is finished, placing the mouse into an ice box for standby, placing the mouse into an anesthesia glass tank, adding 2ml of Isoflurane to perform anesthesia, positioning a puncture part after anesthesia (the position 2mm below the included angle between the xiphoid process and the left costal margin of the mouse is inserted into the right side of the mouse at an angle of 15-30 degrees, the depth of the inserted needle is 5-10mm, slowly pushing the needle after the hand breaks through, standing for 10-20s after the pushing is finished, taking out the needle after matrigel is solidified, and indicating that the injection is successful when the head of the needle meets blood indication).

6.2.3 grouping and administration

On day 15 post inoculation, 12 nude mice were transferred to IVIS imaging system and laboratory for intraperitoneal injection of D-lucferin into each nude mouse, waiting 15-30min, imaged and recorded. After the mice had developed tumors, 12 nude mice were randomly divided into 2 groups according to the random number table, which were: SNU-449 model control group, SNU-449LT group. Wherein the model control group was intravenously injected with an equivalent amount of LNP-coated equivalent amount of double distilled water 2 times per week for 3 weeks, and the LT group was intravenously injected at 50mg/kg tail vein 1 time per week. A total of 4 doses were taken, and fluorescence images were measured once a week.

6.2.4 bioluminescence assay experiments

A15 mg/ml fresh solution of Luciferin reagent in DPBS was prepared and filtered through a 0.2um filter. The injection amount was 150mg luciferin/kg body weight per mouse. The Luciferin solution was administered by intraperitoneal injection. IVIS imaging can be used after waiting for 10-15 min.

6.3 Observation indicators and results

6.3.1 general Condition observations of mice

All 12 nude mice had tumors, and none of them died by the first review of the images after the last treatment period. All the mice in each group have no abnormality in foraging and drinking water, are free to move, and have no obvious change in weight.

6.3.2 comparison of tumor dynamic growth and survival

The tumor-bearing growth status of the mice is dynamically measured, and a tumor fluorescence signal dynamic curve is drawn by using a Prism 8 program. The average value plus or minus standard deviation of the measurement data obtained from each group is adopted

Showing that SPSS16.0 software is used for processing, a one-factor variance test is adopted for multi-group data comparison, and a t test is adopted for inter-group mean comparison. The test level α is 0.05, and p <0.05 is considered statistically significant.

As shown in fig. 13-14, tumor fluorescence signals decreased in tumor-bearing mice 4 weeks after LT treatment, while tumors in the control group progressed or stabilized; the tumor growth rate of the mice in the treatment group is respectively obviously lower than that of the mice in the control group (P is less than 0.01).

The present invention has been further described with reference to specific embodiments, but it should be understood that the detailed description should not be construed as limiting the spirit and scope of the present invention, and various modifications made to the above-described embodiments by those of ordinary skill in the art after reading this specification are within the scope of the present invention.

Claims

1. Application of tribuloside in preparing medicine for treating hepatocellular carcinoma is provided.

2. A pharmaceutical composition for treating hepatocellular carcinoma is characterized in that the pharmaceutical composition is a tribuloside liposome nanoparticle complex, and comprises tribuloside and liposome, wherein the tribuloside is coated in the liposome.

3. The pharmaceutical composition of claim 2, wherein the tribuloside and the liposome nanoparticles are mixed in equal volume, centrifuged, concentrated, and left to stand to obtain the tribuloside-coated liposome nanoparticles.

4. The pharmaceutical composition of claim 2 or 3, wherein the starting components of the liposomal nanoparticles comprise 1, 2-dioleol-3-dimethylamino-propane, L- α -phosphatidylcholine, cholesterol, and cholesterol-polyethylene glycol.

5. The pharmaceutical composition of claim 4, wherein the molar ratio of 1, 2-dioleyl-3-dimethylamino-propane, L- α -phosphatidylcholine, cholesterol, and cholesterol-polyethylene glycol is (9-10): (3-4): (7-8): (1-2).

6. The pharmaceutical composition of claim 5, wherein 1, 2-dioleyl-3-dimethylamino-propane, L- α -phosphatidylcholine, cholesterol, and cholesterol-polyethylene glycol are dissolved in water bath at 60 ℃ to form alcohol solutions, and the alcohol solutions are mixed according to the molar ratio of (9-10): 3-4): 7-8): 1-2, and slowly dropped into the rapidly stirred 4-hydroxyethylpiperazine ethanesulfonic acid solution to obtain a mixed solution containing 35% alcohol concentration, and then alcohol in the mixed solution is dialyzed and removed, and filtered to obtain the liposome nanomaterial solution.

7. The pharmaceutical composition according to claim 6, wherein 20mM HEPES is used as the dialysis solution, and the dialysis solution is changed every 2 hours during dialysis for 8-12 h.

8. The pharmaceutical composition of claim 2, wherein the pharmaceutical composition is used for treating hepatocellular carcinoma cells in which the liver cancer cells are Hep3B, SNU-449.

9. Use of a pharmaceutical composition according to any one of claims 2 to 8 in the manufacture of a medicament for the treatment of hepatocellular carcinoma.