Medicine for treating liver cancer or reversing drug resistance of sorafenib, double-drug liposome and application

Abstract

The invention relates to the technical field of nano-drugs, in particular to a drug for treating liver cancer or reversing drug resistance of sorafenib, a double-drug liposome and application. The medicine is prepared by mixing ginsenoside CK and sorafenib according to the mass ratio of (5-8): 1. Tests prove that the combined use of ginsenoside CK and sorafenib can reverse the drug resistance of sorafenib, and meanwhile, the effects of enhancing the curative effect and reducing the toxicity are achieved. The drug is further prepared into a double-drug liposome, so that the maximum encapsulation efficiency of CK and the maximum encapsulation efficiency of SOR reach 87% and 83% respectively, the defects of poor water solubility and low bioavailability of existing drugs are overcome, and the drug has a wide prospect in the aspect of combined treatment of liver cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanomedicine, and particularly relates to a drug for treating liver cancer or reversing sorafenib drug resistance, a double-drug liposome and application. BACKGROUND

[0002] Sorafenib is a multi-kinase inhibitor and is the only drug approved by the US Food and Drug Administration (FDA) for the treatment of hepatocellular carcinoma. It inhibits tumor proliferation and neovascularization by inhibiting the Raf / MAPK / ERK signaling pathway, but at the same time, it activates the PI3K / Akt signaling pathway and its key downstream factors, and forms a cross-talk with the Raf / MAPK / ERK signaling pathway, resulting in acquired drug resistance of liver cancer cells to sorafenib. Therefore, its long-term application is limited.

[0003] In the prior art, CN202510870985.1 discloses a sorafenib-paclitaxel combined prodrug and a preparation method and application thereof. Sorafenib and paclitaxel are covalently connected by a disulfide bond to successfully prepare SPTX drug. Then the drug can self-assemble into stable nanoparticles in a water solution system containing 1% anhydrous ethanol and 1% polyoxyethylene castor oil, relying on hydrophobic interaction, van der Waals force and the special chemical properties of the disulfide bond itself. Although the combination of sorafenib and paclitaxel shows a powerful strategy in the treatment of certain diseases, however, the toxicity brought by this combination cannot be ignored, and their toxicity to normal cells is significantly higher than that when either of the drugs is used alone. Therefore, when considering using this combination therapy, doctors and patients must carefully weigh the potential benefits and risks.

[0004] CN201810227715.9 discloses a preparation method of sorafenib nanomicelles, which successfully improves the solubility of sorafenib in water through a specific process. However, this method does not effectively solve the problem of drug resistance of sorafenib in actual application and the problem of side effects that may be caused during use, which still puzzles the improvement of clinical application and treatment effect; CN202011199694.8 discloses a nanometer preparation of emodin combined with sorafenib. In the in vitro experiment on HepG2 cells, when the concentration of the nanometer preparation reaches 50 μM, more than 20% of the cells still survive, which indicates that the nanometer preparation still has certain deficiencies in cell inhibition effect and still has a large optimization space, which needs to be further researched and improved to improve its inhibition effect and clinical application potential.

[0005] Ginsenoside CK is one of the key components of pharmacological activity of ginseng. Researches show that rare ginsenoside CK can induce tumor cell apoptosis and cell cycle arrest by inhibiting PI3K / Akt and other signaling pathways, and has the advantages of high biological activity, mild effect, no toxic and side effects. However, there is no report in the prior art about the combination of ginsenoside CK and sorafenib for treating liver cancer and reversing sorafenib resistance. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides a medicine for treating liver cancer or reversing sorafenib resistance. Experiments have proved that the combination of ginsenoside CK and sorafenib can reverse sorafenib resistance, and at the same time achieve the effects of enhancing efficacy and reducing toxicity.

[0007] To achieve the above object, the technical scheme adopted by the present application is as follows: The first object of the present application is to provide a medicine for treating liver cancer or reversing sorafenib resistance, which is a combination of ginsenoside CK and sorafenib.

[0008] As a preferred embodiment of the present application, the mass ratio of ginsenoside CK and sorafenib is 5-8:1.

[0009] As a preferred embodiment of the present application, the medicine comprises a pharmaceutically acceptable excipient.

[0010] Further preferably, the medicine is a liposome.

[0011] The second object of the present application is to provide a double-drug liposome, which is in a spherical or spheroid nanovesicle structure and is prepared according to the following steps: Dissolve ginsenoside CK, sorafenib, cholesterol and egg yolk lecithin in an ethanol solution to obtain a mixed solution; Uniformly drop the mixed solution into water under stirring, stir, and dialyze the obtained liposome suspension to obtain the double-drug liposome.

[0012] The hydrophobic drug (ginsenoside CK, sorafenib SOR) molecules are combined with the hydrophobic tails of the liposome bilayer by hydrophobic force and stably embedded in the membrane structure. Cholesterol can reduce the fluidity and arrangement disorder of lipid molecules, especially can resist the destruction of temperature changes (such as phase transition temperature) to the membrane structure, and enhance the stability of the liposome in storage and in vivo circulation.

[0013] As a preferred embodiment of the present application, the amount ratio of ginsenoside CK, sorafenib, cholesterol, egg yolk lecithin and ethanol is 4.38-8.75 mg:0.6-1.25 mg:5.33-20 mg:80 mg:2-2.2 mL.

[0014] As a preferred embodiment of the present application, when the mixed solution is dropped into water at a constant speed, the stirring speed of the water is 300-900 rpm, and the stirring time of the obtained mixed system is 4-5 h.

[0015] As a preferred embodiment of the present application, the water is deionized water, and the amount of the water is 18-20 mL.

[0016] As a preferred embodiment of the present application, the dialysis is that the liposome suspension is loaded into a dialysis bag with a specification of 14 kDa, and is dialyzed in 800-1000 mL of pure water for 24-36 h.

[0017] As a preferred embodiment of the present application, the preparation of the double-drug liposome further comprises a process of adding deoxycholic acid to the liposome suspension. After the double-drug liposome is added with deoxycholic acid, the double-drug liposome has obvious stability in simulated gastric juice and slow release in simulated intestinal juice, so that the two drugs can be slowly released.

[0018] Further preferably, after the deoxycholic acid is added to the liposome suspension, stirring and dialysis are performed, and the double-drug liposome is obtained.

[0019] Further preferably, in the mixed system of the liposome suspension and the deoxycholic acid, the concentration of the deoxycholic acid is 0.4-0.8 mg / mL.

[0020] More preferably, the average particle size of the double-drug liposome is 88.99-101.73 nm, and the PDI is 0.17-0.18.

[0021] The third object of the present application is to provide the use of the double-drug liposome in the preparation of an antitumor drug.

[0022] Compared with the prior art, the present application has the following beneficial effects: 1. The ginsenoside CK and sorafenib are innovatively combined in the present application, and it is found that the combination can reverse sorafenib resistance, and can enhance the therapeutic effect and reduce the toxicity. When 30 μM of CK and 6 μM of sorafenib are combined for 48 h, the survival rate of HepG2 is less than 10%, the synergistic effect is verified to be more than 1.15, and the excellent synergistic effect is indicated.

[0023] 2、Ginsenoside CK and sorafenib are both strong in lipid solubility and low in bioavailability, so the present application provides a ginsenoside CK-sorafenib double-drug liposome, which dissolves ginsenoside CK, sorafenib, cholesterol (cholesterol can change the fluidity and polarity of the liposome, reduce the membrane permeability and thus reduce drug leakage), egg yolk lecithin in ethanol solution to a transparent state to obtain a transparent and clear solution of ginsenoside CK, and then the clear solution is dropped into water stirred at a specific speed under room temperature (if the stirring speed is too high, the large shear force will destroy the membrane structure of the liposome, resulting in leakage of the drug already encapsulated in the phospholipid layer, and the encapsulation rate is reduced. If the stirring speed is too low, the contact and combination opportunities between the drug molecules and the liposome are reduced, some drugs cannot enter the liposome, and the encapsulation rate is reduced.), and the ginsenoside CK and sorafenib double-drug liposome is obtained after dialysis. The ginsenoside CK-sorafenib liposome prepared in the present application changes the solubility and stability of the drug, the preparation method adopted is mature, reproducible, easy to realize large-scale production, the product quality is stable, and meets the requirements of pharmaceutical preparation industrialization.

[0024] 3、The present application explores the influence law of the preparation process of carrier phospholipid and drug mass ratio, phospholipid and cholesterol mass ratio, volume ratio of organic phase and water phase, and electromagnetic stirring speed on the embedding property of the liposome, and the encapsulation rates of ginsenoside CK and sorafenib reach 87% and 83% respectively under the optimal conditions.

[0025] 4、The ginsenoside CK-sorafenib double-drug liposome prepared in the present application has significant stability and sustained release in simulated gastrointestinal fluid after adding deoxycholic acid, and the cumulative release of ginsenoside CK and sorafenib is only 9% and 7% respectively within 2 hours; the release amounts reach 92% and 91% respectively within 48 hours. The leakage rate of the liposome is less than 10% after being stored at 4°C for 28 days.

[0026] 5、The present application verifies the synergistic inhibitory effect of ginsenoside CK and sorafenib on HepG2 liver cancer cells and the safety of the blank liposome through MTT experiment. The cell uptake experiment shows that the ginsenoside CK-sorafenib double-drug liposome can be quickly absorbed by cells and gradually accumulated in the cells over time. The quantitative apoptosis experiment shows that the positive cell rate of the ginsenoside CK-sorafenib double-drug liposome is significantly improved to different degrees compared with free CK, free SOR and free double-drug. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a transmission electron microscope graph of the prepared ginsenoside CK-sorafenib double-drug liposome.

[0028] Figure 2 It is a Fourier infrared spectrum graph of the prepared ginsenoside CK-sorafenib double-drug liposome.

[0029] Figure 3 Figure is the influence diagram of the lipid-drug ratio (a), the phospholipid-drug ratio (b), the organic phase and the aqueous phase (c), and the rotation speed (d) on the encapsulation efficiency of ginsenoside CK-sorafenib double-drug liposome.

[0030] Figure 4 Figure is the particle size distribution diagram of the prepared ginsenoside CK-sorafenib double-drug liposome.

[0031] Figure 5 Figure is the drug release characteristics of the ginsenoside CK-sorafenib double-drug liposome prepared in Example 1 under different DCA content coating conditions; a, the release characteristics diagram of ginsenoside CK; b, the release characteristics diagram of sorafenib; c, the cumulative release amount of ginsenoside CK and sorafenib within 48 hours; d, the stability of CK and SOR at different temperatures.

[0032] Figure 6 Figure (a) is the influence of HepG2 cell viability on free SOR, free CK, CK-SOR and Lipo-CK-SOR, and (b) is the safety verification of different concentrations of blank liposome.

[0033] Figure 7 Figure is the HepG2 cell apoptosis effect diagram of ginsenoside CK-sorafenib double-drug liposome.

[0034] Figure 8 Figure is the influence diagram of ginsenoside CK-sorafenib double-drug liposome on the migration ability of HepG2 cells, a, 24h; b, 48h. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described clearly and completely below in combination with the data in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] It should be noted that the professional terms used in the present application are only for the purpose of describing the specific embodiments and are not intended to limit the scope of protection of the present application. Unless otherwise specified, the various raw materials, reagents, instruments and equipment used in the following embodiments of the present application can be purchased from the market or prepared by the existing method.

[0037] Ginsenoside CK is one of the key components of the pharmacological activity of ginseng, and research shows that rare ginsenoside CK can induce tumor cell apoptosis and cell cycle arrest by inhibiting multiple signaling pathways such as PI3K / Akt, and has the advantages of high biological activity, mild effect, no toxic side effects, etc. However, there is no report in the prior art that ginsenoside CK is used in combination with sorafenib to treat liver cancer, and whether ginsenoside CK can reverse the drug resistance of sorafenib needs further study.

[0038] In view of the problems of sorafenib drug resistance and large side effects in the prior art, the present application innovatively uses ginsenoside CK in combination with sorafenib, CK inhibits the PI3K / Akt pathway, reverses the drug resistance of sorafenib, and at the same time achieves the effects of enhancing efficacy and reducing toxicity. Under the combined use of 30 μM of CK and 6 μM of sorafenib for 48 h, the survival rate of HepG2 is less than 10%, and the synergistic effect is verified to be more than 1.15, indicating excellent synergistic effect.

[0039] To solve the solubility, stability and drug resistance problems of the above-mentioned drug combination, the present application further prepares a new type of double-drug liposome, and the preparation method comprises the following steps: accurately weighing ginsenoside CK, sorafenib, cholesterol and egg yolk lecithin into a test tube, dissolving in an appropriate amount of ethanol solution, and fully dissolving each component under vortex and ultrasonic conditions to make the solution transparent to obtain a mixed clear solution; place a small beaker containing deionized water on a magnetic stirrer, put a magnet and set a certain speed; drop the mixed clear solution into the deionized water at a certain speed, stir at room temperature for 4-5 h to obtain a liposome suspension; then put the liposome suspension into a dialysis bag, put it into a beaker containing 800-1000 mL of pure water, and stir and dialyze at room temperature for 24-36 h to obtain a ginsenoside CK-sorafenib double-drug liposome suspension.

[0040] In view of the problems of complex preparation process, tedious operation and low efficiency in the prior art, the present application greatly reduces the technical threshold and production cost by using the ethanol injection method, significantly simplifies the preparation process compared with the traditional process, and the operation steps are more intuitive and easy to understand, making the whole preparation process more efficient and convenient. The average particle size of the prepared ginsenoside CK-sorafenib double-drug liposome is less than 100 nm, and the encapsulation efficiency of ginsenoside CK and sorafenib is 87% and 83% respectively, meeting the basic drug requirements.

[0041] In view of the problem that the liposome is easily digested by oral gastric acid in the prior art, the present application coats deoxycholic acid (DCA) on the periphery of the liposome to make the liposome stable in simulated gastric juice, thereby slowly releasing the drug. When the final concentration of DCA-coated liposome is 0.6 mg / mL, ginsenoside CK and sorafenib are only released by 9% and 7% respectively in artificial gastric juice (pH=1.2) within 2 hours.

[0042] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below in combination with specific embodiments.

[0043] Embodiment 1 A ginsenoside CK-sorafenib double-drug liposome, a preparation method thereof comprising the following steps: S1, 7 mg of ginsenoside CK (CK), 1 mg of sorafenib (SOR), 16 mg of cholesterol and 80 mg of egg yolk lecithin are precisely weighed into a plastic centrifuge tube, dissolved in 2 mL of an ethanol solution, and each component is fully dissolved under vortex and ultrasonic conditions until the solution is transparent to obtain a mixed solution.

[0044] S2, a small beaker containing 18 mL of deionized water is placed on a magnetic stirrer, and the rotation speed is set to 700 rpm. The mixed clear solution obtained in S1 is taken, and the clear solution is uniformly dropped into the beaker containing 18 mL of deionized water, and stirred at room temperature for 4 h to obtain a liposome suspension.

[0045] S3, the liposome suspension obtained in S2 is placed into a dialysis bag with a specification of 14 kDa, and placed into a beaker containing 1000 mL of pure water, and stirred at a rotation speed of 700 r under normal temperature conditions, and dialyzed for 24 h to obtain a ginsenoside CK-sorafenib double-drug liposome (labeled as Lipo-CK-SOR, the same below).

[0046] S4, 10 mL of Lipo-CK-SOR obtained in S3 is taken, 6 mg of deoxycholic acid (abbreviated as DCA) is added, and stirring is continued for 30 min, then it is packaged with a 14 kDa dialysis bag and dialyzed in pure water for 12 h to obtain a deoxycholic acid-coated Lipo-CK-SOR.

[0047] Embodiment 2 A ginsenoside CK-sorafenib double-drug liposome, a preparation method thereof differs from that of Embodiment 1 only in that the mass of ginsenoside CK and sorafenib in S1 is replaced by 4.38 mg and 0.6 mg respectively.

[0048] Embodiment 3 A ginsenoside CK-sorafenib double-drug liposome, a preparation method thereof differs from that of Embodiment 1 only in that the mass of ginsenoside CK and sorafenib in S1 is replaced by 5.8 mg and 0.73 mg respectively.

[0049] Embodiment 4 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the mass of cholesterol in S1 step is replaced from 16 mg to 5.33 mg.

[0050] Example 5 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the mass of cholesterol in S1 step is replaced from 16 mg to 6.7 mg.

[0051] Example 6 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the mass of cholesterol in S1 step is replaced from 16 mg to 20 mg.

[0052] Example 7 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the volume of deionized water in S2 step is replaced from 18 mL to 22 mL.

[0053] Example 8 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the volume of deionized water in S2 step is replaced from 18 mL to 14 mL.

[0054] Example 9 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the volume of deionized water in S2 step is replaced from 18 mL to 10 mL.

[0055] Example 10 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the stirring speed in S2 step is replaced from 700 rpm to 300 rpm.

[0056] Example 11 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the stirring speed in S2 step is replaced from 700 rpm to 500 rpm.

[0057] Example 12 A ginsenoside CK-sorafenib double-drug liposome, the difference between the preparation method of which and that of Example 1 is that the stirring speed in S2 step is replaced from 700 rpm to 900 rpm.

[0058] Example 13 A ginsenoside CK-sorafenib double-drug liposome, a preparation method thereof comprising the following steps: S1, precisely take 7 mg of ginsenoside CK (CK), 1 mg of sorafenib (SOR), 16 mg of cholesterol and 80 mg of egg yolk lecithin into a plastic centrifuge tube, dissolve in 2 mL of ethanol solution, and fully dissolve each component under vortex and ultrasonic conditions until the solution is transparent to obtain a mixed solution.

[0059] S2, place a small beaker containing 18 mL of deionized water on a magnetic stirrer, and set the rotation speed to 700 rpm. Take the mixed clear solution obtained in S1, and drop the clear solution into the beaker containing 18 mL of deionized water at a constant speed, and stir at room temperature for 4 h to obtain a liposome suspension.

[0060] S3, place the liposome suspension obtained in S2 into a dialysis bag with a specification of 14 kDa, and place it into a beaker containing 1000 mL of pure water, and stir at a rotation speed of 700 rpm at room temperature, and dialyze for 24 h to obtain a ginsenoside CK-sorafenib double-drug liposome.

[0061] Comparative Example 1 A ginsenoside CK-sorafenib double-drug liposome, a preparation method thereof and Example 1 are different only in that no ginsenoside CK and sorafenib are added in S1 step to obtain and record as a blank liposome.

[0062] Comparative Example 2 Free ginsenoside CK is used as Comparative Example 2.

[0063] Comparative Example 3 Free sorafenib is used as Comparative Example 3.

[0064] Comparative Example 4 Free mixed 7 mg of ginsenoside CK and free 1 mg of sorafenib are used as Comparative Example 4.

[0065] The Lipo-CK-SOR prepared in Example 1 is subjected to morphology characterization by transmission electron microscopy, and it is observed that Figure 1 It is concluded that the Lipo-CK-SOR as a whole is in a spherical concave form, the surface is smooth, the particles are obviously not adhered, and the size is relatively uniform. The average particle size of the liposome is measured and counted to be <100 nm.

[0066] The Lipo-CK-SOR prepared in Example 1 is subjected to characterization by a Fourier transform infrared absorption spectrometer. It is observed that Figure 2 It is concluded that the ginsenoside CK spectrum is located at 1075 cm -1 to 1080 cm -1The characteristic peaks are vibrational peaks of α-glycosidic bonds, caused by the stretching vibration of the CO bond in the glycosidic bond. In the sorafenib spectrum, the stretching vibration of the NH bond is located at 3300–3400 cm⁻¹. -1 The Lipo-CK-SOR spectrum simultaneously showed signal peaks for both functional groups, but because the drugs were encapsulated in liposomes, the intensity of the characteristic peaks was significantly weaker than that of the pure drugs. This proves that both drugs have been successfully loaded into liposomes.

[0067] observe Figure 3 As shown in equation (a), the liposome encapsulation efficiency first increases and then decreases as the lipid-to-drug ratio decreases. This may be because when the amount of drug encapsulated in the liposome phospholipid layer reaches saturation, free lipid-soluble drugs can no longer be encapsulated in the phospholipid layer.

[0068] observe Figure 3 As shown in Figure b, the encapsulation efficiency of liposomes initially increases and then decreases with increasing cholesterol ratio. When the phospholipid ratio changes from 15:1 to 5:1, and then to 4:1, the encapsulation efficiency of CK and SOR first increases and then decreases. This is because when fewer cholesterol molecules are inserted into the gaps between phospholipid molecules, the degree of freedom of phospholipid molecule movement increases significantly, leading to excessive membrane fluidity. Therefore, the affinity between hydrophobic drugs and the membrane decreases, resulting in a decrease in encapsulation efficiency.

[0069] observe Figure 3 The results showed that the encapsulation efficiency of liposomes initially increased and then decreased with decreasing ratios. The encapsulation efficiencies of CK and SOR reached their peaks at an organic-to-water ratio of 9:1, at 70% and 78%, respectively. This is because at higher ratios, sufficient volume of the aqueous phase accelerates the dilution of the organic solvent, promoting lipid self-assembly and thus increasing the encapsulation efficiency. However, at lower ratios, high concentrations of ethanol interfere with the hydrophobic interactions between lipid molecules, weakening the aggregation force of the hydrophobic tail chains of phospholipids, ultimately leading to a decrease in encapsulation efficiency. Stirring speed may regulate the mixing efficiency of the organic and aqueous phases, the local microenvironment, and the kinetics of lipid assembly, directly affecting the structural integrity of liposomes and drug encapsulation efficiency. Therefore, this invention also investigated the effect of stirring speed on encapsulation efficiency.

[0070] like Figure 3 As shown in Figure d, drug encapsulation initially increases and then decreases with increasing stirring speed. This is because lower stirring speeds reduce the contact and binding opportunities between drug molecules and liposomes. Some drugs cannot enter the liposomes but remain free in solution, reducing the encapsulation efficiency. When the stirring speed is too high, the liposome membrane structure is easily damaged. Excessive shear force can destroy the liposome membrane structure, leading to leakage of drugs already encapsulated within the phospholipid layer, thus decreasing the encapsulation efficiency.

[0071] observe Figure 4The average particle size of Lipo-CK-SOR was found to be 88.99 nm, and the polydispersity index was 0.18 < 0.3, indicating that the Lipo-CK-SOR prepared by the optimal method has uniform particle size and good dispersibility.

[0072] like Figure 5 As shown in Figures a-b, compared to the original liposome suspension, liposomes with a final concentration of 0.4 mg / mL deoxycholic acid added and stirred for 30 min after obtaining the final product exhibited a significant sustained-release effect. In a gastric acid environment at pH 1.2 for 2 hours, the release rate further decreased to 9% and 7% respectively when the final concentration of deoxycholic acid was 0.6 mg / mL. However, at 0.8 mg / mL, the stability enhancement effect did not continue to improve. Therefore, adding a concentration of deoxycholic acid greater than 0.6 mg / mL is beneficial for protecting the sustained-release performance of liposomes and the drug in an acidic environment. Liposomes without deoxycholic acid coating (Example 13) released rapidly in simulated gastric juice, with a release rate exceeding 40% within 2 hours, which is detrimental to sustained release and human absorption. Figure 5 As shown in Figure c, the cumulative release of CK and SOR within 48 hours reached 92% and 91%, respectively. In summary, the strategy of coating deoxycholic acid gives liposome suspensions sustained-release properties, providing a feasible approach for drug delivery through gastric juice.

[0073] like Figure 5 As shown in Figure d, at 25°C, the leakage rates of both CK and SOR were above 30% within 28 days and continued to decline rapidly. In contrast, the leakage rates of all samples stored at 4°C were less than 10%. This indicates that liposomes are stable at low temperatures such as 4°C. This is because low temperatures promote tighter arrangement of lipid molecules, enhancing structural stability; conversely, higher temperatures increase the mobility of phospholipid molecules, leading to a looser structure.

[0074] The following tests were conducted on the in vitro biosafety and proliferation inhibition effects of Lipo-CK-SOR from Example 1 of this invention, as well as Comparative Examples 1 and 2, on HepG2 liver cancer cells. The specific application methods and results are as follows: Application method: a. The in vitro biocompatibility of the Lipo-CK-SOR prepared in Example 1 was evaluated using the MTT assay, and the specific procedures are as follows: HepG2 cells were introduced at a rate of 1 × 10⁻⁶ per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and allowed to adhere for 16 hours. The old culture medium was discarded, and the cells were incubated with the aforementioned groups for 48 hours. The concentration gradients of CK (coal oxidase) were 10, 20, 30, 40, and 50 μM, and the concentration gradients of SOR (sulfuric acid) were 2, 4, 6, 8, and 10 μM. 20 μL of MTT (5 mg / mL) solution was added to each well, and after 4 hours, the solution was discarded using a disposable syringe. Finally, 150 μL of DMSO was added, and the plates were shaken at low speed for 10 minutes to dissolve the methylzanol crystals. The absorbance was measured at 490 nm using a microplate reader (model 680, USA).

[0075] HepG2 cell viability was calculated using the following formula: ; Among them, A490 experimental group represents the absorbance of the experimental group (including cells, MTT, sample solution, and human HepG2 cell culture medium) at 490 nm; A490 zeroing well represents the absorbance of the zeroing well (MTT and human HepG2 cell culture medium) at 490 nm; A490 control group represents the absorbance of the control group (including cells, MTT, and human HepG2 cell culture medium) at 490 nm.

[0076] observe Figure 6 The results showed that cell viability gradually decreased with increasing CK and SOR drug concentrations. The inhibitory effects of free single and free dual drugs on HepG2 cell proliferation were dose-dependent. Figure 6 As shown, the cell viability of cells co-incubated with Comparative Example 4 was significantly lower than that of Comparative Examples 2 and 3, indicating a synergistic effect of the two drugs used in combination. Furthermore, Figure 6 This indicates that Example 1 inhibited the proliferation of HepG2 liver cancer cells more effectively than Comparative Example 4. This enhanced efficacy can be attributed to the phospholipid carrier, which promotes concentrated drug uptake by cells.

[0077] b. The apoptosis assays of the Lipo-CK-SOR prepared in Example 1 and Comparative Examples 2, 3, and 4 were performed using the eBioscience™ Annexin V apoptosis detection kit. The specific procedures are as follows: Cells were seeded in 6-well plates at appropriate concentrations. Control group, Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 were set up respectively. Incubate for 48 hours. Discard the culture medium, wash the cells with PBS three times. Digest each well with 0.5 mL 0.25% trypsin without EDTA. Then add 1 mL culture medium to each well to stop the digestion. After centrifugation, discard the supernatant, wash twice with PBS, and then separate 1 / 6 from the group with more cells to set up BLANK and FITC, PI single staining tubes. The experimental group is washed twice with Binding buffer, then labeled with FITC (5 μL) and PI (10 μL) after adding 100 μL of Binding buffer, and incubated for 15 minutes. Add 400 μL of Binding buffer to each group, and complete the detection within 1 hour using a flow cytometer (Beckman FC500, USA).

[0078] Observation Figure 7 The apoptosis experiment quantification showed that the positive cell rate of Example 1 was increased by 27.85%, 17.92%, 12.07% and 6.03% compared with the control group, Comparative Example 2, Comparative Example 3 and Comparative Example 4 respectively, proving the effectiveness of Example 1.

[0079] c. The Lipo-CK-SOR prepared in Example 1 and Comparative Example 2, Comparative Example 3 and Comparative Example 4 were used to explore the ability to inhibit cell migration, and the specific operation was as follows: HepG2 cells in logarithmic growth phase were seeded in a 6-well plate, and a uniform horizontal line was drawn at an appropriate density. After the cells adhered and grew to 80%, a vertical scratch was made on the cell layer with a 200 μL pipette tip. Then, the cells were washed twice with PBS to remove the cells that had fallen and floated. Comparative Example 2, Comparative Example 3 and Comparative Example 4 were used for co-culture with the cells. The basic medium without the addition of drugs was used as a control, and three repeated holes were set up in parallel. The horizontal line drawn on the back of the 6-well plate was wiped off. Then, photographs were taken under a microscope at 0h, 24h and 48h respectively. The scratch area was calculated using Image J software, and the cell migration rate was calculated using the following formula.

[0080] Observation Figure 8 It was found that the Lipo-CK-SOR prepared in Example 1 was used for HepG2 cell migration experiment, and it was found that the healing speed of the scratch was slowed down, so the migration ability of cancer cells was significantly slowed down, and Example 1 could effectively inhibit the proliferation of cancer cells. The migration rate of the control group was 51.2% at 48 hours, while the migration rates of Comparative Example 4 and Example 1 were 14.9% and 11.8% respectively, indicating that both combinations could inhibit the horizontal migration of HepG2 cells, but the inhibitory effect of Example 1 on cell migration was more significant.

[0081] It should be understood that the numerical ranges herein are to be understood as being inclusive of both the numerical values recited and any number between the two endpoints, unless otherwise indicated. Since many embodiments of the application can be made without departing from the spirit and essential characteristics of the application, the application resides in the claims hereinafter appended.

Claims

1. A medicament for treating liver cancer or reversing sorafenib resistance, characterized in that, It is obtained by mixing ginsenoside CK and sorafenib in a mass ratio of 5-8:

1.

2. The medicament according to claim 1, characterized in that, The medicine comprises pharmaceutically acceptable excipients.

3. The medicament according to claim 2, characterized in that, The medicine is a liposome.

4. The medicament according to claim 3, characterized in that, It is prepared according to the following steps: Ginsenoside CK, sorafenib, cholesterol and egg yolk lecithin are dissolved in an ethanol solution to obtain a mixed solution; The mixed solution is dropped into water under stirring at a constant speed, and the obtained liposome suspension is dialyzed to obtain the double-drug liposome.

5. The medicament according to claim 4, characterized in that, The use amount ratio of ginsenoside CK, sorafenib, cholesterol, egg yolk lecithin and ethanol is 4.38-8.75 mg:0.6-1.25 mg:5.33-20 mg:80 mg:2-2.2 mL.

6. The medicine of claim 4, wherein, The mixed solution is dropped into deionized water at a constant speed, and the stirring speed of the deionized water is 300-900 rpm; The dialysis is that the liposome suspension is loaded into a dialysis bag with a specification of 14 kDa, and dialyzed in pure water for 24-36 h.

7. The medicament according to claim 4, characterized in that, It also includes the process of adding deoxycholic acid to the liposome suspension.

8. The medicament according to claim 7, characterized in that, After adding deoxycholic acid to the liposome suspension, stirring and dialysis are performed to obtain the double-drug liposome.

9. The medicament according to claim 8, characterized in that, In the mixed system of the liposome suspension and deoxycholic acid, the concentration of deoxycholic acid is 0.4-0.8 mg / mL.

10. The medicament according to claim 9, characterized in that, The average particle size of the double-drug liposome is 88.99-101.73 nm, and the PDI is 0.17-0.18.

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