An antitumor composition of arsenic trioxide-artemether, double-drug nano-liposome and its preparation method and use

Abstract

The application discloses an antitumor composition of arsenic trioxide and artesunate for enhancing the drug efficacy of arsenic trioxide, and discloses GA modified pH responsive arsenic trioxide-artesunate double-drug nanoliposome including the composition, the double-drug nanoliposome including glycyrrhetinic acid-polyethylene glycol-hydrazone-distearyphosphatidyl ethanolamine polymer, cholesterol, artesunate and calcium arsenate nanoparticles; the glycyrrhetinic acid-polyethylene glycol-hydrazone-distearyphosphatidyl ethanolamine and cholesterol form nanoliposome, and the nanoliposome is loaded with artesunate and calcium arsenate nanoparticles; the calcium arsenate nanoparticles contain arsenic trioxide, and a preparation method and application in preparing antitumor drugs are disclosed. The application first co-loads the highly toxic traditional Chinese medicine arsenic trioxide and artesunate to play a synergistic effect, realizes the attenuation and efficiency enhancement of arsenic trioxide through targeted modification delivery and tumor microenvironment response release, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antitumor composition of arsenic trioxide and artesunate, as well as GA-modified pH-responsive arsenic trioxide-artesunate dual-drug-carrying nanoliposomes, their preparation methods, and uses. Background Technology

[0002] Arsenic trioxide (As2O3) is the main component of the traditional Chinese medicine arsenic trioxide (arsenic), and modern medicine uses it to treat acute promyelocytic leukemia with significant efficacy. Some scholars have found that As2O3 also inhibits growth and induces apoptosis in various solid tumor cells, exhibiting broad-spectrum anticancer activity. However, due to the lack of specificity in the distribution of As2O3 in vivo, it often produces severe adverse reactions in other normal tissues when reaching effective concentrations. Furthermore, the short half-life, rapid elimination after administration, strong gastrointestinal reactions, and unclear metabolic processes of As2O3 limit its application in solid tumors.

[0003] Synergistic effects and reduced toxicity are currently a hot topic in As2O3 anti-tumor research. Artesunate (ART), a sesquiterpene lactone polymer containing an endoperoxide group extracted from the traditional Chinese medicine Artemisia annua, is one of the commonly used antimalarial drugs. ART also shows good efficacy in anti-HCC and can synergistically enhance the effects of other drugs, reversing multidrug resistance. For example, the combination of ART and sorafenib can increase the sensitivity of tumor cells to sorafenib, improve efficacy, and reduce the dosage. Combined with cisplatin, it can increase the sensitivity of ovarian cancer to sorafenib, improve clinical efficacy, and reduce adverse reactions. Meanwhile, studies have reported that the combination of artemisinin and As2O3 has a synergistic inhibitory effect on HepG2 liver cancer cells. Since ART is a derivative of artemisinin, the combination of ART and As2O3 may also have potential synergistic effects. Therefore, designing a novel drug delivery system to simultaneously target and deliver As2O3 and ART to tumor cells could produce synergistic effects while reducing toxic side effects. Therefore, the combined application of As2O3 and ART will produce a synergistic effect, achieving enhanced efficacy and reduced toxicity of As2O3.

[0004] The key to achieving targeted and effective drug release at tumor sites is exerting anti-tumor effects. In previous studies, the applicant synthesized a glycyrrhetinic acid (GA)-modified polyethylene glycol-poly(lactic-co-glycolic acid) block copolymer (PEG-PLGA)—GA-PEG-PLGA—and used it to prepare nanomicelles, obtaining relatively ideal targeting results. However, intracellular drug release remained unsatisfactory. The introduction of pH-sensitive bonds can control intracellular drug release through a "stimulus-response" mechanism. Hydrazone bonds, formed by the reaction of aldehyde and hydrazine groups, are among the most studied acid-labile bonds, easily hydrolyzed by acidification in weakly acidic environments. Therefore, if hydrazone bonds can be combined to synthesize polymers with dual targeting and intracellular pH responsiveness, and nanoliposomes can be prepared to simultaneously encapsulate arsenic trioxide and artesunate, targeted drug release within tumor cells could be achieved, reducing toxicity and enhancing efficacy. Currently, there are no reports in this area. Summary of the Invention

[0005] To address the key issues of safe delivery and efficient efficacy of arsenic trioxide, this invention provides a GA-modified pH-responsive arsenic trioxide / artesunate dual-drug-carrying nanoliposome. Through the synergistic effect of As2O3 and ART, targeted delivery, and site-specific release, As2O3 is reduced in toxicity and enhanced in efficacy, thus promoting its clinical application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] An antitumor composition comprising arsenic trioxide and artesunate; wherein the molar ratio of arsenic trioxide to artesunate is 1:1 to 1:20, preferably 1:1 to 1:15, more preferably 1:5 to 10, and even more preferably 1:8 to 9.

[0008] The combination of arsenic trioxide and artesunate provided by this invention has a significant synergistic effect on tumor cells, especially solid tumor cells. Previous studies investigated the combined effect of ART and As2O3. The strength of drug combination is generally expressed by the combination index (CI). According to the Soriano method (0.9 ≤ CI ≤ 1.1 indicates additiveness, 0.8 ≤ CI < 0.9 indicates low synergy, 0.6 ≤ CI < 0.8 indicates moderate synergy, 0.4 ≤ CI < 0.6 indicates high synergy, and 0.2 ≤ CI < 0.4 indicates significant synergy), the results show that the combination of ART and As2O3 has a moderate to high synergistic effect on various HCC cell types, especially showing a significant synergistic effect on Huh-7 cells (CI < 0.2469).

[0009] The present invention also provides the use of the composition comprising arsenic trioxide and artesunate in the preparation of anti-solid tumor drugs, particularly for the preparation of anti-liver cancer drugs.

[0010] This invention also provides an arsenic trioxide-artesunate dual-drug-loaded nanoliposome, comprising the aforementioned antitumor composition of arsenic trioxide and artesunate, utilizing nanoliposomes to provide dual targeting and intracellular pH responsiveness, thereby reducing toxicity and enhancing efficacy. The arsenic trioxide-artesunate dual-drug-loaded nanoliposome comprises glycyrrhetinic acid-polyethylene glycol-hydrazone bond-distearate phosphatidylethanolamine polymer (GA-PEG-hyd-DSPE), cholesterol, artesunate (ART), and calcium arsenic nanoparticles; GA-PEG-hyd-DSPE and cholesterol form nanoliposomes, within which ART and calcium arsenic nanoparticles are encapsulated; the calcium arsenic nanoparticles contain arsenic trioxide.

[0011] The GA-PEG-hyd-DSPE is prepared by the following method:

[0012] A condensation reaction is performed between a tert-butyloxycarbonyl-amino-polyethylene glycol-amino polymer (Boc-NH-PEG-NH2) and glycyrrhetinic acid (GA) to generate a glycyrrhetinic acid-polyethylene glycol-amino-tert-butyloxycarbonyl-protected amino polymer, denoted as GA-PEG-NH-Boc. The tert-butyloxycarbonyl group is then removed from GA-PEG-NH-Boc to generate a glycyrrhetinic acid-polyethylene glycol-amino polymer, denoted as GA-PEG-NH2. GA-PEG-NH2 then reacts with a hydroxyl-hydrazone-carboxyl compound (HO-hyd-CO). The reaction of glycyrrhetinic acid (GA) with succinic anhydride (SGA) produces a glycyrrhetinic acid-polyethylene glycol-hydrazone-hydroxyl polymer, denoted as GA-PEG-hyd-OH. GA-PEG-hyd-OH reacts with succinic anhydride to produce a glycyrrhetinic acid-polyethylene glycol-hydrazone-carboxyl polymer, denoted as GA-PEG-hyd-COOH. GA-PEG-hyd-COOH is then reacted with distearylphosphatidylethanolamine (DSPE) to produce a glycyrrhetinic acid-polyethylene glycol-hydrazone-distearylphosphatidylethanolamine polymer, denoted as GA-PEG-hyd-DSPE.

[0013] Furthermore, the calcium arsenic nanoparticles are prepared from arsenic trioxide. Further, the calcium arsenic nanoparticles are prepared by reacting arsenic trioxide (As₂O₃) with calcium stearate.

[0014] This invention also provides a method for preparing arsenic trioxide-artesunate dual-drug-carrying nanoliposomes, the method comprising the following steps:

[0015] (1) Synthesis of glycyrrhetinic acid-modified amino polyethylene glycol

[0016] A condensation reaction is carried out between tert-butyloxycarbonyl-amino-polyethylene glycol-amino polymer (Boc-NH-PEG-NH2) and glycyrrhetinic acid (GA) to generate glycyrrhetinic acid-polyethylene glycol-amino-tert-butyloxycarbonyl-protected amino polymer, denoted as GA-PEG-NH-Boc. GA-PEG-NH-Boc is deprotected by tert-butyloxycarbonyl to generate glycyrrhetinic acid-polyethylene glycol-amino polymer, denoted as GA-PEG-NH2.

[0017] (2) Synthesis of GA-PEG-hyd-COOH

[0018] GA-PEG-NH2 is reacted with a hydroxyl-hydrazone-carboxyl compound (HO-hyd-COOH) to generate a glycyrrhetinic acid-polyethylene glycol-hydrazone-hydroxyl polymer, denoted as GA-PEG-hyd-OH. GA-PEG-hyd-OH is then reacted with succinic anhydride to generate a glycyrrhetinic acid-polyethylene glycol-hydrazone-carboxyl polymer, denoted as GA-PEG-hyd-COOH.

[0019] (3) Synthesis of GA-PEG-hyd-DSPE

[0020] GA-PEG-hyd-COOH was reacted with distearylphosphatidylethanolamine (DSPE) to generate a glycyrrhetinic acid-polyethylene glycol-hydrazone bond-distearylphosphatidylethanolamine polymer, denoted as GA-PEG-hyd-DSPE.

[0021] (4) Arsenic trioxide (As2O3) reacts with calcium stearate to generate calcium arsenic nanoparticles;

[0022] (5) Nanoliposomes were prepared by using GA-PEG-hyd-DSPE, cholesterol, artesunate (ART) and calcium arsenic nanoparticles to obtain arsenic trioxide-artesunate dual-drug-loaded nanoliposomes that simultaneously encapsulate calcium arsenic nanoparticles and artesunate.

[0023] HO-hyd-COOH is shown in the following formula.

[0024]

[0025] The reaction pathway from step (1) to step (3) is shown in the following equation:

[0026]

[0027] Furthermore, in the method, the molar ratio of arsenic trioxide to artesunate is 1:1 to 1:20, preferably 1:1 to 1:15, more preferably 1:5 to 10, and even more preferably 1:8 to 9.

[0028] In step (1), the molecular weight of Boc-PEG-NH2 is 2000-5000 Da, preferably 2000-3000 Da.

[0029] Further, step (1) is preferably carried out according to the following steps: in chloroform solvent, Boc-NH-PEG-NH2 and glycyrrhetinic acid (GA) undergo a condensation reaction under the action of condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and catalyst 4-dimethylaminopyridine (DMAP), and the resulting reaction solution a is post-treated to obtain GA-PEG-NH-Boc;

[0030] GA-PEG-NH-Boc was reacted with trifluoroacetic acid in chloroform solvent to remove the Boc protecting group. The resulting reaction solution b was then treated to obtain glycyrrhetinic acid-polyethylene glycol-amino polymer, denoted as GA-PEG-NH2.

[0031] In step (1), the molar ratio of Boc-NH-PEG-NH2, GA, EDC and DMAP is 1:1 to 4:1 to 5:0.1 to 2, preferably 1:1 to 1.5:2 to 2.5:0.1 to 0.5, and more preferably 1:1.1:2:0.1.

[0032] The condensation reaction in step (1) is preferably carried out in a chloroform solvent.

[0033] The volumetric amount of chloroform used is 10–50 mL / g, calculated by mass of Boc-NH-PEG-NH2.

[0034] The condensation reaction was carried out at room temperature for 10–20 hours.

[0035] In step (1), the reaction temperature for removing the Boc protecting group is 0-5°C (preferably carried out in an ice bath), and the reaction time is preferably 1-3 hours.

[0036] The reaction to remove the Boc protecting agent is preferably carried out in a chloroform solvent, wherein the volume of chloroform used is 4 to 10 mL / g based on the mass of GA-PEG-Boc.

[0037] The volumetric dosage of trifluoroacetic acid, based on the mass of GA-PEG-NH-Boc, is 1.5–2 mL / g.

[0038] The preferred post-treatment step for reaction solution a is as follows: after the reaction is completed, reaction solution a is concentrated under reduced pressure, then poured into a large amount of ice-cold ether to precipitate, filtered, and the filter cake is dried to obtain GA-PEG-NH-Boc.

[0039] The preferred post-treatment steps for reaction solution b are as follows: after the reaction is completed, reaction solution b is washed with water, dried, and the filtrate is concentrated under reduced pressure. The concentrate is poured into a large amount of ice-cold ether to precipitate, filtered, and the filter cake is dried to obtain GA-PEG-NH2.

[0040] Furthermore, step (2) is preferably performed as follows:

[0041] In chloroform solvent, GA-PEG-NH2 and HO-hyd-COOH undergo a condensation reaction under the action of condensing agent EDC and catalyst DMAP. The resulting reaction solution c is then treated to obtain GA-PEG-hyd-OH.

[0042] GA-PEG-hyd-OH was dissolved in chloroform solvent, succinic anhydride was added, and the reaction was heated. The resulting reaction solution was then treated to obtain GA-PEG-hyd-COOH.

[0043] The molar ratio of GA-PEG-NH2, HO-hyd-COOH, EDC and DMAP is 1:1 to 4:1 to 5:0.1 to 2, preferably 1:1 to 1.5:2 to 2.5:0.1 to 0.5, and more preferably 1:1.1:2:0.1.

[0044] The condensation reaction in step (2) is preferably carried out in a chloroform solvent.

[0045] The volumetric amount of chloroform used is 4 to 20 mL / g, calculated by the mass of GA-PEG-NH2.

[0046] The condensation reaction in step (2) is carried out at room temperature for 10 to 20 hours.

[0047] The preferred post-treatment step for the reaction solution c is as follows: after the reaction is completed, the reaction solution c is concentrated under reduced pressure, poured into a large amount of ice-cold ether to precipitate, filtered, and the filter cake is dried to obtain GA-PEG-hyd-OH.

[0048] In step (2), the molar ratio of GA-PEG-hyd-OH to succinic anhydride is 1:1 to 5, preferably 1:2.

[0049] The reaction temperature of GA-PEG-hyd-OH and succinic anhydride is 55-70℃, preferably 60-65℃; the reaction time is 2-5h, preferably 3-4h.

[0050] The preferred reaction solvent for GA-PEG-hyd-OH and succinic anhydride is chloroform, and the volume of chloroform used is 4 to 20 mL / g based on the mass of GA-PEG-hyd-OH.

[0051] The preferred post-treatment step for the reaction solution d is as follows: after the reaction is completed, the reaction solution d is concentrated under reduced pressure, poured into a large amount of ice-cold ether to precipitate, filtered, and the filter cake is dried to obtain GA-PEG-hyd-COOH.

[0052] Furthermore, step (3) is preferably performed as follows:

[0053] In chloroform solvent, GA-PEG-hyd-COOH and DSPE undergo a condensation reaction under the action of condensing agent EDC and catalyst DMAP. The resulting reaction solution is then post-treated to obtain GA-PEG-hyd-DSPE.

[0054] The molar ratio of GA-PEG-hyd-COOH, DSPE, EDC and DMAP is 1:1 to 5:1 to 6:0.1 to 1, preferably 1:1:2:0.1.

[0055] The condensation reaction in step (3) is preferably carried out in a chloroform solvent.

[0056] The volumetric amount of chloroform used is 5 to 30 mL / g, calculated by the mass of GA-PEG-hyd-COOH.

[0057] The reaction temperature of the condensation reaction in step (3) is 30-50°C, preferably 40°C; the reaction time is 10-20 hours.

[0058] The preferred post-treatment step for the reaction solution e is as follows: after the reaction is completed, the reaction solution e is concentrated under reduced pressure, poured into a large amount of ice-cold ether to precipitate, filtered, and the filter cake is dried to obtain GA-PEG-hyd-DSPE.

[0059] Furthermore, step (4) is preferably performed as follows:

[0060] Calcium stearate and oleic acid were added to toluene solvent, heated and stirred to form a transparent solution, and then arsenic trioxide (As2O3) solution was added dropwise while stirring. The reaction was heated and stirred, and then anhydrous ethanol was added to form calcium arsenic nanoparticle precipitate. The precipitate was collected by ultracentrifugation to obtain calcium arsenic nanoparticles.

[0061] The calcium stearate and oleic acid are added to toluene solvent, and the heating and stirring temperature is 60-100°C, preferably 80-90°C.

[0062] After adding arsenic trioxide (As₂O₃) solution, the reaction is heated and stirred at a temperature of 40–80°C, preferably 50–70°C. The stirring time is 2–30 h, preferably 10–20 h.

[0063] The ultracentrifugation speed is 15,000 to 30,000 rpm, and the ultracentrifugation time is preferably 15 to 30 minutes;

[0064] The volumetric amount of oleic acid used is 15-30 mL / g based on the mass of calcium stearate, preferably 20 mL / g;

[0065] The volumetric amount of toluene used is 150-300 mL / g based on the mass of calcium stearate;

[0066] The mass ratio of calcium stearate to arsenic trioxide is 1:0.1 to 0.3, preferably 1:0.15 to 0.2.

[0067] The concentration of the arsenic trioxide solution is preferably 3-5 mM.

[0068] Furthermore, step (5) is preferably performed as follows:

[0069] GA-PEG-hyd-DSPE, cholesterol, and ART were dissolved in an organic solvent, the organic solvent was removed by rotary evaporation, and then a dispersion of calcium and arsenic nanoparticles was added. The mixture was heated to carry out a hydration reaction, and then ultrasonically processed by a probe to obtain arsenic trioxide-artesunate dual-drug-loaded nanoliposomes that simultaneously encapsulate calcium and arsenic nanoparticles and artesunate.

[0070] The organic solvent is one or more of acetone, dichloromethane, chloroform, and diethyl ether, preferably acetone or dichloromethane.

[0071] The ratio of the total mass of ART and calcium arsenic nanoparticles to the total mass of GA-PEG-hyd-DSPE and cholesterol, i.e., the drug-lipid ratio, is 0.1 to 1.0:1, preferably 0.2 to 0.5:1;

[0072] The mass ratio of GA-PEG-hyd-DSPE to cholesterol is 1 to 10:1, preferably 2 to 6:1.

[0073] The drug encapsulated, calculated as As and ART, has a molar ratio of As2O3 to ART of 1:1 to 20, preferably 1:5 to 15.

[0074] The volume of the organic solvent is 150–500 mL / g, based on the mass of DSPE-hyd-PEG-GA.

[0075] In the dispersion of the calcium and arsenic nanoparticles, the mass concentration of the calcium and arsenic nanoparticles is 0.5–1 mg / mL, preferably 0.8 mg / mL.

[0076] The hydration reaction is carried out at a temperature of 20–60°C, preferably 30–50°C.

[0077] The hydration reaction takes 10 to 60 minutes, preferably 30 to 50 minutes.

[0078] The concentration of the calcium arsenic nanoparticle dispersion is 0.5–1 mg / mL.

[0079] The ultrasonic treatment time for the probe is 3 to 5 minutes, preferably 4 minutes.

[0080] The present invention also provides arsenic trioxide-artesunate dual-drug-loaded nanoliposomes prepared by the above method.

[0081] The arsenic trioxide-artesunate dual-drug-loaded nanoliposomes provided by this invention have an encapsulation efficiency of 10-90%.

[0082] The arsenic trioxide-artesunate dual-drug-loaded nanoliposomes provided by this invention have a particle size of 60-150 nm.

[0083] Arsenic trioxide-artesunate dual-drug-carrying nanoliposomes exhibit pH responsiveness, with a higher drug release rate under acidic conditions than under neutral conditions.

[0084] This invention also provides the application of the arsenic trioxide-artesunate dual-drug-loaded nanoliposomes in the preparation of antitumor drugs. Further, in this application, the antitumor drug is preferably an anti-solid tumor drug.

[0085] More preferably, the application of the arsenic trioxide-artesunate dual-drug-loaded nanoliposomes in the preparation of anti-liver cancer drugs.

[0086] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0087] This invention is the first to discover that the combination of ART and As2O3 has a moderate to high synergistic effect on a variety of HCC cells, especially showing a significant synergistic effect on Huh-7 cells. Therefore, the combination of ART and As2O3 can be used to prepare anti-tumor drugs, especially anti-solid tumor drugs, and more preferably anti-liver cancer drugs.

[0088] However, in order to reduce the toxicity of combination drugs to normal cells, it is necessary to improve the drug formulation, enhance the targeting of tumor cells, and reduce toxicity while increasing efficacy.

[0089] The DSPE-hyd-PEG-GA polymer provided by this invention has good targeting properties, significantly increases uptake by tumor cells, and after uptake, the drug is mainly distributed on the cytoplasm and cell membrane. The active targeting ability of GA and the drug release-promoting effect of hydrazone bonds significantly improve its toxicity to tumor cells.

[0090] Furthermore, GA-modified pH-responsive dual-drug-loaded liposomes outperformed other liposomes in inducing oxidative damage in tumor cells. GA-modified pH-responsive dual-drug-loaded liposomes exhibited a significant cell cycle arrest effect on tumor cells. Due to their better affinity for tumor cells and their promoting effect on intracellular release, they showed greater potential for inducing tumor cell apoptosis.

[0091] This invention leverages the synergistic effect of co-loading As2O3 and ART to enhance the anti-tumor activity of As2O3. By modifying GA and designing hydrazone bonds, a DSPE-hyd-PEG-GA polymer with dual targeting and intracellular pH responsiveness is synthesized. This polymer is then used to prepare nanoliposomes, which simultaneously encapsulate As2O3 and ART, enabling the liposomes to achieve targeted delivery and site-specific release, thereby reducing toxicity.

[0092] Specifically, the drug delivery system targets tumor sites via GA ligand-receptor. After entering tumor cells, the drug undergoes a first-order pH response within the lysosome, resulting in the cleavage of hydrazone bonds and the release of ART and CaAs NP. The CaAs NP then undergoes a second-order pH response, dissolving and releasing As2O3. The entire lysosome also ruptures due to the gradual increase in osmotic pressure, ultimately achieving targeted release of ART and As2O3 within tumor cells. This synergistic effect reduces the toxicity of As2O3 and enhances its efficacy, providing a new approach for the better clinical application of As2O3 in anti-tumor therapy and demonstrating promising clinical application prospects. Attached Figure Description

[0093] Figure 1 Example 1: Synthetic route for preparing DSPE-hyd-PEG-GA.

[0094] Figure 2 GA-PEG-NH2 and GA 1 H NMR spectrum.

[0095] Figure 3 DSPE-Hyd-PEG-GA 1 HNMR spectrum.

[0096] Figure 4 FT-IR spectra of DSPE-Hyd-PEG-GA and NH2-PEG-NH2.

[0097] Figure 5 CaAs NP TEM scan.

[0098] Figure 6 TEM scan of DSPE-hyd-PEG-GA@ART / CaAs NPs

[0099] Figure 7 CaAs NP elemental analysis diagram.

[0100] Figure 8 XPS plot of CaAs NP.

[0101] Figure 9 XRD pattern of CaAs NP.

[0102] Figure 10FT-IR spectra of CaAs NP and DSPE-hyd-PEG-GA.

[0103] Figure 11 In vitro release curves of ART and As from DSPE-hyd-PEG-GA@ART / CaAs NPs in pH 7.4 and 4.5 buffer solutions.

[0104] Figure 12 Figures A and B show the fluorescence intensity of Huh-7 cells after incubation with Nile Red-labeled drugs for 0.5, 1, 2, and 4 hours; Figures C and D show laser confocal scanning electron microscopy and flow cytometry results of Huh-7 cells after incubation with Nile Red-labeled drugs for 4 hours; Figures E and F show the experimental results of the uptake mechanism of DSPE-hyd-PEG-GA@NR in Huh-7 cells. ** p<0.01.

[0105] Figure 13 Figure A shows the cell viability of Huh-7 cells after treatment with each group of drugs; Figure B shows the IC50 results of Huh-7 cells after treatment with each group of drugs.

[0106] Figure 14 Figure A shows the flow cytometry results of Huh-7 cells after treatment with various drugs; Figure B shows the apoptosis statistics; Figures C and D show the cell cycle statistics.

[0107] Figure 15 Figure A shows the ROS laser confocal scan of Huh-7 cells after treatment with each drug group; Figure B shows the flow cytometry results; Figures C and D show the mitochondrial membrane potential flow cytometry results of Huh-7 cells after treatment with each drug group. * p<0.05, ** p<0.01. Detailed Implementation

[0108] An exemplary embodiment of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0109] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0110] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0111] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0112] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0113] Example 1

[0114] (1) Synthesis of glycyrrhetinic acid-modified amino polyethylene glycol

[0115] Weigh 1g of Boc-NH-PEG 2000 -NH2 was dissolved in 15 mL of chloroform, and GA (1.1 eq.), EDC (2.0 eq.), and DMAP (0.1 eq.) were added and dissolved completely. The reaction was carried out at room temperature for 15 h. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice-cold diethyl ether to precipitate, filtered to collect the product, and dried under vacuum to obtain the product GA-PEG. 2000 -NH-Boc. Weigh 2g of GA-PEG. 2000 -NH-Boc was dissolved in 9 mL of chloroform. 3 mL of trifluoroacetic acid was added under ice bath conditions, and the mixture was stirred for 1 hour under ice bath conditions. The reaction solution was washed three times with water, dried over anhydrous sodium sulfate, and the solution was concentrated under reduced pressure. The precipitate was then poured into a large amount of ice-cold diethyl ether, filtered, and dried under vacuum to obtain the product GA-PEG. 2000 -NH2.

[0116] (2) GA-PEG 2000 Synthesis of -hyd-COOH

[0117] Weigh 2g of GA-PEG 2000-NH2 was dissolved in 10 mL of chloroform, and HO-hyd-COOH (1.1 eq.), EDC (2.0 eq.), and DMAP (0.1 eq.) were added and dissolved completely. The reaction was carried out at room temperature for 12 h. The reaction solution was concentrated under reduced pressure, poured into a large amount of ice-cold diethyl ether to precipitate, filtered to collect the product, and dried under vacuum to obtain the product GA-PEG. 2000 -hyd-OH. Weigh 2g of GA-PEG. 2000 -hyd-OH was dissolved in 10 mL of chloroform, and succinic anhydride (2.0 eq.) was added until completely dissolved. The reaction was carried out at 60 °C for 3 h. The reaction solution was concentrated under reduced pressure, and the precipitate was poured into a large amount of ice-cold diethyl ether. The product was collected by filtration and dried under vacuum to obtain the product GA-PEG. 2000 -hyd-COOH.

[0118] (3) GA-PEG 2000 -hyd-DSPE synthesis

[0119] Weigh 1g of GA-PEG 2000 -hyd-COOH was dissolved in 10 mL of chloroform, and DSPE (1.0 eq.), EDC (2.0 eq.), and DMAP (0.1 eq.) were added and dissolved completely. The reaction was carried out at 40 °C for 12 h. The reaction solution was concentrated under reduced pressure, and the precipitate was poured into a large amount of ice-cold diethyl ether. The product was collected by filtration and dried under vacuum to obtain the product GA-PEG. 2000 -hyd-DSPE, or denoted as DSPE-Hyd-PEG 2000 -GA.

[0120] (4) GA-PEG 2000 -hyd-DSPE structure confirmed

[0121] use 1 ¹H NMR was used to determine the structure and actual molar composition percentage of the synthesized product. Fourier transform infrared spectroscopy (FT-IR) was used to characterize the structure of the copolymer. GA-PEG 2000 -NH2 and GA 1 H NMR spectrum as follows Figure 2 As shown, DSPE-Hyd-PEG 2000 -GA 1 H NMR spectrum as follows Figure 3 As shown, NH2-PEG 2000 -NH2 and GA-PEG 2000 The FT-IR spectrum of -hyd-DSPE is as follows: Figure 4 As shown. The results indicate that both the GA and hydrazone bonds are correctly connected, GA-PEG 2000 -hyd-DSPE was successfully synthesized, and can also be written as DSPE-hyd-PEG. 2000 -GA.

[0122] Example 2

[0123] Screening for the optimal ratio of arsenic trioxide / artesunate

[0124] Human liver cancer cells HepG2, Hep3b, HCCLM3, Huh-7, and SMMC-7721 were treated with two drugs at concentrations of ART (0, 2.6013, 13.01, 26.013, 65.033, 130.066, 260.13, 520.26 μM) and As2O3 (0, 0.1, 0.3, 1, 3, 10, 30, 100 μM), respectively. The IC50 of each cell line was calculated. Based on the results, the three most sensitive cell lines were selected and treated with free or mixed drugs at concentrations of 0.125, 0.25, 0.5, and 1 times the IC50 of ART and As2O3, respectively, to determine the combination index (CI). The CI value for selecting the optimal ratio of ART to As2O3 was calculated using CompuSyn software. The ART / As2O3 ratio with the lowest CI value was selected for further experiments.

[0125] The synergistic effect of drug combinations is generally expressed by the synergistic index (CI). According to the Soriano method (0.9 ≤ CI ≤ 1.1 indicates additiveness, 0.8 ≤ CI < 0.9 indicates low synergy, 0.6 ≤ CI < 0.8 indicates moderate synergy, 0.4 ≤ CI < 0.6 indicates high synergy, and 0.2 ≤ CI < 0.4 indicates significant synergy), the results show that the combination of As2O3 and ART has a moderate to high synergistic effect on various HCC cell types, especially showing a significant synergistic effect on Huh-7 cells (CI < 0.2469). Therefore, co-loading As2O3 and ART will produce a significant synergistic effect, as shown in Table 1.

[0126] Table 1. CI values ​​of combined ART and As2O3 on Huh-7 cells

[0127]

[0128] Example 3

[0129] Preparation and characterization of calcium arsenic nanoparticles

[0130] 100 mg of calcium stearate and 2 mL of oleic acid were added to 20 mL of toluene. The mixture was magnetically stirred at 80 °C to form an optically transparent solution. After cooling the reaction system to room temperature, 20 mL of As₂O₃ solution (4 mM) was added dropwise under magnetic stirring. After stirring at 50 °C for 20 hours, a large amount of anhydrous ethanol was added to precipitate CaAs NP. The precipitate was then collected by ultracentrifugation (15000 rpm, 20 min), washed three times with purified water, and then redispersed with purified water.

[0131] A suitable amount of sample was placed in a cuvette, and the particle size, PDI, and potential were measured by DLS. The morphology of the drug delivery system was observed by TEM, the molecular structure and chemical bonds of CaAs NP were detected by XPS, and the infrared characteristics of CaAs NP were characterized by FT-IR.

[0132] TEM scan images of CaAs NPs are as follows: Figure 5 As shown, the elemental analysis diagram is as follows: Figure 7 As shown, the XPS spectrum of CaAs NP is as follows: Figure 8 As shown, the XRD pattern is as follows Figure 9 As shown, the FT-IR spectrum is as follows Figure 10 As shown.

[0133] Drug loading and encapsulation efficiency were determined. 0.5 mL of a 2.7 mg / mL CaAs NP solution was accurately measured, ultrafiltered, and centrifuged. 100 μL of the aqueous phase was taken, diluted to 10 mL with 5% nitric acid solution, and filtered through a 0.22 μm filter membrane. The free arsenic content (W) was then determined using ICP-based assay. F Similarly, 100 μL of a 2.7 mg / mL CaAs NP solution was added, along with 50 μL of perchloric acid, and ultrasonically destroyed. The solution was then diluted to 10 mL with 5% dilute nitric acid, filtered through a 0.22 μm filter, and the total content (W) was measured. T The ICP instrument test parameters were set as follows: RF power 1100W, plasma volumetric flow rate 50L / min, auxiliary gas volumetric flow rate 0.5L / min, nebulizing gas volumetric flow rate 0.3L / min, pump speed 50L / min, stabilization delay 5s, cleaning time 30s, carrier gas high-purity argon, and arsenic elemental analysis spectrum 189nm. The drug loading (DL) and encapsulation efficiency (EE) of As were determined according to the following formulas (1) and (2): (1) DL% = [(W T -W F ) / W C ]×100%;(2)EE%=[(W T -W F ) / W D ]×100%, of which, W D W represents the amount of drug added. C The total mass of the drug delivery system.

[0134] The results showed that CaAs NP was successfully prepared. Pharmaceutical properties studies revealed that the nanoparticles were spherical with uniform particle size, averaging 142.39 ± 21.50 nm. The elemental proportions of As, Ca, and O were 2.17%, 2.83%, and 12.26%, respectively, and they were uniformly distributed within the nanoparticles. The arsenic loading and encapsulation efficiency in the sample were 27.28 ± 1.26% and 51.49 ± 2.12%, respectively.

[0135] Example 4

[0136] Preparation and characterization of arsenic trioxide / artesunate dual-drug-carrying nanoliposomes

[0137] DSPE-hyd-PEG 2000 -GA 0.0986g, cholesterol 0.03874g, and ART 0.0246g were dissolved in 20mL of dichloromethane. The organic solvent was removed by rotary evaporation at 40℃ under negative pressure for 30min. 5mL of CaAsNP dispersion (containing 4mg CaAsNP) was added, and the mixture was hydrated at 40℃ for 35min. The mixture was then sonicated with a probe for 4min to obtain arsenic trioxide / artesunate dual-drug-loaded nanoliposomes, denoted as DSPE-hyd-PEG. 2000 -GA@CaAs NP / ART.

[0138] A suitable amount of sample was placed in a cuvette, and the particle size, PDI, and potential were determined by DLS. The morphology of the drug delivery system was observed by TEM. A suitable amount of sample was diluted with PBS, dropped onto a copper grid, and allowed to air dry. The sample was then observed under TEM (accelerating voltage 75 kV, magnification 20000x) and photographed. 0.5 mL of the drug delivery system solution was accurately measured, ultrafiltered, and centrifuged. 100 μL of the external aqueous phase was taken, diluted to 10 mL with 5% nitric acid solution, filtered through a 0.22 μm filter membrane, and the free arsenic content (W) was determined by ICP. F Similarly, 100 μL of a 2.7 mg / mL CaAs NP solution was added, along with 50 μL of perchloric acid, and ultrasonically destroyed. The solution was then diluted to 10 mL with 5% dilute nitric acid, filtered through a 0.22 μm filter, and the total content (W) was measured. T The ICP instrument test parameters were set as follows: RF power 1100W, plasma flow rate 50L / min, auxiliary gas flow rate 0.5L / min, nebulizing gas flow rate 0.3L / min, pump speed 50L / min, stabilization delay 5s, cleaning time 30s, carrier gas high-purity argon, and arsenic elemental analysis spectral line 189nm. The centrifuged aqueous phase was placed in a 10mL volumetric flask and diluted to the mark with acetonitrile. The content of free ART, i.e., W, was determined by high-performance liquid chromatography (HPLC). F Similarly, take 0.5 mL of the drug delivery system solution and place it in a 10 mL volumetric flask. Add an appropriate amount of acetonitrile to break the emulsion. After sonication, wait for the solution to cool to room temperature, then dilute to the mark with acetonitrile. Filter through a 0.45 μm microporous membrane. Take the filtrate and determine the total ART content in the delivery system, i.e., W, using HPLC. T The encapsulation efficiency (EE) of As and ART was determined using the following formula: EE% = [(W T -W F ) / W D ]×100%, of which, W D W represents the amount of drug added. CThe total mass of the drug delivery system.

[0139] FT-IR characterization of DSPE-hyd-PEG 2000 Infrared characteristics of -GA@ART / CaAs NP. The dialysis bag method was used to investigate DSPE-hyd-PEG. 2000 -GA@ART / CaAs NP in vitro release behavior. DSPE-hyd-PEG 2000 -GA@ART / CaAs NP solution was placed in a dialysis bag (MWCO = 3500 Da) and then placed in PBS buffer at pH 7.4 and 4.5, respectively. Samples were taken at 0.25, 0.5, 1, 2, 4, 8, 10, 12, 24, 48, and 72 h, and the release of As and ART was determined by ICP-MS and HPLC.

[0140] DSPE-hyd-PEG 2000 - GA@CaAs NP / ART TEM scan images such as Figure 6 As shown, the FT-IR spectrum is as follows Figure 10 As shown, DSPE-hyd-PEG 2000 In vitro release profiles of ART and As from -GA@ART / CaAs NPs in pH 7.4 and 4.5 buffer solutions are shown below. Figure 11 As shown.

[0141] The results showed that the GA-modified pH-responsive dual-drug-loaded liposomes prepared in this invention were spherical with a particle size of 100.91±39.31 nm. The encapsulation efficiencies of As and ART were 21.30±0.98% and 33.03±0.86%, respectively. The in vitro release results showed that the release rate at pH 4.5 was significantly higher than that at pH 7.4, indicating good pH responsiveness.

[0142] Example 5

[0143] Cellular uptake and intracellular localization

[0144] To evaluate the liver cancer targeting ability of GA-modified liposomes, the fluorescent probe Nile Red NR was loaded into the liposomes to track its uptake.

[0145] Following the steps in Example 4, DSPE-hyd-PEG 2000 -GA should be replaced with DSPE-PEG. 2000 DSPE-hyd-PEG 2000 DSPE-hyd-PEG 2000 -GA; replace ART with NR, and replace 5 mL of CaAs NP dispersion with 5 mL of water to prepare DSPE-PEG. 2000 @NR,DSPE-hyd-PEG2000 @NR and DSPE-hyd-PEG 2000 -GA@NR (NR 150μM).

[0146] Huh-7 cells with free NR, DSPE-PEG 2000 @NR,DSPE-hyd-PEG 2000 @NR and DSPE-hyd-PEG 2000 Cells were incubated with GA@NR (NR 150 μM) and other drugs for 0.5 h, 1 h, or 4 h, and the fluorescence intensity in the cells was analyzed by flow cytometry. After 4 h of incubation, the cell membrane was stained with 10 μM green fluorescent probe (DIO), and the cell nucleus was stained with 4',6-diamidinyl-2-phenylindole (DAPI). Fluorescence images were captured by laser confocal scanning microscopy.

[0147] The fluorescence intensity curves of Huh-7 cells after incubation with Nile Red-labeled drugs for 0.5, 1, 2, and 4 hours are shown in the figure below. Figure 12 As shown in Figure A (the three figures in the upper left corner), Figure 12 Figure B is a bar chart showing the contrast of fluorescence intensity. ** This means p < 0.01.

[0148] Laser confocal scanning electron microscopy images of Huh-7 cells after incubation with Nile Red-labeled drugs for 4 hours are shown below. Figure 12 As shown in Figure C, the flow cytometry results are as follows: Figure 12 As shown in Figure D.

[0149] The results showed that GA-modified pH-responsive dual-drug-loaded liposomes significantly increased cellular uptake. Figure 12 A, B), after cellular uptake, the drug is mainly distributed in the cytoplasm and cell membrane. Figure 12 C, D).

[0150] Example 6

[0151] Intake pathway research

[0152] Inhibitors were used to determine the uptake pathway at the following concentrations: free GA 10 μM, 2-deoxyglucose 20 mM, chlorpromazine 10 μM, vortexin 10 μM, genistein 50 μM, and methyl-β-cyclodextrin 5 mM. Each group of Huh-7 cells was pre-incubated with the specified inhibitors for 1 hour, followed by administration of DSPE-hyd-PEG. 2000 -GA@NR (NR concentration 150 μM), incubate for 4 h, then wash three times with cold PBS, and measure by flow cytometry (FCM).

[0153] DSPE-hyd-PEG 2000The uptake mechanism of GA@NR in Huh-7 cells is as follows: Figure 12 As shown in the E and F diagrams. ** This represents p < 0.01. (Compared to DSPE-hyd-PEG) 2000 Compared to the GA@NR group, the fluorescence intensity of cells pre-incubated with GA, 2-deoxyglucose, Watermanin, genistein, and methyl-β-cyclodextrin was significantly reduced, with the methyl-β-cyclodextrin group showing the most significant decrease. The chlorpromazine group showed no significant change. Pre-incubation with 2-deoxyglucose significantly reduced uptake, indicating that cellular uptake is related to energy-dependent endocytosis. Pre-incubation with Watermanin, genistein, and methyl-β-cyclodextrin significantly inhibited cellular uptake of DSPE-hyd-PEG. 2000 The uptake of GA@NR (p<0.01) indicates that the internalization pathway for cellular uptake is mediated by macropinocytosis and caverin. In contrast, the uptake of DSPE-hyd-PEG-GA@NR in the chlorpromazine pre-incubation group showed no significant change, indicating that the clathrin-mediated pathway is not the uptake pathway. Therefore, macropinocytosis and caverin-mediated endocytosis are the internalization pathways for GA-modified liposomes. Furthermore, pretreatment with free GA significantly reduced the uptake of GA-modified liposomes by competitively binding to GA receptors on the cell membrane surface. During pretreatment, free GA saturates these binding sites, preventing GA-modified NPs from using them. These results suggest that the uptake of GA-modified liposomes by Huh7 cells largely depends on these GA binding sites.

[0154] The results showed that cellular uptake of GA-modified pH-responsive dual-drug-carrying liposomes was mainly mediated by macropinocytosis and caveolin pathways. Figure 12 E, F).

[0155] Example 7

[0156] Cytotoxicity detection

[0157] The MTT assay was used to study the in vitro cytotoxicity of liposomes on Huh-7 cells. Huh-7 cells were co-incubated with each drug group (DSPE-PEG). 2000 @ART / CaAs NP,DSPE-hyd-PEG 2000 @ART / CaAs NP, and DSPE-hyd-PEG 2000 -GA@ART / CaAs NP) was continuously incubated for 48 h at As concentrations of 0, 2.5, 25, 50, 100, 150, and 200 μM. After discarding the supernatant, 20 μL of MTT solution (5 mg / mL) was added to the cells. -1Incubate for another 4 hours. Then, add 150 μL of dimethyl sulfoxide solution of methyl sulfoxide crystals and shake on a shaker for 10 minutes. After the crystals are completely dissolved, measure the absorption at a wavelength of 570 nm and calculate the IC50 for each group.

[0158] Prepared according to the steps of Example 4, except that DSPE-hyd-PEG is added. 2000 -GA replaced with DSPE-PEG 2000 DSPE-PEG@ was prepared 2000 ART / CaAs NP.

[0159] Prepared according to the steps of Example 4, except that DSPE-hyd-PEG is added. 2000 - Replace GA with SPEE-hyd-PEG 2000 DSPE-hyd-PEG was prepared. 2000 @ART / CaAs NP.

[0160] The cell activity of Huh-7 cells after treatment with various drugs is as follows: Figure 13 As shown in Figure A, the IC50 results are as follows: Figure 13 As shown in Figure B.

[0161] The results showed that the cytotoxicity of GA-modified pH-responsive dual-drug-loaded liposomes increased with increasing As concentration; GA and hydrazone modification significantly improved the cytotoxicity of the liposomes due to the active targeting ability of GA and the drug-releasing effect of hydrazone.

[0162] Example 8

[0163] Apoptosis-promoting effects and related mechanisms

[0164] ①Pro-apoptotic effect

[0165] Huh-7 cells were cultured with each drug group (ART / CaAs NP, DSPE-PEG@ART / CaAs NP, DSPE-hyd-PEG@ART / CaAs NP, and DSPE-hyd-PEG-GA@ART / CaAs-NPs) at an As concentration of 150 μM for 48 hours. Cells were then harvested, resuspended in 500 μL of binding buffer, and stained with 5 μL annexin-FITC and 5 μL PI (propidium iodide) solution in the dark at room temperature for 15 minutes. Finally, measurements were performed by FCM.

[0166] The flow cytometry results of Huh-7 cells after treatment with various drugs are shown in the figure below. Figure 14 As shown in Figure A, the apoptosis statistics are as follows: Figure 14 As shown in Figure B.

[0167] The results showed that the apoptosis rate of the GA-modified pH-responsive dual-drug-loaded liposome treatment group was significantly higher than that of other groups. Due to its better affinity for tumor cells and its promoting effect on intracellular release, it showed greater potential to induce apoptosis in Huh-7 cells compared to other groups.

[0168] ② Cell cycle arrest

[0169] Huh-7 cells were incubated with each drug group for 72 h, then collected and fixed overnight in 70% ethanol at 4 °C. Huh-7 cells were harvested and treated with 50 μg / mL ethanol at 37 °C. -1 RNase staining for 0.5 h, then in the dark with 65 μg·mL⁻¹ -1 The PI was stained in an ice bath for 0.5 h and analyzed by FCM.

[0170] The statistical chart of cell cycle results of Huh-7 cells after treatment with different groups of drugs is shown below. Figure 14 As shown in Figures C and D.

[0171] The results showed that, compared with other groups, GA-modified pH-responsive dual-drug-loaded liposomes exhibited a significant cell cycle arrest effect on Huh-7 cells.

[0172] ③ Measurement of intracellular reactive oxygen species

[0173] The density in each pore is 1.5 × 10⁻⁶ 5 Huh-7 cells were incubated overnight in four-cell confocal culture dishes. Cells were incubated with each drug group for 6 h and then treated with a ROS detection kit. Fluorescence was observed and images were captured using a CLSM (laser confocal scanning microscope) with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0174] ROS laser confocal scanning images of Huh-7 cells after treatment with various drugs are shown below. Figure 15 Figure A shows the flow cytometry results. Figure 15 As shown in Figure B. * p<0.05, ** p<0.01.

[0175] The results showed that all groups treated with the drugs and formulations resulted in higher ROS levels compared to the control group (p<0.01). Furthermore, the ROS levels in the GA-modified pH-responsive dual-drug-loaded liposome treatment group were higher than those in the ART / CaAs NPs treatment group (p<0.01). Increased ROS levels led to cell growth arrest and apoptosis. These results indicate that GA-modified pH-responsive dual-drug-loaded liposomes are superior to other liposomes in inducing oxidative damage in Huh-7 cells.

[0176] ④ Mitochondrial potential measurement

[0177] After culturing for 12 hours, Huh-7 cells were washed with PBS and cultured continuously for 72 hours with each group of drugs. After washing and mixing with JC-1 staining working solution, the cells were measured by FCM.

[0178] The flow cytometry results of mitochondrial membrane potential in Huh-7 cells after treatment with various drugs are as follows: Figure 15 As shown in Figures C and D. * p<0.05, ** p<0.01.

[0179] Mitochondrial membrane potential is a key parameter of mitochondrial function, and its decrease is generally considered to promote apoptosis. An increase in the green fluorescence ratio indicates mitochondrial depolarization. Figure 15 As shown in C and D, compared with the ART / CaAs NP group, the level of JC-1 green ratio in Huh-7 cells was significantly increased after treatment with GA-modified pH-responsive dual-drug-loaded liposomes (p<0.01), which may be one of the mechanisms by which it induces apoptosis.

[0180] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An arsenic trioxide-artesunate dual-drug-loaded nanoliposome, wherein the arsenic trioxide-artesunate dual-drug-loaded nanoliposome comprises a glycyrrhetinic acid-polyethylene glycol-hydrazone-distearate phosphatidylethanolamine polymer, cholesterol, artesunate, and calcium arsenic nanoparticles; the glycyrrhetinic acid-polyethylene glycol-hydrazone-distearate phosphatidylethanolamine polymer and cholesterol form the nanoliposome, and artesunate and calcium arsenic nanoparticles are loaded within the nanoliposome; the calcium arsenic nanoparticles are prepared by reacting arsenic trioxide with calcium stearate, and the calcium arsenic nanoparticles contain arsenic trioxide; wherein, The glycyrrhetinic acid-polyethylene glycol-hydrazone-distearate phosphatidylethanolamine polymer is prepared by the following method: a condensation reaction is performed between the tert-butyloxycarbonyl-amino-polyethylene glycol-amino polymer and glycyrrhetinic acid to generate a glycyrrhetinic acid-polyethylene glycol-amino-tert-butyloxycarbonyl-protected amino polymer, denoted as GA-PEG-NH-Boc. The tert-butyloxycarbonyl protection of GA-PEG-NH-Boc is removed to generate a glycyrrhetinic acid-polyethylene glycol-amino polymer, denoted as GA-PEG-NH2. GA-PEG-NH2 then reacts with a hydroxyl-hydrazone... The reaction of glycyrrhetinic acid with carboxyl groups produces a glycyrrhetinic acid-polyethylene glycol-hydrazone-hydroxyl polymer, denoted as GA-PEG-hyd-OH. GA-PEG-hyd-OH reacts with succinic anhydride to produce a glycyrrhetinic acid-polyethylene glycol-hydrazone-carboxyl polymer, denoted as GA-PEG-hyd-COOH. GA-PEG-hyd-COOH is then reacted with distearylphosphatidylethanolamine to produce a glycyrrhetinic acid-polyethylene glycol-hydrazone-distearylphosphatidylethanolamine polymer, denoted as GA-PEG-hyd-DSPE.

2. The arsenic trioxide-artesunate dual-drug-carrying nanoliposomes as described in claim 1, characterized in that... The encapsulation efficiency of the arsenic trioxide-artesunate dual-drug-carrying nanoliposomes is 20-90%, and the particle size is 60-150 nm, wherein the molar ratio of arsenic trioxide to artesunate is 1:1 to 1:

20.

3. The method for preparing arsenic trioxide-artesunate dual-drug-carrying nanoliposomes according to any one of claims 1 to 2, characterized in that... The preparation method includes the following steps: (1) Synthesis of glycyrrhetinic acid-modified amino polyethylene glycol The tert-butyloxycarbonyl-amino-polyethylene glycol-amino polymer is condensed with glycyrrhetinic acid to generate glycyrrhetinic acid-polyethylene glycol-amino-tert-butyloxycarbonyl-protected amino polymer, denoted as GA-PEG-NH-Boc. The tert-butyloxycarbonyl protection of GA-PEG-NH-Boc is removed to generate glycyrrhetinic acid-polyethylene glycol-amino polymer, denoted as GA-PEG-NH2. (2) Synthesis of GA-PEG-hyd-COOH GA-PEG-NH2 is reacted with a hydroxy-hydrazone-carboxyl compound to generate a glycyrrhetinic acid-polyethylene glycol-hydrazone-hydroxy polymer, denoted as GA-PEG-hyd-OH. GA-PEG-hyd-OH is then reacted with succinic anhydride to generate a glycyrrhetinic acid-polyethylene glycol-hydrazone-carboxyl polymer, denoted as GA-PEG-hyd-COOH. (3) Synthesis of GA-PEG-hyd-DSPE GA-PEG-hyd-COOH was reacted with distearylphosphatidylethanolamine to generate a glycyrrhetinic acid-polyethylene glycol-hydrazone bond-distearylphosphatidylethanolamine polymer, denoted as GA-PEG-hyd-DSPE; (4) Arsenic trioxide reacts with calcium stearate to form calcium arsenic nanoparticles; (5) Nanoliposomes were prepared using GA-PEG-hyd-DSPE, cholesterol, artesunate, and calcium arsenic nanoparticles to obtain arsenic trioxide-artesunate dual-drug-loaded nanoliposomes simultaneously loaded with calcium arsenic nanoparticles and artesunate; the hydroxyl-hydrazone-carboxyl group compounds are shown in the following formula. ； The reaction equations for steps (1) to (3) are shown below: 。 4. The method as described in claim 3, characterized in that... The step (1) is carried out in the following steps: Boc-NH-PEG-NH2 and glycyrrhetinic acid GA are condensed in chloroform solvent under the action of condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC and catalyst 4-dimethylaminopyridine DMAP. The resulting reaction solution a is then treated to obtain GA-PEG-NH-Boc. GA-PEG-NH-Boc was reacted with trifluoroacetic acid in chloroform solvent to remove the Boc protecting group. The resulting reaction solution b was then treated to obtain glycyrrhetinic acid-polyethylene glycol-amino polymer, denoted as GA-PEG-NH2. Step (2) was carried out according to the following steps: In chloroform solvent, GA-PEG-NH2 and hydroxy-hydrazone-carboxyl compound HO-hyd-COOH undergo a condensation reaction under the action of condensing agent EDC and catalyst DMAP. The resulting reaction solution c is then treated to obtain GA-PEG-hyd-OH. GA-PEG-hyd-OH was dissolved in chloroform solvent, succinic anhydride was added, and the reaction was heated. The resulting reaction solution was then treated to obtain GA-PEG-hyd-COOH. Step (3) is performed as follows: In chloroform solvent, GA-PEG-hyd-COOH and distearate phosphatidylethanolamine undergo a condensation reaction in the presence of condensing agent EDC and catalyst DMAP. The resulting reaction solution is then post-treated to obtain GA-PEG-hyd-DSPE.

5. The method as described in claim 3, characterized in that... Step (4) is performed as follows: Calcium stearate and oleic acid were added to toluene solvent, heated and stirred to form a transparent solution, and then arsenic trioxide solution was added dropwise while stirring. The reaction was heated and stirred, and anhydrous ethanol was added to form calcium arsenic nanoparticle precipitate. The precipitate was collected by ultracentrifugation to obtain calcium arsenic nanoparticles. Step (5) is performed as follows: GA-PEG-hyd-DSPE, cholesterol, and artesunate were dissolved in an organic solvent, the organic solvent was removed by rotary evaporation, and then a dispersion of calcium and arsenic nanoparticles was added. The mixture was heated to carry out a hydration reaction, and then ultrasonically treated with a probe to obtain arsenic trioxide-artesunate dual-drug-loaded nanoliposomes that simultaneously encapsulate calcium and arsenic nanoparticles and artesunate.

6. The method as described in claim 3, characterized in that... The ratio of the total mass of artesunate and calcium arsenic nanoparticles to the total mass of GA-PEG-hyd-DSPE and cholesterol was 0.1–1.0:

1.

7. The use of the arsenic trioxide-artesunate dual-drug-loaded nanoliposomes as described in any one of claims 1 to 2 in the preparation of anti-liver cancer drugs.

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