CN109364024B

CN109364024B - Preparation method of targeting polymer, targeting liposome, preparation method of liposome targeting preparation and application

Info

Publication number: CN109364024B
Application number: CN201811319139.7A
Authority: CN
Inventors: 武敬亮; 姜虹; 高志芹; 张波; 田桂香; 李志鹏; 连波
Original assignee: Weifang Medical University
Current assignee: Weifang Medical University
Priority date: 2018-11-07
Filing date: 2018-11-07
Publication date: 2020-11-06
Anticipated expiration: 2038-11-07
Also published as: CN109364024A

Abstract

The invention discloses a target molecule distearoylphosphatidylethanolamine-polyethylene glycol-glycyrrhetinic acid, which is used for preparing a liposome, wherein the liposome comprises the following components of DSPE-PEG-GA, lecithin and cholesterol, and the introduction of the GA molecule improves the accuracy of the liposome for delivering drugs, so that the drugs are delivered to a target part for release, and the anti-tumor effect is better exerted, therefore, the liposome is used for loading CUR and CA4P, and the CUR & CA4P/GA-LPs is obtained. The invention combines curcumin and combretastatin disodium phosphate, achieves the effect of synergistic anti-tumor by inhibiting tumor cell proliferation and anti-tumor angiogenesis, and solves the problem of tumor drug resistance caused by long-term treatment of a single drug.

Description

Preparation method of targeting polymer, targeting liposome, preparation method of liposome targeting preparation and application

Technical Field

The invention relates to a targeted drug system, in particular to a targeted polymer used in the targeted drug system, a preparation method of the targeted polymer, a targeted liposome, a liposome targeted preparation and a preparation method thereof.

Background

Hepatocellular carcinoma is one of the highest morbidity and mortality malignancies in the world. Chemotherapy is one of the main strategies for the clinical treatment of liver cancer. However, various clinical studies have shown that the clinical efficacy of conventional cytotoxic chemotherapeutic agents, such as doxorubicin, paclitaxel, etc., is limited due to systemic toxicity, lack of selectivity and drug resistance. Therefore, the need to find a new treatment to solve such problems is very urgent. Clinically, the effect of the combined treatment of two or more drugs by different anti-tumor methods is more and more obvious, and anti-angiogenesis is a promising anti-tumor strategy by blocking the development of tumor vessels. Therefore, the combination of chemotherapeutic drugs and anti-angiogenesis drugs can improve the treatment effect more effectively.

Curcumin (CUR) is a diphenol compound extracted from turmeric rhizome, has potent effects against prostate, breast and colon cancers, and is considered as a third generation anticancer drug. It can regulate signaling pathways by inhibiting proliferation, thereby inhibiting cancer cell growth and inducing apoptosis. The hydrophilic medicine combretastatin disodium phosphate is a novel anti-tumor compound taking blood vessels as targets, and is a prodrug of soluble phosphate combretastatin. The combretastatin disodium phosphate ((CA4P) mainly acts on vascular endothelial cells of tumors to cause the dysfunction of tumor vasoconstriction, so that tumor cells die due to insufficient blood supply, but both the two have certain limitations in clinical application, for example, curcumin belongs to a fat-soluble medicament, has low solubility in water, and the combretastatin disodium phosphate has short half-life in vivo and influences the exertion of curative effect.

Drug Delivery Systems (DDS) offer an opportunity to achieve effective co-delivery of multiple drugs and have been marketed and subjected to clinical trials. Among the different nanocarriers, liposomes (lipopomes, LPs) have received increasing attention, and their structure comprises a hydrophilic core and a hydrophobic shell, and have significant encapsulation strength for hydrophilic, hydrophobic and amphoteric drugs, solving the solubility problem of drugs. In addition, liposomes have significant advantages over other drug delivery vehicles, including reduced systemic toxicity, appropriate particle size to enhance specific accumulation of solid tumors, flexibility of structure to enhance sustained release of drug, etc. Moreover, the liposome has passive targeting property and is easily phagocytized by mononuclear cells represented by macrophages. Intravenous administration can be selectively focused on a mononuclear phagocyte system, 70-89% of the intravenous administration is focused on liver and spleen, and therefore, the intravenous administration has advantages for treating liver tumor and preventing tumor metastasis.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides a preparation method of a targeting polymer. It is another object of the present invention to provide targeted liposomes prepared using the targeting polymers. The invention also provides a preparation method and application of the liposome targeting preparation.

The technical scheme is as follows: in order to solve the above problems, the present invention provides, in a first aspect, a method for preparing a targeting polymer, comprising the steps of: (1a) dissolving glycyrrhetinic acid in an organic solvent, adding a condensing agent, stirring for 1-2h, removing the organic solvent, adding the obtained solid into ethylenediamine, stirring at room temperature for 12-24h, removing the ethylenediamine, and purifying to obtain ethylenediamine-modified glycyrrhetinic acid; (1b) and (2) dissolving DSPE-PEG-NHS and the modified glycyrrhetinic acid obtained in the step (1a) in an organic solvent according to a molar ratio of 1:1-10, reacting for 36-48h at room temperature under the protection of nitrogen, and after the reaction is finished, purifying reactants to obtain a target polymer distearoylphosphatidylethanolamine-polyethylene glycol-glycyrrhetinic acid (DSPE-PEG-GA).

Preferably, in the step (1a), the condensing agent is DMT-MM, and the mass ratio of the glycyrrhetinic acid, the condensing agent and the ethylenediamine is 1-1.5:0.5-1.5: 5-30.

Preferably, in the step (1b), a pH regulator and a catalyst are added into the organic solvent, wherein the pH regulator is triethylamine, and the catalyst is EDC. The reaction principle of the targeting polymer of the present invention is shown in FIG. 1, and the preparation of the targeting polymer of the present invention comprises the following steps:

as shown in fig. 1A, firstly dissolving Glycyrrhetinic Acid (GA) in an organic solvent methanol, adding a condensing agent 4- (4, 6-dimethoxytriazine) -4-methylmorpholine hydrochloride (DMT-MM), placing the mixture in a reaction vessel for reaction, stirring the mixture at room temperature, performing rotary evaporation to remove methanol, slowly adding the obtained solid into ethylenediamine, stirring the mixture at room temperature overnight, performing rotary evaporation to remove ethylenediamine, dissolving the rotary evaporated solid in ethanol, performing ultrasonic treatment, adding silica gel powder, performing rotary evaporation to remove ethanol to obtain a powdery solid, and performing separation and purification to obtain ethylenediamine-modified glycyrrhetinic acid (GA-N) powder, wherein the specific steps of separation and purification are as follows: dissolving silica gel powder in ethyl acetate, introducing into a column, and depositing silica gel to form a compact silica gel column; loading: slowly pouring GA-N powder into the column, and adding ethyl acetate to make the liquid level not lower than the powder; and (3) elution: adding ethyl acetate and ethanol with the volume ratio of 2:1 into a column as developing agents for elution; collecting: the sample is collected when the sample is added, and TLC detection is carried out during the collection until the sample point appears.

As shown in fig. 1B, the modified glycyrrhetinic acid is dissolved in dimethyl sulfoxide, then DSPE-PEG-NHS powder is added, EDC is added for catalytic reaction according to the reaction condition, a small amount of triethylamine as a pH regulator is added to the reaction system to adjust the pH, the reaction is carried out under the protection of nitrogen, the obtained reaction product is dialyzed, and the solid DSPE-PEG-GA is obtained by freeze drying.

The second aspect of the invention provides the application of the targeting polymer prepared in the way in a liver targeting drug delivery system.

In a third aspect, the invention provides a targeted liposome prepared by using the prepared targeted polymer, wherein the targeted liposome comprises the targeted polymer, phospholipid and cholesterol in a mass ratio of 1-10:3-30: 2-20. The introduction of GA molecules enhances the active targeting property of the whole liposome system, and a specific receptor of GA exists on a liver cell membrane and is specifically combined with GA. Therefore, compared with unmodified liposomes, the GA modified liposomes have more ideal active targeting effect on liver cancer cells and tissues, which is beneficial to further improving the anti-tumor curative effect.

The phospholipid in the invention can be selected from natural phospholipid or synthetic phospholipid, preferably, the phospholipid is selected from one or more of soybean phospholipid, lecithin, distearoyl phosphatidylcholine, 1, 2-dioleoyl phosphatidylcholine, dioleoyl phosphatidylethanolamine and distearoyl phosphatidylethanolamine-polyethylene glycol.

Furthermore, the phospholipids in the invention can be all selected from natural phospholipids, such as one or more of soybean phospholipids and lecithin; the phospholipids of the invention may also be derived entirely from synthetic phospholipids, such as one or more of Distearoylphosphatidylcholine (DSPC), 1, 2-Dioleoylphosphatidylcholine (DOPC), Dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG).

The targeted liposome of the present invention is prepared by a membrane dispersion method, and preferably, the medium used for preparing the targeted liposome is a PBS buffer solution with the pH of 7.4, which acts to emulsify phospholipid and cholesterol to form nanoliposomes.

The fourth aspect of the invention provides a liposome targeting preparation, in particular to a targeted liposome loaded with drugs curcumin and combretastatin disodium; the liposome targeting preparation comprises liposome, curcumin and combretastatin disodium; the mass ratio of the curcumin to the phospholipid is 1: 20-60; the mass ratio of the curcumin to the combretastatin disodium is 10-1: 1-10.

The fifth aspect of the invention provides a preparation method of a liposome targeting preparation, which comprises the following steps: (2a) dissolving phospholipid, cholesterol and DSPE-PEG-GA in an organic solvent, adding curcumin for dissolving, removing the organic solvent by rotary evaporation, and adding a combretastatin disodium aqueous solution for hydration when a layer of uniform film is formed, wherein the hydration time is 30-60 min; (2b) and (3) carrying out ultrasonic treatment on the hydrated solution in the step (2a) for 5-10min, and then removing unencapsulated free drugs to obtain the liposome targeting preparation.

In the step (2a), the mass ratio of the DSPE-PEG-GA to the phospholipid to the cholesterol is 1-10:3-30: 2-20; the mass ratio of the curcumin to the phospholipid is 1: 20-60; the mass ratio of the curcumin to the combretastatin disodium is 10-1: 1-10.

Further, the organic solvent in step (2a) may be chloroform (chloroform) or diethyl ether, and the conditions of rotary evaporation in a rotary evaporator may be: the rotating speed is 15-60rpm, and the water bath temperature is 30-40 ℃. After a uniform film is formed, adding combretastatin disodium phosphate PBS aqueous solution for hydration, wherein the water bath temperature is 35-60 ℃, and the hydration time is 30-60 min.

Further, the method for removing free drug is selected from dialysis and G-50 chromatography column.

The free CUR was removed as follows: (1) adding distilled water into the G-50 powder for soaking, performing column packing after soaking, filling a dead zone at the lower end of a sintered plate of the chromatographic column with distilled water, slowly filling the swelled gel into the chromatographic column, and adding the sample after column packing is completed; (2) before sample adding, opening an outlet to allow water to flow out, closing the water outlet after only a very thin layer of water is left, and vertically and slowly adding a sample into the column; (3) and (3) elution: reserving a water layer to ensure that the surface of the chromatographic column is not dried, and eluting with PBS; collecting a sample: the collection was started as soon as the sample was put on the gel column, and a sample from which free CUR was removed was obtained.

The removal method of free CA4P was as follows: and (3) putting the prepared sample into a dialysis bag, wherein the dialysis medium is PBS solution.

Further, after the preparation is finished, the particle size of the liposome is controlled by sequentially passing the liposome through a polyether sulfone membrane method with membrane aperture of 450nm and 220nm to obtain the targeted co-loaded liposome with the particle size range of 120-240 nm, and a series of physicochemical property tests are carried out on the targeted co-loaded liposome, wherein the entrapment rate is 60-90%, and the drug-loading rate is 0.7-6%.

The obtained targeting preparation carrying curcumin and combretastatin disodium phosphate together is applied to the aspect of liver cancer treatment.

In the present invention, "%" is a mass percentage unless otherwise specified.

Has the advantages that: (1) the GA molecules are introduced into the targeting polymer, the specific receptor of GA exists on the hepatic cell membrane, and the GA molecules are specifically combined with GA, compared with unmodified liposome, the GA-modified liposome has more ideal active targeting effect on liver cancer cells and tissues, so that the accuracy of liposome delivery drugs is improved, the drugs are delivered to a target part for release, and the anti-tumor effect is better played; (2) according to the targeted liposome prepared by the targeted polymer, the liposome is used as a drug loading system, so that on one hand, the problem of drug solubility is solved, and the CUR which is difficult to dissolve in water is encapsulated in the hydrophobic layer of the liposome and is conveyed into cells to play a role; on the other hand, the slow release of the medicines can be realized, and simultaneously, the two medicines show a slow release trend in a certain time, so that the action time is prolonged: the change of the blood vessel parameters induced by CA4P is transient, the blood flow of KHT tumor is reduced by CA4P after 4 hours of treatment, but the blood flow returns to the baseline level after 20 hours, and after being encapsulated by liposome, the medicine is released in a slow and continuous mode, so that the circulation time in the body is increased, and the effective treatment effect is achieved; (3) the invention combines curcumin and combretastatin disodium phosphate, combines the anti-tumor cell proliferation medicament and the anti-tumor angiogenesis medicament, achieves the effect of synergistic anti-tumor by inhibiting the tumor cell proliferation and the anti-tumor angiogenesis, and solves the problem of tumor drug resistance generated by long-term treatment of a single medicament. The liposome is used as a drug carrier to solve the solubility problem of curcumin, and simultaneously, the two drugs show a slow release trend within a certain time, so that the action time is prolonged.

Drawings

FIG. 1 is a schematic diagram of the reaction of a targeting polymer according to the present invention;

FIG. 2 shows the results of DSPE-PEG-NHS, GA-N and DSPE-PEG-GA in the present invention¹H nuclear magnetic resonance spectrum, wherein the peak value of the a is 0.7-1.5ppm of GA-N, and the peak value of the b is 3.6ppm of DSPE-PEG-NHS.

FIG. 3 is a schematic representation of CA 4P/GA-LPs. The drug loading system consisted of a hydrophobic layer, a hydrophilic core and a linker attached to the table DSPE-PEG-GA. Loading hydrophilic CA4P and hydrophobic CUR into the water inner core and hydrophobic layer, respectively;

figure 4 is a characterization of liposome performance: wherein A is blank liposome TEM image, B is CUR & CA4P/GA-LPs TEM image, and C is CUR & CA4P/GA-LPs particle size distribution diagram;

FIG. 5 is a graph of the in vitro release of CUR and CA4P from CUR & CA 4P/GA-LPs;

FIG. 6 is a fluorescence micrograph of cells ingesting targeted co-carried liposomes, wherein A is a fluorescence micrograph of CUR & CA4P/LPs incubated BEL-7402 cells and B is a fluorescence micrograph of CUR & CA4P/GA-LPs incubated BEL-7402 cells;

FIG. 7 is the results of evaluation of liposome cytotoxicity, wherein A is the cell survival rate of BEL-7402 cells treated with blank liposomes, free CUR, physically mixed CUR + CA4P, CUR & CA4P/LPs and CUR & CA4P/GA-LPs for 24 hours, B is the cell survival rate of B16 cells treated with blank liposomes, free CUR, physically mixed CUR + CA4P, CUR & CA4P/LPs and CUR & CA4P/GA-LPs for 24 hours, and C is the cell survival rate of different samples treated with BEL-7402 for 24 hours, 48 hours and 72 hours; d is the cell survival rate of different samples treated for 24h, 48h and 72h on B16 cells;

FIG. 8 is the results of evaluation of tumor cell migration inhibition, wherein A is the results of migration inhibition of BEL-7402 cells by different samples, B is the results of migration inhibition of B16 cells by different samples, C is the results of migration (%) calculated by measuring the average width of scar from 0h to 24h of BEL-7402 cells by Image-Pro6.0, and D is the results of migration (%) calculated by measuring the average width of scar from 0h to 24h of B16 cells by Image-Pro6.0;

FIG. 9 is a NIR image of H22 tumor-bearing mice injected with free DiR and DiR/GA-LPs;

figure 10 is the results of the evaluation of in vivo antitumor efficacy of targeted co-loaded liposomes: wherein A is the body weight of the mouse and B is the tumor tissue stripped from the killed mouse; c is the anti-tumor effect of H22 tumor-bearing mice after intravenous injection by normal saline and various pharmaceutical preparation groups, and D is the tumor growth inhibition rate of different pharmaceutical preparations;

FIG. 11 is a histological analysis of tumor tissue.

Detailed Description

The present invention will be further described with reference to the following examples.

Raw materials and instruments

1.1 sources of raw materials

Curcumin (CUR), lecithin, Cholesterol (CHOL), Sephadex TMG-50, MTT, DAPI, RPMI-1640 medium, Fetal Bovine Serum (FBS), cyan chain diabody, trypsin, hematoxylin, eosin, from Solarbio, Inc. of Beijing;

combretastatin disodium phosphate (CA4P), available from Innochem technologies ltd, beijing;

glycyrrhetinic Acid (GA) from feiche pharmaceuticals, inc;

4- (4, 6-Dimethoxytriazine) -4-methylmorpholine hydrochloride (DMT-MM) from Medpep, Inc. of Shanghai

1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) from Aladdin reagent database;

fluorescent probes (DiR) from peking panbo biochemicals ltd;

1, 2-distearoyl-SN-glycerol-3-phosphoethanolamine-N-hydroxysuccinimide-polyethylene glycol (DSPE-PEG-NHS) from Western Anrixi scientific Co., Ltd;

human hepatoma cell BEL-7402 and melanoma cell B16 from the Collection center of Life of Wuhan university;

murine hepatoma cells H22, from Kyoto Biotech, Inc., Wuhan Kayki.

1.2 instruments

Rotary evaporator, Shanghai Yangrong Biochemical instruments factory;

electronic balance, shanghai shunhui scientific instruments ltd;

gas bath constant temperature oscillator, jin Tan City medical instrument factory;

a biological tissue embedding machine, a computer ultra-thin microtome, Kedi instruments and equipments, Jinhua, Zhejiang;

inverted fluorescence microscope, japan nikon;

upright fluorescence microscopy, olympus, japan;

ultrasonic cell crusher, Ningbo Xinzhi Biotech GmbH;

malvern Zetasizer Nano ZS 90 laser granulometer, Malvern, uk;

nuclear magnetic resonance apparatus, JEOL, japan.

Second, sample preparation

Example 1: synthesis of targeting molecule DSPE-PEG-GA

(1) Synthesis of GA-N

1.41g of GA is precisely weighed and dissolved in 30ml of methanol, 0.976g of condensing agent DMT-MM is added, the system is placed in a 100ml eggplant-shaped bottle for reaction, the mixture is stirred for 1 hour at room temperature, TLC detects the generation of an intermediate product GA-ES, methanol is removed by rotary evaporation at 37 ℃, the obtained solid is slowly added into 30ml of ethylenediamine, and the mixture is stirred at room temperature overnight. Rotary steaming at 60 ℃ to remove ethylenediamine, dissolving the rotary steamed solid with ethanol, performing ultrasonic treatment, adding silica gel powder, rotary steaming to remove ethanol to obtain powdery solid, and separating and purifying: dissolving silica gel powder in ethyl acetate, introducing into the column to deposit silica gel to form a compact silica gel column; loading: slowly pouring the powder into the column, and adding ethyl acetate to make the liquid surface not lower than the powder; and (3) elution: adding ethyl acetate and ethanol with the volume ratio of 2:1 into a column as developing agents for elution; collecting: and starting to collect the sample when the sample is added, carrying out TLC detection in the collection process until a sample point appears, and finally carrying out rotary evaporation to obtain GA-N powder.

(2) Synthesis of DSPE-PEG-GA

Accurately weighing 15mg of GA-N in a 50ml eggplant-shaped bottle, fully dissolving in 3ml of dimethyl sulfoxide, adding 20mg of DSPE-PEG-NHS powder (the molar ratio of DSPE-PEG-NHS to GA-N is 1:3), adding 1.58mg of EDC for catalytic reaction, adding 20 mu l of triethylamine in a reaction system to adjust the pH value, reacting at room temperature for 48h under the protection of nitrogen, dialyzing for 72h, and freeze-drying to obtain the DSPE-PEG-GA solid. The NMR spectra were determined by dissolving GA-N, DSPE-PEG-NHS and DSPE-PEG-GA in chloroform, respectively, and the results are shown in FIG. 2.

As can be seen from FIG. 2, the targeting material DSPE-PEG-GA was synthesized by coupling aminated GA to DSPE-PEG-NHS. The characteristic peak of DSPE-PEG-NHS at 3.6ppm (PEG group) is shown, the characteristic peak of GA-N is shown at 0.7-1.5ppm, and the characteristic peak of DSPE-PEG-GA is shown at 0.7-1.3ppm (imidazole ring). These results indicate that the successful introduction of GA-N into DSPE-PEG-NHS synthesized DSPE-PEG-GA with characteristic peaks of 3.6 and 0.7-1.3ppm, respectively.

Example 2: preparation of blank Targeted liposomes (GA-LPs)

120mg of lecithin, 30mg of cholesterol and 20mg of DSPE-PEG-GA (the mass ratio is 12:3:2) are fully dissolved in 5ml of trichloromethane and placed in a 100ml eggplant-shaped bottle, and the trichloromethane is removed by rotary evaporation by using a rotary evaporator, wherein the rotary evaporation conditions are as follows: 35 ℃ C, 18 rpm. And when a uniform thin film is formed, adding 5ml of PBS (pH 7.4) aqueous solution for hydration, wherein the hydration time is 40min, carrying out ultrasonic treatment for 5min, and sequentially passing through a polyether sulfone film with the film aperture of 450nm and the film aperture of 220nm to prepare the blank targeted liposome.

Example 3: preparation of Targeted Co-loaded CUR and CA4P liposomes

120mg of lecithin, 30mg of cholesterol and 20mg of DSPE-PEG-GA (mass ratio of 12:3:2) are fully dissolved in 5ml of trichloromethane, the obtained solution is placed in a 100ml eggplant-shaped bottle, 3mg of CUR (mass ratio of CUR to phospholipids is 1:40) is added for full dissolution, and the trichloromethane is removed by rotary evaporation through a rotary evaporator, wherein the rotary evaporation conditions are as follows: 35 ℃ C, 18 rpm. When a uniform film is formed, adding 5ml of 3mg/ml aqueous solution of CA4P PBS (pH 7.4) (mass ratio of CUR to CA4P is 1:5) for hydration, wherein the hydration time is 40min, performing ultrasonic treatment for 5min, and removing unencapsulated free drug by G-50 sephadex column and dialysis:

and (3) removing the CUR: gel preparation: weighing 10G of G-50 powder into a 250ml conical flask, adding 100ml of distilled water, uniformly stirring, soaking for 24 hours, intermittently stirring in the midway, removing small particles suspended on the surface by an inclined method, and repeatedly rinsing for 2-3 times; column assembling: filling the dead zone at the lower end of the sintered plate of the chromatographic column with distilled water, slowly filling the swelled gel into the chromatographic column along a glass rod, and repeatedly balancing with distilled water when the gel column is deposited to about 10 cm; sample adding: before sample adding, opening an outlet to allow water to flow out, closing the water outlet after only a very thin layer of water is left, and vertically and slowly adding 1ml of sample into the column by using a pipette; and (3) elution: during elution, the surface of the chromatographic column is not dried, a water layer of 0.2cm is reserved, and elution is carried out by PBS; collecting a sample: the collection was started as soon as the sample was put on the gel column, and a sample from which free CUR was removed was obtained.

Removal of free CA 4P: 2ml of the prepared sample is put into a dialysis bag, and the dialysis medium is PBS solution for 1 to 2 days.

Sequentially passing the liposome processed by the steps through a polyether sulfone membrane with membrane aperture of 450nm and 220 nm. The particle size and zeta potential of CUR & CA4P/GA-LPs were measured using a Malvern particle sizer and the drug Loading (LE) and Encapsulation Efficiency (EE) were calculated according to the following formulas.

Determining the encapsulation rate of the CUR to be 75.33 percent and the drug loading rate to be 2.07 percent; the encapsulation rate of CA4P is 87.23%, and the drug loading is 5.17%. The average particle size was determined to be 168.9 nm.

As shown in fig. 3, liposomes consist of a hydrophilic core and a hydrophobic shell. The hydrophobic drug CUR was loaded into the hydrophobic membrane, CA4P was wrapped in the hydrophilic core, and DSPE-PEG-GA was embedded in the outer membrane.

The blank liposomes prepared in example 2 and the targeted liposomes prepared in example 3 were further observed by scanning electron microscopy for morphological features, as shown in fig. 4A and 4B, CUR & CA4P/GA-LPs had a circular bubble structure with uniform size, similar to the blank liposomes, as shown in fig. 4C, CUR & CA4P/GA-LPs had an average particle size of about 168.9nm and a narrow particle size distribution (PDI ═ 0.23).

Example 4: preparation of Targeted Co-loaded CUR and CA4P liposomes

The preparation of CUR & CA4P/GA-LPs was carried out by a membrane dispersion method: 120mg of lecithin, 40mg of cholesterol and 20mg of DSPE-PEG-GA (mass ratio of 12:3:2) are fully dissolved in 5ml of trichloromethane, the obtained solution is placed in a 100ml eggplant-shaped bottle, 3mg of CUR (mass ratio of CUR to phospholipids is 1:40) is added for full dissolution, and the trichloromethane is removed by rotary evaporation through a rotary evaporator, wherein the rotary evaporation conditions are as follows: 35 ℃ C, 18 rpm. When a uniform film is formed, 5ml of a 1.8mg/ml aqueous solution of CA4P PBS (pH 7.4) (the mass ratio of the CUR to the CA4P is 1:3) is added for hydration, and the hydration time is 40 min. And (3) performing ultrasonic treatment for 5min after hydration, removing unencapsulated free drugs by a G-50 sephadex column and a dialysis method, and controlling the particle size of the liposome by sequentially passing through polyether sulfone membranes with membrane pore diameters of 450nm and 220 nm.

Measuring the encapsulation rate of the CUR by 71 percent and the drug loading rate by 1.5 percent; 80.6 percent of CA4P entrapment rate and 4.37 percent of drug loading. The average particle size was determined to be 161.8 nm.

Example 5: preparation of Targeted Co-loaded CUR and CA4P liposomes

120mg of lecithin, 40mg of cholesterol and 20mg of DSPE-PEG-GA (mass ratio of 12:3:2) are fully dissolved in 5ml of trichloromethane, the obtained solution is placed in a 100ml eggplant-shaped bottle, 3mg of CUR (mass ratio of CUR to phospholipids is 1:40) is added for full dissolution, and the trichloromethane is removed by rotary evaporation through a rotary evaporator, wherein the rotary evaporation conditions are as follows: 35 ℃ C, 18 rpm. When a uniform thin film is formed, adding 5ml of 0.2mg/ml aqueous solution of CA4P PBS (pH is 7.4) (the mass ratio of the CUR to the CA4P is 3:1) for hydration, wherein the hydration time is 40min, carrying out ultrasonic treatment for 5min, removing unencapsulated free drugs through a G-50 sephadex column and a dialysis method, and sequentially passing through a polyether sulfone film with the film pore diameter of 450nm and the film diameter of 220 nm.

Measuring the encapsulation rate of the CUR by 64.3 percent and the drug loading rate by 0.4 percent; 82% of CA4P entrapment rate and 4.2% of drug loading. The average particle size was determined to be 196.2 nm.

Example 6: determining the Effect of different Components on Liposome Properties

Following the procedure for preparing liposomes in example 3, the following liposomes were prepared, respectively:

blank GA-LPs: lecithin 120mg, cholesterol 40mg, DSPE-PEG-GA 20 mg;

CUR-LPs: lecithin 120mg, cholesterol 40mg, CUR 3 mg;

CA 4P-LPs: lecithin 120mg, cholesterol 40mg, CA4P15 mg;

CUR & CA 4P/LPs: lecithin 120mg, cholesterol 40mg, CUR 3mg, CA4P15 mg;

CUR & CA 4P/GA-LPs: lecithin 120mg, cholesterol 40mg, DSPE-PEG-GA 20mg, CUR 3mg, CA4P15 mg;

the results of measuring the particle diameter (DLS, nm), dispersion index (PDI), Potential (. zeta. -Potential, mV), encapsulation efficiency (EE,%), and drug loading (LE,%) of the liposomes are shown in Table 1.

TABLE 1 results of the Performance measurements of different liposome samples

The drug-loaded liposome can be easily accumulated in a tumor microenvironment by enhancing the permeability and retention (EPR) effect, and the surface charge is a sign of liposome stability and interaction with cells, and the results in Table 1 show that the potential of CUR & CA4P/LPs and CUR & CA4P/GA-LPs zeta is about-25 mV, so that the stability is better.

Example 7: determining the influence of different ratios of the two drugs on the liposome performance, and screening the drug-loading ratio

120mg of lecithin, 40mg of cholesterol, 20mg of DSPE-PEG-GA (the mass ratio is 12:3:2), 3mg of CUR (the mass ratio of the CUR to the phospholipid is 1:40), the dosage of CA4P is adjusted according to the ratio of the CUR, the preparation conditions are consistent, and the average particle size, zeta potential, EE and LE of each liposome formula are shown in Table 2.

As can be seen from the results in table 2, LE and EE were measured when the ratio of CUR to CA4P was between 1:1 and 10:1, and the third group (CUR: CA4P ═ 1:5) used further experiments with the liposomes prepared according to example 3 as drug-loaded liposomes, since the values of LE and EE were more favorable than those of the other groups.

TABLE 2 characterization of liposomes for different drug ratios

Example 8: the liposomes prepared by the method of example 3 were further measured for their change in properties at various times, and the results are shown in Table 3.

TABLE 3 CUR & CA4P/GA-LPs liposomes Performance Change results at different times

As can be seen from the results in Table 3, after 2 weeks of stability testing of the liposome preparation, there was no significant change in the physicochemical properties of the CUR & CA 4P/GA-LPs.

Example 9: in vitro drug release of targeted co-loaded liposome

The release of CUR and CA4P from liposomes was investigated using dialysis. PBS (pH 7.4) was used as release medium, and 1% Tween80 and 20% absolute ethanol were added. 1ml of CUR & CA4P/GA-LPs was transferred to a dialysis bag (molecular weight cut-off 3.5kDa) and placed in an erlenmeyer flask containing 50ml of release medium. The system was placed in a gas bath constant temperature shaker at 100rpm and 37 ℃. At specific time points 0.5, 1,2, 4, 8, 12, 24, 36, 48h, 4ml of medium were taken while supplementing the same volume with the release medium. The absorbance of CUR and CA4P was measured at 424nm and 288nm, respectively, using an ultraviolet spectrophotometer, and the cumulative drug release was calculated. The cumulative CUR and CA4P release rates (Er) were calculated using the following formula:

wherein M is_dIs the amount of drug in the liposome, V₀To release the bulk volume of the liquid, C_iTo release the concentration of CUR or CA4P in the fluid, V_rIs the volume of the replacement fluid.

As shown in fig. 5, no significant burst release occurred for both drugs. In contrast, both drugs were released continuously over 48 hours, and it is clear that the cumulative release rates of both drugs were almost the same during the first 10 hours of the experiment, while the release rate of CA4P after 10 hours was significantly faster than CUR. Approximately 60% of CA4P was released within 24 hours, while less than 50% of CURs were released at the same time. The cumulative release of CA4P and CUR at 48h was 65% and 50%, respectively, probably because the release rate of hydrophilic CA4P was faster than that of hydrophobic CUR in water-soluble PBS.

Example 10: targeted co-loaded liposome cellular uptake

The uptake of the liposome preparation by the liver cancer cell BEL-7402 was evaluated by a qualitative method. The density is 6 multiplied by 10³Cells were seeded in 6-well plates at 37 ℃ in CO₂Incubate in the incubator for 24 hours until adherent cell growth is observed. Using the CURs separately&CA4P/LPs，CUR&CA4P/GA-LPs (10. mu.g/mL CUR) was incubated for 1h, all reagents discarded, washed three times with cold PBS, fixed with 4% paraformaldehyde for 10min, DAPI stained (0.1. mu.g/mL, diluted with PBS) in CO₂Incubate in incubator for 10 min. Finally, the plate was washed 3 times with PBS, observed with a fluorescent inverted microscope and photographed. Images were synthesized using Image Pro plus 6.0 software.

Cell uptake of CUR & CA4P/LPs and CUR & CA4P/GA-LPs was observed using an inverted fluorescence microscope. As shown in FIG. 6, FIG. 6A is a fluorescence micrograph of the CUR & CA4P/LPs treatment, FIG. 6B is a fluorescence micrograph of the CUR & CA4P/GA-LPs treatment, with DAPI (blue) as a fluorescent marker of the nucleus of BEL-7402. Green fluorescence in the cytoplasm indicates uptake of CUR by the cells. In the combined images, blue and green fluorescence co-localisation indicates that the liposomes are entering the cells by endocytosis. As can be seen from the figure, the fluorescence micrograph of the incubated BEL-7402 cells, CUR & CA4P/GA-LPs, has a stronger green fluorescence than CUR & CA 4P/LPs. The CUR & CA4P/GA-LPs can be taken up by cancer cells through GA receptor-mediated endocytosis, and the introduction of the GA molecules promotes the liver cancer cells to take up the liposome, so that more drugs are accumulated in cytoplasm.

Example 11: in vitro cytotoxicity assay and migration assay of targeted co-loaded liposomes

1. In vitro cytotoxicity assay

The MTT method was used to determine the cytotoxicity of different dosage forms in vitro. Will be about 5X 10³B16 and BEL-7402 cells were seeded in 96-well plates at 37 deg.C in the presence of CO₂Incubate in incubator for 24 hours. Firstly, a cell compatibility test is carried out, blank GA-LPs liposome is prepared according to the method and the dosage of the embodiment 3, and blank GA-LPs with different concentrations (0.01-10 mu g/ml) is respectively added to treat cells for 24h, 48h or72 h. Add 10. mu. MTT reagent (5mg/mL) to each well and incubate for 4h in the dark. After the time comes, approximately 150. mu.l of DMSO was added to each well, and the mixture was shaken for 10min to dissolve formazan crystals. The detection was carried out using a microplate reader, with the wavelength set at 490 nm. According to the above method, different concentrations of CUR + CA4P and CUR are used&CA4P/LPs、CUR&CA4P/GA-LPs treated cells, cultured for 24, 48 and 72 h.

2. Cell migration assay

Cell migration assay five groups were set up, free CUR, physically mixed CUR + CA4P, CUR&CA4P/LPs,CUR&CA4P/GA-LPs was evaluated by a wound healing assay. Specifically, the density was 1X 10⁴B16 and BEL-7402 cells were seeded in 6-well plates, respectively, at 37 ℃ in CO₂Incubate in incubator for 24 hours. When the cells grew adherently to fill each well, three lines were drawn in parallel in each well using a 200. mu.l tip of a sterile pipette, the cells floated by the drawing were washed with PBS, and the scratch state of the cells was recorded for 0h under a microscope and the field of view was marked. Cells were incubated with different drug formulations, the solvent of the diluted drug stock was RPMI-1640 medium containing 1.5% serum, and the invasion of cells into the scratched area was recorded by photographing after 24h of drug incubation. The influence of the drug on cell migration was evaluated by calculating the mobility, which was calculated as follows:

where Wn and W0 represent the average width of the scratch at n h and the average width of the scratch at 0h, respectively.

FIGS. 7A and B show that the cell viability of B16 and BEL-7402 after 24h treatment with blank GA-LPs is over 86%, which indicates that blank GA-LPs has no obvious toxicity under experimental conditions and can be used as a drug delivery carrier.

As shown in fig. 7A, free CUR, physically mixed CUR + CA4P, CUR & CA4P/LPs, and CUR & CA4P/GA-LPs expressed dose-dependent cytotoxic effects after 24h incubation on BEL-7402 cells, similar to the B16 cell treatment results shown in fig. 7B, and as can be seen from fig. 7A and 7B, the binding of CUR and CA4P was more cytotoxic to BEL-7402 or B16 cells, indicating that the chemotherapeutic drug in combination with the antiangiogenic drug was cytotoxic.

As shown in fig. 7C and 7D, similar to the physical combination of the two drugs, the liposome combination exhibited time-dependent cytotoxicity at 24h, 48h and 72h for both cell lines, and as can be seen in fig. 7C and 7D, the co-loaded liposomes exhibited stronger cytotoxicity than the mixture of free CUR and CA 4P. Indicating that the drug-loaded liposome is internalized into cells through membrane fusion or endocytosis, thereby inhibiting drug efflux mediated by glycoprotein. CUR & CA4P/LPs was more cytotoxic than CUR & CA4P/LPs, with significance (p <0.05), indicating that the introduction of GA increased cellular uptake of the drug-loaded liposomes, resulting in more drug accumulation in tumor cells.

To further confirm the cytotoxic effects of the various preparations, wound healing assays were performed in BEL-7402 and B16 cells to evaluate tumor cell migration inhibition. The ability of the cells to migrate and migrate to the scarred area was measured after 24h and photographed at specific time points as shown in fig. 8, fig. 8A is the result of the BEL-7402 wound healing test, fig. 8B is the wound healing test of B16, the mobility (%) calculated by measuring the average width of the scar from 0h to 24h by Image-pro6.0 and the results are shown in fig. 8C and 8D, from which it can be seen that the migration and invasion ability of the tumor cells after treatment with four different drug formulations are different, the scarred of the control group almost disappeared within 24h of culture, the mobility of CUR was 63.85% and 45.68%, respectively, and the migration inhibition effect of CUR + CA4P was better than that of the control group, 47.28% and 38.29%, respectively. The results show that the combination has higher migration inhibition capability than the single drug group. The inhibition effect of the co-carried liposome group on cell migration is stronger than that of physical mixed CUR + CA 4P; CUR & CA4P/GA-LPs was more potent in inhibiting cell migration than CUR & CA4P/LPs, with mobilities of 15.74% and 15.69% in the two cell lines, respectively. The cell migration test result is consistent with the cell toxicity test result, the cell killing capability of the group CUR & CA4P/GA-LPs is strongest, and P is less than 0.01.

Example 12: in vivo biodistribution of targeted co-loaded liposomes

A DiR-entrapped GA-LPs (DiR/GA-LPs) was prepared by NIRF observation of the biodistribution of the GA-LPs formulation in vivo, and GA-LPs consisted of 120mg lecithin, 40mg cholesterol, and 20mg DSPE-PEG-GA, and was used to monitor the distribution of liposomes. When the tumor reaches 100mm³At this time, DiR/Blank LPs were injected into the tail vein of mice. Free DiR served as a control. Mice were anesthetized with 10% chloral hydrate and NIRF images were monitored in real time using an excitation wavelength of 745nm and an emission wavelength of 835 nm. Results were analyzed using Living Image 3.1 software.

As shown in FIG. 9, the results indicate that the NIRF signal of free DiR is much lower than that of the DiR/blankLPs group, monitored from 1h to 48 h. At 2h, no accumulation of fluorescent signal in the tumor area was observed in the free DiR group. In contrast, DiR/Blank LPs showed an accumulation of fluorescence signal in the tumor area and the fluorescence signal persisted at 48h post-injection. The Blank LPs drug delivery system increased the accumulation of DiR in the tumor.

Example 13: in vivo antitumor efficacy of targeted co-loaded liposomes

1. In vivo antitumor efficacy

To evaluate the CUR&CA4P/GA-LPs has antitumor effect in H22 tumor-bearing BALB/c male mice. Firstly, establishing a tumor-bearing mouse model, which comprises two steps: first 0.3ml of 5X 10⁴Liver cancer cells H22 were injected into the abdominal cavity of mice, and five cells were injected. Two weeks later, the mice developed ascites tumors. Second, ascites from the mice were extracted, centrifuged to collect H22 cells, diluted with physiological saline at a density of 4X 10⁶One per ml. The mice were inoculated subcutaneously into the right hind limb of the back with 0.2ml of a cell suspension. When the tumor volume grows to about 100mm³On the left and right, mice were divided into (1) physiological saline (control group); (2) free CUR group; (3) CUR + CA4P group; (4) CUR&CA4P/LPs group; (5) CUR&CA4P/GA-LPs group. Each group of mice was treated with a different drug formulation, and an equal volume of 0.2mL was injected intravenously every other day. The pharmaceutical formulation was injected at a dose of 5mg/kg (calculated as CUR). During treatment, mouse body weight was monitored and tumor diameter was measured. According to the formula: (L.times.W)²) Tumor volume was calculated where L is the longest tumor diameter and W is the shortest tumor diameter (mm). After two weeks of treatment, mice were sacrificed and tumors were denuded. Measuring tumorsWeight, calculating the tumor growth inhibition rate:

tumor growth inhibition rate (1-tumor weight of drug-treated group/tumor weight of control group) × 100%.

2. Histochemical staining

In vivo anti-tumor studies, tissue chemical staining was performed after drug administration, tumor tissues were stripped, fixed in 4% formaldehyde for over 24 hours, and tissues were embedded with paraffin.

Embedding:

(1) preparation work: cutting the fixed tumor tissue into 0.5cm³Placing the small blocks into an embedding box, washing the small blocks overnight by running water to remove paraformaldehyde;

(2) and (3) dehydrating: putting 70% ethanol (1h) → 80% ethanol (1h) → 95% ethanol (1h) → 100% ethanol (0.5h) into the embedding box in sequence for operation;

(3) and (3) transparency: treating in dimethylbenzene (I) for 0.5h → taking out, treating in dimethylbenzene (II) for 15min, observing tissue transparency and hardness, and preventing and treating tissue breakage;

(4) wax dipping: treating in molten paraffin (I) at 60 ℃ for 1h → taking out, and then continuing treating in molten paraffin (II) for 2 h;

(5) embedding: paraffin wax is injected into a wax block box by using a biological tissue embedding machine at the temperature of 60 ℃, the tissue is placed in the target position of the wax box, and the tissue is placed on a freezing table of the embedding machine for solidification and marking;

(6) slicing: solidifying the wax block overnight, and then cutting the wax block into slices of 4 mu m by using a computer ultrathin slicer;

(8) exhibition of slices: cutting out a complete slice, then placing the slice in a water bath kettle at 45 ℃, unfolding until the tissue has no folds, fishing out the slice by using a dry adhesive glass slide, and sucking excess water by using filter paper to make a mark;

(9) sheet sticking: baking the slices in a thermostat at 60 deg.C for 2 h.

Subsequent histochemical staining was performed:

(1) dewaxing: treating in xylene (I) for 10min, taking out, adding into xylene (II), and treating for 10 min;

(2) entering water: 100% ethanol (10min) → 95% ethanol (3-5min) → 80% ethanol (1-3min) → 70% ethanol (1-2min), washing off dissolved paraffin and xylene, gradually reducing ethanol concentration, and preventing flaking;

(3) washing with water: washing with distilled water for 2 times, and gently washing off ethanol;

(4) hematoxylin staining: dropping hematoxylin dye solution on the tissue with a pipette, treating for 10-15min, and cleaning with distilled water;

(5) differentiation with 0.5% hydrochloric acid-ethanol for 5-10 s: preparing 0.5% hydrochloric acid-ethanol by adding 0.5-1ml hydrochloric acid

95% ethanol to 10 ml;

(6) washing with distilled water for 5-10min, and washing with gradient ethanol after the flakes are seen to be blue by naked eyes: 70% ethanol (10min) → 80% ethanol (10 min);

(7) after 5-10s of eosin staining, the following treatments were performed: 95% ethanol (2min) → 95% ethanol (1-2min) → 100% ethanol (10-15min) → xylene (10-15min) → neutral gum sealing → microscopic observation and photographic recording.

As shown in fig. 10A, the free CUR group showed a significant weight loss trend compared to the control group, indicating that free curcumin may produce some systemic toxicity. As can be seen, there was no substantial weight loss in the liposome group, which was on the same upward trend as the control group, indicating that the liposome formulation could reduce systemic toxicity. In fig. 10B, the mice treated with saline had a rapid increase in tumor size during the experiment, and the mice treated with the four drug formulations had a slight increase in tumor size, indicating that these drug formulations all inhibited tumor development. In addition, the combination of CUR and CA4P was more effective in antitumor therapy than monotherapy, and the two drugs achieved additive effects through pro-apoptotic and anti-angiogenic activities. As shown in fig. 10B, 10C and 10D, the two liposome formulations had stronger antitumor effects than the physically mixed CUR + CA4P, consistent with the MTT method results, with synergistic effects. From the above results, it can be seen that liposomes enhance the accumulation of CUR and CA4P in the tumor region by the EPR effect, thereby enhancing the inhibition efficiency. In addition, as shown in FIG. 10D, the tumor suppression rate of CUR & CA4P/GA-LPs was 90.5%, which was higher than that of CUR & CA 4P/LPs. The CUR & CA4P/GA-LPs is shown to be absorbed by tumor cells through GA receptor-mediated endocytosis, and the antitumor effect is increased through active liver targeting drug delivery.

The tumor tissue sections were stained with H & E and further evaluated for anti-tumor effects. As shown in FIG. 11, the normal saline control group showed no significant necrosis of tumor cells, while the nuclei remained intact, and the tumor cells were treated with the drug, which resulted in a reduction in nuclei, low cell density, significant morphological changes, indicating that the development of tumor cells was inhibited. The physically mixed group produced more nuclear shrinkage of tumor cell nuclei than the free CUR group, suggesting that the binding of CUR and CA4P could enhance the anti-tumor effect through pro-apoptotic and anti-angiogenic activity. In addition, the tumor cells treated with the liposome formulation had lower cell density and greater nuclear shrinkage than the physically mixed CUR + CA4P group, indicating that more drug was accumulated in the tumor region under the liposome formulation treatment. The therapeutic effect of CUR & CA4P/GA-LPs was the best among all the prescriptions. The results show that the GA-LPs can effectively transfer the drug to the tumor microenvironment through the EPR effect, and the absorption amount of the drug by tumor cells is increased through GA-receptor mediated endocytosis.

Claims

1. A method for preparing a liposome targeting preparation is characterized by comprising the following steps:

(1) preparation of targeting polymers

(1a) Dissolving glycyrrhetinic acid in an organic solvent, adding a condensing agent, stirring for 1-2h, removing the organic solvent, adding the obtained solid into ethylenediamine, stirring for 12-24h at room temperature, removing the ethylenediamine, and purifying to obtain ethylenediamine-modified glycyrrhetinic acid, wherein the condensing agent is DMT-MM, and the mass ratio of the glycyrrhetinic acid, the condensing agent and the ethylenediamine is 1-1.5:0.5-1.5: 5-30;

(1b) dissolving DSPE-PEG-NHS and the modified glycyrrhetinic acid obtained in the step (1a) in an organic solvent according to a molar ratio of 1:1-10, reacting for 36-48h at room temperature under the protection of nitrogen, and after the reaction is finished, purifying reactants to obtain a target polymer DSPE-PEG-GA; adding a pH regulator and a catalyst into the organic solvent, wherein the pH regulator is triethylamine, and the catalyst is EDC;

(2) preparation of liposome targeting preparation

(2a) Dissolving phospholipid, cholesterol and DSPE-PEG-GA in an organic solvent, adding curcumin for dissolving, removing the organic solvent by rotary evaporation, and adding a combretastatin disodium aqueous solution for hydration when a layer of uniform film is formed, wherein the hydration time is 30-60 min; the mass ratio of the DSPE-PEG-GA to the phospholipid to the cholesterol is 1-10:3-30: 2-20; the mass ratio of the curcumin to the phospholipid is 1: 20-60; the mass ratio of the curcumin to the combretastatin disodium is 10-1: 1-10;

(2b) and (3) carrying out ultrasonic treatment on the hydrated solution in the step (2a) for 5-10min, and then removing unencapsulated free drugs to obtain the liposome targeting preparation.

2. The method for preparing a liposome-targeted formulation according to claim 1, wherein in step (2a), the phospholipid is selected from one or more of soybean phospholipid, lecithin, distearoylphosphatidylcholine, 1, 2-dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, and distearoylphosphatidylethanolamine-polyethylene glycol.

3. Use of the targeted liposome preparation of claim 1 or 2 in the preparation of a medicament for the treatment of liver cancer.