CN104844730B - Low molecular heparin-glycyrrhetinic acid polymer and synthetic method and application thereof - Google Patents

Abstract

The invention relates to a low-molecular-weight heparin-glycyrrhetinic acid polymer and its synthesis method and application. First, low-molecular-weight heparin-adipic acid dihydrazide is synthesized, and then glycyrrhetinic acid succinate is synthesized to prepare glycyrrhetinic acid succinate Active ester solution, and finally synthesize low molecular weight heparin-glycyrrhetinic acid polymer. Heparin was used as the water-soluble framework material, and glycyrrhetinic acid was used to carry out hydrophobic modification on the water-soluble framework through succinic anhydride and adipic dihydrazide as linking arms to prepare glycyrrhetinic acid-mediated nanocarrier materials with targeting ability. The heparin-glycyrrhetinic acid polymer of the invention has good biocompatibility, biodegradability and non-immunogenicity. The prepared liver-targeted drug-loaded nano-micelle has high encapsulation efficiency and drug loading capacity, has excellent sustained-release properties, can enhance drug therapeutic effect, and reduce non-specific toxic and side effects, and has good due prospects.

Description

Low molecular weight heparin-glycyrrhetinic acid polymer and its synthesis method and application

technical field

The present invention relates to the synthesis of a nano-polymer self-assembled micelle material (low molecular weight heparin-glycyrrhetinic acid polymer), in particular to the synthesis of low molecular weight heparin-glycyrrhetinic acid polymer and the preparation of the polymer as a biological material Application method of antitumor drug nano-micelle preparation. The invention belongs to the fields of biomedical materials and nano drug preparations.

Background technique

Over the past few decades, the incidence and mortality of malignant tumors have shown an obvious upward trend, becoming one of the frequently-occurring diseases that seriously threaten human life and health. Some studies predict that by 2050, the number of new cases and deaths of malignant tumors in the Asia-Pacific region alone will reach 7.3 million and 5.5 million, which is more than twice that of 2000. Among them, liver cancer is called "the king of cancers", with a low cure rate and a high mortality rate. However, my country is a high-incidence area of liver cancer in the world, and the mortality rate of liver cancer has increased by 41.7% in the past 20 years. About 1.25 million people die of liver cancer in the world every year, and nearly half of them occur in my country. The early symptoms of liver cancer are hidden and difficult to detect, but once obvious symptoms appear, the degree of malignancy is high and the development is rapid. If the treatment is not timely or the treatment plan is not selected properly, the average survival time is only half a year. At present, for the clinical treatment of liver cancer, in order to make up for the incompleteness of surgical treatment and the shortcomings of easy-to-induce metastasis, chemotherapy still occupies a very important position as an adjuvant treatment. However, traditional chemotherapeutic drugs based on cytotoxicity have strong cytotoxicity to normal organs of the human body while obtaining therapeutic effects through systemic administration, and then cause serious toxic side effects. For example, paclitaxel is a natural anti-cancer active ingredient extracted from Taxus genus plants. As a highly effective cytotoxic anticancer drug, its clinical preparation Taxol Injection has been used as a first-line drug for breast cancer, ovarian cancer, prostate cancer and acute leukemia for a long time. However, its solvent polyoxyethylene castor oil is prone to toxic side effects such as neurotoxicity, cardiotoxicity, nephrotoxicity, and allergic reactions. In addition, the systemic distribution of paclitaxel can cause severe cardiotoxicity and nephrotoxicity. Using mild and tolerable chemotherapy to reduce the potential danger of chemotherapy is an urgent problem that researchers in the field of medicine need to solve.

Nano drug delivery system is a research hotspot in the field of formulation development and delivery of anticancer drugs in recent years. It wraps or disperses chemical drugs in a nanoscale matrix through nano-carrier materials, effectively improves the solubility of insoluble drugs, prolongs the circulation time of drugs in the blood, changes the distribution of drugs in vivo, and improves the bioavailability of drugs. Has a certain degree of targeting. These advantages can effectively reduce the non-specific adverse reactions in the course of chemotherapy, improve the efficacy of treatment, and are especially suitable for the treatment of tumors. Among them, the polymer micelle system has attracted the attention of scholars at home and abroad in the field of solubilization and delivery of poorly soluble drugs due to its low toxicity and good stability. It is a self-assembled structure with a typical core-shell structure formed spontaneously by amphiphilic polymers in water through intramolecular and intermolecular forces of hydrophobic groups. This micellar system consists of a tight hydrophobic core and a hydrophilic shell, because the core can contain some poorly soluble drugs through hydrophobic forces, so this system is considered to be a promising delivery of poorly soluble drugs carrier. However, these nano drug delivery systems do not have the ability to escape from the reticuloendothelial system, and are easily phagocytized and cleared by mononuclear macrophages in vivo. On the other hand, the passive targeting achieved by the nano-scale particle size of the preparation still has the phenomenon of insufficient uptake of autopolymers at the tumor site, and even leads to multidrug resistance, thereby reducing the curative effect. In order to complete the targeted drug delivery of tumor nanosystems, it has become a relatively mature targeting strategy to couple target molecules on the surface of nanocarriers by using the specific molecular recognition mechanism in vivo.

As early as the early 1990s, Negishi confirmed that rat liver cell membrane fractions contained a large number of glycyrrhetinic acid binding sites, which were highly specific. Subsequently, carrier materials modified with glycyrrhetinic acid as the target molecule were reported by many scholars at home and abroad, for example, glycyrrhetinic acid modified hyaluronic acid polymer and glycyrrhetinic acid as the hydrophobic Acid-modified sulfated chitosan polymer; Glycyrrhetinic acid-modified PEG–b-poly(γ-benzyl L-glutamate) polymer with glycyrrhetinic acid as a targeting molecule distributed on the polymer surface via water-soluble PEG long chains Micelles and glycyrrhetinic acid modified chitosan/PEG nanoparticles, etc. The results show that this type of carrier material has remarkable liver targeting ability. Therefore, using glycyrrhetinic acid as the liver-targeting mediating group can research and develop a new type of glycyrrhetinic acid-mediated liver-targeting nano drug delivery system. Chinese patent CN101254308A discloses a glycyrrhetinic acid-polyethylene glycol/chitosan liver-targeted compound drug delivery system and its preparation method, and shows that the targeted drug delivery system has a strong binding ability to liver cancer cells; Chinese patent CN101642573A discloses a chitosan-based liver-targeted nanoparticle drug delivery system and a preparation method thereof. Glycyrrhetinic acid is used as a target molecule while being coupled to sulfate chitosan or carboxymethyl chitosan as a hydrophobic group on, thereby constructing and forming a liver-targeting nano drug delivery system; Chinese patent CN102336802A discloses glycyrrhetinic acid-modified lipids, liver-targeting liposomes, micelles and complexes and a preparation method, and glycyrrhetinic acid is mixed with phospholipids or cholesterol in Glycyrrhetinic acid-modified lipids were prepared under condensing agent conditions, and glycyrrhetinic acid-mediated liver-targeting liposomes and micellar carriers were prepared using them as basic materials. It can be seen that the use of glycyrrhetinic acid as a targeting group in the construction of a liver-targeted drug delivery system has attracted the attention of many scholars.

Heparin is mainly an animal mucopolysaccharide sulfate extracted from the mucous membrane of the bovine lung or small intestine of pigs. It was named after it was first found in the liver. Heparin is mainly a water-soluble long-chain macromolecule composed of structural units such as glucosamine, L-iduroside, N-acetylglucosamine and D-glucuronic acid, with an average molecular weight of about 12000Da. Heparin has been used in clinical research as a blood anticoagulant as early as 1939 because of its unique ability to bind to antithrombin. However, the clinical application of heparin is also easy to cause bleeding and thrombocytopenia, which limits its further application in the field of medicine. Low molecular weight heparin is a degradation product of unfractionated heparin. Compared with unfractionated heparin, low molecular weight heparin has similar or better anticoagulant, anti-inflammatory, anti-angiogenic and anti-tumor properties, and its possible adverse reactions are significantly reduced. Therefore, low molecular weight heparin is safer and more reliable for the treatment of related diseases. Low-molecular-weight heparin has better water solubility due to its small molecular weight and reduced skeleton rigidity. In addition, the heparin skeleton contains a large number of active and modifiable free groups, which can endow it with new properties through chemical modification. Therefore, in addition to being used as an anticoagulant active ingredient, the nano drug delivery system based on water-soluble low-molecular-weight heparin has attracted the attention of many pharmaceutical preparation researchers.

In recent years, the hydrophobic modification of the low molecular weight heparin skeleton and the construction of polymer micelles in aqueous media have attracted the research interests of many experts and scholars in the field of medicine. The amphiphilic polymer was constructed by chemically introducing small hydrophobic molecules into the heparin skeleton and induced to self-assemble and polymerize into core-shell micelles with a hydrophobic inner core. However, under normal circumstances, these nanomicelle systems do not have the ability to escape from the reticuloendothelial system, and are easily phagocytized and cleared by mononuclear macrophages in vivo. On the other hand, the passive targeting achieved by the nano-scale particle size of the preparation still has the phenomenon of insufficient uptake of autopolymers at the tumor site, and even leads to multidrug resistance, thereby reducing the curative effect. In order to complete the targeted drug delivery of tumor nanosystems, it has become a relatively mature targeting strategy to use the specific molecular recognition mechanism in vivo to couple target molecules during the construction of nanocarriers.

In summary, low molecular weight heparin was used as the water-soluble framework material to construct polymer micelles, and glycyrrhetinic acid was introduced into the low molecular weight heparin molecule for hydrophobic modification, so that it had the ability to self-assemble and aggregate in aqueous solution and utilize its The specific binding ability to liver cancer parenchymal cells enables liver-targeted drug delivery of nanomicelles. This design method increases drug solubility, changes drug distribution in the body, prolongs drug residence time in vivo, and at the same time exerts the targeting performance of glycyrrhetinic acid modified materials. The design method has great research in tumor diagnosis and treatment. Development prospects. This design has not been reported yet. In addition, anticancer drug sensitizers are used in combination with first-line anticancer drugs to reverse the multidrug resistance caused by single long-term drug use and enhance the therapeutic effect of anticancer drugs.

Contents of the invention

Aiming at the above-mentioned prior art, the present invention provides a low-molecular-weight heparin-glycyrrhetinic acid polymer, its synthesis method, and its application as a biodegradable material for the preparation of anti-tumor drug liver-targeting nano-preparations .

The present invention is achieved through the following technical solutions:

The synthetic method of low-molecular-weight heparin-glycyrrhetinic acid polymer comprises steps as follows:

(1) Synthesis of low-molecular-weight heparin-adipate dihydrazide: Dissolve low-molecular-weight heparin in distilled water, add adipate dihydrazide, 1-(3-dimethylaminopropyl)-3- Ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, adjust the pH of the reaction system to 6-7 with sodium hydroxide, and react at room temperature for 20-30 hours to obtain low molecular weight heparin-adipate dihydrazide ;

(2) Synthesis of glycyrrhetinic acid succinate: dissolve glycyrrhetinic acid in anhydrous tetrahydrofuran, add succinic anhydride and 4-dimethylaminopyridine, stir at room temperature in the dark for 20-30 hours, and then remove by rotary evaporation organic solvent, then add ethyl acetate to dissolve, wash and purify to obtain glycyrrhetinic acid succinate;

(3) Preparation of glycyrrhetinic acid succinate active ester solution: dissolve glycyrrhetinic acid succinate in N,N dimethylformamide, then add 1-(3-dimethylaminopropyl)- 3-Ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were stirred at room temperature for 3-5 hours, and then an organic base was added to obtain a glycyrrhetinic acid succinate active ester solution;

(4) Synthesis of low-molecular-weight heparin-glycyrrhetinic acid polymer: dissolve the low-molecular-weight heparin-adipate dihydrazide obtained in step (1) in distilled water, stir to make it fully swell and dissolve, and then add N, Dilute with N dimethylformamide and set aside; drip the glycyrrhetinic acid succinate active ester solution obtained in step (3) into the above-mentioned low-molecular-weight heparin-adipate dihydrazide solution under strong stirring, and stir the reaction at room temperature After 40-50 hours, a reactant solution is obtained, and the reaction solution is purified and freeze-dried to obtain a low-molecular-weight heparin-glycyrrhetinic acid polymer.

In the above synthesis method, low molecular weight heparin, distilled water, adipic acid dihydrazide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 1 The mass ratio of -hydroxybenzotriazole is 0.5-0.6:100:6-7:0.9-1:0.6-0.7. The molecular weight of low molecular weight heparin is 3kD～6kD. The substitution degree of adipate dihydrazide of low molecular weight heparin-adipate dihydrazide is 43%.

In step (2), the mol ratio of glycyrrhetinic acid, succinic anhydride, and 4-dimethylaminopyridine is 1:4:0.1; the consumption of anhydrous tetrahydrofuran is to add 25 milliliters per gram of glycyrrhetinic acid; anhydrous tetrahydrofuran and ethyl acetate The volume ratio of esters is 1:2.

The mol ratio of glycyrrhetinic acid succinate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, organic base in step (3) It is 1:2:2:2. The organic base is triethylamine, N,N,N',N'-tetramethylethylenediamine or N,N-diisopropylethylamine. The dosage of N,N dimethylformamide is 70-300 milliliters per gram of glycyrrhetinic acid succinate.

In step (4), the mass ratio of low molecular weight heparin-adipate dihydrazide to glycyrrhetinic acid succinate active ester is 1:0.2-0.6. The volume ratio of distilled water to N,N dimethylformamide is 1:3, add 100ml of distilled water to every gram of low molecular weight heparin-adipate dihydrazide. The degree of substitution of glycyrrhetinic acid ranges from 3% to 10% (adjust the required degree of substitution according to the physical and chemical properties of the self-assembled system required).

The low molecular weight heparin-glycyrrhetinic acid polymer prepared by the above method.

The application of the low-molecular-weight heparin-glycyrrhetinic acid polymer as a carrier material for insoluble antitumor drugs in the preparation of receptor-mediated liver-targeted drug-loaded nanometer self-assembled micelles. In specific application, the preparation method is as follows: the low molecular weight heparin-glycyrrhetinic acid polymer is ultrasonically dispersed in deionized water; Slowly add it dropwise to the above-mentioned low molecular weight heparin-glycyrrhetinic acid polymer aqueous solution, continue to stir vigorously at room temperature, use probe type ultrasonic treatment, transfer the solution to a dialysis bag for water dialysis after the ultrasonic treatment, and then centrifuge the obtained solution, In order to remove unencapsulated drugs, the supernatant is then passed through a 0.8 μm filter membrane to obtain nanomicelle preparations of antitumor drugs.

The preferred preparation method is as follows: Weigh 50 mg of low-molecular-weight heparin-glycyrrhetinic acid polymer, ultrasonically disperse it in 5 mL of deionized water for later use; in addition, dissolve 5-40 mg of antineoplastic drugs, or antineoplastic drugs and antineoplastic drug sensitizers in 1 mL In the organic solvent, slowly add it dropwise to the above-mentioned low molecular weight heparin-glycyrrhetinic acid polymer aqueous solution under the condition of strong stirring, after continuing to stir vigorously at room temperature for 4 hours, use the probe type ultrasonic treatment under the power condition of 120W three times, each time for 2 minutes, the temperature At 4°C to 8°C, pulse on for 2s and stop for 4s. After ultrasonication, transfer the solution to a dialysis bag for dialysis against water for 24 hours, and then centrifuge the resulting solution at 4000r/min for 20min to remove unencapsulated drugs. The supernatant is then passed through a 0.8 μm filter membrane to obtain a nanomicelle preparation of antitumor drugs, which is stored at 4° C. or freeze-dried to obtain a freeze-dried powder of drug-loaded nanomicelles.

The insoluble antitumor drugs are paclitaxel, doxorubicin, docetaxel, etc.; the antitumor drug sensitizers are curcumin, quercetin, etc.

The organic solvent is tetrahydrofuran, N,N dimethylformamide or dimethyl sulfoxide.

The low-molecular-weight heparin-glycyrrhetinic acid polymer of the present invention uses water-soluble low-molecular-weight heparin as the basic skeleton, and carries out targeted hydrophobic modification on it, and the obtained amphiphilic polymer can be formed by self-assembly in an aqueous medium Targeted nano micelles, which have the following advantages:

1. The carrier material low molecular heparin-glycyrrhetinic acid polymer prepared by the present invention has excellent biocompatibility, degradability and non-immunogenicity, and the preparation process is simple and the conditions are mild. It is an excellent liver-targeting nano Drug carrier.

2. The drug-loaded nano-micelle prepared by the present invention has a rounded shape, a uniform spherical shape, a small particle size, a high encapsulation efficiency and drug-loading capacity, and good stability.

3. During the self-assembly and construction process of the nanomicelle prepared by the present invention, glycyrrhetinic acid as a hydrophobic group forms a stable inner core and endows the carrier material with potential liver-targeting properties. The design idea is reasonable and the operation is easy.

4. The drug-loaded nanomicelle prepared by the present invention overcomes the defect of poor water solubility of insoluble drugs, greatly improves the solubility of insoluble antitumor drugs, and provides an ideal new carrier for its targeted therapy.

5. The drug-loaded nanomicelle preparation prepared by the present invention simultaneously encapsulates insoluble antitumor drugs and their sensitizers in the micelle preparation, which can effectively control the generation of multidrug resistance, reduce the dosage, and provide drug treatment effect.

Description of drawings

Figure 1: H NMR spectrum of the carrier material low molecular weight heparin-glycyrrhetinic acid polymer.

Figure 2: Particle size distribution of paclitaxel-loaded low molecular weight heparin-glycyrrhetinic acid nanomicelles.

Figure 3: Electron micrographs of paclitaxel-loaded low molecular weight heparin-glycyrrhetinic acid nanomicelles.

Figure 4: Zeta potential diagram of paclitaxel-loaded low molecular weight heparin-glycyrrhetinic acid nanomicelles.

detailed description

The present invention will be further described below in conjunction with embodiment. The following examples are not intended to limit the scope of the present invention.

Embodiment 1: Synthesis of low molecular weight heparin-glycyrrhetinic acid polymer

(1) Synthesis of low-molecular-weight heparin-adipate dihydrazide: Weigh 0.5g low-molecular-weight heparin and dissolve it in 100mL distilled water, stir to make it fully swell and dissolve, and then add 6.53g adipic acid dihydrazide to the solution in turn. Hydrazide, 0.96g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.68g 1-hydroxybenzotriazole, adjust the reaction system pH value with sodium hydroxide 6.8, react at room temperature for 24 hours, dialyze with distilled water for three days, and freeze-dry to obtain the intermediate product low molecular weight heparin-adipate dihydrazide.

(2) Synthesis of glycyrrhetinic acid succinate: Weigh 200 mg of glycyrrhetinic acid and ultrasonically dissolve it in 5 mL of anhydrous tetrahydrofuran, add 4 times the molar amount of succinic anhydride and 0.1 times the molar amount of 4-dimethylaminopyridine, After stirring at room temperature in the dark for 24 hours, the organic solvent was removed by rotary evaporation. Then add 10 mL of ethyl acetate to dissolve, wash with 1% hydrochloric acid solution and ultrapure water three times respectively, dry with anhydrous sodium sulfate overnight, and evaporate the organic solvent. The product was purified by silica gel column chromatography (eluent: ethanol:ethyl acetate (11:3; v/v) to obtain glycyrrhetinic acid succinate.

(3) Weigh 20 mg of glycyrrhetinic acid succinate and dissolve it in 3 mL of N,N dimethylformamide, then add 1-(3-dimethylamino Propyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide equivalent to 2 times the molar amount of glycyrrhetinic acid succinate, after stirring at room temperature for 4 hours, add the equivalent of glycyrrhetinic acid succinate N,N,N',N'-tetramethylethylenediamine with twice the molar amount of succinate to obtain a solution of glycyrrhetinic acid succinate active ester.

(4) Synthesis of low-molecular-weight heparin-glycyrrhetinic acid polymer: Weigh 100 mg of low-molecular-weight heparin-adipate dihydrazide obtained in step (1) and dissolve it in 10 mL of distilled water, stir to make it fully swell and dissolve, and then Add 3 times the volume of N,N dimethylformamide to dilute and set aside. The glycyrrhetinic acid succinate active ester solution obtained in step (3) was dripped into the above-mentioned low molecular weight heparin-adipate dihydrazide solution under strong stirring, and stirred and reacted at room temperature for 48 hours to obtain a reactant solution. The reaction solution was dialyzed against a mixed solution of methanol and water (volume ratio 4:1) for three days to remove free glycyrrhetinic acid succinate and by-products therein, and then dialyzed against distilled water for three days to remove the organic solvent. Low molecular weight heparin-glycyrrhetinic acid polymer can be obtained by freeze-drying. ¹ H NMR quantitative analysis showed that the substitution degree of glycyrrhetinic acid was 6.8%.

The ¹ H NMR structure spectra of low molecular weight heparin (HEP) and the resulting low molecular weight heparin-adipate dihydrazide (HEP-ADH) and low molecular weight heparin-glycyrrhetinic acid polymer (HEP-GA) are shown in Figure 1, Compared with HEP, in the HEP-ADH NMR spectrum, the new characteristic peaks located at 1.0-1.6ppm and 2.1-2.5ppm belong to the proton peak of the branched chain of adipic acid dihydrazide, which also confirms that adipic acid Dihydrazides have been successfully coupled to the HEP backbone. As for HEP-GA, the chemical shift is 0.5-1.4ppm, and the new proton peak at 5.52ppm is the characteristic proton peak on the proton of the glycyrrhetinic acid skeleton ring. The above results proved that glycyrrhetinic acid was successfully coupled to low molecular weight heparin through adipate dihydrazide.

Embodiment 2: Synthesis of low molecular weight heparin-glycyrrhetinic acid polymer

(1) Synthesis of low-molecular-weight heparin-adipate dihydrazide: Weigh 0.5g low-molecular-weight heparin and dissolve it in 100mL distilled water, stir to make it fully swell and dissolve, and then add 6.53g adipic acid dihydrazide to the solution in turn. Hydrazide, 0.96g 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.68g 1-hydroxybenzotriazole, adjust the reaction system pH value with sodium hydroxide 6.8, react at room temperature for 24 hours, dialyze with distilled water for three days, and freeze-dry to obtain the intermediate product low molecular weight heparin-adipate dihydrazide.

(2) Synthesis of glycyrrhetinic acid succinate: Weigh 200 mg of glycyrrhetinic acid and ultrasonically dissolve it in 5 mL of anhydrous tetrahydrofuran, add 4 times the molar amount of succinic anhydride and 0.1 times the molar amount of 4-dimethylaminopyridine, After stirring at room temperature in the dark for 24 hours, the organic solvent was removed by rotary evaporation. Then add 10 mL of ethyl acetate to dissolve, wash with 1% hydrochloric acid solution and ultrapure water three times respectively, dry with anhydrous sodium sulfate overnight, and evaporate the organic solvent. The product was purified by silica gel column chromatography (eluent: ethanol:ethyl acetate (11:3; v/v) to obtain glycyrrhetinic acid succinate.

(3) Weigh 30 mg of glycyrrhetinic acid succinate and dissolve it in 3 mL of N,N dimethylformamide, then add 1-(3-dimethylamino Propyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide equivalent to 2 times the molar amount of glycyrrhetinic acid succinate, after stirring at room temperature for 4 hours, add the equivalent of glycyrrhetinic acid succinate Glycyrrhetinic acid succinate active ester solution was obtained by adding N,N-diisopropylethylamine with 2 times the molar amount of succinate.

(4) Synthesis of low-molecular-weight heparin-glycyrrhetinic acid polymer: Weigh 100 mg of low-molecular-weight heparin-adipate dihydrazide obtained in step (1) and dissolve it in 10 mL of distilled water, stir to make it fully swell and dissolve, and then Add 3 times the volume of N,N dimethylformamide to dilute and set aside. The glycyrrhetinic acid succinate active ester solution obtained in step (3) was dripped into the above-mentioned low molecular weight heparin-adipate dihydrazide solution under strong stirring, and stirred and reacted at room temperature for 48 hours to obtain a reactant solution. The reaction solution was dialyzed against a mixed solution of methanol and water (volume ratio 4:1) for three days to remove free glycyrrhetinic acid succinate and by-products therein, and then dialyzed against distilled water for three days to remove the organic solvent. Low molecular weight heparin-glycyrrhetinic acid polymer can be obtained by freeze-drying. ¹ H NMR quantitative analysis showed that the substitution degree of glycyrrhetinic acid was 8.5%.

Example 3: Preparation of paclitaxel-loaded low molecular weight heparin-glycyrrhetinic acid polymer nanomicelles

Disperse 50 mg of low-molecular-weight heparin-glycyrrhetinic acid (prepared in Example 1) ultrasonically in 5 mL of deionized water, and set aside; another 20 mg of paclitaxel was dissolved in 1 mL of tetrahydrofuran, and slowly added dropwise to the above-mentioned low-molecular-weight heparin under vigorous stirring. -In the aqueous solution of glycyrrhetinic acid polymer, after continuing to stir vigorously at room temperature for 4 hours, use probe ultrasonic treatment at 120W power for three times, each time for 2 minutes, keep the temperature at 4°C-8°C, pulse on for 2s and stop for 4s, after the ultrasonication is completed The solution was transferred to a dialysis bag and dialyzed against water for 24 hours, and then the resulting solution was centrifuged at 4000 r/min for 20 minutes to remove unencapsulated drugs. Then, the supernatant was passed through a 0.8 μm filter membrane to obtain a paclitaxel-loaded nanomicelle preparation, and freeze-dried to obtain a paclitaxel-loaded nanomicelle lyophilized powder. The detection results of high performance liquid chromatography showed that the drug loading capacity was 20.6%, and the encapsulation efficiency was 72.0%.

After diluting the prepared paclitaxel-loaded nano-micelle solution to an appropriate multiple, measure the particle size and Zeta potential of the micelles with a Malvern Zetasizer Nano-ZS laser potential particle size analyzer, the average particle size of the micelles measured is 90.2nm, more than The dispersion coefficient PI of the system is 0.23, and the Zeta potential value is -25.2mV. Figure 2 is the particle size distribution diagram of paclitaxel-loaded low molecular weight heparin-glycyrrhetinic acid nanomicelles. The surface morphology of the micelles was observed with a H-7000 transmission electron microscope. As shown in Figure 3, the prepared drug-loaded micelles were round in shape, uniform in size, and small in adhesion. Figure 4 is the Zeta potential diagram of the drug-loaded nanomicelle.

Example 4: Preparation of low molecular weight heparin-glycyrrhetinic acid polymer nanomicelles loaded with paclitaxel and curcumin

50 mg of low molecular weight heparin-glycyrrhetinic acid polymer (prepared in Example 1) was ultrasonically dispersed in 5 mL of deionized water, and set aside; another 20 mg of paclitaxel and 20 mg of curcumin were dissolved in 1 mL of tetrahydrofuran, and slowly dripped under strong stirring. Add it to the above-mentioned low-molecular-weight heparin-glycyrrhetinic acid polymer aqueous solution, continue to stir vigorously at room temperature for 4 hours, then use probe-type ultrasonic treatment at 120W power three times, each time for 2 minutes, keep the temperature at 4°C-8°C, and pulse for 2s After stopping for 4 seconds, the solution was transferred to a dialysis bag for dialysis against water for 24 hours, and then the resulting solution was centrifuged at 4000 r/min for 20 minutes to remove unencapsulated drugs. The supernatant was then passed through a 0.8 μm filter membrane to obtain a nanomicelle preparation co-loaded with paclitaxel and curcumin, and freeze-dried to obtain a lyophilized powder of paclitaxel-loaded nanomicelles. The results of high performance liquid chromatography showed that the drug loading and encapsulation efficiency of paclitaxel were 17.2% and 63.5%, respectively, and the drug loading and encapsulation efficiency of curcumin were 14.9% and 55.0%, respectively.

Example 5: Preparation of low molecular weight heparin-glycyrrhetinic acid polymer nanomicelles loaded with docetaxel and quercetin

50mg of low molecular weight heparin-glycyrrhetinic acid polymer (prepared in Example 2), ultrasonically dispersed in 5mL of deionized water, set aside; another 20mg of docetaxel and 20mg of quercetin were dissolved in 1mL of tetrahydrofuran, under vigorous stirring Slowly add it dropwise to the above-mentioned low molecular weight heparin-glycyrrhetinic acid polymer aqueous solution, and continue to stir vigorously at room temperature for 4 hours, then use probe-type ultrasonic treatment at 120W power three times, each time for 2 minutes, and keep the temperature at 4 ° C ~ 8 ° C, The pulse was turned on for 2 seconds and stopped for 4 seconds. After the ultrasound was completed, the solution was transferred to a dialysis bag for dialysis against water for 24 hours, and then the resulting solution was centrifuged at 4000r/min for 20 minutes to remove unencapsulated drugs. The supernatant was then passed through a 0.8 μm filter membrane to obtain a nanomicelle preparation co-loaded with paclitaxel and curcumin, and freeze-dried to obtain a lyophilized powder of paclitaxel-loaded nanomicelles. The results of high performance liquid chromatography showed that the drug loading and encapsulation efficiency of docetaxel were 18.9% and 66.1%, respectively, and the drug loading and encapsulation efficiency of quercetin were 14.4% and 53.1%, respectively.

Claims (10)

1. the synthetic method of Low molecular heparin-enoxolone polymer, is characterized in that, comprise the following steps that

(1) synthesis of Low molecular heparin-adipic dihydrazide: Low molecular heparin is dissolved in distilled water, it is sequentially added into adipic dihydrazide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, 1-hydroxy benzo triazole wherein, reaction system pH=6-7 is regulated with sodium hydroxide, room temperature reaction 20-30 hour, obtains Low molecular heparin-adipic dihydrazide；

(2) synthesis of enoxolone succinate: enoxolone is dissolved in anhydrous tetrahydro furan, add succinic anhydride and DMAP, after stirring 20-30 hour under lucifuge room temperature, rotation is evaporated off organic solvent, being subsequently adding acetic acid ethyl dissolution, washing, purification obtain enoxolone succinate；

(3) preparation of enoxolone succinate active ester solution: enoxolone succinate is dissolved in N, in N-dimethylformamide, it is subsequently adding 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, after being stirred at room temperature 3-5 hour, add organic base, obtain enoxolone succinate active ester solution；

(4) synthesis of Low molecular heparin-enoxolone polymer: be dissolved in distilled water by the Low molecular heparin-adipic dihydrazide of gained in step (1), stirring makes its most swelling, dissolving, is subsequently adding DMF dilution, standby；Gained enoxolone succinate active ester solution in step (3) is instilled in above-mentioned Low molecular heparin-adipic dihydrazide solution under vigorous stirring, reaction 40-50 hour is stirred at room temperature, obtaining reactant solution, reaction solution purification, lyophilization i.e. obtain Low molecular heparin-enoxolone polymer.

The synthetic method of Low molecular heparin the most according to claim 1-enoxolone polymer, it is characterized in that, Low molecular heparin described in step (1), distilled water, adipic dihydrazide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, the mass ratio of 1-hydroxy benzo triazole are 0.5-0.6:100:6-7:0.9-1:0.6-0.7.

The synthetic method of Low molecular heparin the most according to claim 1-enoxolone polymer, is characterized in that, described in step (1), the molecular weight of Low molecular heparin is 3kD～6kD.

The synthetic method of Low molecular heparin the most according to claim 1-enoxolone polymer, is characterized in that, in step (2), enoxolone, succinic anhydride, the mol ratio of DMAP are 1:4:0.1；The consumption of anhydrous tetrahydro furan is that every gram of enoxolone adds 25 milliliters；Anhydrous tetrahydro furan is 1:2 with the volume ratio of ethyl acetate.

The synthetic method of Low molecular heparin the most according to claim 1-enoxolone polymer, it is characterized in that, in step (3), the mol ratio of enoxolone succinate, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide, organic base is 1:2:2:2.

The synthetic method of Low molecular heparin the most according to claim 1-enoxolone polymer, is characterized in that, described organic base is triethylamine, N, N, N', N'-tetramethylethylenediamine or DIPEA.

The synthetic method of Low molecular heparin the most according to claim 1-enoxolone polymer, it is characterized in that, in step (4), Low molecular heparin-adipic dihydrazide is 1:0.2-0.6 with the mass ratio of enoxolone succinate active ester, distilled water and N, the volume ratio of N-dimethylformamide is 1:3, and every gram of Low molecular heparin-adipic dihydrazide adds 100ml distilled water.

8. Low molecular heparin-enoxolone polymer that the method described in any one of claim 1-7 prepares.

9. the Low molecular heparin described in claim 8-enoxolone polymer as insoluble anti-tumor medicament carrier material receptor-mediated liver target drug-carrying nanoassemble micellar preparation prepare in application.

10. the application described in claim 9, is characterized in that, method is: by Low molecular heparin-enoxolone polymer, in ultrasonic disperse deionized water；By antitumor drug, or antitumor drug and antitumor drug sensitizer are dissolved in organic solvent, it is slowly added dropwise in the case of strong agitation to above-mentioned Low molecular heparin-enoxolone aqueous solutions of polymers, room temperature continues strong agitation, processes with probe type ultrasonic, ultrasonic after be transferred to solution in bag filter water is dialysed, then by gained solution centrifugal, to remove non-encapsulated medicine, supernatant then crosses 0.8 μm filter membrane, obtains the nano-micelle preparations of antitumor drug.