CN106265510A - Multistage target polymer micelle of pH trigger-type release and preparation method thereof in a kind of tumor cell - Google Patents

Abstract

The invention relates to a multi-level targeted polymer micelle for pH-triggered drug release in tumor cells and a preparation method thereof. The micelle is self-assembled in an aqueous medium by a hydrophobized modified polysaccharide polymer having endosomal pH-sensitive properties form. The invention adopts hyaluronic acid-deoxycholic acid-histidine polymer as a carrier, and prepares endosomal pH-sensitive polymer micelles actively targeting tumors by ultrasonic method or dialysis method, and carries insoluble antitumor drugs. The present invention uses EPR-mediated passive targeting, CD44 receptor active targeting, and pH-sensitive targeting strategy synergistic mechanism to carry out drug delivery from four key stages of long-term blood circulation, tumor tissue accumulation, cell uptake, and intracellular drug release. Guided by "whole-process targeting", multi-level targeted drug delivery can be achieved, the intracellular drug concentration can be effectively increased, and a new type of carrier and formulation strategy can be provided for improving the antitumor efficacy of poorly soluble antitumor drugs.

Description

A multi-stage targeting polymer micelle for pH-triggered drug release in tumor cells and its preparation preparation method

technical field

The invention belongs to the technical field of polymer materials and pharmaceutical preparations, and relates to a multi-level targeting polymer micelle for pH-triggered drug release in tumor cells and a preparation method thereof.

technical background

Chemotherapy is an effective method for clinical treatment of tumors, but the multidrug resistance of tumor cells to anticancer drugs is an important reason for the failure of chemotherapy. The classic tumor drug resistance mechanism is the highly expressed drug resistance protein, mainly P-glycoprotein, which "pumps" the anticancer drugs in the cell to the outside of the cell, resulting in a decrease in the level of intracellular drugs and drug resistance. Therefore, using the drug-loading system to deliver targeted drugs into tumor cells and avoid the efflux of P-glycoprotein is the key problem to be solved for reversing multidrug resistance and improving the efficacy of drugs for tumor treatment.

Hyaluronic acid (HA), a targeted polysaccharide with cytokine CD44 receptor targeting effect, was hydrophobized, and the prepared polymer micelles had good biocompatibility, strong drug-loading ability, and good In vivo and in vitro stability and long circulation and other characteristics. The HA micelle system can realize the accumulation of tumor tissue through passive targeting mediated by the high permeability and retention (EPR) effect, and the drug-carrying drug enters the cell through endocytosis mediated by the CD44 receptor, realizing its intumor tissue, tumor Effective accumulation and uptake by cells.

Aiming at the low pH value of endosomes of tumor cells, constructing pH-sensitive micelles with triggered drug release mechanism to deliver anticancer drugs has made a breakthrough in reversing multidrug resistance. The intelligent drug delivery system can trigger drug release in response to the acidic pH of the endosome, and release the drug into the cytoplasm, effectively increase the drug concentration in the cytoplasm, saturate the drug resistance mechanism of tumor cells, and overcome multidrug resistance. Therefore, the combined application of receptor-mediated targeted drug delivery and pH-sensitive targeted drug release strategies in micellar systems shows a good synergistic targeted drug delivery, improving the therapeutic efficiency of anticancer drugs and reducing their toxic side effects. Provide new avenues for targeted therapy of cancer.

Contents of the invention

In view of the hydrophobically modified polysaccharide polymer micelles showing excellent passive targeting, drug loading capacity and long-term circulation characteristics; the tumor targeting ability of the active targeting strategy, and the localized drug release function of the physicochemical targeting strategy, the present invention combines Passive targeting, active targeting and physicochemical targeting mechanisms provide a multi-stage targeting polymer micelle for tumor cell endosome pH-triggered drug release to achieve EPR effect-mediated tumor tissue-targeted accumulation, CD44 Receptor-mediated active targeted uptake into cells and endosome pH-triggered drug release, combined to reverse tumor drug resistance and improve the anti-tumor efficacy of the drug.

Another object of the present invention is to provide a method for preparing the above-mentioned multistage targeting polymer micelles for pH-triggered drug release in endosomes of tumor cells.

In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solutions to achieve:

A multi-stage targeted polymer micelle for pH-triggered drug release in tumor cells, characterized in that the micelle includes a hydrophobically modified polysaccharide polymer with endosomal pH-sensitive properties, and the polymer self-assembles in an aqueous medium form.

The polymer has a hydrophilic segment and a hydrophobic segment, wherein the hydrophilic segment is hyaluronic acid (HA) with CD44 receptor targeting properties; the hydrophobic segment is selected from deoxycholic acid (deoxycholic acid, DOCA), And the endosome pH sensitive unit is connected at its end.

The degree of substitution of the hydrophobic segment is 5%-30%.

The pH sensitive unit is histidine (His) or its structural analogues.

The molecular structure of the histidine or its structural analog contains an imidazole ring, and its pH response range is between 5.0 and 7.4.

The molecular weight of the hyaluronic acid is 1×10 ³ -1×10 ⁷ Da.

The polymer is prepared according to the following process steps:

1) Synthesis of deoxycholic acid-histidine or its structural analog (Trt)

Dissolve deoxycholic acid (DOCA) in the reaction solvent with 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and hydroxysuccinimide (NHS) as activators at room temperature Down activation for 2 to 24 hours;

Dissolve histidine or its structural analogue (Trt) in the reaction solvent, add triethylamine, and then slowly add it dropwise to the above-mentioned deoxycholic acid mixture, stir and react at 30-50°C for 12-48 h , collect the product, and dry it under reduced pressure to obtain deoxycholic acid-histidine or its structural analogue (Trt);

2) Synthesis of hyaluronic acid-deoxycholic acid-histidine or its structural analogues (Trt)

Dissolve hyaluronic acid in the reaction solvent, stir and dissolve at 40-60°C, and cool to room temperature; then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and hydroxysuccinyl imine, activated in ice bath for 2-5 h;

Dissolve the deoxycholic acid-histidine or its structural analog (Trt) obtained in step 1) in the reaction solvent, then slowly add it dropwise to the above hyaluronic acid mixture, and stir the reaction at 40-70°C After 6-12 hours, continue to stir and react at room temperature for 24-48 hours, collect the product, purify the product by dialysis, and freeze-dry to obtain hyaluronic acid-deoxycholic acid-histidine or its structural analogue (Trt);

3) Synthesis of hyaluronic acid-deoxycholic acid-histidine or its structural analogues

Dissolve hyaluronic acid-deoxycholic acid-histidine or its structural analog (Trt) obtained in step 2) in trifluoroacetic acid, add sulfide anisole, stir at room temperature for 2-12 h, collect the product, and dialyze the product Purify and freeze-dry to obtain hyaluronic acid-deoxycholic acid-histidine or its structural analogues.

The reaction solvent is one or more combinations of N,N-dimethylformamide, formamide, ethanol aqueous solution and N,N-dimethylformamide aqueous solution.

In step 1), the molar ratio of deoxycholic acid to histidine or its structural analogues is 1:1-2;

The molar ratio of deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide is 1:1～2:1～2;

The molar ratio of histidine or its structural analog (Trt) to triethylamine is 1:1~2;

In step 2), the molar ratio of hyaluronic acid to deoxycholic acid-histidine or its structural analog (Trt) intermediate is 1:2-5,

The molar ratio of hyaluronic acid to 1-ethyl-(3-dimethylaminopropyl) carbodiimide and hydroxysuccinimide is 1:1～5:1～5,

In step 3), the molar ratio of hyaluronic acid-deoxycholic acid-histidine or its structural analog (Trt) to trifluoroacetic acid and sulfide anisole is 1:1-5:1-5.

The method for preparing the multi-level targeted polymer micelles for pH-triggered drug release in tumor cells is characterized in that: the hydrophobized modified polysaccharide polymer with endosomal pH-sensitive properties is subjected to ultrasound, dialysis or solvent evaporation. Preparation of polymer micelles to load insoluble antitumor drugs.

The concrete steps of described ultrasonic method are:

1) Dissolving the hydrophobized modified polysaccharide polymer with endosome pH-sensitive properties at a concentration of 1-5 mg/mL in phosphate buffer solution (PBS) at pH 7.4;

2) After dissolving the poorly soluble antineoplastic drug in an organic solvent, drop it into the polymer PBS obtained in the above process 1), and perform ultrasonic treatment to prepare drug-loaded micelles with a particle size of 10-1000 nm.

The specific process of the dialysis method is: co-dissolving the hydrophobically modified polysaccharide polymer with endosome pH-sensitive properties and the insoluble antitumor drug in an organic solvent, placing it in a dialysis bag with a molecular weight cut-off of 3500, and immersing it in a pH In the PBS of 7.4, dialyze to remove the organic solvent and free drug, and the liquid in the bag is the prepared drug-loaded micelles with a particle size of 10-1000 nm.

The specific process of the solvent evaporation method is: co-dissolving the hydrophobically modified hyaluronic acid polymer with endosome pH-sensitive properties and insoluble antitumor drugs in an organic solvent, adding PBS with a pH of 7.4, and stirring until the organic solvent The drug-loaded micelles with a particle size of 10-1000 nm were prepared.

The insoluble antineoplastic drug is one or more of paclitaxel, doxorubicin, camptothecin, hydroxycamptothecin, topotecan, dasatinib, 5-fluorouracil, vincristine and cisplatin .

The organic solvent is one or more combinations of ethanol, tetrahydrofuran, dimethyl sulfoxide, and N,N-dimethylformamide.

Through the above technical scheme, the beneficial effects of the present invention are: the micelles can be used for the preparation of drugs for treating tumor diseases, administered through intravenous injection, in physiological environment (pH 7.4) and tumor extracellular fluid (pH 6.5-7.2), The micelles have a complete structure and no or low release of drugs, which can be targeted and accumulated in tumor tissues through EPR effect-mediated tumor blood vessels, and further uptake into tumor cells through the active targeting mechanism of CD44 receptors of HA; finally in endosomes ( (pH 5.0～6.0) depolymerization of micelles, triggering drug release, and at the same time endosomal membrane rupture, releasing drug to cytoplasm, increasing intracellular drug concentration, saturating drug resistance mechanism, overcoming tumor multidrug resistance, and improving drug antitumor efficacy.

In summary, the present invention uses hyaluronic acid-deoxycholic acid-histidine polymer as a carrier to prepare tumor-actively targeted endosomal pH-sensitive polymer micelles through ultrasound, dialysis or solvent evaporation. Encapsulated insoluble antitumor drugs. The present invention utilizes EPR-mediated passive targeting, CD44 receptor active targeting and pH-sensitive targeting strategy synergistic mechanism, from the four key stages of drug delivery: long blood circulation, tumor tissue accumulation, cell uptake and intracellular drug release. "Whole-process targeting" guidance to achieve multi-level targeted drug delivery, providing a new type of carrier and formulation strategy for improving the anti-tumor efficacy of insoluble anti-tumor drugs.

Description of drawings

Fig. 1 is the synthetic roadmap of HA-DOCA-His polymer;

Figure 2 is a particle size distribution diagram of HA-DOCA-His micelles at different pH values;

Figure 3 is the release curve of HA-DOCA-His micelles loaded with paclitaxel in different pH release media;

Figure 4 is a schematic diagram of self-assembly of HA-DOCA-His drug-loaded micelles, targeting to tumor tissue accumulation, tumor cell uptake, and acid-sensitive depolymerization of intracellular endosomes to trigger drug release.

detailed description

In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

Example 1: Synthesis of HA-DOCA-His polymer

0.5 g DOCA was dissolved in 5 mL N,N-dimethylformamide, 0.29 g EDC and 0.18 g NHS were added, and stirred at room temperature for 2 h. 0.37 g of trityl histidine methyl ester hydrochloride (H-His (Trt)-OMe HCl) was dissolved in 20 mL of DMF, 50 µL of triethylamine was added, and slowly added dropwise to the DOCA mixture, the mixture Stir in a water bath at 35 °C for 24 h. NaHCO ₃ solution (pH 9-10) was added to the reaction mixture, the precipitate was obtained by suction filtration, and dried under reduced pressure to obtain DOCA-His (Trt).

Dissolve 0.1 g HA in 5 mL anhydrous formamide, heat to dissolve at 50°C, and cool to room temperature. Add 96 mg EDC and 58 mg NHS, and stir in an ice bath for 2 h. 0.4 g DOCA-His(Trt) was dissolved in 5 mL of anhydrous DMF, slowly added dropwise to the HA mixture, stirred in a water bath at 50 °C for 6 h, and then stirred at room temperature for 24 h. The reaction mixture was dialyzed in distilled water for 2-3 days (dialysis bag molecular weight cut-off: 3500), filtered to remove insoluble impurities, freeze-dried to obtain HA-DOCA-His (Trt) white powder.

Dissolve 0.1 g HA-DOCA-His (Trt) in 0.5 mL DMSO, add 0.5 mL trifluoroacetic acid and 25 µL sulfide anisole, stir at room temperature for 2 h, and dialyze the reaction mixture in alkaline water (pH 9-10) for 2 h d (dialysis bag molecular weight cut-off: 3500), dialyze in distilled water for 2 days, filter to remove insoluble impurities, and freeze-dry to obtain HA-DOCA-His white powder. The reaction scheme is shown in Figure 1.

Example 2: Synthesis of HA-DOCA-His polymer

0.5 g DOCA was dissolved in 5 mL N,N-dimethylformamide, 0.29 g EDC and 0.18 g NHS were added, and stirred at room temperature for 24 h. 0.37 g of trityl histidine methyl ester hydrochloride (H-His (Trt)-OMe HCl) was dissolved in 20 mL of DMF, slowly added dropwise to the DOCA mixture, and the mixture was stirred in a water bath at 35 °C for 24 h. The reaction mixture was spun to remove the organic solvent, and separated by a column to obtain DOCA-His (Trt).

0.1 g HA was dissolved in 5 mL of anhydrous formamide, heated to dissolve at 50 °C, and cooled to room temperature. Add 96 mg EDC and 58 mg NHS, and stir in an ice bath for 2 h. 0.4 g DOCA-His(Trt) was dissolved in 5 mL of anhydrous DMF, slowly added dropwise to the HA mixture, stirred in a water bath at 50 °C for 6 h, and then stirred at room temperature for 24 h. The reaction mixture was dialyzed in distilled water for 2-3 days (dialysis bag molecular weight cut-off: 3500), filtered to remove insoluble impurities, freeze-dried to obtain HA-DOCA-His (Trt) white powder.

Dissolve 0.1 g HA-DOCA-His (Trt) in 0.5 mL DMSO, add 0.5 mL trifluoroacetic acid and 25 µL sulfide anisole, stir at room temperature for 2 h, and dialyze the reaction mixture in distilled water for 2 d (dialysis bag molecular weight cut-off: 3500), dialyzed in distilled water for 2 days, filtered to remove insoluble impurities, and freeze-dried to obtain HA-DOCA-His white powder.

Example 3: Synthesis of HA-DOCA-His polymer

0.5 g DOCA was dissolved in 5 mL N,N-dimethylformamide, 0.29 g EDC and 0.18 g NHS were added, and stirred at room temperature for 24 h. 0.37 g of trityl histidine methyl ester (H-His(Trt)-OH) was dissolved in 20 mL of DMF, slowly added dropwise to the DOCA mixture, and the mixture was stirred in a water bath at 35 °C for 24 h. The reaction mixture was rotary evaporated to remove the organic solvent, and separated by column to obtain DOCA-His(Trt).

0.1 g HA was dissolved in 5 mL anhydrous formamide, heated to dissolve at 60 °C, and cooled to room temperature. Add 192 mg EDC and 116 mg NHS, and magnetically stir in ice bath for 5 h. 0.8 g DOCA-His (Trt) was dissolved in 5 mL of anhydrous DMF, slowly added dropwise to the HA mixture, and stirred at room temperature for 48 h. Ethanol was added to the reaction mixture to precipitate and remove impurities to obtain HA-DOCA-His (Trt) white powder.

Dissolve 0.1 g HA-DOCA-His (Trt) in 0.5 mL DMSO, add 0.5 mL trifluoroacetic acid and 50 µL sulfide anisole, stir at room temperature for 12 h, and dialyze the reaction mixture in distilled water for 2 days (dialysis bag molecular weight cut-off: 3500), dialyzed in distilled water for 2 days, filtered to remove insoluble impurities, and freeze-dried to obtain HA-DOCA-His white powder.

The ¹ HNMR spectrum of HA-DOCA-His is shown in Figure 2. The chemical shift of -CH ₃ on the NHCOCH ₃ of HA is 1.95 ppm, and the chemical shift of hydrogen on the 2, 3, 4 and 5 carbons is 3.0-4.0 ppm. The chemical shift of hydrogen on the 6-position carbon is 4.35-4.45 ppm, 0.8-2.0 ppm belongs to the characteristic peaks of -CH ₃ and -CH ₂ - in DOCA, and 7.45 ppm and 8.82 ppm belong to -N=CH in His The characteristic peaks of - and -N=CH=C- confirmed that DOCA-His was successfully synthesized on HA.

Example 4: Preparation of paclitaxel-loaded HA-DOCA-His micelles by ultrasonic method

HA-DOCA-His was dissolved in PBS with a pH of 7.4 at a concentration of 1 to 5 mg/mL, stirred by magnetic force until completely dissolved, paclitaxel (10% of the amount of HA-DOCA-His added) dissolved in absolute ethanol (methanol, tetrahydrofuran) %, 20% and 30%) were slowly added dropwise into the above polymer solution, stirred magnetically for 24-48 h, ultrasonicated in an ice bath (ultrasonic power 100-400 W, working 2 s, intermittent 3 s) for 2-20 min, micelles The solution was filtered through a 0.45 µm filter membrane to obtain drug-loaded micelles, which were freeze-dried to obtain drug-loaded micelles powder.

Example 5: Preparation of HA-DOCA-His micelles loaded with doxorubicin by dialysis

Weigh 5 mg of doxorubicin hydrochloride and disperse it in 1 mL of DMSO (tetrahydrofuran), add 7 µL of triethylamine, stir until the doxorubicin dissolves, and prepare a doxorubicin DMSO (tetrahydrofuran) solution (5 mg/mL) . HA-DOCA-His was dissolved in DMSO (10 mg/mL), and doxorubicin (10%, 20% and 30% of the added amount of HA-DOCA-His) was added to DMSO (tetrahydrofuran) solution, and magnetically stirred at room temperature to make it Mix well, place in a dialysis bag (molecular weight cut-off: 3500), immerse in PBS with pH 7.4 and dialyze for 3 days, the liquid in the dialysis bag is filtered through a 0.45 µm filter membrane to obtain drug-loaded micelles, which can be obtained by lyophilization Micellar powder.

Example 6: Preparation of HA-DOCA-His micelles loaded with doxorubicin by solvent evaporation

Weigh 5 mg of doxorubicin hydrochloride and disperse it in 1 mL of methanol, add 7 µL of triethylamine, stir until the doxorubicin dissolves, and prepare a doxorubicin methanol solution (5 mg/mL). Dissolve HA-DOCA-His in tetrahydrofuran (10 mg/mL), add adriamycin (10%, 20% and 30% of the amount of HA-DOCA-His added) methanol solution, stir magnetically to mix, drop into PBS with pH 7.4 was stirred magnetically for 24-48 h, and the micellar solution was filtered through a 0.45 µm filter membrane to obtain drug-loaded micelles, which were then freeze-dried to obtain drug-loaded micelles powder.

Example 7: HA-DOCA-His Micellar pH Sensitivity Assay

HA-DOCA-His polymer was dissolved in PBS pH 7.4 at a concentration of 1 mg/mL. Measure the average particle size and PI of the micelles with a particle size analyzer, adjust the pH to 6.5, 6.0, and 5.0 in turn with 0.01 mol/L HCl solution, and stir the sample solution magnetically for 30 min and let stand for 10 min for each pH value adjusted. min, measure the particle size, repeat three times, and observe the change of particle size. The results are shown in Figure 3. The particle size of the micelles did not change significantly at pH 7.4 and 6.5, but the particle size and PI increased sharply at pH 6.0 and pH 5.0, indicating that HA-DOCA-His micelles have endosome pH (5.0-6.0) sensitivity.

Example 8: In vitro release of paclitaxel-loaded HA-DOCA-His micelles

The in vitro drug release of the drug-loaded micelles prepared in Example 4 was investigated by dialysis. Precisely measure 3 mL of paclitaxel-loaded HA-DOCA-His micelles, place them in a dialysis bag (molecular weight cut-off: 3500), immerse in 80 mL of 2% Cremophor EL (w/v), pH 7.4, 6.5, 6.0 , 5.0 in PBS, shake at 37°C and 100 r/min, sample 3 mL at 1, 2, 4, 6, 8, 12, 16, 24, 36 h and 48 h, and supplement the same pH value and volume and temperature of fresh media. The samples were filtered through a 0.45 µm filter membrane, the primary filtrate was discarded, the paclitaxel content was determined by HPLC, and the cumulative drug release percentage was calculated. The results are shown in Figure 4. In the release media of pH 7.4, 6.5, 6.0, and 5.0, the cumulative drug release of paclitaxel-loaded HA-DOCA-His micelles was 12.2%, 13.8%, 29.9% and 55.3%, respectively, indicating that HA-DOCA -His micelles are stable in both physiological environment (pH 7.4) and tumor extracellular fluid (>pH6.5), and can trigger drug release when entering the acidic environment (pH 5.0-6.0) of tumor cell endosomes.

Claims (17)

1. the multistage target polymer micelle of pH trigger-type release in a tumor cell, it is characterised in that this micelle is by having The hydrophobization modified polysaccharide polymer of endosome pH sensitivity characteristic is self-assembly of in aqueous medium.

2., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 1, its feature exists Having hydrophilic segment and hydrophobic segment in described polymer, wherein hydrophilic segment is the hyalomitome with CD44 receptor target characteristic Acid；Hydrophobic segment is selected from deoxycholic acid, and connects endosome pH sensing unit at its end.

3., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 2, its feature exists Substitution value in described polymer hydrophobic segment is 5%～30%.

4., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 2, its feature exists PH sensing unit in described polymer is histidine or its analog.

5., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 4, its feature exists Containing imidazole ring in the molecular structure of described histidine or its analog, its pH response range between 5.0～7.4 it Between.

6., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 2, its feature exists Molecular weight in described hyaluronic acid is 1 × 10³～1 × 10⁷ Da。

7., according to the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 1, its feature exists Prepare according to following processing step in described polymer:

1) deoxycholic acid-histidine or the synthesis of its analog

Deoxycholic acid is dissolved in reaction dissolvent, with 1-ethyl-(3-dimethylaminopropyl) carbodiimide and hydroxysuccinimidyl acyl Imines is activator, activates 2～24 h under room temperature；

Histidine or its analog are dissolved in reaction dissolvent, add triethylamine, be then slowly dropped to above-mentioned deoxidation gallbladder In acid mixed liquor, under 30～50 DEG C of temperature conditionss, stirring reaction 12～48 h, collect product, drying under reduced pressure, obtain deoxidation gallbladder Acid-histidine or its analog；

2) hyaluronic acid-deoxycholic acid-histidine or the synthesis of its analog

Hyaluronic acid is dissolved in reaction dissolvent, stirring and dissolving under 40～60 DEG C of temperature conditionss, is cooled to room temperature；It is subsequently adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide, activate 2～5 h under ice bath；

Deoxycholic acid-the histidine or its analog (Trt) that step 1) are obtained are dissolved in reaction dissolvent, the most slowly drip Being added in above-mentioned hyaluronic acid mixed liquor, under 40～70 DEG C of temperature conditionss, stirring reaction 6～12 h, continue stirring at room temperature Reaction 24～48 h, collect product, product through dialysis purification, lyophilization, obtain hyaluronic acid-deoxycholic acid-histidine or Its analog (Trt)；

3) hyaluronic acid-deoxycholic acid-histidine or the synthesis of its analog

By step 2) gained hyaluronic acid-deoxycholic acid-histidine or its analog (Trt) be dissolved in trifluoroacetic acid, add Thioanisole, stirs 2～12 h, collects product under room temperature, product, through dialysis purification, lyophilization, obtains hyaluronic acid-deoxidation Cholic acid-histidine or its analog.

8. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that institute Stating reaction dissolvent is in N, N-dimethylformamide, Methanamide, ethanol water and N, N-dimethylformamide in water One or more combinations.

9. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that step Rapid 1), in, described deoxycholic acid is 1:1～2 with the mol ratio of histidine or its analog；

The mol ratio of deoxycholic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-Hydroxysuccinimide be 1:1～ 2:1～2；

The mol ratio of histidine or its analog (Trt) and triethylamine is 1:1～2.

10. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that Step 2) in, described hyaluronic acid is 1:2 with the mol ratio of deoxycholic acid-histidine or its analog (Trt) intermediate ～5,

Hyaluronic acid and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, the mol ratio of N-Hydroxysuccinimide be 1:1～ 5:1～5.

11. according to the multistage target polymer of pH trigger-type release in the tumor cell described in claim 7, it is characterised in that In step 3), hyaluronic acid-deoxycholic acid-histidine or its analog (Trt) and trifluoroacetic acid, thioanisole mole Ratio is 1:1～5:1～5.

The multistage target polymer glue of pH trigger-type release in 12. 1 kinds of tumor cells as described in any one of claim 1 ~ 11 The preparation method of bundle, it is characterised in that: the hydrophobization with endosome pH sensitivity characteristic is modified hyaluronic acid polymer and uses Ultrasonic method, dialysis or solvent evaporation method prepare polymer micelle, and bag carries insoluble anti-tumor medicament.

13. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12 Method, it is characterised in that concretely comprising the following steps of described ultrasonic method:

1) the described hydrophobization with endosome pH sensitivity characteristic is modified hyaluronic acid polymer by the concentration of 1～5 mg/mL It is dissolved in the phosphate buffer of pH 7.4；

2), after being dissolved with organic solvent by insoluble anti-tumor medicament, said process 1 is instilled) the polymer micelle solution of gained In, sonicated, prepared particle diameter is the carrier micelle of 10～1000 nm.

14. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12 Method, it is characterised in that the detailed process of described dialysis is: the described hydrophobization with endosome pH sensitivity characteristic is modified thoroughly Bright matter acid polymer and insoluble anti-tumor medicament are codissolved in organic solvent, are placed in the bag filter that molecular cut off is 3500, Immersing in the phosphate buffer of pH 7.4, dialysis removes organic solvent and free drug, and in bag, liquid is prepared particle diameter It it is the carrier micelle of 10～1000 nm.

15. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12 Method, it is characterised in that the detailed process of described solvent evaporation method is: the described hydrophobization with endosome pH sensitivity characteristic is repaiied Decorations hyaluronic acid polymer and insoluble anti-tumor medicament are codissolved in organic solvent, add the phosphate buffer of pH 7.4, stir Mixing and fling to organic solvent, prepared particle diameter is the carrier micelle of 10～1000 nm.

16. according to the preparation side of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 12 Method, it is characterised in that described insoluble anti-tumor medicament be paclitaxel, amycin, camptothecine, hydroxy camptothecin, topotecan, More than the one or two kinds of in Dasatinib, 5-fluorouracil, vincristine and cisplatin.

17. according to the system of the multistage target polymer micelle of pH trigger-type release in the tumor cell described in claim 13-15 Preparation Method, it is characterised in that described organic solvent is ethanol, oxolane, dimethyl sulfoxide, N, in N-dimethylformamide One or more combinations.