Polymer with pH responsiveness and active targeting property as well as preparation method and application thereof

Abstract

The invention discloses a polymer with pH responsiveness and active targeting as well as a preparation method and application of the polymer. The polymer has the following chemical structural formula: # imgabs0 #, in the formula, the value of n is 37, and the value range of m is 20-43. The polymer not only can realize pH responsiveness and active targeting at the same time, but also has good biocompatibility and biodegradability, can be self-assembled in an aqueous medium to form a polymer micelle with a core-shell structure to be used as a targeting carrier of an antitumor drug, and has excellent drug loading performance. Therefore, the active targeting release of the anti-tumor drug is realized, so that the drug toxicity is reduced, and the targeting treatment effect is improved.

Description

Technical Field

[0001] The present invention relates to a polymer with pH responsiveness and active targeting, a preparation method and application thereof, and belongs to the technical field of medicinal targeting carrier materials. Background Art

[0002] Chemotherapy, a primary clinical treatment for cancer, often produces numerous side effects, such as premature drug release into the bloodstream, resulting in insufficient drug concentration at the tumor site, and adverse effects on normal cells. In recent years, nanoparticle-based drug delivery systems have been increasingly recognized as a promising strategy for overcoming these adverse effects in cancer therapy. Since the initial introduction of drug delivery vehicles, polymeric micelles have garnered increasing attention as a versatile nanoscale drug platform for cancer therapy.

[0003] The polymer micelles are nano-micelles with a special "core-shell" structure formed by self-assembly of amphiphilic block polymers in an aqueous medium. The hydrophobic core can be used to load hydrophobic drugs to improve their solubility, and the hydrophilic shell can prevent the drugs from being taken up in large quantities by the mononuclear phagocyte system (MPS) and the endothelial reticular system (RES), thereby prolonging the drug's blood circulation time and enhancing the therapeutic effect. In addition, their nano-particle size can avoid being recognized and phagocytosed by macrophages, and through the EPR effect of tumor tissue, they can enhance passive targeting and reduce adverse drug reactions. They are considered to be one of the most promising anti-tumor drug delivery systems. However, conventional polymer micelles have two major defects: one is that the micelles release drugs too early in the systemic circulation, increasing systemic toxic side effects; the other is that the concentration of drugs released or the accumulation of drugs at the tumor site is insufficient, and the effective therapeutic concentration cannot be reached. Intelligent polymer micelle delivery systems that can trigger release in response to external stimuli (such as temperature, pH, ultrasound, enzymes, etc.) have obvious advantages in this regard and have become a research hotspot for anti-tumor drug preparations. Among them, pH-sensitive polymer micelles that release drugs in a dependent manner are the most attractive. This is because: on the one hand, the pH of the extracellular matrix of solid tumors is 6.5-7.2, which is lower than the pH of normal tissues at 7.4, and the cells contain endosomes (pH = 5.5-6.0) and lysosomes (pH = 4.5-5.0) with even lower pH; on the other hand, polymer micelles are mainly taken up by cells through endocytosis, and the cells to which the micelles reach are The intracellular site is the endosome / lysosome. The micelles undergo a pH gradient change from extracellular internalization to intracellular entry and the subsequent process. When the pH of the external environment is higher than the pKa of the block polymer forming the polymer micelles, the polymer micelles encapsulate the drug in a compact and complete form and basically do not release the drug. When the pH of the external environment is lower than the pKa of the block polymer forming the polymer micelles, the micelles swell rapidly or even disaggregate, and the drug is released rapidly. Therefore, at the tumor site or after entering the cell, the micelles respond to changes in their pH environment to accelerate drug release. This property can be used to achieve targeted drug delivery to specific sites in the body (tumors) or within cells (endosomes, lysosomes, cytoplasm). However, in the disclosed prior art, due to the limitations of biocompatibility and biodegradability, the hydrophilic segments of the pH-sensitive block polymers that form pH-sensitive polymer micelles are generally limited to polyethylene glycol (PEG), and the PEG on the hydrophilic surface of the micelles may hinder the cellular uptake of the micelles and subsequent intracellular processes. In addition, pH-sensitive polymer micelles generally have hydrophobic segments that show pH sensitivity, and the pH-sensitive region that forms the micelles is in the core of the micelles. Therefore, their response to the stimulation of the external environment pH is relatively slow, resulting in weak active targeting of tumor cells.

[0004] Chinese patent application number CN202311143263.3 discloses a "folate-modified reduction-responsive polymer, its preparation method, and application." This application utilizes dextran and stearic acid as the hydrophilic and hydrophobic ends of the micelle, respectively, with cystamine as a linker. The polymer responds to high intracellular GSH (intracellular reduced glutathione) concentrations within tumor cells via disulfide bonds to achieve reduction-responsive drug release within the cells. The polymer is then modified with FA to impart specific tumor cell targeting. While the polymer described in this application can serve as a carrier for poorly soluble drugs and, to a certain extent, achieves the goals of solubilizing poorly soluble drugs, tumor targeting, and precise drug release at the tumor site, the applicant's research has found that when plasma GSH concentrations reach approximately 2 to 20 μM, the cystamine disulfide bond prematurely breaks in the systemic circulation, leading to premature drug leakage and the risk of toxicity to normal tissues. Furthermore, as the stearic acid grafting rate increases, a significant steric hindrance effect occurs, causing the folic acid grafting rate to decrease, thereby compromising its active targeting of tumor cells. Summary of the Invention

[0005] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a polymer with pH responsiveness and active targeting, and a preparation method and application thereof.

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

[0007] A pH-responsive and actively targeted polymer having the following chemical structure: ;

[0008] In the formula, the value of n is 37, and the value range of m is 20 to 43.

[0009] A method for preparing a polymer with pH responsiveness and active targeting properties comprises first connecting a hydrophilic segment dextran (Dex) and a hydrophobic segment polybenzyloxycarbonyllysine (PZLL) through an amide bond to prepare an amphiphilic block polymer Dex-b-PZLL, and then utilizing an esterification reaction to graft folic acid (FA) onto the hydrophilic end of the amphiphilic block polymer to obtain a polymer FA-Dex-b-PZLL.

[0010] In one embodiment, the preparation method comprises the following steps:

[0011] a) Dex-NH2 was synthesized by reductive amination of the terminal end of dextran (Dex);

[0012] b) Using Dex-NH2 as an initiator, ring-opening polymerization of N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) was induced to synthesize the amphiphilic block polymer Dex-b-PZLL;

[0013] c) Folic acid (FA) was grafted onto the hydrophilic end of an amphiphilic block polymer (Dex-b-PZLL) via an esterification reaction to synthesize the polymer FA-Dex-b-PZLL.

[0014] In one implementation scheme, step a) includes the following specific operations:

[0015] First, dextran and an amination reagent are subjected to a condensation reaction in a solvent at 50-70°C. After the condensation reaction is monitored to be completed, a reducing agent is added to the reaction system, and the reduction reaction is continued at 50-70°C. After the reduction reaction is monitored to be completed, the reaction solution is poured into methanol for precipitation, and the solid is collected by filtration. The obtained solid is purified and then freeze-dried to obtain Dex-NH2.

[0016] In a preferred embodiment, the solvent is dimethyl sulfoxide, the amination reagent is ethylenediamine, and the reducing agent is sodium cyanoborohydride.

[0017] In one implementation scheme, step b) includes the following specific operations:

[0018] Under nitrogen protection, Dex-NH2 and N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) are dissolved in an anhydrous organic solvent and then reacted at room temperature. After the reaction is completed, the reaction solution is poured into methanol for precipitation. The precipitate is collected by centrifugation, washed with deionized water, and then freeze-dried. The resulting solid is Dex-b-PZLL.

[0019] In a preferred embodiment, the anhydrous organic solvent is anhydrous dimethyl sulfoxide.

[0020] In a preferred embodiment, the molar ratio of Dex-NH2 to N6-benzyloxycarbonyl-L-lysine cyclic anhydride is 1:(10-30).

[0021] In one implementation scheme, step c) includes the following specific operations:

[0022] Under nitrogen protection, folic acid (FA), N'N-dicyclohexylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) are stirred in an anhydrous organic solvent at 20-40°C in the dark for 20-40 minutes, and then Dex-b-PZLL is added and reacted at 20-40°C in the dark. After the reaction, the reaction solution is first dialyzed against phosphate buffered saline (PBS) with a pH of 7.4, and then dialyzed against deionized water. The collected dialyzate is then centrifuged, and the collected precipitate is freeze-dried to obtain FA-Dex-b-PZLL.

[0023] In a preferred embodiment, the anhydrous organic solvent is anhydrous dimethyl sulfoxide.

[0024] In a preferred embodiment, the molar ratio of folic acid (FA) to N'N-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) is 1:(1-3):(1-3), and the mass ratio of Dex-b-PZLL to folic acid (FA) is (2-10):1.

[0025] The polymer with pH responsiveness and active targeting of the present invention can be used to prepare drug-loaded polymer micelles encapsulating antitumor drugs.

[0026] In one embodiment, the preparation of the anti-tumor drug-loaded polymer micelles comprises the following steps:

[0027] The pH-responsive and actively targeted polymer FA-Dex-b-PZLL of the present invention and an anti-tumor drug are first dissolved in an organic solvent miscible with water, and then the organic solvent is removed by dialysis to obtain the obtained product.

[0028] In one embodiment, the polymer with pH responsiveness and active targeting properties described in the present invention is used to prepare drug-loaded polymer micelles encapsulating letrozole.

[0029] In one embodiment, the preparation of the drug-loaded polymer micelles containing letrozole comprises the following steps:

[0030] At room temperature, the pH-responsive and actively targeted polymer FA-Dex-b-PZLL and letrozole described in the present invention are respectively dissolved in a water-miscible organic solvent and ultrasonically mixed to achieve uniform mixing. The mixed solution is then slowly added dropwise to deionized water under stirring. After the addition is completed, stirring is continued for 1 to 4 hours. Finally, the solution is transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500Da and dialyzed to remove the organic solvent. After the dialysis is completed, the resulting dialysate is filtered through a filter with a pore size of 0.45μm. The resulting filtrate is the drug-loaded polymer micelles encapsulating letrozole.

[0031] In a preferred embodiment, the mass ratio of polymer FA-Dex-b-PZLL to letrozole is (3-5):1.

[0032] Compared with the prior art, the present invention has the following significant beneficial effects:

[0033] The present invention prepares an amphiphilic block polymer by first linking a hydrophilic segment of dextran and a hydrophobic segment of poly(benzyloxycarbonyllysine) via an amide bond. Then, folic acid is grafted onto the hydrophilic end of the amphiphilic block polymer via an esterification reaction. As a result, the polymer of the present invention has both a pH-sensitive amide bond and a folic acid ligand that can specifically recognize folate receptors highly expressed on the surface of tumor cells, achieving both pH responsiveness and active targeting. Furthermore, the polymer can self-assemble in an aqueous medium to form core-shell polymer micelles for use as targeting carriers for anti-tumor drugs. The formed polymer micelles have good biocompatibility and biodegradability. Furthermore, by selecting poly(benzyloxycarbonyllysine) as the hydrophobic segment and an amide bond as the pH-sensitive bond, the present invention not only avoids the risk of premature release of the drug-loaded nanomicelles, but also achieves high substitution of folic acid (substitution degree can be as high as 13.56%). The anti-breast cancer drug letrozole can be loaded at a maximum of 22.65% and an encapsulation efficiency of 91.37%, demonstrating excellent drug loading performance. Therefore, compared with the prior art, the present invention not only produces significant progress, but also has significant value in achieving active targeted release of anti-tumor drugs to reduce drug toxicity and improve targeted therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention provides a synthetic route for preparing a polymer with pH responsiveness and active targeting.

[0035] Figure 2 This is a schematic diagram showing that the polymer with pH responsiveness and active targeting according to the present invention can be used as a drug-loaded polymer micelle;

[0036] Figure 3 is the infrared spectra of Dex, Dex-NH2, Lys(Z)-NCA, Dex-b-PZLL, FA and FA-Dex-b-PZLL in Example 1;

[0037] Figure 4a is the Dex in Example 1 1 H-NMR spectrum;

[0038] Figure 4b is the Dex-NH2 in Example 1 1 H-NMR spectrum;

[0039] Figure 4c is the Dex-b-PZLL in Example 1 1 H-NMR spectrum;

[0040] Figure 4d is the FA-Dex-b-PZLL in Example 1 1 H-NMR spectrum;

[0041] Figure 5 The polymer FA-Dex-b-PZLL prepared in Example 1-3 10 、FA-Dex-b-PZLL 20 and FA-Dex-b-PZLL 30 GPC elution curve;

[0042] Figure 6 is the critical micelle concentration curve of the blank polymer micelles prepared in Example 4;

[0043] Figure 7 is a particle size distribution diagram of the blank polymer micelles prepared in Example 4;

[0044] Figure 8 is a scanning electron microscope (SEM) photograph of the blank polymer micelle prepared in Example 4;

[0045] Figure 9 is a particle size distribution diagram of the drug-loaded polymer micelles prepared in Example 5;

[0046] Figure 10 is a scanning electron microscope (SEM) photograph of the drug-loaded polymer micelles prepared in Example 5;

[0047] Figure 11 is the standard curve of absorbance (Abs) versus concentration (C) of letrozole described in Example 5;

[0048] Figure 12 The drug-loaded polymer micelle Letrozole / FA-Dex-b-PZLL prepared in Example 5 10 Drug release curves at pH = 7.4, pH = 6.5, and pH = 4.5;

[0049] Figure 13 is a graph showing the biocompatibility test results of the blank polymer micelles prepared in Example 4;

[0050] Figure 14 The drug-loaded polymer micelle Letrozole / FA-Dex-b-PZLL prepared in Example 5 10 Diagram of the inhibitory effect on breast cancer cells. DETAILED DESCRIPTION

[0051] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The reagents, methods, and equipment used in the following examples, unless otherwise specified, are conventional reagents, methods, and equipment in the art and can be obtained by commercial purchase.

[0052] The present invention provides a polymer with pH responsiveness and active targeting, which has the following chemical structure:

[0053] ;

[0054] In the formula, the value of n is 37, and the value range of m is 20 to 43.

[0055] Figure 1 The present invention provides a synthetic route for preparing the polymer having pH responsiveness and active targeting, which comprises Figure 1 As shown, the preparation method of the polymer of the present invention may include the following specific steps:

[0056] a) Dex-NH2 was synthesized by reductive amination reaction of the terminal aldehyde group of dextran with ethylenediamine;

[0057] b) Using the amino group of Dex-NH2 as an initiator, the ring-opening polymerization of N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) was induced to synthesize the amphiphilic block polymer Dex-b-PZLL;

[0058] c) Folic acid (FA) is grafted onto the hydrophilic end (side chain hydroxyl group of the dextran chain segment) of an amphiphilic block polymer (Dex-b-PZLL) through an esterification reaction to synthesize an amphiphilic block polymer FA-Dex-b-PZLL with a folic acid ligand.

[0059] Depend on Figure 2As shown, the polymer described herein comprises a hydrophobic segment, a hydrophilic segment, and a folate ligand. The hydrophilic segment is dextran (Dex), and the hydrophobic segment is polybenzyloxycarbonyllysine (PZLL). The hydrophilic and hydrophobic segments are connected by an amide bond. The folate ligand is grafted onto the hydrophilic end of the amphiphilic block polymer (Dex-b-PZLL) (the hydroxyl groups of the dextran segment). This polymer self-assembles in water to form polymer micelles, which can be used to encapsulate anti-tumor drugs. The resulting drug-loaded polymer micelles maintain good integrity during blood circulation and can utilize the active targeting properties of the folate ligand to enter tumor cells. Once inside the tumor cells, the amide bonds cleave in the slightly acidic environment of the tumor cells, disrupting the core-shell structure of the drug-loaded polymer micelles and releasing the encapsulated anti-tumor drug, achieving a targeted therapeutic effect. It can be seen that using the polymer micelles of the present invention as carriers of anti-tumor drugs can not only reduce the toxic side effects of anti-tumor drugs on normal tissues and normal cells, but also improve the targeted therapeutic effect on tumor cells, and has significant application value.

[0060] Example 1: Preparation of the polymer FA-Dex-b-PZLL of the present invention

[0061] a) Synthesis of Dex-NH2

[0062] 3 g of dextran (Dex, 0.5 mmol) and 30 mL of dimethyl sulfoxide (DMSO) were added to a 100 mL round-bottom flask and stirred thoroughly to dissolve. 1.32 g of ethylenediamine (21.96 mmol) was then added, and a condensation reaction was carried out at 60°C. After monitoring the completion of the condensation reaction (approximately 48 hours), 0.3 g of sodium cyanoborohydride (NaBH3CN, 4.77 mmol) was added to the reaction system as a reducing agent and the reduction reaction was continued at 60°C. After monitoring the completion of the reduction reaction (approximately 48 hours), the reaction was terminated, the reaction solution was poured into methanol for precipitation, and the solid was collected by filtration. The collected solid was then purified using water as the good solvent and methanol as the poor solvent. The purified product was then dissolved in deionized water and dialyzed in deionized water using a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours. The retentate after dialysis was freeze-dried to obtain 2.37 g of a fluffy white solid, which was Dex-NH2.

[0063] b) Synthesis of amphiphilic block polymer Dex-b-PZLL

[0064] Under nitrogen protection, Dex-NH2 (0.45 g, 0.1 mmol) and N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) (0.306 g, 1 mmol) were dissolved in 5 mL of anhydrous DMSO. The molar ratio of the initiator Dex-NH2 to Lys(Z)-NCA was 1:10. The reaction was then carried out at room temperature. After the reaction was completed (approximately 72 hours), the reaction solution was poured into methanol for precipitation. The precipitate was collected by centrifugation, washed three times with deionized water, and then freeze-dried to obtain 0.34 g of a white solid, which is Dex-b-PZLL.

[0065] c) Synthetic polymer FA-Dex-b-PZLL

[0066] Under nitrogen protection, 0.03 g of FA (0.068 mmol), 0.028 g of N'N-dicyclohexylcarbodiimide (DCC, 0.136 mmol), 0.017 g of 4-dimethylaminopyridine (DMAP, 0.136 mmol) and 20 mL of anhydrous DMSO were added to a 100 mL three-necked flask, and then stirred at 30 ° C in the dark for 30 minutes to activate the carboxyl group of FA, and then 0.3 g of Dex-b-PZLL was added. FA, DCC and DMAP were mixed. The molar ratio of 1:2:2 was 1:2:2, and the reaction was continued with stirring at 30°C in the dark. After the reaction was completed (about 24 hours), the reaction was terminated, and the reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da. It was first dialyzed in phosphate buffered saline (PBS) with a pH of 7.4, and then dialyzed in deionized water. The collected dialysate was centrifuged (10000 rpm, 10 min, room temperature), the supernatant was discarded, and the precipitate collected by centrifugation was freeze-dried to obtain 0.23 g of yellow solid, which was FA-Dex-b-PZLL.

[0067] Figure 3 The infrared spectra of Dex, Dex-NH2, Lys(Z)-NCA, Dex-b-PZLL, FA and FA-Dex-b-PZLL in this embodiment, wherein: (a) is a comparison of the infrared spectra of the raw materials dextran (Dex) and N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) used in the embodiment and the synthesized intermediates Dex-NH2 and Dex-b-PZLL; (b) is a comparison of the infrared spectra of the FA-Dex-b-PZLL polymer prepared in the embodiment and Dex-b-PZLL and folic acid (FA); (c) is a partial enlarged view of (b); Figure 3 As shown in the infrared spectrum of the raw material Lys(Z)-NCA, 1856 cm -1 and 1775cm -1 The two C=O stretching vibration absorption peaks of cyclic anhydride; 749 cm-1 and 695 cm -1 is the out-of-plane bending vibration peak of the CH on the benzene ring; compared with the characteristic peaks of the raw materials Dex and Lys(Z)-NCA, it can be found in the infrared spectrum of Dex-b-PZLL that 1856cm -1 and 1775 cm -1 The characteristic peak of cyclic anhydride at 900-650 cm -1 The characteristic peak of benzene ring appeared, and at 1627 cm -1 and 1535 cm -1 New characteristic peaks appeared at 1570 cm-1, which belonged to the amide I and amide II peaks on the polyamino acid peptide chain, proving the successful preparation of Dex-b-PZLL block copolymer. Compared with the infrared absorption peak of Dex-b-PZLL, FA-Dex-b-PZLL showed a strong infrared absorption peak at 1570 cm-1. -1 、1311 cm -1 、1193 cm -1 、840 cm -1 、640 cm -1 New characteristic peaks appeared at the positions of the IR spectra, which were consistent with the infrared characteristic peaks of folic acid, proving the successful grafting of folic acid.

[0068] Figure 4a is the Dex in this embodiment 1 H-NMR spectrum, Figure 4b is the Dex-NH2 in this embodiment 1 H-NMR spectrum, Figure 4c is the Dex-b-PZLL of this embodiment 1 H-NMR spectrum, Figure 4d is the FA-Dex-b-PZLL of this embodiment 1 H-NMR diagram; by comparison Figure 4a and Figure 4b Visible: In Figure 4b Dex-NH2 1 In the H-NMR spectrum, the characteristic peak of the terminal proton of the terminal glucose pyranose unit of dextran completely disappeared, indicating that the terminal group functionalization of dextran with ethylenediamine was successful. Figure 4c The amphiphilic block copolymer Dex-b-PZLL shown 1In the H NMR diagram: 7.31 ppm is the proton absorption peak on the benzyl ring; 4.96 ppm is the methylene proton absorption peak on the benzyloxycarbonyl group; 3.76 ppm is the methine proton absorption peak of the hydrophobic block polylysine; chemical shifts near 1-1.68 ppm and at 2.94 ppm are the methylene proton absorption peaks of the hydrophobic block polylysine; some absorption peaks with chemical shifts between 3-5 ppm are proton absorption peaks on dextran; Figure 4c The amphiphilic block copolymer Dex-b-PZLL shown 1 Compared with the H NMR spectrum, Figure 4d FA-Dex-b-PZLL shown 1 In the H-NMR spectrum, three new proton signal peaks appeared at δ = 8.62 ppm, 6.63 ppm, and 4.54 ppm, which were attributed to the pyrazine and NH proton signal peaks of folic acid. These new proton signal peaks proved that folic acid had been successfully grafted.

[0069] By analyzing the 1 The corresponding proton hydrogen integration in the H-NMR spectrum is calculated according to the following formula:

[0070]

[0071] Where, I A is the integral of the CH protons on the pyrazine ring of folic acid, I 2-6 is the integral of protons between 3.0 and 3.7 ppm in dextran;

[0072] The degree of substitution of folic acid (DS FA );

[0073] According to calculations, the degree of substitution of folic acid in this embodiment is approximately 7.39%, that is, each glucan molecular chain contains approximately 1.08 folic acid molecules.

[0074] Example 2: Preparation of the polymer FA-Dex-b-PZLL of the present invention

[0075] a) Synthesis of Dex-NH2

[0076] 3 g of dextran (Dex, 0.5 mmol) and 30 mL of dimethyl sulfoxide (DMSO) were added to a 100 mL round-bottom flask and stirred thoroughly to dissolve. 1.98 g of ethylenediamine (32.95 mmol) was then added, and a condensation reaction was carried out at 60°C. After monitoring the completion of the condensation reaction (approximately 48 hours), 0.45 g of sodium cyanoborohydride (NaBH3CN, 7.16 mmol) was added to the reaction system as a reducing agent and the reduction reaction was continued at 60°C. After monitoring the completion of the reduction reaction (approximately 48 hours), the reaction was terminated, the reaction solution was poured into methanol for precipitation, and the solid was collected by filtration. The collected solid was then purified using water as the good solvent and methanol as the poor solvent. The purified product was then dissolved in deionized water and dialyzed in deionized water using a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours. The retentate after dialysis was freeze-dried to obtain 2.58 g of a fluffy white solid, which is Dex-NH2.

[0077] b) Synthesis of amphiphilic block polymer Dex-b-PZLL

[0078] Under nitrogen protection, Dex-NH2 (0.45 g, 0.1 mmol) and N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) (0.612 g, 2 mmol) were dissolved in 5 mL of anhydrous DMSO. The molar ratio of the initiator Dex-NH2 to Lys(Z)-NCA was 1:20. The reaction was then carried out at room temperature. After the reaction was completed (approximately 72 hours), the reaction solution was poured into methanol for precipitation. The precipitate was collected by centrifugation, washed three times with deionized water, and then freeze-dried to obtain 0.56 g of a white solid, which is Dex-b-PZLL.

[0079] c) Synthetic polymer FA-Dex-b-PZLL

[0080] Under nitrogen protection, 0.06 g of FA (0.136 mmol), 0.056 g of N'N-dicyclohexylcarbodiimide (DCC, 0.272 mmol), 0.034 g of 4-dimethylaminopyridine (DMAP, 0.272 mmol) and 20 mL of anhydrous DMSO were added to a 100 mL three-necked flask, and then stirred at 30 ° C in the dark for 30 minutes to activate the carboxyl group of FA, and then 0.3 g of Dex-b-PZLL was added. FA, DCC and DMAP were mixed. The molar ratio of 1:2:2 was 1:2:2, and the reaction was continued with stirring at 30°C in the dark. After the reaction was completed (about 24 hours), the reaction was terminated, and the reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da. It was first dialyzed in phosphate buffered saline (PBS) with a pH of 7.4, and then dialyzed in deionized water. The collected dialysate was centrifuged (10000 rpm, 10 min, room temperature), the supernatant was discarded, and the precipitate collected by centrifugation was freeze-dried to obtain 0.29 g of yellow solid, which was FA-Dex-b-PZLL.

[0081] By analyzing the FA-Dex-b-PZLL polymer obtained in this example 1 Based on the H-NMR spectrum and the calculation formula described in Example 1, it was calculated that the degree of substitution of folic acid in this example was approximately 10.07%, that is, each glucan molecular chain contained approximately 1.37 folic acid molecules.

[0082] Example 3: Preparation of the polymer FA-Dex-b-PZLL of the present invention

[0083] a) Synthesis of Dex-NH2

[0084] 3 g of dextran (Dex, 0.5 mmol) and 30 mL of dimethyl sulfoxide (DMSO) were added to a 100 mL round-bottom flask and stirred thoroughly to dissolve. 3.3 g of ethylenediamine (54.91 mmol) was then added, and the condensation reaction was carried out at 60°C. After monitoring the completion of the condensation reaction (approximately 48 hours), 0.75 g of sodium cyanoborohydride (NaBH3CN, 11.93 mmol) was added to the reaction system as a reducing agent and the reduction reaction was continued at 60°C. After monitoring the completion of the reduction reaction (approximately 48 hours), the reaction was terminated, the reaction solution was poured into methanol for precipitation, and the solid was collected by filtration. The collected solid was then purified using water as the good solvent and methanol as the poor solvent. The purified product was then dissolved in deionized water and dialyzed in deionized water using a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours. The retentate after dialysis was freeze-dried to obtain 2.79 g of a fluffy white solid, which is Dex-NH2.

[0085] b) Synthesis of amphiphilic block polymer Dex-b-PZLL

[0086] Under nitrogen protection, Dex-NH2 (0.45 g, 0.1 mmol) and N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) (0.918 g, 3 mmol) were dissolved in 5 mL of anhydrous DMSO. The molar ratio of the initiator Dex-NH2 to Lys(Z)-NCA was 1:30. The reaction was then carried out at room temperature. After the reaction was completed (approximately 72 hours), the reaction solution was poured into methanol for precipitation. The precipitate was collected by centrifugation, washed three times with deionized water, and then freeze-dried to obtain 0.71 g of a white solid, which is Dex-b-PZLL.

[0087] c) Synthetic polymer FA-Dex-b-PZLL

[0088] Under nitrogen protection, 0.15 g of FA (0.34 mmol), 0.14 g of N'N-dicyclohexylcarbodiimide (DCC, 0.68 mmol), 0.085 g of 4-dimethylaminopyridine (DMAP, 0.68 mmol) and 20 mL of anhydrous DMSO were added to a 100 mL three-necked flask, and then stirred at 30 ° C in the dark for 30 minutes to activate the carboxyl group of FA, and then 0.3 g of Dex-b-PZLL was added. The molar ratio of FA, DCC and DMAP was 0. The mixture was stirred at a 30°C dark-proof temperature with a 1:2:2 ratio. After the reaction was completed (approximately 24 hours), the reaction was terminated. The reaction solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da in phosphate buffered saline (PBS) at pH = 7.4, and then dialyzed in deionized water. The collected dialysate was centrifuged (10000 rpm, 10 min, room temperature), the supernatant was discarded, and the precipitate collected by centrifugation was freeze-dried to obtain 0.34 g of yellow solid, which was FA-Dex-b-PZLL.

[0089] By analyzing the FA-Dex-b-PZLL polymer obtained in this example 1 Based on the H-NMR spectrum and the calculation formula described in Example 1, it was calculated that the degree of substitution of folic acid in this example was approximately 13.56%, that is, each glucan molecular chain contained approximately 1.84 folic acid molecules.

[0090] In Examples 1-3, the amphiphilic block polymers with different hydrophobic chain ratios were prepared by changing the molar ratio of the initiator Dex-NH2 to Lys(Z)-NCA to 1:10, 1:20, and 1:30. Therefore, the pH-responsive and active targeting polymers prepared in Examples 1-3 were respectively designated as FA-Dex-b-PZLL. 10 (Example 1), FA-Dex-b-PZLL 20 (Example 2), FA-Dex-b-PZLL 30(Example 3); Figure 5 The polymer FA-Dex-b-PZLL prepared in Examples 1-3 10 、FA-Dex-b-PZLL 20 and FA-Dex-b-PZLL 30 The GPC elution curve of Figure 5 As shown, all three GPC curves show a unimodal distribution and a narrow molecular weight distribution, indicating that no unreacted raw materials were detected in the polymer and the reaction process was relatively complete. According to the GPC test principle, the polymer with a larger molecular weight flows out first, so the molecular weight is larger on the left side of the figure. The GPC curves show that Examples 1-3 have successfully prepared the polymers with pH responsiveness and active targeting according to the present invention.

[0091] Example 4: Preparation of FA-Dex-b-PZLL blank polymer micelles

[0092] FA-Dex-b-PZLL prepared in Example 1 10 FA-Dex-b-PZLL was prepared by dialysis using polymer as raw material. 10 Blank polymer micelles, the specific operation is:

[0093] 50 mg of polymer FA-Dex-b-PZLL 10 The polymer was dissolved in 500 μL DMSO solution and evenly dispersed in DMSO by ultrasound. The solution was then slowly added dropwise to 20 mL deionized water while stirring at room temperature and continued to stir for 4 h. Finally, the solution was transferred to a dialysis bag (MWCO = 3500 Da) and dialyzed with deionized water for 48 h. After the dialysis, the dialysate was filtered with a filter with a pore size of 0.45 μm. The filtrate was the blank polymer micelle solution. The blank polymer micelle solution was freeze-dried to obtain a blank polymer micelle solid.

[0094] Using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a hydrophobic dye, the critical micelle concentration (CMC) of blank micelles in water was determined by the spectral dye solubilization method. The specific operation is as follows:

[0095] First, a blank micellar aqueous dispersion stock solution with a concentration of 1 mg / mL was prepared. Then, 12 samples with a concentration gradient (0.001-0.5 mg / mL) were prepared by serial dilution. Then, 40 μL of a 0.4 mmol / L 1,6-diphenyl-1,3,5-hexatriene (DPH) methanol solution was added to 4 mL of the micellar dispersion with increasing concentrations to make the final DPH concentration reach 4 × 10 -6mol / L; the sample was then incubated at room temperature in the dark for 24 hours to ensure that DPH was completely distributed into the hydrophobic core of the micelles; finally, a TU-1901 UV-visible spectrophotometer was used to collect the UV-visible absorption spectrum of DPH in the range of 300-500 nm, and the maximum absorbance value at a wavelength of 350 nm was recorded. The logarithm of the blank micelle concentration was used as the horizontal axis and the UV absorbance of DPH at 350 nm was used as the vertical axis to plot the graph. The inflection point when the UV absorbance suddenly changed was the CMC value of the polymer.

[0096] Figure 6 The blank polymer micelle FA-Dex-b-PZLL prepared in this example 10 The critical micelle concentration curve is given by Figure 6 As shown: blank polymer micelle FA-Dex-b-PZLL prepared in this example 10 The critical micelle concentration (CMC) is 0.04 mg / mL, and it has excellent stability.

[0097] Figure 7 is the particle size distribution diagram of the blank polymer micelles prepared in this example, Figure 7 As shown, the average particle size of the prepared blank polymer micelles is less than 100 nm, and the particle size is unimodal and the PDI is very low; Figure 8 is a scanning electron microscope (SEM) photograph of the blank polymer micelles prepared in this example. Figure 8 As shown, the prepared blank polymer micelles present a uniform spherical structure in aqueous solution, have clear boundaries, and are well dispersed.

[0098] Example 5: Preparation of drug-loaded polymer micelles containing anticancer drugs

[0099] FA-Dex-b-PZLL prepared in Example 1 10 The polymer was used as a carrier. 30 mg of polymer FA-Dex-b-PZLL was first added. 10 Dissolved in 500 μL DMSO solution and 10 mg of anticancer drug letrozole dissolved in 200 μL acetonitrile, letrozole and polymer FA-Dex-b-PZLL 10 The mass ratio of the two was 1:3, and then the two were mixed evenly by ultrasound. The mixed solution was then slowly added dropwise to 20 mL of deionized water at room temperature while stirring. After the addition was completed, stirring was continued for 4 h. Finally, the solution was transferred to a dialysis bag (MWCO = 3500 Da) and dialyzed with deionized water for 48 h. After the dialysis was completed, the dialysate was filtered through a filter with a pore size of 0.45 μm. The filtrate obtained was the drug-loaded polymer micelles encapsulating the anticancer drug letrozole, which is abbreviated as:

[0100] Letrozole / FA-Dex-b-PZLL 10 , and freeze-dried to obtain drug-loaded polymer micelle solid.

[0101] Figure 9 is the particle size distribution diagram of the drug-loaded polymer micelles prepared in this example, Figure 9 As shown in the figure, the average particle size of the prepared drug-loaded polymer micelles is larger than that of the blank polymer micelles, but is still nanoparticles below 200 nm, and the particle size still presents a unimodal distribution and the PDI is very low; Figure 10 is a scanning electron microscope (SEM) photograph of the drug-loaded polymer micelles prepared in this example. Figure 10 As shown, the prepared drug-loaded polymer micelles still present a uniform spherical structure in aqueous solution, have clear boundaries, and good dispersion.

[0102] In order to determine the content of letrozole in the drug-loaded polymer micelles, a standard curve of letrozole concentration (C) and absorbance (Abs, measured at a wavelength of 240 nm) was first drawn, as shown in Figure 11 shown.

[0103] Next, 10 mg of the drug-loaded polymer micelle solid prepared in this example was weighed, 1 mL of DMSO was added, and ultrasonic treatment was performed for 10 min. Then, 5 mL of physiological phosphate buffer (PBS, pH = 7.4) was added. After removing the organic solvent by bubbling nitrogen, 2 mL of PBS solution was added and vigorously mixed by ultrasonication for 30 seconds. Finally, the solution was filtered through a 0.45 μm filter. Finally, the absorbance (Abs) of the filtrate was measured at a wavelength of 240 nm using a UV-visible spectrophotometer. The absorbance (Abs) was taken into account. Figure 11 The mass of letrozole drug entrapped in the drug-loaded polymer micelles was calculated, and the corresponding drug loading and encapsulation efficiency were calculated by the following formula:

[0104]

[0105] The drug loading capacity of the drug-loaded polymer micelles prepared in this example was calculated to be 15.97% and the encapsulation efficiency was 82.20% by the above method.

[0106] Example 6: Preparation of drug-loaded polymer micelles containing anticancer drugs

[0107] FA-Dex-b-PZLL prepared in Example 1 10 The polymer was used as a carrier. 40 mg of the polymer FA-Dex-b-PZLL was first added. 10 Dissolved in 500 μL DMSO solution and 10 mg of anticancer drug letrozole dissolved in 200 μL acetonitrile, letrozole and polymer FA-Dex-b-PZLL 10The mass ratio of the two is 1:4, and then the two are mixed evenly by ultrasound. The mixed solution is then slowly added dropwise into 20 mL of deionized water while stirring at room temperature. After the addition is completed, stirring is continued for 4 h. Finally, the solution is transferred to a dialysis bag (MWCO=3500Da) and dialyzed with deionized water for 48 h. After the dialysis is completed, the dialysate is filtered through a filter with a pore size of 0.45 μm. The filtrate obtained is the drug-loaded polymer micelles, which is freeze-dried to obtain drug-loaded polymer micelle solids.

[0108] Using the calculation method described in Example 5, it was calculated that the drug loading of the drug-loaded polymer micelles prepared in this example was 18.23% and the encapsulation efficiency was 88.45%.

[0109] Example 7: Preparation of drug-loaded polymer micelles containing anticancer drugs

[0110] FA-Dex-b-PZLL prepared in Example 1 10 The polymer was used as a carrier. 50 mg of the polymer FA-Dex-b-PZLL was first added. 10 Dissolved in 500 μL DMSO solution and 10 mg of anticancer drug letrozole dissolved in 200 μL acetonitrile, letrozole and polymer FA-Dex-b-PZLL 10 The mass ratio of the drug and the polymer was 1:5, and then the two were mixed evenly by ultrasound. The mixed solution was then slowly added dropwise into 20 mL of deionized water while stirring at room temperature. After the addition was completed, stirring was continued for 4 h. Finally, the solution was transferred to a dialysis bag (MWCO = 3500 Da) and dialyzed with deionized water for 48 h. After the dialysis was completed, the dialysate was filtered through a filter with a pore size of 0.45 μm. The filtrate obtained was the drug-loaded polymer micelles, which was freeze-dried to obtain the drug-loaded polymer micelle solid.

[0111] Using the calculation method described in Example 5, it was calculated that the drug loading of the drug-loaded polymer micelles prepared in this example was 22.65% and the encapsulation efficiency was 91.37%.

[0112] Example 8: In vitro release experiment of drug-loaded polymer micelles

[0113] This example simulates the drug release behavior of the drug-loaded polymer micelle Letrozole / FA-Dex-b-PZLL described in the present invention under the normal physiological environment of the human body (PBS, pH 7.4), the external microenvironment of the tumor (pH 6.5), and the lysosomal environment of tumor cells (pH 4.5). The specific experimental process is as follows:

[0114] 3 mL of the drug-loaded polymer micelle solution prepared in Example 5 was respectively taken and placed in a dialysis bag, and then immersed in 47 mL of PBS solution with a pH value of 4.5, 6.5, and 7.4, respectively. The mixture was shaken in a constant temperature shaker at 37°C (200 rpm), and 3 mL of dissolution medium was quantitatively taken at the specified time points (1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 12 h, 14 h, 24 h, 30 h, 36 h, 48 h, 54 h, 60 h, 72 h). After sampling, an isothermal and equal volume of PBS solution with the corresponding pH was immediately added. The absorbance of the extracted solution was measured using a double-beam UV-visible spectrophotometer, and the amount of letrozole released from the drug-loaded polymer micelles Letrozole / FA-Dex-b-PZLL at each time point was calculated according to the letrozole standard curve described in Example 5, thereby plotting the in vitro release curve of the drug-loaded polymer micelles.

[0115] The cumulative release of letrozole was calculated using the following formula:

[0116]

[0117] Where: V e = 3 mL (buffer replacement volume), V0 = 50 mL (buffer total volume), C i is the concentration of letrozole in the sample at the time of sampling, m drug is the mass of letrozole in the drug-loaded polymer micelles, and n is the total number of samplings.

[0118] Figure 12 The drug-loaded polymer micelle Letrozole / FA-Dex-b-PZLL prepared in Example 5 10 The drug release curves at pH = 7.4, pH = 6.5 and pH = 4.5 are shown in Figure 2. Figure 12 As shown, the drug-loaded polymer micelles prepared in the present invention are sensitive to acidity, and the release rate at pH = 4.5 is significantly greater than the release rate under the normal physiological environment of the human body at pH = 7.4.

[0119] Example 9: Biocompatibility test of blank polymer micelles

[0120] Human breast cancer cells (MDA-MB-231) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum albumin in a cell culture incubator at 37°C and 5% CO2. Mouse fibroblasts (L929) were cultured in DMEM medium containing 10% fetal bovine serum albumin and 1% streptomycin-penicillin in a cell culture incubator at 37°C and 5% CO2.

[0121] When the monolayer coverage of MDA-MB-231 cells and L929 cells reached 80%, they were digested with appropriate amounts of trypsin and the digested MDA-MB-231 cells and L929 cells were plated at 1×10 4 / well were seeded in 96-well plates, and then all 96-well plates were placed in a constant temperature incubator at 37°C containing 5% CO2 and incubated for 24 hours; next, the culture medium was removed, and 100 μL of fresh culture medium or culture medium containing different concentrations of blank polymer micelles (prepared in Example 4) were added (the concentrations of blank polymer micelles were 0 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL, respectively), with 7 replicates for each concentration, and incubated for another 48 hours; the culture medium was removed, each well was washed twice with PBS solution, and then 100 μL of CCK8 solution (i.e., a solution obtained by diluting 10 times 10 μL of CCK8 stock solution with culture medium) was added to each well, and the cells were incubated in the incubator for another 1 hour; finally, the optical density (OD) of each well was measured at 450 nm using a microplate reader, and the cell viability was calculated according to the following formula:

[0122] ;

[0123] Where: A sample is the absorbance value of the sample well, A blank is the absorbance value of the blank well (containing neither cells nor polymer micelles), A control is the absorbance value of the control group (containing cells but not polymer micelles).

[0124] Depend on Figure 13 The results show that even when the concentration of the blank polymer micelles reached the maximum (200 μg / mL), the survival rates of normal cells (L929 cells) and cancer cells (MDA-MB-231 cells) were still above 90%, indicating that the polymer micelles of the present invention had no inhibitory effect on cell growth. In other words, the polymer micelles of the present invention were non-cytotoxic and had good biocompatibility.

[0125] Example 10: Inhibitory effect of drug-loaded polymer micelles on breast cancer cells

[0126] The drug-loaded polymer micelle solid freeze-dried in Example 5 was reconstituted with physiological saline to prepare a storage solution of a certain concentration, and a free letrozole storage solution of the same drug concentration was prepared; human breast cancer cells (MDA-MB-231) were cultured at a density of 1×10 4The cells were seeded at a density of 100 μg / well in a 96-well plate and incubated in a culture incubator for 24 h. Then, the cells were exposed to different concentrations of free letrozole stock solution and drug-loaded polymer micelles Letrozole / FA-Dex-b-PZLL. 10 After incubation for 48 h in the storage solution, the supernatant was discarded, and the cells were washed with PBS. The cell viability was subsequently detected using the standard CCK-8 method.

[0127] The experimental results are as follows Figure 14 As shown by Figure 14 As shown: With the drug-loaded polymer micelles Letrozole / FA-Dex-b-PZLL 10 With the increase of the concentration of free letrozole, the cell survival rate decreased significantly. However, in comparison, the inhibitory effect of free letrozole on MDA-MB-231 cells was significantly higher than that of drug-loaded polymer micelles Letrozole / FA-Dex-b-PZLL. 10 This may be because it takes a certain amount of time to release letrozole from the drug-loaded polymer micelles compared to free letrozole, which is consistent with the in vitro drug release behavior. Although free letrozole has a significantly higher inhibitory effect on MDA-MB-231 cells than drug-loaded polymer micelles in in vitro experiments, free letrozole usually faces problems such as rapid drug degradation, systemic toxicity, and poor solubility in vivo. The polymer micelles of the present invention can simultaneously achieve pH responsiveness and active targeting, and have good biocompatibility and biodegradability. Using the polymer micelles of the present invention as carriers of anti-tumor drugs can significantly improve the targeting of tumor cells, effectively solve the toxic and side effects on normal tissues and cells caused by the systemic distribution of free drugs, and the problem that the concentration of drugs released at the tumor site or the accumulation of drugs does not reach an effective therapeutic concentration. It has significant application value in improving the active targeted release of anti-tumor drugs and improving the targeted therapeutic effect.

[0128] Finally, it should be pointed out that the above are only some preferred embodiments of the present invention and should not be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above contents of the present invention fall within the scope of protection of the present invention.

Claims

1. A polymer with pH responsiveness and active targeting, characterized in that: It has the following chemical structure: ; In the formula, the value of n is 37, and the value range of m is 20 to 43.

2. A method for preparing the polymer having pH responsiveness and active targeting according to claim 1, characterized in that: First, the hydrophilic segment dextran and the hydrophobic segment polybenzyloxycarbonyllysine are connected through an amide bond to prepare an amphiphilic block polymer, and then folic acid is grafted onto the hydrophilic end of the amphiphilic block polymer through an esterification reaction.

3. The preparation method according to claim 2, characterized in that The steps include: a) Dex-NH2 was synthesized by reductive amination of the terminal end of dextran (Dex); b) Using Dex-NH2 as an initiator, ring-opening polymerization of N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) was induced to synthesize the amphiphilic block polymer Dex-b-PZLL; c) Folic acid (FA) was grafted onto the hydrophilic end of an amphiphilic block polymer (Dex-b-PZLL) via an esterification reaction to synthesize the polymer FA-Dex-b-PZLL.

4. The preparation method according to claim 3, characterized in that Step a) includes the following specific operations: First, dextran and an amination reagent are subjected to a condensation reaction in a solvent at 50-70°C. After the condensation reaction is monitored to be completed, a reducing agent is added to the reaction system, and the reduction reaction is continued at 50-70°C. After the reduction reaction is monitored to be completed, the reaction solution is poured into methanol for precipitation, and the solid is collected by filtration. The obtained solid is purified and then freeze-dried to obtain Dex-NH2.

5. The preparation method according to claim 3, characterized in that Step b) includes the following specific operations: Under nitrogen protection, Dex-NH2 and N6-benzyloxycarbonyl-L-lysine cyclic anhydride (Lys(Z)-NCA) are dissolved in an anhydrous organic solvent and then reacted at room temperature. After the reaction is completed, the reaction solution is poured into methanol for precipitation. The precipitate is collected by centrifugation, washed with deionized water, and then freeze-dried. The resulting solid is Dex-b-PZLL.

6. The preparation method according to claim 3, characterized in that Step c) includes the following specific operations: Under nitrogen protection, folic acid (FA), N'N-dicyclohexylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) are stirred in an anhydrous organic solvent at 20-40°C in the dark for 20-40 minutes, and then Dex-b-PZLL is added and reacted at 20-40°C in the dark. After the reaction, the reaction solution is first dialyzed against phosphate buffered saline (PBS) with a pH of 7.4, and then dialyzed against deionized water. The collected dialyzate is then centrifuged, and the collected precipitate is freeze-dried to obtain FA-Dex-b-PZLL.

7. Use of the pH-responsive and active-targeting polymer according to claim 1 in the preparation of drug-loaded polymer micelles for encapsulating antitumor drugs.

8. The use according to claim 7, characterized in that: The preparation of the drug-loaded polymer micelles encapsulating the antitumor drug is firstly dissolving the pH-responsive and actively targeted polymer and the antitumor drug in a water-miscible organic solvent, and then removing the organic solvent by dialysis.

9. Use of the pH-responsive and actively targeted polymer according to claim 1 in the preparation of drug-loaded polymer micelles encapsulating letrozole.

10. The use according to claim 9, characterized in that The preparation of drug-loaded polymer micelles containing letrozole comprises the following steps: At room temperature, the pH-responsive and actively targeted polymer according to claim 1 and letrozole are respectively dissolved in a water-miscible organic solvent and ultrasonically mixed to uniformly mix them; the mixed solution is then slowly added dropwise to deionized water under stirring. After the addition is completed, stirring is continued for 1 to 4 hours; finally, the solution is transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3500Da for dialysis to remove the organic solvent therein. After the dialysis is completed, the obtained dialysate is filtered through a filter with a pore size of 0.45μm, and the obtained filtrate is the drug-loaded polymer micelles encapsulating letrozole.

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