Highly efficient mucous penetrating amphiphilic polymer micelles, and methods of making and using the same

Abstract

The application discloses a high-efficiency mucus-penetrating amphiphilic polymer micelle, and a preparation method thereof, which comprises the following steps: (1) CB-tBu synthesis; (2) synthesis of an NHS-ATRP initiator; (3) synthesis of NHS-PCB-tBu; (4) synthesis of PHABA; (5) synthesis of NHS-Azo; (6) synthesis of NHS-Azo-PCB-tBu; (7) synthesis of DSPE-Azo-PCB; and (8) preparation of the high-efficiency mucus-penetrating amphiphilic polymer micelle. The high-efficiency mucus-penetrating amphiphilic polymer micelle DSPE-Azo-PCB prepared by the application has a stronger mucus-penetrating effect and can efficiently cross the lung mucosal barrier; the micelle has a lower critical micelle concentration, and can effectively improve the structure dissociation problem of micelles caused by a large amount of blood dilution at present, and has huge social and economic benefits.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a high-efficiency mucus-penetrating amphiphilic polymer micelles, a preparation method and application thereof. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD), acute lung injury (ALI) / acute respiratory distress syndrome (ARDS) and lung cancer and other lung diseases are the leading causes of death, thus reflecting the limitations of existing treatment methods. Simple traditional dosage forms have not been able to meet the needs of many disease treatments due to short half-life and poor targeting. In order to solve this problem, nano drug delivery systems have been developed, which have unique advantages such as increasing drug solubility, achieving precise treatment through active / passive targeting, prolonging blood circulation time, and providing opportunities for the development of stimulus-responsive intelligent nano drug delivery systems.

[0003] The current mainstream drug delivery method is still intravenous or oral administration, but it is often difficult to achieve an effective concentration in the lungs. Inhalation therapy locally delivers chemotherapeutic drugs to the lungs, increasing the concentration of drugs retained in the lungs, thereby improving their efficacy and reducing their systemic side effects. In addition, inhalation therapy avoids first-pass metabolism and also increases patient compliance. However, pulmonary inhalation drug delivery for lung disease treatment still faces many obstacles, among which the mucus barrier is the first barrier to entering the lung tissue, and the mucus-cilia clearance is the main defense mechanism of the upper respiratory tract. Secreted mucus traps particles, and coughing or swallowing can remove nanoparticles that have a strong binding ability with mucin in mucus. Mucus itself has high viscoelasticity, so the surface properties of nano-preparations can be adjusted to facilitate mucus penetration. Currently, polyethylene glycol-modified nanoparticles are commonly used to penetrate the mucus layer, but polyethylene glycol materials are not completely biodegradable and can cause immunogenicity in the body, and also reduce cell uptake of nanoparticles.

[0004] Zwitterionic material betaine has been proven to have good biocompatibility and good resistance to non-specific protein adsorption. Unlike polyethylene glycol, which is hydrated only through hydrogen bonds, zwitterionic materials have strong ion-induced hydration. In addition, it has been found that polycarboxyl betaine zwitterionic polymers also have the ability to escape macrophage clearance and can be efficiently taken up by alveolar epithelial cells.

[0005] Polymeric micelles are self-assembled in water from hydrophilic one end and hydrophobic one end of polymer material, which is a microparticle drug delivery system with core-shell structure, and the hydrophobic core can be used as a reservoir for poorly soluble drugs, and the solubility of lipid-soluble drugs can be improved. Compared with small molecule surfactants, polymeric micelles have low critical micelle concentration, good dilution resistance, strong solubilization, good thermodynamic stability and other characteristics, and are generally considered to be one of the most potential drug delivery systems. However, there is no public report so far on the combination of zwitterionic polymers and micelles to solve the problems of difficult penetration of mucus barrier and low targeting. SUMMARY

[0006] In view of the above situation, in order to overcome the defects of the prior art, the purpose of the present application is to provide a high-efficiency mucus-penetrating zwitterionic polymer micelle and its preparation method and application, which can effectively solve the problems of difficult penetration of polymer micelles into mucus barrier and low targeting.

[0007] To achieve the above purpose, the technical solution solved by the present application is a high-efficiency mucus-penetrating zwitterionic polymer micelle, the structure general formula of the micelle is DSPE-Azo-PCB, and the structure formula is as follows:

[0008]

[0009] Among them, DSPE is distearoyl phosphatidyl ethanolamine, Azo is azobenzene bond, and the molecular weight of PCB is 2-5kDa.

[0010] The preparation method of the high-efficiency mucus-penetrating zwitterionic polymer micelle comprises the following steps:

[0011] (1) Synthesis of CB-tBu: 2-(dimethylamino) ethyl methacrylate and tert-butyl bromoacetate are co-dissolved in anhydrous acetonitrile at a molar ratio of 1:1-2, stirred at 50°C under nitrogen protection for 12-24h, then 50-100ml of cold ether is added, white solid is precipitated, separated by suction filtration and vacuum dried for 12-24h to obtain CB-tBu monomer;

[0012] (2) Synthesis of NHS-ATRP initiator: 9.8mmol of N-hydroxysuccinimide is dissolved in dichloromethane, 8.3mmol of 2-bromopropionic acid is added dropwise under ice bath, to obtain a first solution, 8.2mmol of dicyclohexyl carbodiimide is dissolved in anhydrous dichloromethane solution, and the first solution is added dropwise under ice bath, and reacted at room temperature for 12-24h, then filtered twice, the filtrate is rotary evaporated under reduced pressure for 30-60min to remove the solvent, dissolved in ethyl acetate and ultrasonicated for 10-30min, filtered after ultrasonication, the filtrate is rotary evaporated for 30-60min, concentrated, and purified by recrystallization to obtain NHS-ATRP;

[0013] (3) Synthesis of NHS-PCB-tBu: Anhydrous N,N-dimethylformamide was bubbled with nitrogen for 1-2 h, a double-neck flask, a round-bottom flask, a magnetic stirrer and a condenser were dried in an oven at 110 °C for 30-60 min, CuBr was deoxygenated and used as deoxygenated CuBr; CB-tBu monomer and NHS-ATRP initiator were added into a first double-neck flask at a molar ratio of 1:1-1.5, deoxygenated by circulation between nitrogen and vacuum for three times, 2-5 ml of anhydrous N,N-dimethylformamide was added into the first double-neck flask after deoxygenation, deoxygenated again, and a monomer / initiator solution was obtained; 0.49 mmol of deoxygenated CuBr monomer was added into a second double-neck flask, deoxygenated by circulation between nitrogen and vacuum for three times; 0.49 mmol of HMTETA was added into the second double-neck flask, deoxygenated again, 5-10 ml of anhydrous N,N-dimethylformamide was added again for deoxygenation, and the catalyst solution was obtained by stirring for 30-60 min under nitrogen protection; the catalyst solution was then added into the monomer / initiator solution in the first double-neck flask, deoxygenated by circulation between nitrogen and vacuum for three times, heated at 60 °C with a condenser tube refluxing, stirred for 12-24 h under nitrogen protection, 50-100 ml of ether was then added into the reaction mixture for precipitation, and the mixture was placed in a refrigerator for standing, the supernatant was discarded, 5-10 ml of acetone was added at 0 °C, and the precipitate was ultrasonically precipitated for 2-3 times, the supernatant was removed, and the precipitate was dried in vacuum for 12-24 h to obtain NHS-PCB-tBu;

[0014] (5) Synthesis of NHS-Azo: 2.06 mmol of PHABA was dissolved in anhydrous N,N-dimethylformamide; 2.88 mmol of N-hydroxysuccinimide, 2.88 mmol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.412 mmol of 4-dimethylaminopyridine were dissolved in anhydrous N,N-dimethylformamide, the two were mixed and stirred at room temperature for 5-10 h, washed with hydrochloric acid, filtered, washed with water, and dried in vacuum for 12-24 h to obtain NHS-Azo;

[0015] (6) Synthesis of NHS-Azo-PCB-tBu: 0.67 mmol of NHS-PCB-tBu is dissolved in anhydrous N,N-dimethylformamide, and 0.32 mmol of NHS-Azo ester is dissolved in anhydrous N,N-dimethylformamide, and then 0.29 mmol of DMAP is added, and the mixture is stirred at 60 DEG C under nitrogen protection and refluxed for 12-24 h, and then stirred at room temperature for 40-48 h, and then precipitated with 50-100 ml of ether, and washed with ethyl acetate, and dried in vacuum for 12-24 h to obtain NHS-Azo-PCB-tBu;

[0016] (7) Synthesis of DSPE-Azo-PCB: 0.5 mmol of NHS-Azo-PCB-tBu is dissolved in anhydrous N,N-dimethylformamide, and 0.48 mmol of DSPE is dissolved in 45 ml of chloroform, and then 0.9 mmol of triethylamine is added dropwise, and then stirred at room temperature for 5-10 d, and then rotary evaporated at 50 DEG C for 30-60 min, and then precipitated in ether, and then filtered to remove the filtrate, and then resuspended in acetonitrile, and then rotary evaporated at 60 DEG C for 30-60 min, and then 4 ml of TFA is added, and then stirred at room temperature for 4-8 h, and then precipitated in ether, and then filtered, and then dried in vacuum for 12-24 h, and then neutralized with 20 mM of hydroxylamine in PBS at pH = 8 to dissolve the uncoupled PCB, and then ultrafiltered in PBS, and then ultrafiltered twice in water, and then obtained a DSPE-Azo-PCB solution, and then freeze-dried in pure water for 12-24 h to obtain the dried polymer DSPE-Azo-PCB;

[0017] (8) Preparation of high-efficiency mucus-penetrating amphiphilic polymer micelles: the polymer DSPE-Azo-PCB is dissolved in trifluoroethanol, and then rotary evaporated under reduced pressure for 15-20 min to form a dense film layer, and then vacuumed for 0.5-2 h, and then hydrated with deionized water or PBS, and then stirred at room temperature for 10-60 min to obtain high-efficiency mucus-penetrating amphiphilic polymer micelles.

[0018] The high-efficiency mucus-penetrating amphiphilic polymer micelles are used for preparing a targeted drug carrier.

[0019] The high-efficiency mucus-penetrating amphiphilic polymer micelles DSPE-Azo-PCB prepared by the method have stronger mucus-penetrating effect, can efficiently cross the lung mucosal barrier, have lower critical micelle concentration, and can effectively improve the structure dissociation problem of micelles caused by massive dilution of blood, and have huge social and economic benefits. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a synthesis route diagram of the application.

[0021] Figure 2 is a nuclear magnetic resonance spectrum of the polymer micelles DSPE-Azo-PCB of the application.

[0022] Figure 3 is a mucin penetration assay graph of the polymer micelles of the present application.

[0023] Figure 4 is a mucin adsorption assay graph of the polymer micelles of the present application. DETAILED DESCRIPTION

[0024] The specific embodiments of the present application are described in detail below with reference to the accompanying drawings and specific cases.

[0025] EMBODIMENT

[0026] A high-efficiency mucin penetration type amphiphilic polymer micelle, the structural general formula of the micelle is DSPE-Azo-PCB, and the structural formula is:

[0027]

[0028] In the formula, DSPE is distearoyl phosphatidyl ethanolamine, Azo is an azobenzene bond, and the molecular weight of PCB is 2-5 kDa.

[0029] The preparation method of the high-efficiency mucin penetration type amphiphilic polymer micelle, the synthesis route is as shown in Figure 1 The preparation method of the high-efficiency mucin penetration type amphiphilic polymer micelle, the synthesis route is as shown in

[0030] (1) Synthesis of betaine monomer (CB-tBu): 0.032 mol of 2-(dimethylamino) ethyl methacrylate and 0.055 mol of tert-butyl bromoacetate were taken into a 100 mL round-bottom flask, 30 mL of anhydrous acetonitrile was added and ultrasonically mixed, and then stirred at 50°C under nitrogen protection for 24 h. Then 100 mL of cold ether was added, a large amount of white solid was precipitated, and the solid was separated by suction filtration and vacuum dried for 12-24 h to obtain the betaine monomer CB-tBu;

[0031] (2) Synthesis of NHS ester type ATRP initiator (NHS-ATRP): 9.8 mmol of N-hydroxysuccinimide (NHS) and 8.3 mmol of 2-bromopropionic acid were taken into a 500 mL round-bottom flask, 300 mL of anhydrous dichloromethane was added and ultrasonically dissolved to obtain a first solution. The first solution was cooled to 0°C in an ice water bath, and 8.2 mmol of dicyclohexyl carbodiimide (DCC) was added dropwise in 20 mL of anhydrous dichloromethane (DCM), and the reaction was carried out at room temperature for 24 h. The reaction mixture was filtered twice, the filtrate was rotary evaporated under reduced pressure for 50 min to remove the solvent, and then ultrasonically dissolved in ethyl acetate for 20 min to wash away the unremoved impurities. After filtration, the filtrate was rotary evaporated for 50 min and then concentrated. Further purification by recrystallization with isopropyl alcohol was carried out to obtain the NHS ester type ATRP initiator NHS-ATRP;

[0032] (3) Synthesis of NHS-PCB-tBu: Anhydrous DMF was deoxygenated by bubbling nitrogen for 1 h, the double-neck flask, Erlenmeyer flask, magnetic stirrer, and condenser were dried in an oven at 110 °C for 30 min, CuBr was deoxygenated in advance, that is, 50 mL of glacial acetic acid was added to a 100 mL Erlenmeyer flask, 1 g of CuBr was stirred at room temperature for 1 h, and then filtered to separate the residue, and the residual glacial acetic acid was washed away by adding anhydrous ethanol 2-3 times to obtain yellow-white CuBr solid, which was quickly transferred to an Erlenmeyer flask, and deoxygenated CuBr was obtained by pumping vacuum for 2 h under nitrogen protection, and was ready for use; 3.7 mmol of CB-tBu monomer and 0.5 mmol of NHS-ATRP initiator were added to a first double-neck flask, and deoxygenated by circulating between nitrogen and vacuum for 3 times, and then 5 mL of anhydrous N,N-dimethylformamide was added to the first double-neck flask, and deoxygenated again, to obtain a monomer / initiator solution; 0.49 mmol of deoxygenated CuBr monomer was added to a second double-neck flask, and deoxygenated by circulating between nitrogen and vacuum for 3 times; 0.49 mmol of HMTETA was then added to the second double-neck flask, and deoxygenated again, and then 10 mL of anhydrous N,N-dimethylformamide was added and deoxygenated again, and stirred for 30 min under nitrogen protection, to obtain a catalyst solution, which was then added to the monomer / initiator solution in the first double-neck flask, and deoxygenated by circulating between nitrogen and vacuum for 3 times, and heated at 60 °C with a reflux condenser, and stirred for 24 h under nitrogen protection, and then 60 mL of ether was added to the reaction mixture to precipitate, and the green oily precipitate was placed in a refrigerator, and the supernatant was discarded, and then 10 mL of acetone was added at 0 °C, and the precipitate was ultrasonically precipitated 3 times, and the supernatant was removed, and the precipitate was dried in a vacuum oven for 24 h, to obtain NHS-PCB-tBu;

[0033] (4) Synthesis of p-hydroxyazobenzoic acid (PHABA): 13.05 mmol of p-aminobenzoic acid was weighed, and 20 mL of hydrochloric acid aqueous solution was added, and stirred in an ice water bath, and mixed and dissolved, and 15.8 mmol of sodium nitrite aqueous solution was added dropwise, and then stirred in an ice water bath for 30 min to obtain a reaction solution, and the reaction solution was transferred to a 500 mL round-bottom flask, and 15.86 mmol of solid phenol was dissolved in 100 mL of saturated sodium bicarbonate aqueous solution, and slowly added dropwise to the reaction solution, and the reaction was continued to stir in an ice water bath for 1 h. The reaction solution was adjusted to pH 3 with dilute hydrochloric acid aqueous solution, and filtered, and the filter cake was recrystallized in an ethanol aqueous solution (ethanol: water = 15:2), and dried in a vacuum oven, to obtain p-hydroxyazobenzoic acid PHABA;

[0034] (5) Synthesis of NHS-Azo: Take PHABA 2.06 mmol dissolved in 20 mL of anhydrous DMF, and take N-hydroxysuccinimide (NHS) 2.88 mmol, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) 2.88 mmol, 4-dimethylaminopyridine (DMAP) 0.412 mmol, and add 10 mL of anhydrous DMF to stir and mix uniformly to dissolve, and add the above mixture to a flask with PHABA at room temperature for 5 h to terminate the reaction. Wash with hydrochloric acid aqueous solution, filter, wash with water, and dry in vacuum for 24 h to obtain NHS-Azo ester NHS-Azo;

[0035] (6) Synthesis of NHS-Azo-PCB-tBu initiator: Take 0.67 mmol of NHS-PCB-tBu dissolved in 20 mL of anhydrous DMF, and take 0.32 mmol of NHS-Azo ester dissolved in 10 mL of anhydrous DMF, mix the two, and add 0.29 mmol of DMAP, heat and stir under nitrogen protection at 60°C to reflux for 24 h, and stir at room temperature for another 48 h, add 100 mL of ether to precipitate, wash with ethyl acetate, and dry in vacuum for 24 h to obtain NHS-Azo-PCB-tBu initiator;

[0036] (7) Synthesis of DSPE-Azo-PCB: Take 0.5 mmol of NHS-AZO-PCB-tBu dissolved in 6.2 mL of anhydrous DMF, and take 0.48 mmol of DSPE dissolved in 45 mL of chloroform (trichloromethane), mix the two, and add 0.9 mmol of triethylamine (TEA) dropwise, stir at room temperature for 5 days, then spin at 50°C, and precipitate in ether; filter, resuspend with acetonitrile, and extract, filter to remove unbound DSPE in the filtrate, spin at 60°C for 60 min, evaporate the acetonitrile, and add 4 mL of TFA (trifluoroacetic acid) to stir and treat for 4 h; then precipitate with ether, filter, and dry in vacuum for 12-24 h, and obtain DSPE-Azo-PCB and unbound AZO-PCB in the product, which is neutralized with 20 mM of hydroxylamine in phosphate buffer (pH = 8) to dissolve the unbound PCB, then ultrafiltrate in PBS, repeat the ultrafiltration in water twice to obtain a DSPE-Azo-PCB solution, and then freeze-dry in pure water for 24 h to obtain DSPE-Azo-PCB, whose 1H-NMR spectrum is shown in Figure 2

[0037] ​(8) Preparation of high efficient mucous penetrating zwitterionic polymer micelles: 10 mg of polymer DSPE-Azo-PCB was dissolved in 5 ml of trifluoroethanol, rotary evaporation under reduced pressure for 15 min to form a dense thin film layer, vacuumized for 0.5-2 h, hydrated with 4 ml of deionized water or PBS, stirred at room temperature for 10-60 min to obtain high efficient mucous penetrating zwitterionic polymer micelles.

[0038] The high efficient mucous penetrating zwitterionic polymer micelles DSPE-Azo-PCB prepared by the examples of the present application have stronger mucous penetrating effect and can efficiently cross the lung mucosal barrier. The specific data are as follows:

[0039] I. Mucous penetrating ability

[0040] 120 mg of mucin was ultrasonically dissolved in 4 ml of phosphate buffer, and 1 ml was taken by a pipette gun and added to four NMR tubes, respectively, and named a, b, c and d. 10 mg of zwitterionic polymer DSPE-Azo-PCB was ultrasonically dissolved in 5 ml of trifluoroethanol in a flask, and coumarin 6 (concentration of 1 mg / mL) was dissolved in anhydrous methanol, and 10 μl of coumarin 6 solution was added to the flask and ultrasonically mixed. Then, rotary evaporation was performed at 45°C for 30 min to obtain a dense and uniform thin film layer. Then, vacuumization was performed at room temperature for 2 h with a diaphragm pump to remove the residual trifluoroethanol and methanol in the flask; after adding 4 ml of PBS buffer solution, stirring was performed at 600 rpm / min, and after 20 min of reaction, a clear and transparent coumarin 6 fluorescent labeled DSPE-Azo-PCB micelle solution was obtained. The preparation was transferred to a 10 mL EP tube, and sonication was performed under ice water bath at a power of 200 W for 5 min, with a working interval of 3 s, and repeated for 60 times, and then stopped, and then filtered with a 0.22 μm microporous filter. DSPE-PCB / C6 micelles, Tween 80 / C6 micelles and F-127 / C6 micelles were prepared according to the same process. 200 μl of Tween 80 / C6 micelles was slowly added to the a NMR tube, 200 μl of F-127 / C6 micelles was slowly added to the b NMR tube, 200 μl of DSPE-PCB / C6 micelles was slowly added to the c NMR tube, and 200 μl of DSPE-Azo-PCB / C6 micelles was slowly added to the d NMR tube, and then placed in a 37°C environment for 2 h, and then the penetrating depth was observed.

[0041] The results are as follows: Figure 3As shown, it can be clearly seen that the Tween 80 / C6 micelles and F-127 / C6 micelles formulations in NMR tubes a and b have very weak penetration ability in mucus, while the DSPE-PCB / C6 micelles formulation in NMR tube c has a relatively strong penetration ability in mucus, but it is diffusely distributed throughout the mucus layer. The DSPE-Azo-PCB / C6 micelles formulation in NMR tube d has the strongest penetration ability in mucus, and most of the formulation does not remain in the mucus layer but completely penetrates the mucus layer, indicating that DSPE-Azo-PCB has the strongest penetration ability in mucus.

[0042] II. Mucin Adsorption Capacity

[0043] 50 μL of each of the following formulations—DSPE-Azo-PCB micelles, DSPE-PCB micelles, Tween 80 micelles, and F-127 micelles—was thoroughly mixed with 2 mL of mucin (1 mg / mL), and then incubated in a shaker (37 °C, 100 rpm) for 2 h. The mixture was then centrifuged at 10,000 rpm for 2 min, and the supernatant was collected. The amount of free mucin remaining in the supernatant was measured using a mucin detection kit (the amount of mucin adsorbed onto the nanoparticles was calculated as the difference between the total amount of added mucin and the amount of free mucin in the supernatant).

[0044] The results are as follows Figure 4 As shown, Tween 80 micelles adsorbed 0.235 mg of mucin, F-127 micelles adsorbed 0.230 mg of mucin, DSPE-PCB micelles adsorbed 0.128 mg of mucin, and DSPE-Azo-PCB micelles adsorbed 0.108 mg of mucin. This indicates that DSPE-Azo-PCB micelles adsorbed the least amount of mucin, proving that it adsorbs less mucin in vivo and can help to quickly penetrate the mucus layer.

[0045] As can be clearly seen from the above, the present invention has the following beneficial effects compared with the prior art:

[0046] 1. The product prepared by this invention is a highly efficient mucus-penetrating amphoteric polymer micelle. It uses polycarboxybenzene with good biocompatibility as the hydrophilic group, DSPE as the hydrophobic group, and Azo as the bridging bond. The azobenzene bond can be oxidized and reduced in hypoxic lung disease tissue, thereby enabling targeted and controlled release of the encapsulated drug, providing technical support for the preparation of targeted drug carriers.

[0047] 2. A highly efficient mucus-penetrating amphoteric polymer micelle, DSPE-Azo-PCB, was synthesized using a thin-film dispersion method. Compared with traditional micelles, it has a stronger mucus-penetrating effect and can efficiently cross the lung mucosal barrier, effectively solving the problems of polymer micelles being unable to efficiently penetrate the mucus barrier and having low targeting.

[0048] 3. Using biocompatible polycarboxybetaine as the hydrophilic group and DSPE as the hydrophobic group, it has a low critical micelle concentration, which can effectively improve the structural dissociation problem of micelles caused by large-scale blood dilution, resulting in huge social and economic benefits.

[0049] Unless otherwise specified in the embodiments of this invention, the conditions shall be performed according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products that can be purchased commercially. The above are merely preferred embodiments of this invention and are not intended to limit the invention in any way. Any person skilled in the art who can make changes or modifications to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the technical solution of this invention shall fall within the protection scope of this invention.

Claims

1. A method for preparing highly efficient mucus-penetrating amphoteric polymer micelles, characterized in that, Includes the following steps: (1) Synthesis of CB-tBu: 2-(dimethylamino)ethyl methacrylate and tert-butyl bromoacetate were co-dissolved in anhydrous acetonitrile at a molar ratio of 1:1-2. The mixture was stirred at 50°C for 12-24 h under nitrogen protection. Then, 50-100 ml of cold diethyl ether was added, and a white solid precipitated. The solid was separated by filtration and dried under vacuum for 12-24 h to obtain the CB-tBu monomer. (2) Synthesis of NHS-ATRP initiator: 9.8 mmol of N-hydroxysuccinimide was dissolved in dichloromethane, and 8.3 mmol of 2-bromopropionic acid was added dropwise under ice bath to obtain the first solution. 8.2 mmol of dicyclohexylcarbodiimide was dissolved in anhydrous dichloromethane solution and added dropwise to the first solution under ice bath. After reacting at room temperature for 12-24 h, the solution was filtered twice. The filtrate was evaporated under reduced pressure for 30-60 min to remove the solvent. The solution was dissolved in ethyl acetate and sonicated for 10-30 min. After sonication, the solution was filtered. The filtrate was evaporated under reduced pressure for 30-60 min and concentrated. After recrystallization and purification, NHS-ATRP was obtained. (3) Synthesis of NHS-PCB-tBu: Anhydrous N,N-dimethylformamide was bubbled with nitrogen for 1-2 h. The double-necked flask, flask, magnetic initiator, and condenser were dried in an oven at 110℃ for 30-60 min. CuBr was deoxygenated to obtain deoxygenated CuBr for later use. 3.7 mmol CB-tBu monomer and 0.5 mmol NHS-ATRP initiator were added to the first double-necked flask and deoxygenated by circulating nitrogen and vacuum three times. After deoxygenation, 2-5 ml of anhydrous N,N-dimethylformamide was added to the first double-necked flask and deoxygenated again to obtain monomer / initiator solution. 0.49 mmol of deoxygenated CuBr monomer was added to the second double-necked flask and deoxygenated by circulating nitrogen and vacuum three times. 0.49 mmol HMTETA was added to the second double-necked flask and deoxygenated again. 5-10 ml of HMTETA was added to the second double-necked flask. Anhydrous N,N-dimethylformamide was deoxygenated again, and stirred under nitrogen protection for 30-60 min to obtain a catalyst solution. The catalyst solution was then added to the monomer / initiator solution in the first double-necked flask, and deoxygenation was performed by circulating between nitrogen and vacuum three times. The mixture was heated to 60°C and refluxed in a condenser, and stirred under nitrogen protection for 12-24 h. Then, 50-100 ml of diethyl ether was added to the reaction mixture to precipitate the precipitate, and the mixture was placed in a refrigerator to stand. The upper mixed solution was discarded. Then, 5-10 ml of acetone was added at 0°C, and the mixture was sonicated to precipitate 2-3 times. The supernatant was discarded, and the precipitate was dried under vacuum overnight for 12-24 h to obtain NHS-PCB-tBu. (4) Synthesis of PHABA: 13.05 mmol of p-aminobenzoic acid was dissolved in hydrochloric acid aqueous solution and stirred under ice bath. 15.8 mmol of sodium nitrite aqueous solution was added dropwise and stirred under ice bath for 30-60 min to obtain the reaction solution. Then, 15.86 mmol of solid phenol was dissolved in saturated sodium bicarbonate aqueous solution and slowly added dropwise to the reaction solution. The mixture was stirred under ice bath for 1-2 h. The pH of the reaction solution was adjusted to 3 with dilute hydrochloric acid aqueous solution. After filtration, the solution was recrystallized and dried under vacuum for 12-24 h to obtain PHABA. (5) Synthesis of NHS-Azo: 2.06 mmol PHABA was dissolved in anhydrous N,N-dimethylformamide; 2.88 mmol N-hydroxysuccinimide, 2.88 mmol 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 0.412 mmol 4-dimethylaminopyridine were dissolved together in anhydrous N,N-dimethylformamide. The two were mixed and stirred at room temperature for 5-10 h. The mixture was washed with hydrochloric acid aqueous solution, filtered, washed with water, and dried under vacuum for 12-24 h to obtain NHS-Azo. (6) Synthesis of NHS-Azo-PCB-tBu: 0.67 mmol NHS-PCB-tBu was dissolved in anhydrous N,N-dimethylformamide, and 0.32 mmol NHS-Azo was dissolved in anhydrous N,N-dimethylformamide. After mixing the two, 0.29 mmol DMAP was added. The mixture was stirred and refluxed at 60°C under nitrogen protection for 12-24 h, and then stirred at room temperature for 40-48 h. 50-100 ml of diethyl ether was added to precipitate the precipitate, which was washed with ethyl acetate and dried under vacuum for 12-24 h to obtain NHS-Azo-PCB-tBu. (7) Synthesis of DSPE-Azo-PCB: 0.5 mmol NHS-Azo-PCB-tBu was dissolved in anhydrous N,N-dimethylformamide, and 0.48 mmol DSPE was dissolved in 45 mL chloroform. After mixing the two, 0.9 mmol triethylamine was added dropwise. The mixture was stirred at room temperature for 5-10 days, then rotary evaporated at 50℃ for 30-60 min, and precipitated in ether. After filtering to remove the filtrate, the precipitate was resuspended in acetonitrile and filtered. After rotary evaporation at 60℃ for 30-60 min, 4 mL TFA was added and stirred at room temperature for 4-8 h. The precipitate was filtered and dried under vacuum overnight for 12-24 h. Then, the uncoupled PCB was neutralized in PBS at pH=8 with 20 mM hydroxylamine until dissolved. The PCB was then ultrafiltered in PBS and the ultrafiltration was repeated twice in water to obtain a DSPE-Azo-PCB solution. The solution was then freeze-dried in pure water for 12-24 h to obtain the dried polymer DSPE-Azo-PCB. (8) Preparation of high efficiency mucus-penetrating amphoteric polymer micelles: Dissolve polymer DSPE-Azo-PCB in trifluoroethanol, evaporate under reduced pressure for 15-20 min until a dense thin film layer is formed, vacuum for 0.5-2 h, add deionized water or PBS for hydration, stir at room temperature for 10-60 min to obtain high efficiency mucus-penetrating amphoteric polymer micelles.

2. The method for preparing high-efficiency mucus-penetrating amphoteric polymer micelles according to claim 1, characterized in that, The solvent used for recrystallization in step (2) is isopropanol.

3. The method for preparing high-efficiency mucus-penetrating amphoteric polymer micelles according to claim 1, characterized in that, The deoxygenation method of CuBr after deoxygenation in step (3) is as follows: place glacial acetic acid and CuBr in a flask at a ratio of 50:1, with glacial acetic acid in ml and CuBr in g, stir at room temperature for 1 h, filter and separate, then add anhydrous ethanol 2-3 times to wash away the residual glacial acetic acid, and obtain yellowish-white solid CuBr. Evacuate the solid CuBr under nitrogen protection for 2 h to obtain the final product.

4. The method for preparing high-efficiency mucus-penetrating amphoteric polymer micelles according to claim 1, characterized in that, The solvent used for recrystallization in step (4) is a mixture of ethanol and water in a volume ratio of 15:2.

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