A triblock polymer with long circulation and long-acting anti-protein stability, and its preparation and application

Abstract

The present invention discloses a triblock polymer with long circulation and long-acting anti-protein stability, and its preparation and application. The polymer is [PPMA x -b-POPMA y -b-PhGMA z , where x = 15 - 50, y = 15 - 50, and z = 15 - 50. The monomer molecules of this polymer are easy to synthesize, and the polymer blocks are easy to regulate, and can self-assemble to form micelles with different stabilities. The polymer of the present invention can form pH and GSH dual-responsive crosslinked micelles, which can effectively load water-insoluble drugs and have good in vivo circulation ability, drug release performance and good biocompatibility. The micelles of the present invention enhance the stability of the micelles in the physiological environment through block ratio regulation and crosslinking strategy, and have good pH-responsive performance, and can effectively release drugs in the tumor microenvironment, improving the drug delivery efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical polymer materials, and particularly relates to a triblock polymer with long circulation and long-lasting anti-protein stability, a preparation method thereof, and a biological application based on a pH and GSH dual-responsive crosslinked micelle. Background Art

[0002] In recent years, the harm of cancer to human health has received increasing attention. Among many cancer treatment strategies, chemotherapy is a mainstream treatment method. However, traditional chemotherapeutic drugs have disadvantages such as poor water solubility and strong toxic and side effects. Using a drug carrier material to deliver drugs can effectively make up for these defects. Among the currently developed drug-loaded materials, polymer micelles are widely used for the encapsulation and delivery of water-insoluble drugs due to their excellent biocompatibility, easy chemical modification, etc. Although the current research on polymer micelles often has a rather cumbersome preparation process.

[0003] In fact, during the drug delivery process in vivo, micelles often become unstable due to adsorbing substances such as proteins and lipids in the blood, ultimately resulting in low delivery efficiency. At the same time, the shear force and dilution effect in blood circulation will also reduce the micelle concentration below the critical micelle concentration (CMC) to cause micelle instability and disintegration, thus seriously affecting the drug delivery effect. Therefore, when designing a polymer micelle drug delivery system, in addition to paying attention to the drug-loading performance and therapeutic ability of the carrier, the kinetic stability, thermodynamic stability, and anti-protein adsorption stability of micelles in blood circulation should also be fully considered. At present, the research on the long-lasting anti-protein adsorption stability performance of polymer micelles is less and the performance still needs to be improved, which greatly limits the application potential of micelles in actual tumor treatment. In addition, although the current research on the influencing strategies for the long-circulation stability performance of polymer micelles is relatively extensive, the problems are often viewed singly, and the influence of these stability enhancement strategies has not been explored synergistically. Finally, there has been no research on the long circulation and long-lasting anti-protein stability of polymer micelles with a hydrophilic chain segment having a hydroxyl functional group so far. Summary of the Invention

[0004] In order to overcome the problems of complex polymer structure, insufficient long-lasting anti-protein performance, and difficult regulation of stability enhancement strategies in the above-mentioned prior art, and to meet the requirements such as the physiological stability and tumor treatment ability of the above-mentioned micelles, the primary object of the present invention is to provide a triblock polymer with long circulation and long-lasting anti-protein stability based on pH and GSH-responsive crosslinking bonds.

[0005] The triblock polymer described in the present invention has the synergy of stability enhancement strategies such as Π-Π conjugation and hydrogen bonding, as well as hydroxyl-protein repulsion, endowing its micelles with good long-circulation stability and drug encapsulation ability. By combining the regulation of the polymer block ratio and the crosslinking strategy, the kinetic, thermodynamic, and anti-protein stabilities of the micelles can be further enhanced simultaneously, and pH and GSH-responsive bond cleavage can be achieved in the acidic tumor microenvironment with a high GSH concentration, realizing the long-term and stable release of drugs. Therefore, the triblock polymer described in the present invention is a drug delivery material with good application potential.

[0006] Another object of the present invention is to provide a preparation method of the above-mentioned triblock polymer based on pH- and GSH-responsive crosslinking bonds and having long-circulation and long-term anti-protein stability.

[0007] In the method of the present invention, three kinds of block monomers with the same methacrylic acid-2-hydroxypropyl ester (HPMA) main chain structure are first prepared, namely: (1) hydrophilic block (glyceryl methacrylate, hGMA); (2) crosslinking site block (2-oxopropyl methacrylate, OPMA); (3) hydrophobic block (2-hydroxy-3-phenoxypropyl methacrylate, PMA). Then, the atom transfer radical polymerization (ARGET ATRP) method is used to polymerize the hydrophobic block PMA, the crosslinking site block OPMA, and the hydrophilic block hGMA in sequence using a small molecule initiator to prepare triblock polymers with different block ratios.

[0008] The polymer preparation process of the present invention is simple and can accurately and quickly regulate the block ratio of the amphiphilic block polymer, thereby enhancing the stability, anti-protein adsorption ability, and drug delivery performance of the micelles formed by the polymer.

[0009] Another object of the present invention is to provide the application of the above-mentioned triblock polymer based on pH- and GSH-responsive crosslinking bonds and having long-circulation and anti-protein stability in the preparation of drugs, which is applied to the field of loading water-insoluble drugs.

[0010] The object of the present invention is achieved by the following technical solutions:

[0011] A triblock polymer with long-circulation and long-term anti-protein stability, its structure is: poly(2-hydroxy-3-phenoxypropyl methacrylate)-b-poly(2-oxopropyl methacrylate)-b-poly(glyceryl methacrylate) (PPMA x -b-POPMA y -b-PhGMA z ), and the block ratio x:y:z of poly(2-hydroxy-3-phenoxypropyl methacrylate), poly(2-oxopropyl methacrylate), and poly(glyceryl methacrylate) is 15-50:15-50:15-50.

[0012] Preferably, the block ratio x:y:z of poly(2-hydroxy-3-phenoxypropyl methacrylate), poly(2-oxopropyl methacrylate) and poly(glycerol methacrylate) is 15 to 35:15 to 35:15 to 35.

[0013] Preferably, the degrees of polymerization of poly(2-hydroxy-3-phenoxypropyl methacrylate), poly(2-oxopropyl methacrylate) and poly(glycerol methacrylate) are x = 15 to 50, y = 15 to 50 and z = 15 to 50, respectively.

[0014] The preparation method of the above triblock polymer with long circulation and long-acting anti-protein stability comprises the following steps:

[0015] Under an inert gas atmosphere, after mixing poly(2-hydroxy-3-phenoxypropyl methacrylate) (PMA), ligand, catalyst, reducing agent and solvent evenly, add a small molecule initiator, and carry out a heating reaction. After the complete conversion of poly(2-hydroxy-3-phenoxypropyl methacrylate) (PMA), add poly(2-oxopropyl methacrylate) (OPMA) and continue the reaction. After the complete conversion, add poly(glycerol methacrylate) (hGMA) and continue the reaction, and then purify to obtain the amphiphilic triblock polymer (PPMA x -b-POPMA y -b-PhGMA z ).

[0016] Preferably, the molar ratio of poly(2-hydroxy-3-phenoxypropyl methacrylate) (PMA), poly(2-oxopropyl methacrylate) (OPMA), poly(glycerol methacrylate) (hGMA), ligand, catalyst, reducing agent and small molecule initiator is: 15 to 50:15 to 50:15 to 50:0.018 to 0.75:0.018 to 0.062:0.181 to 0.89:0.36 to 1.

[0017] Preferably, the ligand is at least one of 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA), tripyridylmethylamine (TPMA) and bipyridine (bpy).

[0018] Preferably, the catalyst is at least one of copper bromide (CuBr2), iron chloride and copper chloride.

[0019] Preferably, the reducing agent is at least one of stannous octoate (Sn(Oct)2) and ascorbic acid.

[0020] Preferably, the small molecule initiator is at least one of ethyl 2-bromoisobutyrate (EBriB), azobisisobutyronitrile and 1,1,2,2-tetraphenylethylene glycol.

[0021] Preferably, the solvent in the preparation method of the triblock polymer with long circulation and long-acting anti-protein stability is at least one of anisole, toluene and acetonitrile.

[0022] Preferably, the temperature of the heating reaction in the preparation method of the triblock polymer with long circulation and long-acting anti-protein stability is 50-70 °C; the heating reaction time of 2-hydroxy-3-phenoxypropyl methacrylate (PMA) is 8-12 h; the heating reaction time of 2-oxopropyl methacrylate (OPMA) is 24-72 h; the heating reaction time of glycerol methacrylate (hGMA) is 72-120 h.

[0023] Preferably, the 2-hydroxy-3-phenoxypropyl methacrylate (PMA) is prepared by the following method: under an inert gas atmosphere, methacrylic acid, a polymerization inhibitor, an acid-binding agent, and 1,2-epoxy-3-phenoxypropane are dissolved in a solvent, heated for reaction, and purified to obtain 2-hydroxy-3-phenoxypropyl methacrylate.

[0024] More preferably, the molar ratio of methacrylic acid, the polymerization inhibitor, 1,2-epoxy-3-phenoxypropane and the acid-binding agent is 1:0.00033-0.00136:1-1.1:0.10-0.13.

[0025] More preferably, the reaction temperature in the preparation method of the 2-hydroxy-3-phenoxypropyl methacrylate (PMA) is 50-70 °C, and the time is 18-24 h.

[0026] More preferably, the polymerization inhibitor is at least one of dibutylhydroxytoluene (BHT) and methoxyphenol (MEHQ); the acid-binding agent is at least one of triethylamine and diisopropylethylamine.

[0027] More preferably, in the preparation method of the 2-hydroxy-3-phenoxypropyl methacrylate (PMA), methacrylic acid and the polymerization inhibitor are first added to the solvent for dispersion and then the acid-binding agent and 1,2-epoxy-3-phenoxypropane are added dropwise.

[0028] More preferably, the purification refers to gradient elution by silica gel column chromatography (mobile phase: n-hexane:ethyl acetate 20:1, 15:1, 10:1).

[0029] Preferably, the 2-oxopropyl methacrylate (OPMA) is prepared by the following method: under an inert gas atmosphere and ice bath conditions, methacryloyl chloride and hydroxyacetone are dissolved in a solvent and reacted for 1.5-2.5 h, then an acid-binding agent is added and the reaction is continued at room temperature for 9-12 h, and purified to obtain 2-oxopropyl methacrylate.

[0030] More preferably, the molar ratio of the methacryloyl chloride, hydroxyacetone and acid-binding agent is 1:0.78 - 0.84:1.03 - 1.09.

[0031] More preferably, the solvent in the preparation method of the 2-oxopropyl methacrylate (OPMA) is at least one of dichloromethane, toluene and carbon tetrachloride; the concentration of the methacryloyl chloride in the solvent is 0.5 - 1.5 mol / L, and most preferably 1.033 mol / L.

[0032] More preferably, the normal temperature refers to 15 - 40 °C.

[0033] More preferably, the acid-binding agent is at least one of triethylamine and diisopropyl ethylamine.

[0034] More preferably, in the preparation method of the 2-oxopropyl methacrylate (OPMA), the methacryloyl chloride is added first and then the hydroxyacetone is added dropwise.

[0035] More preferably, the purification refers to gradient elution by silica gel column chromatography (mobile phase: n-hexane:ethyl acetate 15:1, 10:1, 4:1).

[0036] Preferably, the glycerol methacrylate (hGMA) is prepared by the following method: under atmospheric reflux and heating conditions, glycidyl methacrylate is hydrolyzed in water and purified to obtain glycerol methacrylate (hGMA).

[0037] More preferably, in the hydrolysis system of glycidyl methacrylate, the volume fraction of the aqueous solution is 8 - 12% w / w.

[0038] More preferably, the temperature of the hydrolysis of glycidyl methacrylate is 70 - 80 °C, and the time is 9 - 12 h.

[0039] More preferably, the purification refers to gradient elution by silica gel column chromatography (mobile phase: n-hexane:ethyl acetate 8:1, 4:1, 0:1).

[0040] Preferably, the purification method is as follows: after the conversion of the glycerol methacrylate (hGMA) is completed, it is cooled, tetrahydrofuran (THF) is added to the product mixture to terminate the reaction, and then the catalyst is removed by passing through a neutral alumina chromatography column. After rotary evaporation and concentration, it is dropped into 8 - 12 times the volume of ice-cold n-hexane for precipitation. The rotary evaporation - precipitation is repeated 1 - 5 times, and the purified product is obtained after vacuum drying.

[0041] The application of the above-mentioned triblock polymer with long circulation and long-term anti-protein stability in drug loading.

[0042] Preferably, the application is as follows: A triblock polymer with long circulation and long-acting anti-protein stability and a water-insoluble drug are dissolved in a solvent, the pH is adjusted to 4-5, and stirred for 12-24 hours. Then it is dialyzed in a phosphate buffer solution, and a catalyst and a crosslinking agent are added. After reacting at room temperature, it is dialyzed with deionized water again to obtain a pH and GSH-responsive crosslinked micelle system loaded with the water-insoluble drug and having long circulation and long-acting anti-protein stability performance.

[0043] More preferably, the mass ratio of the triblock polymer with long circulation and long-acting anti-protein stability to the water-insoluble drug is 1:0.5-1.

[0044] More preferably, the dialysis in the phosphate buffer solution means first dialyzing in a phosphate buffer solution with a pH of 4.0-4.5 for 8 hours, then dialyzing in a phosphate buffer solution with a pH of 5.0-5.5 for 8 hours, and finally dialyzing in a phosphate buffer solution with a pH of 6.5 for 8 hours; the concentration of the phosphate buffer solution is 0.5 mol / l in all cases.

[0045] More preferably, the catalyst is 2-amino-5-methoxybenzoic acid; the crosslinking agent is dithiodihydrazide; the concentration of the catalyst in the dialysis solution is 8-12 mmol / L; the molar ratio of the crosslinking agent to 2-oxopropyl methacrylate in the triblock polymer is 1.25-1.30:1.

[0046] More preferably, the water-insoluble drug refers to a drug with a solubility of less than or equal to 1 g in 1 L of water. Further preferably, the water-insoluble drug is a water-insoluble anti-cancer drug, such as curcumin (Cur).

[0047] More preferably, the solvent is dimethyl sulfoxide (DMSO); the mass-volume ratio of the triblock polymer with long circulation and long-acting anti-protein stability to the solvent is 0.3-0.5 mg / mL, and the final micelle concentration obtained is 0.1-0.25 mg / mL.

[0048] More preferably, the reaction time at room temperature is 12-24 h.

[0049] More preferably, the dialysis time with deionized water is 24 h; the dialysis solution is changed every two hours for the first 12 hours, and then every 6 hours.

[0050] More preferably, the dialysis uses a dialysis bag with a molecular weight cut-off (MWCO) of 3.5 kDa.

[0051] The obtained pH- and GSH-responsive cross-linked micelle system loaded with water-insoluble drugs has advantages such as good drug retention ability, anti-protein adsorption ability, and long-circulation stability. Moreover, in the tumor acidic (pH 5.0), high GSH concentration (10 mmol / L) microenvironment, the pH- and GSH-responsive cross-linked bonds can be broken, thereby effectively promoting the long-term and stable release of drugs.

[0052] The mechanism of the drug-loading effect of the polymer of the present invention is as follows:

[0053] (1) The electron-donating effect of the β-position hydroxyl group in the benzene ring structure of the polymer hydrophobic block enhances the Π-Π conjugation effect of the system, thereby enhancing the anti-dilution stability and anti-fluid shear ability of the polymer micelles, and ultimately enhancing the physical stability of the system. In addition, in the normal physiological environment, the enhanced π-π stacking effect in the micelle core layer will further improve the drug loading rate and stable loading ability of drugs such as curcumin.

[0054] (2) The ketone carbonyl group of the polymer cross-linking site block can undergo a cross-linking reaction with various cross-linking agents at room temperature to improve the system stability, thereby enhancing the anti-dilution stability and anti-fluid shear ability of the polymer micelles, and ultimately enhancing the physical stability of the system. At the same time, when the polymer micelles are delivered to the tumor acidic (pH 5.0), high GSH concentration (10 mmol / L) microenvironment, the pH- and GSH-responsive cross-linked bonds are broken, resulting in the swelling of the micelle structure and the release of drugs, thereby improving the deficiency of the poor controlled release performance of the system.

[0055] (3) The ketone carbonyl group on the water-insoluble drug (such as curcumin) loaded is also fixed on the polymer micelles through a reaction with the cross-linking agent to form a prodrug system, further enhancing the stability of drug loading in the system. When the polymer drug-loaded cross-linked micelles encounter the above-mentioned tumor microenvironment, the breaking of the reversible cross-linked bonds enables curcumin to be released from the micelle core, achieving the goals of rapid drug release and precise delivery.

[0056] (4) The polyol structure of the polymer hydrophilic block selectively repels opsonin proteins that are not conducive to the long circulation of the system in the blood through the hydroxyl-protein repulsion interaction, thereby reducing the formation of the protein corona in the system, effectively improving the long-term anti-protein stability of the system, and reducing protein adsorption and macrophage recognition and phagocytosis in the blood circulation, thereby enhancing the long-circulation stability of the system.

[0057] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0058] (1) The preparation process flow of the present invention is simple and the conditions are mild. The block composition and degree of polymerization of the triblock polymer can be precisely regulated, and the stability of the formed micelles can be accurately regulated.

[0059] (2) The polymer-based drug-loaded cross-linked micelle system constructed in the present invention simultaneously adopts strategies of enhancing π-π conjugation, and pH- and GSH-responsive reversible cross-linking, which maximally enhances the stability, anti-protein stability, drug retention ability, and long-acting drug release property of the polymer micelles, thereby improving the drug delivery efficiency of the micelles.

[0060] (3) The polymer-based drug-loaded cross-linked micelle system constructed in the present invention also adopts a hydroxyl-protein repulsive interaction strategy. The system has good anti-specific protein adsorption performance, and thus has good macrophage escape ability, enabling long-circulation delivery in vivo, which provides a guarantee for the effective treatment of chemotherapeutic drugs.

[0061] (4) The polymer-based drug-loaded cross-linked micelle system constructed in the present invention has prodrug delivery properties. It has good drug encapsulation and retention abilities under normal physiological conditions. When the drug-loaded cross-linked micelles reach tumor cells, they can respond to the acidic and high-GSH concentration environment in the microenvironment to break the cross-linking bonds, achieving the long-acting release of the anti-cancer drug curcumin and realizing the therapeutic effect of chemotherapeutic drugs. Description of the Drawings

[0062] Figure 1 For the synthetic reaction formulas, 1H NMR spectra, and 13C NMR spectra of the monomers PMA, OPMA, and hGMA in Example 1.

[0063] Figure 2 For the 1H NMR, GPC elution curves, and CMC diagrams of the triblock polymer PPMA x -b-POPMA y -b-PhGMA z in Example 2, with the polymer block ratios of hOP2: 25-25-15, hOP7: 35-15-35, and hOP8: 15-25-35 respectively.

[0064] Figure 3 For the DLS and infrared spectra of the pH- and GSH-responsive cross-linked polymer micelles in Example 3. Among them, the polymer block ratio in the infrared spectrum is 15-25-35.

[0065] Figure 4 For the TEM diagram of the pH- and GSH-responsive cross-linked polymer micelles in Example 3, where the polymer block ratio is 15-25-35.

[0066] Figure 5 For the physical stability, long-acting anti-protein stability, and escape ability diagrams of the pH- and GSH-responsive cross-linked polymer micelles loaded with a model drug in Example 4, where the polymer block ratio is 15-25-35.

[0067] Figure 6DLS, TEM, drug loading and encapsulation efficiency diagrams of the drug-loaded cross-linked micelles in Example 5, where the block ratio of the triblock polymer in the TEM diagram is 15-25-35.

[0068] Figure 7 In vitro release curve of the drug-loaded cross-linked micelles in Example 6.

[0069] Figure 8 Cytotoxicity test diagrams of blank micelles, drug-loaded micelles and free curcumin in Examples 3 and 6.

[0070] Figure 9 Schematic diagram of the preparation process and controlled release of pH- and GSH-responsive cross-linked polymer micelles. Detailed implementation manners

[0071] The present invention will be further described in detail below in conjunction with the examples and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0072] For those not specified in the examples of the present invention, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. Raw materials, reagents, etc. not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0073] Example 1: Preparation of polymer monomers:

[0074] (1) Hydrophobic monomer (PMA)

[0075] A magnetic stir bar was added to a 100 mL dry three-necked flask equipped with a condenser and reflux device. Argon was passed through for 10 minutes, and then the three-necked flask was sealed. 30 mg of BHT (dibutylhydroxytoluene) and 9.039 g of methacrylic acid were injected into the three-necked flask with a syringe. Stirring was carried out at 60 °C for 10 minutes, and then 1.35 g of triethylamine and 15.017 g of 1,2-epoxy-3-phenoxypropane were slowly added dropwise. The reaction was carried out at 60 °C for 24 hours. After the reaction, the crude product was extracted with dichloromethane solution. The solvent was removed by rotary evaporation, and the obtained crude product was eluted by gradient elution on a silica gel column (mobile phase: n-hexane:ethyl acetate 20:1, 15:1, 10:1). Rotary evaporation was carried out again to obtain a pure product in the form of an amber oil. The synthesis reaction formula is shown in A in Figure 1 . The structure of the product was characterized and analyzed by nuclear magnetic resonance hydrogen spectrum and carbon spectrum ( 1 H NMR, 13 C NMR), and the results are shown in B and C in Figure 1 .

[0076] (2) Keto-functionalized monomer (OPMA)

[0077] A magnetic stir bar was added to a 200 mL dry eggplant-shaped flask, and argon was passed through for 10 minutes. Then, the eggplant-shaped flask was sealed, and 10.8 g of methacryloyl chloride and 100 mL of dichloromethane were injected into the flask using a syringe. Next, the flask was placed in an ice bath, and 7.4 g of hydroxyacetone was slowly added. After reacting at 0 °C for 2 hours, 11.1 g of triethylamine was slowly added, and the reaction continued at room temperature for 9 hours. After the reaction, the crude product dichloromethane solution was extracted. The solvent was removed by rotary evaporation, and the obtained crude product was eluted with a gradient on a silica gel column (mobile phase: n-hexane:ethyl acetate 15:1, 10:1, 4:1). Rotary evaporation was performed again to obtain a pure product in the form of a light yellow liquid. The synthetic reaction formula is shown in Figure 1 A. The structure of the product was characterized and analyzed using nuclear magnetic resonance hydrogen spectrum and carbon spectrum ( 1 H NMR, 13 C NMR), and the results are shown in Figure 1 B and C in.

[0078] (3) Hydrophilic monomer (hGMA)

[0079] A magnetic stir bar was added to a 200 mL dry round-bottomed flask, 9.92 g of glycidyl methacrylate and 91.38 mL of deionized water were added, and a condenser reflux device was connected. The reaction was carried out at 80 °C for 9 hours until the solution became clear. After the reaction, the crude product dichloromethane solution was extracted. The solvent was removed by rotary evaporation, and the obtained crude product was eluted with a gradient on a silica gel column (mobile phase: n-hexane:ethyl acetate 8:1, 4:1, 0:1). Rotary evaporation was performed again to obtain a pure product in the form of a transparent viscous liquid. The synthetic reaction formula is shown in Figure 1 A. The structure of the product was characterized and analyzed using nuclear magnetic resonance hydrogen spectrum and carbon spectrum ( 1 HNMR, 13 C NMR), and the results are shown in Figure 1 B and C in.

[0080] Example 2: Preparation of different block triblock copolymers (PPMA x -b-POPMA y -b-PhGMA z ) (x, y, z = 15 - 35)

[0081] The feed amounts of each reactant and the polymerization time are shown in Table 1 below.

[0082] Take a 50 mL dry eggplant-shaped flask, add a magnetic stir bar and the catalyst CuBr2 into it. After sealing the reaction flask, evacuate and refill with argon three times. Then, successively add the solvent anisole anhydride (8 mL), the monomer PMA (3 g, 12.70 mmol), and the ligand HMTETA using a syringe, and stir well for 10 minutes to form the catalyst complex. Next, add the reducing agent Sn(Oct)2 pre-dissolved in 2 mL of anisole anhydride and stir for 10 minutes. After adding the small molecule initiator EBriB using a microsyringe, transfer the mixture to an oil bath at 60 °C and react for 8 - 12 hours. After the PMA reaction is complete, add the monomer OPMA and continue the reaction for 24 - 72 hours. Then, add the monomer hGMA pre-dissolved in 10 mL of anisole anhydride and continue the reaction for 72 - 120 hours. After the reaction is complete, cool the eggplant-shaped flask to room temperature and add THF to terminate the reaction. Then, pass through a neutral alumina column (using THF as the eluent), concentrate by rotary evaporation, and slowly dropwise add to ten times the volume of n-hexane for precipitation, and dry under vacuum at 45 °C and 35 mbar for 24 hours to obtain the product. Using 1 1H NMR and GPC to analyze the composition and structure of the product, Mn = 8.28 - 16.69 kDa, both within 1.3. The results of three representative block ratio polymers are shown as Figure 2 B in

[0083] Table 1

[0084]

[0085]

[0086] Example 3: Preparation of GSH- and pH-responsive crosslinked polymer micelles

[0087] The GSH- and pH-responsive crosslinked polymer micelles were prepared by dialysis. Dissolve the polymer from Example 2 (15 mg) in 30 mL of DMSO and transfer it into a dialysis bag (MWCO = 3.5 kDa), and dialyze with PBS solution (50 mmol / L, pH 6.5) for 24 hours. Change the dialysis solution every two hours for the first 12 hours, and then change the dialysis solution every 6 hours. Subsequently, transfer the micelle solution to a flask, add the catalyst 2-amino-3-methoxybenzoic acid (54.64 mg) at 10 mmol / L and the crosslinker dithiodiacyldrazide (DTP, the dosage is shown in Table 2 below). Stir and react for one day, and then dialyze the micelle solution with deionized water for another 12 hours, changing the dialysis solution every two hours. Finally, lyophilize the crosslinked micelle solution to obtain the crosslinked polymer micelles.

[0088] The particle size, morphology, and crosslinking degree of the crosslinked micelles were characterized by dynamic light scattering (DLS), infrared spectroscopy, and transmission electron microscopy (TEM). The hydrodynamic diameters (Dh) of the three representative block ratio polymer crosslinked micelles were 152.5, 179.7, and 130.9 nm, as shown in Figure 3 A in Figure 3 . The disappearance of the characteristic peaks in the infrared spectrum proved the occurrence of the crosslinking process, as shown in Figure 4 B and C in 35 -b-POPMA 25 -b-PPMA 15 .

[0089] Table 2

[0090]

[0091]

[0092] Example 4: Long-circulating stability and long-acting anti-protein stability experiments

[0093] (1) The crosslinked micelles loaded with the model drug were prepared according to the method for preparing GSH and pH-responsive crosslinked polymer micelles in Example 3. The polymer (15 mg) from Example 2, 150 μL of DiO / DMSO solution (10 μmol / L), and 150 μL of DiI / DMSO solution (10 μmol / L) were dissolved in 30 mL of DMSO, dialyzed, and crosslinked to obtain the corresponding crosslinked micelles loaded with the model drug.

[0094] (2) Take 20 mL of the crosslinked micelles loaded with the model drug in a 50 mL test tube, incubate at 37 °C in a constant temperature shaker at 80 rpm for 14 days, use DLS and a fluorescence spectrometer to measure the corresponding particle size and FRET ratio changes at different time points, and plot the physical stability change curve, as shown in Figure 5 A in

[0095] (3) Take 6 mL of the three representative block ratio polymer crosslinked micelles in a 10 mL test tube, add 270 mg of BSA protein to each, incubate at 37 °C in a constant temperature shaker at 80 rpm for 132 hours, use DLS to measure the corresponding particle size at different time points, and plot the corresponding protein adsorption particle size change curve, as shown in Figure 5 B in Figure 5 . Further use a fluorescence spectrometer to measure the corresponding FRET ratio changes at different time points, and plot the anti-protein stability change curve, as shown in

[0096] C in . The block ratio of the triblock polymer in the figure is 15-25-35.(4) RAW264.7 cells were passaged and cultured in DMEM medium supplemented with 10% FBS, 1% penicillin, and streptomycin at 37 °C and 5% CO₂ concentration. Then, the cells were seeded onto 3.5 cm culture dishes at a density of 500,000 cells per dish and incubated for 24 hours. After aspirating the old medium, 2 mL of medium containing 100 μg / mL blank micelles was added to the culture dishes, and incubation was continued for 1, 2, 3, and 4 hours. Then, the liquid was aspirated and the cells were washed three times, centrifuged, and resuspended in 1 mL of PBS. Flow cytometry was used to test the cells, excited at a wavelength of 488 nm, and the fluorescence emission value at a wavelength of 520 nm was detected to calculate the immune escape rate of the material, as Figure 5 shown in D of

[0097] Example 5: Preparation of cross-linked polymer micelles loaded with the anticancer drug Cur

[0098] The drug-loaded cross-linked micelles were prepared by the dialysis method. 15 mg of the polymer from Example 2 and 15 mg of Cur were dissolved in 30 mL of DMSO, and the pH was adjusted to 4.5 with acetic acid, followed by stirring overnight. Then, the solution was transferred to a dialysis bag (MWCO = 3.5 kDa) and dialyzed against PBS solution (50 mmol / L, pH 4.5) for 12 hours, and then against PBS solution (50 mmol / L, pH 6.5) for 12 hours. The dialysis solution was changed every two hours during the first 12 hours and then every 6 hours. Subsequently, the micelle solution was transferred to a flask, and 10 mmol / L of the catalyst 2-amino-3-methoxybenzoic acid (54.60 mg) and the cross-linking agent dithiodiimide (DTP, dosage as shown in the above table) were added. After stirring and reacting for one day, the micelle solution was dialyzed against deionized water for another 12 hours, with the dialysis solution changed every two hours. Finally, the drug-loaded cross-linked micelle solution was lyophilized to obtain the cross-linked polymer micelles.

[0099] The particle size, drug loading, and morphology of the cross-linked micelles were characterized by dynamic light scattering (DLS), ultraviolet spectrophotometer, and transmission electron microscopy (TEM). The hydrodynamic diameters (Dh) of the drug-loaded polymer cross-linked micelles with three representative block ratios were 175.3, 231.9, and 130.9 nm, respectively, as Figure 6 shown in A of Figure 6 The drug loadings and encapsulation efficiencies were 14.35% / 16.74%, 15.95% / 18.61%, and 10.73% / 12.52%, respectively, as Figure 6 shown in B of

[0100] Example 6: In vitro release of drug-loaded cross-linked micelles

[0101] The drug-loaded crosslinked micelles (3 mg) in Example 5 were separately dispersed in 3 mL of PBS solution (pH 7.4) and 10 mmol / L GSH acetate buffer solution (pH 5.0), and then transferred to a dialysis bag (MWCO = 3.5 kDa), immersed in the corresponding 0.5% Tween buffer solution (47 mL), and dialyzed at 37 °C and 200 rpm. At specific time points, 4 mL of the dialysis external solution was taken and an equal volume of fresh buffer solution (4 mL) was added. The absorbance of the dialysis external solution at 435 nm at different times was measured using a UV-visible spectrophotometer, and an in vitro release curve was plotted, as Figure 7 shown.

[0102] Example 7: Cytotoxicity experiment

[0103] (1) Blank micelles

[0104] L02 cells were subcultured in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin, and streptomycin at 37 °C and 5% CO2 concentration. Then, the cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated for 24 hours. After aspirating the old medium and washing, 150 μL of medium containing 10 μL of blank micelles was added to the wells and incubated for another 24 hours. Then, the liquid was aspirated and the wells were washed three times. Then, 150 μL of medium containing 10 μL of CCK-8 reagent was added and allowed to act for 1 hour. The UV absorbance of the wells was measured at a wavelength of 450 nm, and the cell viability at different material concentrations was calculated, as Figure 8 shown in A and B of

[0105] (2) Drug-loaded micelles and free drugs

[0106] HepG2 cells were subcultured in DMEM medium supplemented with 10% FBS, 1% penicillin, and streptomycin at 37 °C and 5% CO2 concentration. Then, the cells were seeded in a 96-well plate at a density of 5000 cells per well and incubated for 24 hours. After aspirating the old medium and washing, 150 μL of medium containing 10 μL of drug-loaded micelles or free Cur was added to the wells and incubated for 48 hours. Then, the liquid was aspirated and the wells were washed three times. Then, 150 μL of medium containing 10 μL of CCK-8 reagent was added and allowed to act for 1 hour. The UV absorbance of the wells was measured at a wavelength of 450 nm, and the cell viability at different material concentrations was calculated, as Figure 8 shown in C of

[0107] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A triblock polymer with long circulation and long-acting anti-protein stability, characterized in that, Specifically: poly(2-hydroxy-3-phenoxypropyl methacrylate)-b-poly(2-oxopropyl methacrylate)-b-poly(glycerol methacrylate), and the block ratio x:y:z of poly(2-hydroxy-3-phenoxypropyl methacrylate), poly(2-oxopropyl methacrylate) and poly(glycerol methacrylate) is 15-50:15-50:15-50.

2. The triblock polymer with long circulation and long-acting anti-protein stability according to claim 1, wherein The degrees of polymerization of the poly(2-hydroxy-3-phenoxypropyl methacrylate), poly(2-oxopropyl methacrylate) and poly(glycerol methacrylate) are x = 15-50, y = 15-50 and z = 15-50 respectively.

3. The preparation method of a triblock polymer with long circulation and long-acting anti-protein stability according to any one of claims 1 to 2, characterized in that, It includes the following steps: Under an inert gas atmosphere, mix 2-hydroxy-3-phenoxypropyl methacrylate, ligand, catalyst, reducing agent and solvent evenly, then add a small molecule initiator, and carry out a heating reaction. After the 2-hydroxy-3-phenoxypropyl methacrylate is completely converted, add 2-oxopropyl methacrylate and continue the reaction. After complete conversion, add glycerol methacrylate and continue the reaction, and then purify to obtain an amphiphilic triblock polymer.

4. The preparation method of a triblock polymer with long circulation and long-acting anti-protein stability according to claim 3, characterized in that, The molar ratios of the 2-hydroxy-3-phenoxypropyl methacrylate, 2-oxopropyl methacrylate, glycerol methacrylate, ligand, catalyst, reducing agent and small molecule initiator are: 15-50:15-50:15-50:0.018-0.75:0.018-0.062:0.181-0.89:0.36-1; The ligand is at least one of 1,1,4,7,10,10-hexamethyltriethylenetetramine, tripyridylmethylamine and bipyridine; the catalyst is at least one of copper bromide, iron chloride and copper chloride; the reducing agent is at least one of stannous octoate and ascorbic acid; the small molecule initiator is at least one of ethyl 2-bromoisobutyrate, azobisisobutyronitrile and 1,1,2,2-tetraphenylethylene glycol.

5. The preparation method of a triblock polymer with long circulation and long-acting anti-protein stability according to claim 3, characterized in that, In the preparation method of the triblock polymer with long circulation and long-term anti-protein stability, the temperature of the heating reaction is 50-70 °C; the heating reaction time of the 2-hydroxy-3-phenoxypropyl methacrylate is 8-12 h; the heating reaction time of the 2-oxopropyl methacrylate is 24-72 h; the heating reaction time of the glycerol methacrylate is 72-120 h.

6. The preparation method of a triblock polymer with long circulation and long-acting anti-protein stability according to claim 3, characterized in that, The 2-hydroxy-3-phenoxypropyl methacrylate is prepared by the following method: Under an inert gas atmosphere, dissolve methacrylic acid, inhibitor, acid-binding agent and 1,2-epoxy-3-phenoxypropane in a solvent, carry out a heating reaction, and purify to obtain 2-hydroxy-3-phenoxypropyl methacrylate; The 2-oxopropyl methacrylate is prepared by the following method: Under an inert gas atmosphere and ice bath conditions, dissolve methacryloyl chloride and hydroxyacetone in a solvent and react for 1.5-2.5 h, then add an acid-binding agent and continue the reaction at room temperature for 9-12 h, and purify to obtain 2-oxopropyl methacrylate; The glycerol methacrylate is prepared by the following method: Under atmospheric reflux and heating conditions, glycidyl methacrylate is hydrolyzed in water and purified to obtain glycerol methacrylate.

7. The preparation method of a triblock polymer with long circulation and long-acting anti-protein stability according to claim 6, characterized in that, In the preparation method of 2-hydroxy-3-phenoxypropyl methacrylate, the molar ratio of methacrylic acid, inhibitor, 1,2-epoxy-3-phenoxypropane and acid-binding agent is 1: 0.00033-0.00136: 1-1.1: 0.10-0.13; the reaction temperature is 50-70 °C and the time is 18-24 h; the inhibitor is at least one of dibutylhydroxytoluene and p-hydroxyanisole; the acid-binding agent is at least one of triethylamine and diisopropylethylamine; In the preparation method of 2-oxopropyl methacrylate, the molar ratio of methacryloyl chloride, hydroxyacetone and acid-binding agent is 1: 0.78-0.84: 1.03-1.09; the normal temperature refers to 15-40 °C; the acid-binding agent is at least one of triethylamine and diisopropylethylamine; In the preparation method of glycerol methacrylate, the volume fraction of the aqueous solution of glycidyl methacrylate is 8-12% w / w; the hydrolysis temperature is 70-80 °C and the time is 9-12 h.

8. Use of a triblock polymer having long circulation and long-term anti-protein stability according to any one of claims 1-2 in drug loading.

9. Use of a triblock polymer having long circulation and long-acting anti-protein stability according to claim 8 in drug loading, characterized in that, Dissolve a triblock polymer having long circulation and long-term anti-protein stability and a water-insoluble drug in a solvent, adjust the pH to 4-5, stir for 12-24 hours, then dialyze in a phosphate buffer solution, add a catalyst and a crosslinking agent, react at room temperature for 12-24 hours, and then dialyze with deionized water to obtain a pH and GSH-responsive crosslinked micelle system loaded with a water-insoluble drug and having long circulation and long-term anti-protein stability performance.

10. Use of a triblock polymer having long circulation and long-acting anti-protein stability according to claim 9 in drug loading, characterized in that, The mass ratio of the triblock polymer having long circulation and long-term anti-protein stability to the water-insoluble drug is 1: 0.5-1; the water-insoluble drug refers to a drug with a solubility of less than or equal to 1 g in 1 L of water; the water-insoluble drug is a water-insoluble anticancer drug; the water-insoluble anticancer drug is curcumin; The catalyst is 2-amino-5-methoxybenzoic acid; the crosslinking agent is dithiodihydrazide; the concentration of the catalyst in the dialysis solution is 8-12 mmol / L; the molar ratio of the crosslinking agent to 2-oxopropyl methacrylate in the triblock polymer is 1.25-1.30: 1.

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