A polymer carrier, a pharmaceutical composition and their applications

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to a polymer carrier, a pharmaceutical composition and their applications. The polymer carrier has a three-arm structure, and the three arms are respectively a hydrophilic segment, a hydrophobic segment and a responsive segment. The hydrophilic segment is a hydrophilic segment with the ability to resist protein adsorption; the responsive segment is a group that can maintain hydrophobicity and electrical neutrality in the blood while turning into hydrophilicity and positive charge at the lesion site. The three-arm polymer carrier provided by the present invention can induce autonomous transformation of nano-properties such as the size, surface charge and stability of the nano-assembly formed by the polymer due to the transformation of hydrophilic-hydrophobic properties and charge between normal physiological conditions and pathological conditions, significantly enhancing the blood circulation, tumor accumulation, tumor tissue penetration and tumor cell uptake performance of nano-drugs, thereby effectively inhibiting tumor growth and ablating tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine, and particularly relates to a polymer carrier, a pharmaceutical composition and their applications. Background Art

[0002] Cancer has become one of the leading causes of death worldwide. With population growth and aging, the incidence and mortality of cancer are still increasing year by year. So far, for most cancers, no highly curative treatment methods have been found. With the progress of medical development, more and more treatments have been applied clinically, including surgical resection, chemotherapy and radiotherapy. Among them, there are many chemotherapy drugs, including doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin and so on. Due to the complex human body environment, free chemotherapy drugs generally have difficulty in effectively exerting their effects in the body, and the effects of inhibiting tumor growth and ablating tumors are relatively limited. Summary of the Invention

[0003] The first object of the present invention is to overcome the defect that existing tumor chemotherapy drugs cannot effectively inhibit tumor growth and ablate tumors, and to provide a new polymer carrier, which can be used as a loading medium for anti-tumor drugs to effectively inhibit tumor growth and ablate tumors.

[0004] Specifically, the polymer carrier provided by the present invention has a three-arm structure, and the three arms are respectively a hydrophilic segment, a hydrophobic segment and a responsive segment. The hydrophilic segment is a hydrophilic segment with anti-protein adsorption ability; the responsive segment is a group that can maintain hydrophobicity and electrical neutrality in the blood and turn into hydrophilicity and positive electricity at the lesion site.

[0005] The second object of the present invention is to provide a preparation method of the above polymer carrier, which includes obtaining it by respectively initiating the polymerization of hydrophilic monomers, hydrophobic monomers and responsive monomers through a multifunctional initiator by utilizing the difference in the polymerization reaction types of different monomers; and / or, first synthesizing end-group functionalized hydrophilic segment molecules, hydrophobic segment molecules and responsive segment molecules, and then bonding the hydrophilic segment molecules, hydrophobic segment molecules and responsive segment molecules to the same molecular chain through a multifunctional initiator by bioorthogonal chemical reaction or "click" chemical reaction.

[0006] The third object of the present invention is to provide a pharmaceutical composition, which contains a drug and the above polymer carrier, and the drug and the polymer carrier are combined in the form of micelles or the drug is bonded to the polymer carrier.

[0007] The fourth object of the present invention is to provide the application of the above polymer carrier and / or pharmaceutical composition in anti-tumor.

[0008] After in-depth research on the tumor inhibition mechanism and tumor microenvironment, the inventors of the present invention were pleasantly surprised to find that the above-mentioned polymer carrier with a three-arm structure can induce the autonomous transformation of nano-properties such as size, surface charge and stability of the nanoassembly formed by the polymer through the hydrophilic and hydrophobic properties and charge transition between normal physiological conditions and pathological conditions, significantly improving the blood circulation, tumor enrichment, tumor tissue penetration and tumor cell uptake performance of nano-drugs, thereby effectively inhibiting tumor growth and ablating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The Zeta potential and particle size change diagram of the three-arm polymer carrier micelle obtained in Application Example 1-1 at different pH values;

[0010] Figure 2 This is a graph showing the 4T1 cytotoxicity test results corresponding to the drug-loaded three-arm polymer micelles obtained in Application Example 1-1;

[0011] Figure 3 This is the fluorescence result diagram corresponding to the drug-loaded three-arm polymer micelle obtained in Application Example 1-1;

[0012] Figure 4 The effect diagram of inhibiting tumor growth and ablating tumor corresponding to the drug-loaded three-arm polymer micelle obtained in Application Example 1-1;

[0013] Figure 5 This is a graph showing the 4T1 cytotoxicity test results corresponding to the drug-loaded three-arm polymer micelles obtained in Application Example 2-1;

[0014] Figure 6 This is the fluorescence result diagram corresponding to the drug-loaded three-arm polymer micelle obtained in Application Example 2-1;

[0015] Figure 7 The effect diagram of inhibiting tumor growth and ablating tumor corresponding to the drug-loaded three-arm polymer micelle obtained in Application Example 2-1;

[0016] Figure 8 This is a graph showing the 4T1 cytotoxicity test results corresponding to the drug-loaded three-arm polymer micelles obtained in Application Example 3-1;

[0017] Figure 9 This is the fluorescence result diagram corresponding to the drug-loaded three-arm polymer micelle obtained in Application Example 3-1;

[0018] Figure 10 The effect diagram of inhibiting tumor growth and ablating tumor corresponding to the drug-loaded three-arm polymer micelle obtained in Application Example 3-1;

[0019] Figure 11 This is a graph showing the 4T1 cytotoxicity test results corresponding to the drug-loaded three-arm polymer micelles obtained in Application Example 4-1;

[0020] Figure 12 It is the fluorescence result diagram corresponding to the drug-loaded three-armed polymer micelle obtained in Application Example 4-1;

[0021] Figure 13 It is the effect diagram of inhibiting tumor growth and ablating tumors corresponding to the drug-loaded three-armed polymer micelle obtained in Application Example 4-1;

[0022] Figure 14 It is the 4T1 cell cytotoxicity test result diagram corresponding to the drug-loaded three-armed polymer micelle obtained in Application Example 5-1;

[0023] Figure 15 It is the fluorescence result diagram corresponding to the drug-loaded three-armed polymer micelle obtained in Application Example 5-1;

[0024] Figure 16 It is the effect diagram of inhibiting tumor growth and ablating tumors corresponding to the drug-loaded three-armed polymer micelle obtained in Application Example 5-1. Detailed implementation mode

[0025] The polymer carrier provided by the present invention has a three-armed structure, and the three arms are respectively a hydrophilic chain segment, a hydrophobic chain segment and a responsive chain segment. The hydrophilic chain segment is a hydrophilic chain segment with anti-protein adsorption ability; the responsive chain segment is a group that can maintain hydrophobicity and electrical neutrality in the blood and turn into hydrophilicity and positive electricity at the lesion site. Specifically, the structural general formula of the polymer carrier is shown in Formula (I), where A is a hydrophilic chain segment, B is a hydrophobic chain segment, and C is a responsive chain segment.

[0026]

[0027] In a preferred implementation mode, the hydrophilic chain segment is selected from at least one of a polyethylene glycol chain segment, a poly(N-(2-hydroxypropyl)methacrylamide) chain segment, a polyvinylpyrrolidone chain segment, a poly(2-methacryloyloxyethyl phosphorylcholine) chain segment, a poly(methacryloylethyl sulfobetaine) polyoxazoline (POx) chain segment, a polyamino acid (PAA) chain segment, and a polyphosphate chain segment.

[0028] In a preferred implementation mode, the hydrophilic chain segment includes at least one of the following structures:

[0029] m is 20-200;

[0030] m is 20-200,

[0031]

[0032] R` is H or an alkyl group with 1-5 carbon atoms, m is 20-200,

[0033]

[0034] m is from 20 to 200,

[0035]

[0036] m is from 20 to 200,

[0037]

[0038] m is from 20 to 200,

[0039]

[0040] In a preferred embodiment, the hydrophobic segment is selected from at least one of a polyester segment, a polycarbonate segment, a polyphosphate segment, a polyorthoester segment, a polyanhydride segment, a poly(p-dioxanone) segment, a poly(meth)acrylate segment, a poly(meth)acrylamide segment, a polyurethane segment, a polyamino acid and a side-chain functionalized derivative segment thereof.

[0041] In a preferred embodiment, the hydrophobic segment includes at least one of the following structures:

[0042] m is from 10 to 200;

[0043] m is from 10 to 200, and x is from 1 to 4;

[0044] m is from 10 to 200;

[0045] m is from 10 to 200,

[0046]

[0047] m is from 10 to 200,

[0048]

[0049] m is from 10 to 200,

[0050]

[0051]

[0052] R1 and R2 can form a ring by bonding and are selected from at least one of the following structures:

[0053] m is from 10 to 200,

[0054]

[0055] R1 and R2 can form a ring by bonding and are selected from at least one of the following structures:

[0056]

[0057] m is from 10 to 200,

[0058]

[0059] R` is H or an alkyl group having 1 to 5 carbon atoms, and m is from 10 to 200,

[0060] m is from 10 to 200,

[0061]

[0062] In the present invention, the responsive segment is a group that can maintain hydrophobicity and electrical neutrality in the blood and transform into hydrophilicity and positive charge at the lesion site. The polymer carrier can induce autonomous transformation of nano-properties such as the size, surface charge, and stability of the nano-assembly formed by the polymer through the hydrophilic-hydrophobic property and charge transformation of the responsive segment between normal physiological conditions and pathological conditions, thereby effectively enhancing the blood circulation, tumor accumulation, tumor tissue penetration, and tumor cell uptake performance of the nano-drug, and improving the tumor inhibition and / or tumor ablation effect.

[0063] In a preferred embodiment, the responsive segment is at least one of a poly(meth)acrylate segment, a poly(meth)acrylamide segment, a polyurethane segment, a poly-β-amino ester segment, a polyester segment, a polycarbonate segment, a polyphosphate segment, a polypeptide segment, and a polyamino acid segment that can undergo charge and hydrophilic-hydrophobic property transformation under the stimulation of the tumor pathological environment. Among them, the tumor microenvironment stimulation includes pH stimulation, hypoxia stimulation, reduction stimulation, acid stimulation, enzyme stimulation, etc.

[0064] In a preferred embodiment, the responsive segment includes at least one of the following structures:

[0065] m is from 10 to 200, and R` is H or an alkyl group having 1 to 5 carbon atoms,

[0066]

[0067] R1 is an alkyl group of C1-C5, and x = 1-8;

[0068] m = 10-200,

[0069]

[0070] m is 10-200,

[0071]

[0072] m is 10-200,

[0073]

[0074] m is 10-200,

[0075] The preparation method of the polymer carrier provided by the present invention includes obtaining it by respectively initiating the polymerization of hydrophilic monomers, hydrophobic monomers and responsive monomers through a multifunctional initiator by utilizing the difference in the polymerization reaction types of different monomers; and / or, first synthesizing end-group functionalized hydrophilic chain segment molecules, hydrophilic chain segment molecules and hydrophilic chain segment molecules, and then bonding the hydrophilic chain segment molecules, hydrophobic chain segment molecules and responsive chain segment molecules to the same molecular chain through a multifunctional initiator by bioorthogonal chemical reaction or "click" chemical reaction.

[0076] In the preparation process of the above polymer carrier, the first method is to obtain the polymer carrier by respectively initiating the polymerization of hydrophilic monomers, hydrophobic monomers and responsive monomers through a multifunctional initiator by utilizing the difference in the polymerization reaction types of different monomers. Among them, the polymerization reaction types include ring-opening polymerization (ROP), N-carboxyanhydride ring-opening polymerization (NCA-ROP), reversible addition-fragmentation chain transfer radical polymerization (RAFT), nitroxide-mediated radical polymerization (NMP), atom transfer radical polymerization (ATRP), Michael addition polymerization, etc.

[0077] In a preferred embodiment, the multifunctional initiator is selected from at least one of the following compounds:

[0078]

[0079] Correspondingly, the polymerization reaction types corresponding to the above multifunctional initiators are as follows:

[0080]

[0081] In the preparation process of the above polymer carrier, the second method is to first synthesize end-group functionalized hydrophilic chain segment molecules, hydrophobic chain segment molecules and responsive chain segment molecules, and then initiate the hydrophilic chain segment molecules, hydrophobic chain segment molecules and responsive chain segment molecules to be bonded to the same molecular chain via a multifunctional initiator to obtain the polymer carrier. At this time, it is achieved by means of a macromolecular bioorthogonal chemical reaction or a "click" chemical reaction between different functionalized monomers.

[0082] In a preferred embodiment, the multifunctional initiator is selected from at least one of the following compounds:

[0083]

[0084] Correspondingly, the polymerization reaction types corresponding to the above multifunctional initiators are as follows:

[0085]

[0086] The pharmaceutical composition provided by the present invention contains a drug and the above polymer carrier, and the drug and the polymer carrier are combined in the form of micelles or the drug is bonded to the polymer carrier.

[0087] When the drug and the polymer carrier are combined in the form of micelles, the resulting pharmaceutical composition is called a drug-loaded three-armed polymer micelle. At this time, the corresponding preparation process includes dissolving the drug and the polymer carrier in a solvent to obtain a drug / polymer carrier mixed solution, slowly adding the drug / polymer carrier mixed solution to a PBS buffer solution and stirring for 8 to 48 hours, and then dialyzing to obtain the drug-loaded three-armed polymer micelle on the retention side.

[0088] When the drug is bonded to the polymer carrier, the resulting pharmaceutical composition is called a drug-conjugated polymer micelle. At this time, the bonding method can be carried out through various existing chemical reactions, and there is no particular limitation. Those skilled in the art can all understand this and will not be elaborated here.

[0089] In a preferred embodiment, the drug is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, irinotecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, Taltobulin, maytansinol, telanstatin A, auristatin E, auristatin F, Piericidin A, ansamitocin P3, dolastatin 10, β-Amanitin, and responsive prodrugs of the above chemotherapeutic drugs, and the responsive manner is selected from at least one of photo-responsive, radiation-responsive, reactive oxygen species-responsive, hypoxia-responsive, reduction-responsive, pH-responsive, and enzyme-responsive.

[0090] In addition, the present invention also provides the application of the above polymer carrier and / or drug composition in anti-tumor treatment.

[0091] The present invention will be described in detail below through examples.

[0092] To further understand the present invention, the present invention will be specifically described below in conjunction with examples, but the present invention is not limited to these examples or application examples. Changes, substitutions, combinations, simplifications, etc. made by those skilled in the art under the core guiding ideology of the present invention should all be included within the protection scope of the present invention.

[0093] Example 1-1: Synthesis of monomer C7A-MA hydrochloride

[0094]

[0095] Add 2.95 mL of N-(2-hydroxyethyl)hexamethylenediamine (C7A, 0.02 mol) to a round-bottom flask, then add 20 mL of DMF as a solvent, and dropwise add 1.94 mL of methacryloyl chloride (0.02 mol) under ice bath conditions at a dropping rate of 1 drop every 3 seconds. After the addition is complete, remove the ice bath and react at room temperature for 12 h. After the reaction is completed, filter to remove the filtrate, wash three times with ice-cold diethyl ether, and place in a vacuum oven to dry overnight to obtain a white powdery product C7A-MA hydrochloride.

[0096] Chemical shift of C7A-MA: 1 1H-NMR (CDCl3), δ (ppm): 1.2 - 1.4 (NCH2CH2, CCH3); 2.3 - 2.7 (NCH2); 4.3 - 4.5 (COOCH2); 5.3 - 6.0 (CCH2).

[0097] Example 1-2: Synthesis of triblock polymer PDLLA-Ser[P(C7A-MA)]-PEG

[0098]

[0099] Accurately weigh polyethylene glycol (PEG 5k , 5.00 g, 1 mmol) into a Schlenk flask, stop heating after dehydrating for 1 h under vacuum at 110 °C. Add O-[(1,1-dimethylethyl)dimethylsilyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serine (Fmoc-Ser(TBDMS)-OH, 4.42 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar. Under an argon atmosphere, pipette 17 mL of anhydrous DCM into the Schlenk flask, seal it, and react at room temperature for 48 h. Post-reaction treatment: Filter to remove insoluble reaction products, collect the liquid phase and concentrate it to a certain volume, precipitate in excess ether, filter and dry the ether, then dissolve it in ethanol and recrystallize at -20 °C. After suction filtration and drying, obtain a white solid Fmoc-Ser(TBDMS)-PEG (2.86 g, yield 53%).

[0100] Take a dry and clean 25 mL single-necked flask, add 2.00 g of Fmoc-Ser(TBDMS)-PEG and dissolve it in 15 mL of DMF. Add 3 mL of piperidine and stir the reaction at room temperature for 2 h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol and recrystallize twice at -20 °C. Filter to obtain a white solid NH2-Ser(TBDMS)-PEG (1.42 g, yield 74%).

[0101] Take 1.50 g of NH2-Ser(TBDMS)-PEG in a three-necked flask, add 6 mL of DCM and a magnetic stir bar, and dissolve and stir in an ice-water bath. Take triethylamine (TEA, 280 μL, 2 mmol) and 2-bromo-2-methylpropionyl bromide (BIBB, 250 μL, 2 mmol) and dissolve them separately in 2 mL of DCM. Pipette them separately and slowly drip them into the round-bottom flask at a rate of 3 drops / second simultaneously under an ice-bath environment for reaction. After dropping, seal it, remove the ice bath, and react at room temperature for 24 h. After the reaction is completed, filter to remove insoluble substances, wash three times with deionized water, add anhydrous sodium sulfate to remove water, dry for 12 h, then filter to remove sodium sulfate solid, rotary evaporate to remove some solvents to concentrate the product, dissolve it in ethanol and place it in a -20 °C refrigerator for recrystallization. After suction filtration and drying, obtain a yellow solid BIBB-Ser(TBDMS)-PEG (1.06 g, yield 69%).

[0102] Add 1.00 g of BIBB-Ser(TBDMS)-PEG, 2 mL of trifluoroacetic acid (TFA), and 2 mL of dichloromethane to a single-necked flask. Stir the reaction at room temperature for 1 h, rotary evaporate and concentrate the reaction solution, and precipitate with ether three times to obtain a yellow solid BIBB-Ser(OH)-PEG (0.73 g, yield 75%).

[0103] The schlenk flask, glass syringe, and needle used in the reaction were dried in an oven at 120 °C for 2 h to remove water by drying. The vacuum was evacuated and argon was filled three times in a cycle. Under argon protection, add D,L-lactide monomer (DLLA, 7.66 g, 53 mmol), 0.50 g of dried BIBB-Ser-PEG, and a 5% Sn(Oct)2 solution. Absorb 6 mL of freshly distilled toluene with a glass syringe to dissolve, place it in an oil bath preheated to 120 °C, stir until it dissolves, and then react for 48 h. Quench the reaction with liquid nitrogen, add a few drops of dichloromethane to dilute the reaction solution, precipitate and centrifuge it three times in excess methanol, collect the precipitate, and dry it in a vacuum drying oven for 24 h to obtain a white solid, which is the product PEG-PDLLA-BIBB.

[0104] Dry the schlenk flask in an oven at 120 °C. Weigh 0.1 g of PEG-PDLLA-BIBB, 0.2 g of C7A-MA hydrochloride, and 10 μL of PMEDTA into a reaction tube, and add 0.6 mL of DMSO to dissolve. After two cycles of "freezing - evacuating - purging with argon - dissolving", under argon protection, add 3 mg of CuBr, and then after one more cycle of "freezing - evacuating - purging with argon - dissolving", react in a 70 °C water bath in the dark for 24 h. After the reaction is completed, add a small amount of DMSO to dissolve, dialyze with deionized water, and then dry to obtain the product PEG-PDLLA-P(C7A-MA).

[0105] The chemical shift of PEG-PDLLA-P(C7A-MA): 1 1H-NMR(CDCl3), δ(ppm): 0.7 - 1.4 (CH2CH2); 1.4 - 1.7 (CHCH3); 1.7 - 2.2 (BrCCH3); 2.6 - 3.1 (NCH2); 3.3 - 3.4 (OCH3); 3.4 - 4.0 (OCH2CH2O); 4.0 - 4.5 (CH2OH, COOCH2); 4.5 - 4.6 (NHCOCH); 5.0 - 5.4 (COCHO).

[0106] Application Example 1-1

[0107] (1) Charge reversal and particle size change experiment of the three-armed polymer micelle PEG-PDLLA-P(C7A-MA)

[0108] Dissolve the polymer PEG-PDLLA-P(C7A-MA) in 1 mL of DMSO and inject it into 9 mL of PBS buffer to prepare PEG-PDLLA-P(C7A-MA) micelles with different pH values and a concentration of 1 mg / mL. Place the micelles with different pH values in a shaker at 37 °C and 100 rpm overnight. Detect the changes in Zeta potential and particle size at different pH values by DLS. The results are shown in Figure 1 . It can be seen from Figure 1 that compared with the standard physiological pH, the Zeta potential of the polymer turns positive and the particle size decreases in an acidic environment.

[0109] (2) Preparation of the drug-loaded polymer micelle MMAE@PEG-PDLLA-P(C7A-MA) injection

[0110] Prepare the polymer injection according to the equivalent concentration of monomethyl auristatin E (MMAE) at 0.2 mg / kg body weight. Dissolve 17 mg of PEG-PDLLA-P(C7A-MA) and 0.4 mg of MMAE in 1 mL of DMSO to obtain a clear and transparent polymer and drug solution. Slowly inject the polymer and drug solution into 9 mL of PBS. After stirring the solution overnight, dialyze it to obtain the drug-loaded polymer micelle MMAE@PEG-PDLLA-P(C7A-MA).

[0111] (3) Cytotoxicity experiment

[0112] Perform 4T1 cell cytotoxicity tests on MMAE and the polymer injection at different pH values. The results are shown in Figure 2 . It can be seen from Figure 2 that MMAE@PEG-PDLLA-P(C7A-MA) has strong cytotoxicity to 4T1 cells in an acidic environment, which is close to the toxicity of free MMAE, indicating that the micelles are lysed to release the encapsulated MMAE; while the toxicity of MMAE@PEG-PDLLA-P(C7A-MA) in the standard physiological environment is small, indicating that most of the MMAE is embedded in the triblock polymer and not released.

[0113] (4) Pharmacokinetic evaluation

[0114] Take 2 mL of the MMAE@PEG-PDLLA-P(C7A-MA) solution and add 200 μL of the Cy5.5-alykne solution at 1 mg / mL dropwise. Stir the reaction at room temperature for 24 hours. After the reaction, dialyze with methanol for 3 hours first and then with ultrapure water for 24 hours to obtain the Cy5.5 fluorescently labeled polymer solution, denoted as MMAE@PEG-PDLLA-P(C7A-MA) Cy5.5 .

[0115] After the mice got used to the environment, six mice were selected and divided into two groups of three mice each. The Cy5.5 fluorescently labeled polymer solution was injected into the tail vein blood vessels of the mice at a dosing concentration of 5 mg / kg; the serum clearance half-life t 1 / 2 Subsequently, blood was taken from the capillaries around the eyes of the mice, about 50 μL of blood was taken, and it was put into the living body for detection before blood coagulation, and the fluorescence value was measured. The results are as Figure 3 shown. From Figure 3 it can be seen that the t 1 / 2 of MMAE@PEG-PDLLA-P(C7A-MA) is 3.9 h.

[0116] (5) Tumor inhibition experiment

[0117] To study the inhibitory effect of the three-armed polymer encapsulating MMAE drug on the tumors of 4T1 tumor-bearing mice with breast cancer in mice. When the tumors grew to about 100 mm 3 the experiment was started. Prepare free MMAE drug and MMAE@PEG-PDLLA-P(C7A-MA) micelles, and PBS buffer as the blank group. Administer the drug by tail vein injection at a dosing dose of 0.2 mg / kg of MMAE equivalent, and inject 100 μL for each mouse each time. Administer the drug on days 1, 4, 7, 10, and 13, for a total of five administrations. Weigh the mice on days 1, 3, 5, 7, 9, 11, 13, and 15, and measure the tumor volume of the mice. The obtained results are shown in Figure 4 . From Figure 4 it can be seen that compared with the blank group, the free drug MMAE has a weak ability to inhibit tumor growth, while the MMAE@PEG-PDLLA-P(C7A-MA) micelle group has a more obvious inhibitory effect and has a good ability to inhibit tumor growth and ablate tumors.

[0118] Example 2-1: Synthesis of macromolecular chain transfer agent PEG-Lys(NMA)-NH2

[0119]

[0120]

[0121] Accurately weigh polyethylene glycol (PEG 5k, 5.00 g, 1 mmol) into a Schlenk flask, and the heating was stopped after 1 h of water removal under vacuum at 110 °C. Boc-Fmoc-Lysine (Fmoc-Lys(Boc)-OH, 4.68 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar were added. 20 mL of anhydrous DCM was drawn into the Schlenk flask under an argon atmosphere, sealed and reacted at room temperature for 48 h. Post-treatment of the reaction: The insoluble substances in the reaction were removed by filtration, the liquid phase was collected and concentrated to a certain volume, precipitated in excess ether, filtered and dried, and then dissolved in ethanol. Recrystallization was carried out at -20 °C, and after suction filtration and drying, a white solid (Fmoc-Lys(Boc)-PEG) was obtained.

[0122] A dry and clean 25 mL single-necked flask was taken, 2.00 g of Fmoc-Lys(Boc)-PEG was added and dissolved in 15 mL of DMF, 3 mL of piperidine was added, and the reaction was stirred at room temperature for 2 h. The reaction solution was concentrated to a certain volume, dissolved in ethanol and recrystallized twice at -20 °C, and the white solid NH2-Lys(Boc)-PEG was obtained by filtration.

[0123] NH2-Lys(Boc)-PEG (1.00 g, 0.21 mmol) and triethylamine (0.15 g, 1.47 mmol) were dissolved in 10 mL of dichloromethane. After being fully dissolved by magnetic stirring, stirring was continued in an ice-water bath. Then acryloyl chloride (0.14 g, 1.47 mmol) was weighed and dissolved in 2 mL of dichloromethane, placed in a dropping funnel, and slowly dropped into the round-bottom flask at a rate of 3 drops / second for reaction. After the dropping was completed, the reaction was continued overnight. After the reaction was completed, it was washed twice with deionized water, anhydrous sodium sulfate was added to remove water, magnetic stirring and drying were carried out for 12 h, then the sodium sulfate solid was removed by suction filtration, the solvent was removed by rotary evaporation to concentrate the product, dissolved in ethanol and recrystallized twice at -20 °C, and the white solid MNA-Lys(Boc)-PEG was obtained by filtration.

[0124] 1.00 g of MNA-Lys(Boc)-PEG, trifluoroacetic acid (TFA, 2 mL) and 5 mL of dichloromethane were added to a single-necked flask, and the reaction was stirred at room temperature for 1 h. The reaction solution was concentrated by rotary evaporation, dissolved in ethanol and recrystallized twice at -20 °C, and the white solid MNA-Lys(NH2)-PEG was obtained by filtration.

[0125] Dissolve MNA-Lys(NH2)-PEG (50 mg, 0.01 mmol) and (S)-4-isobutyloxazolidine-2,5-dione (50 mg, 0.32 mmol) in DMF (1 mL) in a reaction tube. Add a small amount of LiHMDS catalyst. Stir the reaction mixture at room temperature for 48 hours, then concentrate it by rotary evaporation. After that, dissolve it in chloroform and precipitate with cold ether or methanol. Purify the polymer by centrifugation and dialysis, and obtain the polymer PEG-P(BuAA)-MNA after lyophilization.

[0126] Add PEG-P(BuAA)-MNA (70 mg, 0.007 mmol), ethylene glycol diacrylate (20 mg, 0.118 mmol), 2-(azepan-1-yl)ethylamine (30 mg, 0.176 mmol), and 1 mL DMSO to a reaction tube. After three times of "freezing - evacuating - purging with argon - dissolving", react it at 60 °C in the dark for 24 h. Add N-methylimidazole (2.3 mg, 0.03 mmol) and imidazole (28 mg, 0.0004 mmol) to the flask, and raise the temperature to 70 °C, react in the dark for 24 h. After dialysis of the reaction solution, lyophilize it to obtain PEG-Lys[P(C7A-N-ED)]-P(BuAA).

[0127] Chemical shift of PEG-Lys[P(C7A-N-ED)]-P(BuAA): 1 1H-NMR (CDCl3), δ (ppm): 1.2 - 1.8 (CH(CH3)2); 2.0 - 2.6 (CH2CH2); 2.6 - 3.1 (NCH2); 3.3 - 3.4 (OCH3); 3.4 - 4.0 (NHCH2, OCH2CH2O); 4.0 - 4.2 (CH2OH); 4.5 - 4.6 (NHCOCH, COOCH2, OCOOCH2); 5.6 - 6.5 (NHCOCHCH2); 8.0 - 8.5 (CONH, NH2).

[0128] Application Example 2-1

[0129] (1) Preparation of the drug-loaded polymer micelle PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) injection

[0130] Prepare a polymer injection solution with an equivalent concentration of paclitaxel (PTX) at 10 mg / kg body weight. Dissolve 15 mg of PEG-Lys[P(C7A-N-ED)]-P(BuAA) and 8 mg of PTX in 1 mL of DMSO to obtain a clear and transparent polymer and drug solution. Slowly inject the polymer and drug solution into 9 mL of PBS. After stirring the solution overnight, dialyze it to obtain the drug-loaded polymer micelle PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA).

[0131] (2) Cytotoxicity experiment

[0132] Perform 4T1 cell cytotoxicity tests on PTX and polymer injection solutions at different pH values. The obtained results are shown in Figure 5 . From Figure 5 it can be seen that PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) has strong cytotoxicity to 4T1 cells in an acidic environment, approaching the toxicity of free PTX, indicating that the micelles are lysed to release the encapsulated PTX; while PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) has less toxicity in a standard physiological environment, indicating that most of the PTX is encapsulated in the triblock polymer and not released.

[0133] (3) Pharmacokinetic evaluation

[0134] Take 2 mL of the PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) solution and add 200 μL of a 1 mg / mL Cy5.5-alykne solution dropwise. Stir the reaction at room temperature for 24 hours. After the reaction is completed, dialyze it with methanol for 3 hours first, and then dialyze it with ultrapure water for 24 hours to obtain the Cy5.5 fluorescently labeled polymer solution, denoted as PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) Cy5.5 .

[0135] After the mice are acclimated to the environment, take 6 mice and divide them into two groups of three mice each. Inject the Cy5.5 fluorescently labeled polymer solution into the tail vein blood vessels of the mice at a dosing concentration of 5 mg / kg; after the serum clearance half-life t 1 / 2 take blood from the capillaries around the eyes of the mice. Take about 50 μL of blood and place it in a living body for detection before blood coagulation to measure the fluorescence value. The results are as shown in Figure 6 . From Figure 6 it can be seen that the t 1 / 2 of PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) is 5.1 h.

[0136] (4) Tumor inhibition experiment

[0137] To study the inhibitory effect of a three-armed polymer loaded with PTX drug on tumors in 4T1 tumor-bearing mice with breast cancer in mice, wait until the tumor grows to about 100 mm 3 Start the experiment. Prepare free PTX drug and PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) micelles, and use PBS buffer as the blank group. Administer the drug by tail vein injection at a dose of 10 mg / kg of PTX equivalent, and inject 100 μL for each mouse each time. Administer the drug on the 1st, 4th, 7th, 10th, and 13th days, for a total of five administrations. Weigh the mice on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 15th days, and measure the tumor volume of the mice. The results are shown in Figure 7 . From Figure 7 It can be seen that compared with the blank group, the free drug PTX has a weak ability to inhibit tumor growth, while the PTX@PEG-Lys[P(C7A-N-ED)]-P(BuAA) micelle group has a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0138] Example 3-1: Synthesis of polymer PEG-Glu(Py)-N3

[0139]

[0140]

[0141] Accurately weigh polyethylene glycol (PEG 5k , 5.00 g, 1 mmol) into a schlenk flask, and stop heating after dehydrating in a vacuum environment at 110 °C for 1 h. Add tert-butoxycarbonyl-glutamic acid methyl ester (Boc-Glu(MeO)-OH, 2.61 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stirrer. Pipette 20 mL of anhydrous DCM into the schlenk flask under an argon atmosphere, seal it, and react at room temperature for 48 h. Post-reaction treatment: Filter to remove insoluble reaction substances, collect the liquid phase and concentrate it to a certain volume, precipitate in excess ether, filter and dry the ether, then dissolve it in ethanol, recrystallize at -20 °C, and filter and dry to obtain a white solid Boc-Glu(MeO)-PEG.

[0142] Add 4.00 g of Boc-Glu(MeO)-PEG, trifluoroacetic acid (TFA, 4 mL) and 10 mL of dichloromethane to a single-necked flask, stir and react at room temperature for 2 h, rotary evaporate and concentrate the reaction solution, dissolve it in ethanol and recrystallize twice at -20 °C, and filter to obtain a white solid NH2-Glu(MeO)-PEG.

[0143] Take a dry and clean 25 mL single-necked flask, add NH2-Glu(MeO)-PEG (2.00 g, 0.38 mmol), 3-azidopropionic acid (0.44 g, 3.8 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.73 g, 3.8 mmol), and a small amount of 1-hydroxybenzotriazole (HOBt), dissolve them in 100 mL of dichloromethane, and stir the reaction at room temperature for 24 h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol, and recrystallize it twice at -20 °C. Filter to obtain the white solid N3-Glu(MeO)-PEG.

[0144] Dissolve N3-Glu(MeO)-PEG (1.50 g, 0.285 mmol) in 50 mL of 95% methanol aqueous solution, add 15 g of KOH, and stir the reaction for 4 h. After the reaction is completed, wash it twice with 0.1 M HCl, then wash it twice with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter to remove the sodium sulfate solid, rotary evaporate to remove the solvent and concentrate the product, dissolve it in ethanol, and recrystallize it twice at -20 °C. Filter to obtain the white solid PEG-Glu(OH)-N3.

[0145] Dissolve PEG-Glu(OH)-N3 (1.00 g, 0.19 mmol), propargylamine (0.05 g, 1.9 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 0.37 g, 1.9 mmol), and N-hydroxysuccinimide (NHS, 0.22 g, 1.9 mmol) in 50 mL of dichloromethane, and stir the reaction at room temperature for 24 h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol, and recrystallize it twice at -20 °C. Filter to obtain the white solid PEG-Glu(Py)-N3.

[0146] The chemical shift of PEG-Glu(Py)-N3: 1 H-NMR(CDCl3), δ(ppm): 2.0 - 2.6 (CH2CH2); 2.8 - 3.2 (CCH, N3CH2); 3.3 - 3.4 (OCH3); 3.4 - 4.0 (NHCHC, NHCH2, OCH2CH2O,); 4.5 - 4.6 (NHCOCH); 5.6 - 6.5 (NHCOCHCH2).

[0147] Example 3-2: Synthesis of monomer NDMA

[0148]

[0149] Dissolve N,N - diisopropylethylenediamine (1.00 g, 6.9 mmol) and triethylamine (1.38 g, 13.8 mmol) in 20 mL of dichloromethane. After fully dissolving with magnetic stirring, continue stirring in an ice - water bath. Weigh methacryloyl chloride (0.63 g, 6.9 mmol) and dissolve it in 5 mL of dichloromethane. Place it in a dropping funnel and slowly drop it into the round - bottom flask at a rate of 1 drop every 3 seconds for reaction. After the dropping is completed, continue the reaction overnight. After the reaction is completed, wash it twice with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically for 12 h, then filter off the sodium sulfate solid, rotary - evaporate to remove the solvent and concentrate the product, and obtain the pure product NDMA monomer through column chromatography.

[0150] Chemical shift of PEG - Glu(Py) - N3: 1 1H - NMR(CDCl3), δ(ppm): 1.0 - 1.1 (NCCH3); 1.8 - 2.0 (CCH3); 2.5 - 3.0 (NCH, NCH2); 3.0 - 3.2 (CONHCH2); 5.6 - 6.0 (CCH2).

[0151] Example 3 - 3: Synthesis of polymer DBCO - PNDMA

[0152]

[0153] Add CTA - COOH (10 mg, 0.04 mmol), NDMA (1.07 g, 5 mmol) and a small amount of AIBN into a branched - mouth reaction tube, and ultrasonically dissolve them in 600 μL of DMSO (HPLC grade). After complete dissolution, under the protection of argon gas, freeze - thaw with liquid nitrogen (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring at room temperature for dissolution - argon filling) 3 times to remove air, then place the reaction system in an oil bath at 70 °C for reaction for 24 h. After the reaction is completed, introduce air and quickly cool it in liquid nitrogen to terminate the reaction. The obtained product is precipitated in ethanol, and then dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted monomers, etc. The dialyzed solution is freeze - dried to remove water to obtain the polymer P(NDMA).

[0154] Dissolve 60 mg of P(NDMA) in DMF. Add 4 μL of triethylamine, 200 μL of excessive ethanolamine and a DMF solution containing DBCO - maleimide (4 mg) to the solution. After deoxidizing by bubbling argon for 5 minutes, carry out the reaction at 25 °C for 18 h. Dialyze the reaction mixture with deionized water for 72 h, and then freeze - dry to obtain the solid product DBCO - P(NDMA).

[0155] Chemical shift of DBCO - PNDMA: 11H-NMR (CDCl3), δ (ppm): 1.2 - 1.7 (CCH3, CHCH3); 2.0 - 2.6 (CCH2C, NCH2, CH2CH2); 2.8 - 3.2 (NCOCH2, CH2COOH; NCH, NCOCH2); 3.3 - 3.4 (OCH3); 3.4 - 4.0 (SCHCO, CONCH2, CONHCH2, NHCH2); 4.5 - 5.0 (NCH2); 7.0 - 8.0 (ph).

[0156] Examples 3 - 4: Synthesis of monomer SNMA

[0157]

[0158] Dissolve 1,2 - dithiolan - 3 - pentanoic acid (1.00 g, 4.85 mmol), ethylenediamine (0.58 g, 9.7 mmol), 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (EDC, 1.89 g, 9.7 mmol), and N - hydroxysuccinimide (NHS, 1.12 g, 9.7 mmol) in 50 mL of dichloromethane and stir the reaction at room temperature for 24 h. After the reaction is completed, wash it twice with 1 M NaOH and twice with deionized water. Add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter off the sodium sulfate solid by suction and rotary evaporate to remove the solvent to obtain the intermediate SNN.

[0159] Dissolve SNN (0.8 g, 3.5 mmol) and triethylamine (0.71 g, 7.0 mmol) in 20 mL of dichloromethane. After stirring magnetically to dissolve completely, continue stirring in an ice - water bath. Weigh methylacryloyl chloride (0.73 g, 7.0 mmol) and dissolve it in 5 mL of dichloromethane, place it in a dropping funnel, and slowly drip it into the round - bottom flask at a rate of 3 drops / second for reaction. After the addition is completed, continue the reaction overnight. After the reaction is completed, wash it twice with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter off the sodium sulfate solid by suction and rotary evaporate to remove the solvent to concentrate the product, and obtain the pure product SNMA monomer by column chromatography.

[0160] Chemical shift of SNMA: 1 1H - NMR (CDCl3), δ (ppm): 1.2 - 1.9 (SCHCH2, CCH3, CHCH3); 2.0 - 2.6 (NHCOCH, SCH2, SCH, CCH2C, NCH2, CH2CH2); 2.8 - 3.2 (CH2COOH; NCH); 3.3 - 3.4 (OCH3); 3.4 - 4.0 (NHCH2); 7.0 - 8.0 (ph).

[0161] Examples 3 - 5: Synthesis of Polymer PSNMA - N3

[0162]

[0163] Add CTA - COOH (10 mg, 0.04 mmol), SNMA (1.44 g, 5 mmol) and a small amount of AIBN into a three - neck reaction tube, and ultrasonically dissolve them in 600 μL of DMSO (HPLC grade). After complete dissolution, under the protection of argon gas, freeze - thaw with liquid nitrogen (liquid nitrogen freezing - vacuum pumping for 15 min - stirring and dissolving at room temperature - filling with argon gas) 3 times to remove air, and then place the reaction system in an oil bath at 70 °C for reaction for 24 h. After the reaction is completed, pass air through and quickly cool in liquid nitrogen to terminate the reaction. The obtained product is precipitated in ethanol, and then dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted monomers, etc. The dialyzed solution is freeze - dried to remove water to obtain polymer P(SNMA).

[0164] Dissolve P(SNMA) (50 mg, 0.01 mmol), 2 - azidoethylamine (8.6 mg, 0.1 mmol), 1 - (3 - dimethylaminopropyl) - 3 - ethylcarbodiimide hydrochloride (EDC, 19.5 mg, 0.1 mmol), and N - hydroxysuccinimide (NHS, 11.5 mg, 0.1 mmol) in 1 mL of DMF, and stir - react at room temperature for 24 h. After the reaction is completed, dialyze in deionized water for 1 week (changing water twice a day) to remove the reaction solvent and unreacted monomers, etc. The dialyzed solution is freeze - dried to remove water to obtain polymer P(SNMA) - N3.

[0165] Chemical shift of PSNMA - N3: 1 H - NMR(CDCl3), δ(ppm): 1.2 - 1.9 (SCHCH2, CCH3, CHCH3); 2.0 - 2.6 (NHCOCH, SCH2, SCH, CCH2C, NCH2, CH2CH2); 2.8 - 3.2 (CH2COOH; NCH); 3.3 - 3.4 (N3CH2, OCH3); 3.4 - 4.0 (NHCH2); 7.0 - 8.0 (ph).

[0166] Examples 3 - 6: Synthesis of Three - armed Polymer PEG - PNDMA - PSNMA

[0167]

[0168]

[0169] Weigh PEG-Glu(Py)-N3 (50 mg, 0.01 mmol) and DBCO-P(NDMA) (50 mg, 0.01 mmol), dissolve them in 0.5 mL of DMF, react at 25 °C for 24 h. After the reaction is completed, precipitate with ice-cold diethyl ether three times, dialyze with DMSO and then with deionized water, and lyophilize to obtain the product PEG-P(NDMA)-Py.

[0170] Weigh PEG-P(NDMA)-Py (50 mg, 0.01 mmol), P(SNMA)-N3 (50 mg, 0.01 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol), dissolve them in a mixed solvent of 0.5 mL of DMF and water (DMF:water = 7:3 (V:V)). After evacuating and filling with argon twice, add copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5 (w:w)), then evacuate and fill with argon three times, and react at 45 °C for 48 h. After the reaction is completed, precipitate with ice-cold diethyl ether three times, dialyze with DMSO and then with deionized water, and lyophilize to obtain PEG-P(NDMA)-P(SNMA).

[0171] Chemical shift of PEG-PNDMA-PSNMA: 1 1H-NMR (CDCl3), δ (ppm): 1.2 - 1.7 (CCH3, CHCH3); 2.0 - 2.6 (SCH2, SCH, CCH2C, NCH2, CH2CH2); 2.8 - 3.2 (NCOCH2, CH2COOH; NCH, NCOCH2, CCH, N3CH2); 3.3 - 3.4 (OCH3); 3.4 - 4.2 (OCH2CH2O, NHCHC, NHNCH2, SCHCO, CONCH2, CONHCH2, NHCH2); 4.5 - 5.0 (NHCOCH, NCH2); 5.6 - 6.5 (NHCOCHCH2); 7.0 - 8.0 (ph).

[0172] Application Example 3-1

[0173] (1) Preparation of the drug-loaded polymeric micelle PTX@PEG-P(NDMA)-P(SNMA) injection

[0174] Prepare a polymeric injection at an equivalent concentration of 10 mg / kg body weight of paclitaxel (PTX). Dissolve 15 mg of PEG-P(NDMA)-P(SNMA) and 8 mg of PTX in 1 mL of DMSO to obtain a clear and transparent polymer and drug solution. Slowly inject the polymer and drug solution into 9 mL of PBS, stir the solution overnight and then dialyze to obtain the drug-loaded polymeric micelle PTX@PEG-P(NDMA)-P(SNMA).

[0175] (2) Cytotoxicity experiment

[0176] The cytotoxicity test of PTX with polymer injection solution at different pH values on 4T1 cells was carried out, and the results are shown in Figure 8 . From Figure 8 it can be seen that PTX@PEG-P(NDMA)-P(SNMA) has strong cytotoxicity to 4T1 cells in an acidic environment, which is close to the toxicity of free PTX drug, indicating that the micelles are lysed to release the encapsulated PTX; while PTX@PEG-P(NDMA)-P(SNMA) has less toxicity in a standard physiological environment, indicating that most of the PTX is embedded in the triblock polymer and not released.

[0177] (3) Pharmacokinetic evaluation

[0178] Take 2 mL of PTX@PEG-P(NDMA)-P(SNMA) solution, and add 200 μL of 1 mg / mL Cy5.5-alykne solution dropwise, and stir at room temperature for 24 hours. After the reaction, dialyze with methanol for 3 hours first, and then dialyze with ultrapure water for 24 hours to obtain the Cy5.5 fluorescently labeled polymer solution, denoted as PTX@PEG-P(NDMA)-P(SNMA) Cy5.5 .

[0179] After the mice adapt to the environment, take 6 mice and divide them into two groups, with three mice in each group. Inject the Cy5.5 fluorescently labeled polymer solution into the tail vein blood vessels of the mice, and the dosing concentration is 5 mg / kg; the half-life of serum clearance rate t 1 / 2 After that, take blood from the capillaries around the eyes of the mice, take about 50 μL of blood, and put it into a living body for detection before blood coagulation, and measure the fluorescence value. The results are as Figure 9 shown. From Figure 9 it can be seen that the t 1 / 2 of PTX@PEG-P(NDMA)-P(SNMA) is 5.8 h.

[0180] (4) Tumor inhibition experiment

[0181] Study the inhibitory effect of the triblock polymer encapsulating PTX drug on the tumors of 4T1 tumor-bearing mice with breast cancer in mice. Wait until the tumor grows to about 100 mm 3 and start the experiment. Prepare free PTX drug and PTX@PEG-P(NDMA)-P(SNMA) micelles, and PBS buffer as the blank group. Administer the drug by tail vein injection, and the dosing dose is 10 mg / kg of PTX equivalent, and 100 μL is injected into each mouse each time. Administer the drug on the 1st, 4th, 7th, 10th, and 13th days, for a total of five times. Weigh the mice on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 15th days, and measure the tumor volume of the mice. The results are shown inFigure 10 From Figure 10 It can be seen that, compared with the blank group, the free drug PTX has a relatively weak ability to inhibit tumor growth, while the PTX@PEG-P(NDMA)-P(SNMA) micelle group has a more obvious inhibitory effect and a better ability to inhibit tumor growth and ablate tumors.

[0182] Example 4-1: Synthesis of monomer HPMA

[0183]

[0184] Weigh 25.3 g of anhydrous sodium carbonate into a 250 mL round-bottom flask, add 19.4 g of 2-hydroxypropylamine, a small amount of 4-tert-butylcatechol (inhibitor), and 60 mL of dichloromethane. After stirring magnetically until fully dissolved, continue stirring in an ice-water bath. Then weigh 25 g of methacryloyl chloride and dissolve it in 60 mL of dichloromethane. Place it in a dropping funnel and slowly drop it into the round-bottom flask at a rate of 3 drops / second for reaction. After dropping, continue the reaction overnight. After the reaction is completed, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter to remove the sodium sulfate solid, rotary evaporate to remove part of the solvent to concentrate the product, and place it in a -20 °C refrigerator for freezing crystallization. Filter again to collect the crystals, and wash the crystals with ice-bath-cooled dichloromethane. Finally, dry the product in a vacuum oven.

[0185] Chemical shift of HPMA: 1 1H-NMR(CDCl3), δ(ppm): 1.2 - 1.3 (CCH3); 1.8 - 2.0 (CCH3); 3.0 - 3.2 (COMHCH2); 4.0 - 4.2 (CHOH); 5.4 - 6.0 (CCH2).

[0186] Example 4-2: Synthesis of monomer MA-N-AZO

[0187]

[0188] Weigh 4-hydroxyazobenzene (3.00 g, 15 mmol) into a schlenk flask, add Boc-β-alanine (3.4 g, 18 mmol), dicyclohexylcarbodiimide (DCC, 3.72 g, 18 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stirrer. Under an argon atmosphere, draw 40 mL of anhydrous dichloromethane into the schlenk flask, seal it and react at room temperature for 48 h. Post-reaction treatment: Filter to remove the insoluble reaction products, collect the liquid phase and concentrate it to a certain volume. After separation by column chromatography, rotary evaporate to remove the mobile phase and dry it in a vacuum oven overnight to obtain Boc-Ala-AZO.

[0189] Dissolve Boc-Ala-AZO (1.845 g, 5 mmol) in 10 mL of dichloromethane, add 3 mL of trifluoroacetic acid and stir for 12 h. Rotavapor to remove dichloromethane and trifluoroacetic acid, and dry overnight in a vacuum oven to obtain NH2-Ala-AZO.

[0190] Dissolve NH2-Ala-AZO (1.00 g, 3.7 mmol) and triethylamine (1.87 g, 18.5 mmol) in 15 mL of dichloromethane. After fully dissolving with magnetic stirring, continue stirring in an ice-water bath. Weigh methacryloyl chloride (1.93 g, 18.5 mmol) and dissolve it in 5 mL of dichloromethane. Place it in a dropping funnel and slowly add it dropwise into the round-bottom flask at a rate of 3 drops / second for reaction. After the addition is completed, continue the reaction overnight. After the reaction is completed, wash twice with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter to remove sodium sulfate solid, rotavapor to remove part of the solvent to concentrate the product, separate by column chromatography, rotavapor to remove the mobile phase, and dry overnight in a vacuum oven to obtain MA-N-AZO.

[0191] Chemical shift of MA-N-AZO: 1 1H-NMR (CDCl3), δ (ppm): 1.8 - 2.0 (CCH3); 2.6 - 2.8 (OCOCH2); 3.3 - 3.5 (CONHCH2); 5.0 - 5.5 (CCH2); 7.0 - 8.0 (ph).

[0192] Example 4 - 3: Synthesis of small molecule chain transfer agent BIBB-Ser-CTA

[0193]

[0194] Add O-[(1,1-dimethylethyl)dimethylsilyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-serine (Fmoc-Ser(TBDMS)-OH, 4.42 g, 10 mmol), CTA-OH (4.52 g, 12 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar into a flask. Under an argon atmosphere, pipette 60 mL of anhydrous DCM into a schlenk flask, seal it and react at room temperature for 48 h. After the reaction is completed, filter to remove the insoluble reaction products, collect the liquid phase and concentrate it to a certain volume, and separate by column chromatography to obtain Fmoc-Ser(TBDMS)-CTA.

[0195] Take a dry and clean 25 mL single-necked flask, add 2.00 g of Fmoc-Ser(TBDMS)-CTA and dissolve it in 15 mL of DMF. Add 3 mL of piperidine and stir the reaction at room temperature for 2 h. Rotate the reaction solution to dryness by continuously adding dichloromethane to entrain piperidine and DMF to obtain NH2-Ser(TBDMS)-CTA.

[0196] Take 1.50 g of NH2-Ser(TBDMS)-CTA in a three-necked flask, add 6 mL of DCM and a magnetic stir bar, and dissolve and stir in an ice-water bath. Take triethylamine (TEA, 280 μL, 2 mmol) and 2-bromo-2-methylpropionyl bromide (BIBB, 250 μL, 2 mmol), dissolve them separately in 2 mL of DCM, respectively suck them with a dropper, and slowly drop them into the round-bottom flask at a speed of 3 drops / second simultaneously in an ice-bath environment for reaction. After dropping, seal the flask, remove the ice-bath, and react at room temperature for 24 h. After the reaction is completed, filter off the insoluble matter, wash it three times with deionized water, add anhydrous sodium sulfate to remove water, dry for 12 h, then filter off the sodium sulfate solid, rotate and evaporate to remove part of the solvent to concentrate the product, dissolve it in ethanol and place it in a -20 °C refrigerator for recrystallization, filter and dry to obtain BIBB-Ser(TBDMS)-CTA.

[0197] Add 1.00 g of BIBB-Ser(TBDMS)-CTA, trifluoroacetic acid (TFA, 2 mL) and 2 mL of dichloromethane to a single-necked flask, stir the reaction at room temperature for 1 h, rotate and evaporate to concentrate the reaction solution, and separate and purify it by column chromatography to obtain BIBB-Ser(OH)-CTA.

[0198] Chemical shift of BIBB-Ser(OH)-CTA: 1 1H-NMR(CDCl3), δ(ppm): 0.7 - 1.0 (CH2CH3); 1.2 - 1.5 (CH2CH2); 1.5 - 2.0 (CH2CH2OH, SCH2CH2, CCH3); 2.0 - 2.4 (SCCH2, OCOCH2); 3.2 - 3.4 (SCH2); 3.5 - 4.5 (COCHNH, CHCH2OH, COOCH2).

[0199] Example 4-4: Synthesis of drug precursor DBCO-SS-CPT

[0200]

[0201] Weigh (2-hydroxyethyl) disulfide (0.72 mL, 6 mmol) and DBCO-COOH (2 g, 6 mmol) and add them to a round-bottom flask. Add 40 mL of anhydrous dichloromethane and stir to dissolve under an ice bath. Then dissolve EDC (1 g, 6 mmol) in 10 mL of anhydrous dichloromethane and add it to a dropping funnel. Under nitrogen protection, drop it into the round-bottom flask at a dropping rate of 4 s / drop, and stir at room temperature for 24 hours. After the reaction is completed, filter off the insoluble matter, rotary evaporate and concentrate the filtrate, and perform silica gel column chromatography separation and purification with dichloromethane:methanol = 20:1 (V:V) mobile phase. Collect the product spot, rotary evaporate to remove the mobile phase, and obtain the solid DBCO-SS-OH.

[0202] Under the condition of argon gas passing, add 30 mL of anhydrous dichloromethane to camptothecin (CPT, 1.00 g, 2.87 mmol) and 4-dimethylaminopyridine (DMAP, 1.05 g, 8.62 mmol) and stir well to disperse them evenly. Weigh triphosgene (BTC, 0.28 g, 0.96 mmol) in a round-bottom flask and dissolve it in 10 mL of anhydrous dichloromethane in a fume hood, then drop it into the dichloromethane solution of CPT and stir for 30 minutes. After observing that the solution turns yellowish green, weigh the product DBCO-SS-OH (1.39 g, 3.16 mmol) from the previous step and dissolve it in 15 mL of anhydrous dichloromethane, and slowly drop it into the round-bottom flask and stir at room temperature for 48 hours. After the reaction is completed, the solution turns orange-yellow. Filter off the insoluble by-product salts, rotary evaporate and concentrate, use dichloromethane / methanol 40:1 (V:V) as the mobile phase, perform column chromatography separation and purification, collect the second spot, which is the product spot, rotary evaporate to remove the mobile phase, and obtain the light yellow solid powder DBCO-SS-CPT.

[0203] The chemical shift of DBCO-SS-CPT: 1 1H-NMR (CDCl3), δ (ppm): 0.9 - 1.1 (CH2CH3, CCH3); 1.3 - 1.5 (C(CH3)3); 1.7 - 2.4 (CCH2CH2C, COCCH2CH3, CHCH2, CHCH2CH2); 2.8 - 3.0 (SCH3); 3.2 - 3.6 (NCH2CH3, OCH3); 3.7 - 4.2 (COCH2NH, COOCH2); 4.2 - 4.6 (OCOOCH2, NHCOCH); 5.2 - 5.9 (NCH2C, COOCH2C); 6.0 - 7.0, 7.5 - 8.0 (NHCO); 7.0 - 7.7 (ph).

[0204] Example 4 - 5: Synthesis of drug-conjugated polymer P(CLN-CPT)-Ser[P(MA-N-AZO)]-PHPMA

[0205]

[0206] Add BIBB-Lys(OH)-CTA (25 mg, 0.4 mmol), HPMA (2.00 g, 15.5 mmol) and a small amount of AIBN into a branch port reaction tube, and ultrasonically dissolve them in 600 μL of DMSO (HPLC grade). After complete dissolution, freeze-thaw (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring and dissolution at room temperature - argon filling) 3 times under argon protection to remove air, and then place the reaction system in a 70 °C water bath for reaction for 24 h. After the reaction is completed, introduce air and quickly cool it in liquid nitrogen to terminate the reaction. The obtained product is dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted HPMA monomer, etc. The dialyzed solution is freeze-dried to remove water to obtain the dry macromolecular chain transfer agent BIBB-Lys(OH)-PHPMA.

[0207] The schlenk flask, glass syringe and needles used in the reaction are dried in a 120 °C oven for 2 h to remove water, and the vacuum pumping and argon filling cycle is carried out three times. Under argon protection, add 2-azido-ε-caprolactone (CLN, 0.70 g, 5.6 mmol), 0.50 g of dried BIBB-Lys(OH)-PHPMA and 5% Sn(Oct)2 solution, suck 2 mL of freshly distilled toluene with a glass syringe to dissolve, place it in an oil bath preheated to 110 °C, stir and react for 48 h after dissolution, quench the reaction with liquid nitrogen, add a few drops of dichloromethane to dilute the reaction solution, precipitate it in excess methanol by centrifugation 3 times, and collect the precipitate and dry it in a vacuum drying oven for 24 h to obtain the product PHPMA-PCLN-BIBB.

[0208] Dry the schlenk flask in a 120 °C oven, weigh 0.1 g of PHPMA-PCLN-BIBB, 0.1 g of MA-N-AZO and 10 μL of PMEDTA into a reaction tube, and add 0.6 mL of DMSO to dissolve. After that, after two times of "freezing - vacuum pumping - argon passing - dissolution", under argon protection, add 3 mg of CuBr, and then after another time of "freezing - vacuum pumping - argon passing - dissolution", react in the dark in a 70 °C water bath for 24 h. After the reaction is completed, add a small amount of DMSO to dissolve, dialyze with deionized water and then dry to obtain the product PHPMA-PCLN-P(MA-N-AZO).

[0209] Weigh PHPMA-PCLN-P(MA-N-AZO) (100 mg, 0.01 mmol), DBCO-SS-CPT (73 mg, 0.1 mmol) and 0.5 mL of DMF, react at 25 °C for 48 h. After the reaction is completed, precipitate with ice ether three times, dialyze with DMSO and then with deionized water, and freeze-dry to obtain the product, which is the CPT drug-conjugated hypoxia-responsive triblock polymer PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)].

[0210] The chemical shift of PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)]: 1 1H-NMR (CDCl3), δ (ppm): 0.7 - 1.0 (CH2CH3); 1.2 - 1.5 (CHCH3, CH2CH2); 1.5 - 2.0 (CH2CH2OH, SCH2CH2, CCH3); 2.0 - 2.4 (SCCH2, OCOCH2); 2.5 - 2.8 (CCH2); 3.2 - 3.4 (OCH3, CONHCH2, SCH2); 3.5 - 4.7 (OCH2, N3CH, CHOH, COCHNH, CHCH2OH, COOCH2, OCOOCH2); 5.2 - 5.9 (NCH2C, COOCH2C); 6.5 - 8.0 (CONH, ph).

[0211] Application Example 4-1

[0212] (1) Preparation of the injection of the drug-conjugated polymer micelle PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)]

[0213] Prepare the polymer injection according to the equivalent concentration of camptothecin (CPT) at 10 mg / kg body weight. Dissolve 43 mg of PHPMA-PCLN-P(MA-N-AZO) in 500 μL of DMSO to obtain a clear and transparent polymer and drug solution. Slowly inject the polymer and drug solution into 4.5 mL of PBS, stir the solution overnight and then dialyze to obtain the drug-loaded polymer micelle.

[0214] (2) Cytotoxicity experiment

[0215] Perform cytotoxicity tests on 4T1 cells cultured under normoxia (37 °C, 5% CO2) and hypoxia (37 °C, <0.1% O2). The obtained results are shown in Figure 11 . From Figure 11It can be seen that PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)] has weak cytotoxicity to 4T1 cells under normoxic conditions, while it has strong cytotoxicity under hypoxic conditions, approaching the toxicity of free CPT drug, indicating that CPT is released by micelle cleavage under hypoxic conditions; while most of CPT exists inside the three-arm polymer and is not released under normoxic conditions.

[0216] (3) Pharmacokinetic evaluation

[0217] Take 2 mL of PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)] solution, and add 200 μL of 1 mg / mL Cy5.5-alykne solution dropwise, and stir at room temperature for 24 hours. After the reaction, dialyze with methanol for 3 hours first, and then dialyze with ultrapure water for 24 hours to obtain the Cy5.5 fluorescently labeled polymer solution, denoted as PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)] Cy5.5 .

[0218] After the mice are adapted to the environment, take 6 mice and divide them into two groups, with three mice in each group. Inject the Cy5.5 fluorescently labeled polymer solution into the tail vein blood vessels of the mice, and the dosing concentration is 5 mg / kg; the half-life of serum clearance rate t 1 / 2 Then take blood from the capillaries around the eyes of the mice, take about 50 μL of blood, and put it into the living body for detection before the blood coagulates, and measure the fluorescence value. The results are as Figure 12 shown. From Figure 12 it can be seen that the t 1 / 2 of PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)] is 3.7 h.

[0219] (4) Tumor inhibition experiment

[0220] Study the inhibitory effect of CPT drug-conjugated polymer on the tumors of 4T1 tumor-bearing mice with breast cancer in mice. Wait until the tumor grows to about 100 mm 3 and start the experiment. Prepare free CPT drug and PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)] micelles, and PBS buffer as the blank group. Administer the drug by tail vein injection, and the dosing dose is 10 mg / kg of CPT equivalent, and 100 μL is injected into each mouse each time. Administer the drug on the 1st, 4th, 7th, 10th, and 13th days, for a total of five times. Weigh the mice on the 1st, 3rd, 5th, 7th, 9th, 11th, 13th, and 15th days, and measure the tumor volume of the mice. The obtained results are shown in Figure 13 . From Figure 13It can be seen that, compared with the blank group, the free drug CPT has a relatively weak ability to inhibit tumor growth, while the PHPMA-P(CLN-CPT)-Ser[P(MA-N-AZO)] micelle group shows a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0221] Example 5-1: Synthesis of monomer IPP

[0222]

[0223] Under magnetic stirring at 0 °C, isopropanol (1.68 g, 28.13 mmol) and triethylamine (2.85 g, 28.13 mmol) were dissolved in 100 mL of anhydrous toluene. 2-Chloro-2-oxo-1,3,2-dioxaphospholane (4.01 g, 28.13 mmol) was dissolved in 20 mL of anhydrous toluene and slowly added dropwise to the reaction solution. The mixture was stirred at 0 °C for 24 hours. The precipitate was filtered out, the filtrate was concentrated under vacuum and precipitated in cooled anhydrous diethyl ether, and the IPP monomer was obtained after filtration and drying.

[0224] Chemical shift of IPP: 1 1H-NMR(CDCl3), δ(ppm): 1.1 - 1.2 (CHCH3); 3.5 - 3.6 (PO3CH); 4.2 - 4.3 (PO3CH2).

[0225] Example 5-2: Synthesis of monomer NMZ

[0226]

[0227] 4-Nitroimidazole-1-ethylamine (1.00 g, 6.4 mmol) and triethylamine (1.28 g, 12.8 mmol) were dissolved in 20 mL of dichloromethane. After magnetic stirring for complete dissolution, stirring was continued in an ice-water bath. Then, methacryloyl chloride (0.58 g, 6.4 mmol) was dissolved in 5 mL of dichloromethane, placed in a dropping funnel, and slowly added dropwise into the round-bottom flask at a rate of 3 drops / second for reaction. After the addition was completed, the reaction was continued overnight. After the reaction, it was washed twice with deionized water, anhydrous sodium sulfate was added to remove water, magnetic stirring was carried out for drying for 12 h, then the sodium sulfate solid was removed by filtration, the solvent was removed by rotary evaporation to concentrate the product, and the pure product NMZ monomer was obtained by column chromatography.

[0228] Chemical shift of NMZ: 1 1H-NMR(CDCl3), δ(ppm): 1.9 - 2.0 (CCH3); 3.4 - 4.0 (CONHCH2, NCH2); 5.4 - 5.9 (CCH2); 7.9 - 8.1 (NCH).

[0229] Example 5-3: Synthesis of Small Molecule Chain Transfer Agent CTA-DA-ONP

[0230]

[0231] Add 2,2-bis(hydroxymethyl)acetic acid (1.20 g, 10 mmol), CTA-OH (4.52 g, 12 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar into a Schlenk flask. Under an argon atmosphere, draw 60 mL of anhydrous DCM into the Schlenk flask, seal it and react at room temperature for 48 h. After the reaction is completed, filter to remove the insoluble reaction products, collect the liquid phase and concentrate it to a certain volume, and separate by column chromatography to obtain CTA-DA-OH.

[0232] Add the phosphonium salt initiator SG-COOH (3.12 g, 10 mmol) dissolved in 30 mL of anhydrous DCM into a Schlenk flask, add a magnetic stir bar and place it in an ice bath. Then slowly add CTA-DA-OH (5.19 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), and a small amount of 4-dimethylaminopyridine (DMAP). Stir in the ice bath for 2 h and then react at room temperature overnight. After the reaction is completed, filter to remove the insoluble reaction products, collect the liquid phase and concentrate it to a certain volume, and separate by column chromatography to obtain CTA-DA-ONP.

[0233] Add CTA-DA-ONP (33 mg, 0.4 mmol), N-vinylpyrrolidone (1.72 g, 15.5 mmol) and a small amount of AIBN into a branched reaction tube, and ultrasonically dissolve them in 600 μL of DMSO (HPLC grade). After complete dissolution, freeze-thaw (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring and dissolution at room temperature - argon filling) 3 times under argon protection to remove air, and then place the reaction system in a 70 °C water bath and react for 24 h. After the reaction is completed, pass air through and quickly cool in liquid nitrogen to terminate the reaction. The obtained product is dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted monomers, etc. The dialyzed solution is freeze-dried to remove water to obtain the macromolecular chain transfer agent PVP-DA-ONP.

[0234] Add PVP-DA-ONP (1.00 g, 0.2 mmol), IPP (1.00 g, 6 mmol) and 8 mL of freshly distilled toluene into a dry polymerization tube. Under nitrogen protection, add Al(O iA toluene solution of Pr)3 with a concentration of 0.425 mmol / mL (3 μL, 1.26 μmol) was reacted at 70 °C for 48 h. The resulting solution was inactivated with 1 mol / L acetic acid and precipitated into excess ether. The precipitate was dried under vacuum to obtain the product PVP-DA(ONP)-PIPP.

[0235] PVP-DA(ONP)-PIPP (50 mg, 0.005 mmol), NMZ monomer (50 mg, 0.22 mmol), BPO (2 mg, 0.01 mmol), and 500 μL DMSO were successively added to a polymerization tube. Under the protection of argon, it was frozen and thawed with liquid nitrogen (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring and dissolving at room temperature - argon filling) 3 times to remove air. The reaction was carried out in a constant temperature oil bath at 120 °C for 24 h. After the reaction, it was poured into absolute ethanol for precipitation and centrifuged 3 times, dialyzed in deionized water for 3 days, and freeze-dried to obtain PVP-DA(PNMZ)-PIPP.

[0236] The chemical shift of PVP-DA(PNMZ)-PIPP: 1 H-NMR(CDCl3), δ(ppm): 0.7 - 1.0 (CH2CH3); 1.2 - 1.5 (CCH3, CHCH3, CH2CH2); 1.5 - 2.0 (CH2CH2OH, SCH2CH2, CCH3); 2.0 - 3.0 (SCCH2, OCOCH, NCOCH2); 3.2 - 3.4 (SCH2); 3.5 - 4.5 (CONHCH2, NCH2, CH2OH, PO4CH2, COOCH2); 4.6 - 5.0 (PO4CH); 7.0 - 8.5 (NCHN).

[0237] Application Example 5-1

[0238] (1) Preparation of the drug-loaded polymer micelle DOX@PVP-DA(PNMZ)-PIPP injection

[0239] A polymer injection was prepared according to the equivalent concentration of doxorubicin (DOX) at 5 mg / kg body weight. 15 mg of PVP-DA(PNMZ)-PIPP and 7 mg of DOX were dissolved in 1 mL of DMSO to obtain a clear and transparent polymer and drug solution. The polymer and drug solution was slowly injected into 9 mL of PBS, and the solution was stirred overnight and then dialyzed to obtain the drug-loaded polymer micelle DOX@PVP-DA(PNMZ)-PIPP.

[0240] (2) Cytotoxicity experiment

[0241] Cytotoxicity tests were performed on 4T1 cells cultured under normoxia (37 °C, 5% CO2) and hypoxia (37 °C, <0.1% O2), and the results are shown in Figure 14 . From Figure 14 it can be seen that DOX@PVP-DA(PNMZ)-PIPP has weak cytotoxicity to 4T1 cells under normoxic conditions, while the cytotoxicity is stronger under hypoxic conditions, approaching that of free DOX, indicating that DOX is released by micelle cleavage under hypoxic conditions; while most of the DOX exists inside the three-arm polymer and is not released under normoxic conditions.

[0242] (3) Pharmacokinetic evaluation

[0243] Take 2 mL of DOX@PVP-DA(PNMZ)-PIPP solution, and add 200 μL of 1 mg / mL Cy5.5-alykne solution dropwise, and stir at room temperature for 24 hours. After the reaction, dialyze with methanol for 3 hours first, and then dialyze with ultrapure water for 24 hours to obtain the Cy5.5 fluorescently labeled polymer solution, denoted as DOX@PVP-DA(PNMZ)-PIPP Cy5.5 .

[0244] After the mice are adapted to the environment, take 6 mice and divide them into two groups, with three mice in each group. Inject the Cy5.5 fluorescently labeled polymer solution into the tail vein blood vessels of the mice, and the dosing concentration is 5 mg / kg; the half-life of serum clearance rate t 1 / 2 After that, take blood from the capillaries around the eyes of the mice, take about 50 μL of blood, and put it into a living body for detection before blood coagulation, and measure the fluorescence value. The results are shown in Figure 15 . From Figure 15 it can be seen that the t 1 / 2 of DOX@PVP-DA(PNMZ)-PIPP is 6 h.

[0245] (4) Tumor inhibition experiment

[0246] Study the inhibitory effect of the three-arm polymer loaded with DOX drug on the tumors of 4T1-bearing mice with breast cancer. Wait until the tumor grows to about 100 mm 3 Start the experiment. Prepare free DOX and DOX@PVP-DA(PNMZ)-PIPP micelles, and PBS buffer as the blank group. Administer the drug by tail vein injection, and the dosing dose is 5 mg / kg of DOX equivalent, and 100 μL is injected into each mouse each time. Administer the drug on days 1, 4, 7, 10, and 13, for a total of five times. Weigh the mice on days 1, 3, 5, 7, 9, 11, 13, and 15, and measure the tumor volume of the mice. The results are shown in Figure 16 . From Figure 16It can be seen that, compared with the blank group, the free drug DOX has a weaker ability to inhibit tumor growth, while the DOX@PVP-DA(PNMZ)-PIPP micelle group shows a more obvious inhibitory effect and has a better ability to inhibit tumor growth and ablate tumors.

[0247] Example 6-1: Synthesis of monomer NNM

[0248]

[0249] Imidazole-1-ethylamine (0.71 g, 6.4 mmol) and triethylamine (1.28 g, 12.8 mmol) were dissolved in 20 mL of dichloromethane. After being fully dissolved by magnetic stirring, the mixture was continuously stirred in an ice-water bath. Then, methacryloyl chloride (0.58 g, 6.4 mmol) was dissolved in 5 mL of dichloromethane, placed in a dropping funnel, and slowly dropped into a round-bottom flask at a rate of 3 drops / second for reaction. After the dropping was completed, the reaction was continued overnight. After the reaction, it was washed twice with deionized water, anhydrous sodium sulfate was added to remove water, and it was magnetically stirred and dried for 12 h. Then, the sodium sulfate solid was removed by filtration, the solvent was removed by rotary evaporation to concentrate the product, and the pure product NNM monomer was obtained by column chromatography.

[0250] Chemical shift of NNM: 1 1H-NMR(CDCl3), δ(ppm): 1.5 - 2.0 (CCH3); 3.0 - 4.0 (CONHCH2, NCH2); 5.3 - 6.0 (CCH2); 6.9 - 7.6 (NCH).

[0251] Example 6-2: Synthesis of triblock polymer PNS-PSA-PNNM

[0252]

[0253]

[0254] Accurately weigh CTA-OH (0.4 g, 1 mmol) into a schlenk flask, add O-[(1,1-dimethylethyl)dimethylsilyl]-N-(tert-butoxycarbonyl)-L-serine (Boc-Ser(TBDMS)-OH, 4.2 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar. Under an argon atmosphere, 17 mL of anhydrous DCM was sucked into the schlenk flask, sealed and reacted at room temperature for 48 h. Post-reaction treatment: Filter to remove insoluble reaction products, collect the liquid phase and concentrate it to a certain volume. After column chromatography, the solvent was removed by rotary evaporation to obtain pure Boc-Ser(TBDMS)-CTA.

[0255] Take a dry and clean 25mL single-mouth bottle, add 2.00g Boc-Ser(TBDMS)-CTA and dissolve it in 15mL DCM, add 2mL trifluoroacetic acid, and stir and react at room temperature for 2h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol, and recrystallize it twice at -20℃, and filter to obtain white solid NH2-Ser(OH)-CTA.

[0256] NH2-Ser(OH)-CTA (50 mg, 0.1 mmol) and NB monomer (500 mg, 2 mmol) were dissolved in DMF (5 mL) in a reaction tube. After three cycles of "freeze-vacuum-argon-dissolution", the reaction mixture was stirred at room temperature for 72 hours, then precipitated with cold ether or methanol, and the polymer was purified by centrifugation and dialysis. The purified solution was vacuum dried to obtain the polymer PNB-Ser(OH)-CTA.

[0257] Dissolve PNB-Ser(OH)-CTA with 3mL of dichloromethane. After it is completely dissolved, add 300μL of trifluoroacetic acid under ice bath conditions, stir and react for 1 hour in an ice bath to remove the Boc protecting group. After the reaction is completed, remove dichloromethane and trifluoroacetic acid by rotary evaporation, and then dry in a vacuum oven at room temperature for 4 hours. Add 4mL of ultrapure water to the dried flask and stir to dissolve the obtained product. Adjust the pH to 8.0 with saturated sodium bicarbonate solution to obtain a reaction solution. Add 25mg of propane sultone and 1mL of ultrapure water, stir and react for 24 hours under 256℃ water bath conditions. After the reaction is completed, transfer the reaction solution into a dialysis bag (MWCO=1000Da) and dialyze with ultrapure water for 24 hours to remove unreacted propane sultone, and change the water every 6 hours. Freeze-dry to obtain a solid powder, which is PNS-Ser(OH)-CTA.

[0258] The schlenk flask, glass syringe and needle used in the reaction were dried in an oven at 120°C for 2 h to remove water, and the vacuum was evacuated and filled with argon for three cycles. Salicylic acid monomer (SA, 27.6 mg, 0.2 mmol), dried PNS-Ser(OH)-CTA (55 mg, 0.01 mmol) and pyridine (1 mg, 0.01 mmol) were added under argon protection. 2 mL of anhydrous dichloromethane was drawn into a glass syringe to dissolve it. After stirring until it dissolved, it was reacted at room temperature for 48 h. The reaction solution was precipitated in excess ether and centrifuged three times. The precipitate was collected and dried in a vacuum drying oven for 24 h to obtain a white solid, which was the product PNS-PSA-CTA.

[0259] PNS-PSA-CTA (50 mg, 0.005 mmol), NNM monomer (1.8 mg, 0.01 mmol) and a small amount of AIBN were added into a side-arm reaction tube and dissolved ultrasonically in 200 μL of DMSO (HPLC grade). After complete dissolution, it was frozen and thawed (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring and dissolution at room temperature - argon filling) 3 times under argon protection to remove air, and then the reaction system was placed in a water bath at 70 °C for reaction for 24 h. After the reaction, air was introduced and the reaction was terminated by rapid cooling in liquid nitrogen. The obtained product was dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted NNM monomer, etc. The solution after dialysis was freeze-dried to remove water to obtain the dry product PNS-PSA-PNNM.

[0260] Chemical shift of PNS-PSA-PNNM: 1 1H-NMR (CDCl3), δ (ppm): 0.7 - 1.0 (CH2CH3); 1.2 - 1.5 (CCH3, CHCH3, CH2CH2); 1.5 - 2.0 (SCCH3, SCH2CH2); 2.0 - 3.0 (SCCH2, NCOCH2); 3.2 - 3.4 (SO3CH2, SCH2); 3.5 - 4.5 (CONHCH2, NCH2); 4.6 - 5.0 (OCOCH, phOCH2); 7.0 - 8.0 (NCHCO, ph).

[0261] Example 7-1: Synthesis of monomer MPP

[0262]

[0263] 2,2-Bis(hydroxymethyl)propionic acid (2.00 g, 14.9 mmol) was dissolved in 30 mL of DMF. After it was completely dissolved, KOH (1.00 g, 17.9 mmol) was added to the above solution, and the reaction was carried out in the dark at 100 °C for 2 h. Then benzyl bromide (10.21 g, 59.6 mmol) was slowly added, and the reaction was carried out in the dark at 100 °C for 24 h. After the reaction, it was concentrated by rotary evaporation and transferred to a round-bottom flask with a side arm. Under nitrogen protection, methyl chloroformate (4.20 g, 44.7 mmol) and anhydrous tetrahydrofuran were added, and it was stirred for 0.5 h. Then triethylamine (4.52 g, 44.7 mmol) was added, and the reaction was carried out in an ice bath at 0 °C for 3 h. Then the ice bath was removed and the reaction was carried out at room temperature overnight. After the reaction, the reaction solution was concentrated by rotary evaporation and purified by silica gel column to obtain the product MPP monomer.

[0264] Chemical shift of MPP: 11H-NMR (CDCl3), δ (ppm): 1.2 - 1.4 (CCH3); 3.5 - 4.5 (CONHCH2); 4.6 - 5.0 (OCOCH2, phCH2OCO); 7.0 - 8.0 (ph).

[0265] Example 7 - 2: Synthesis of monomer C5A - MA

[0266]

[0267] Add 1-(3 - aminopropyl)pyrrolidine (C5A, 2.56 g, 0.02 mol) and 2.77 mL of triethylamine (TEA, 0.02 mol) into a round - bottom flask, then add 20 mL of DCM as the solvent. Under ice - bath conditions, add 1.94 mL of methacryloyl chloride (0.02 mol) drop - by - drop at a rate of 1 drop every 3 seconds. After the addition is complete, remove the ice - bath and react at room temperature for 16 h. After the reaction is completed, wash twice with deionized water, then dry with anhydrous Na2SO4, filter by suction, and concentrate the liquid by rotary evaporation. Purify the product C5A - MA through a silica gel column.

[0268] Chemical shift of C5A - MA: 1 1H-NMR (CDCl3), δ (ppm): 1.5 - 2.0 (CCH3, NCH2CH2); 2.0 - 3.0 (CONHCH2); 3.5 - 4.5 (NCH2); 5.3 - 5.6 (CCH2); 7.0 - 8.0 (NCHCO, ph).

[0269] Example 7 - 3: Synthesis of three - armed polymer PNA - PMPP - P(C5A - MA)

[0270]

[0271] Accurately weigh CTA - OH (0.4 g, 1 mmol) into a schlenk flask, add O - [(1,1 - dimethylethyl)dimethylsilyl]-N - (tert - butoxycarbonyl)-L - serine (Boc - Ser(TBDMS)-OH, 4.2 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4 - dimethylaminopyridine (DMAP) and a magnetic stirrer. Under an argon atmosphere, pipette 17 mL of anhydrous DCM into the schlenk flask, seal it, and react at room temperature for 48 h. Post - reaction treatment: Filter to remove insoluble reaction products, collect the liquid phase and concentrate it to a certain volume, remove the solvent by rotary evaporation after column chromatography to obtain pure Boc - Ser(TBDMS)-CTA.

[0272] Take a dry and clean 25 mL single-necked flask, add 2.00 g of Boc-Ser(TBDMS)-CTA and dissolve it in 15 mL of DCM. Add 2 mL of trifluoroacetic acid and stir the reaction at room temperature for 2 h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol and recrystallize twice at -20 °C, and filter to obtain the white solid NH2-Ser(OH)-CTA.

[0273] Dissolve NH2-Ser(OH)-CTA (50 mg, 0.1 mmol), N-ethynylimine (340 mg, 4 mmol), and ammonium dithiocarbamate (11 mg, 0.1 mmol) in DMF (3 mL) in a reaction tube. After three times of "freezing - evacuating - purging with argon - dissolving", stir the reaction mixture at room temperature for 72 h, then precipitate with cold diethyl ether, purify the polymer by centrifugation and dialysis, and vacuum dry the obtained purified solution to obtain the polymer PNA-Ser(OH)-CTA.

[0274] The schlenk flask, glass syringe, and needle used in the reaction were dried in an oven at 120 °C for 2 h to remove water, evacuated and purged with argon three times in a cycle. Under argon protection, add MPP monomer (50 mg, 0.2 mmol), dried PNA-Ser(OH)-CTA (55 mg, 0.01 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.5 mg, 0.01 mmol), dissolve them in 1 mL of DMF, stir until dissolved and react at room temperature for 24 h. The reaction solution was precipitated and centrifuged 3 times in excess diethyl ether, and the precipitate was collected and dried in a vacuum drying oven for 24 h to obtain a white solid, which is the product PNA-PMPP-CTA.

[0275] Add PNA-PMPP-CTA (50 mg, 0.005 mmol), C5A-MA monomer (2 mg, 0.01 mmol), and a small amount of AIBN to a side-arm reaction tube, and ultrasonically dissolve them in 200 μL of DMSO (HPLC grade). After complete dissolution, freeze-thaw (liquid nitrogen freezing - evacuating for 15 min - magnetic stirring and dissolving at room temperature - purging with argon) 3 times under argon protection to remove air, and then place the reaction system in a 70 °C water bath and react for 24 h. After the reaction, introduce air and quickly cool it in liquid nitrogen to terminate the reaction. The obtained product was dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted C5A-MA monomer, etc. The dialyzed solution was freeze-dried to remove water to obtain the dry product PNA-PMPP-P(C5A-MA).

[0276] The chemical shift of PNA-PMPP-P(C5A-MA): 11H-NMR (CDCl3), δ (ppm): 0.7 - 1.0 (CH2CH3); 1.2 - 1.5 (CCH3, CHCH3, CH2CH2); 1.5 - 2.0 (SCCH3, SCH2CH2); 2.0 - 3.0 (NCOCH3, SCCH2); 3.2 - 3.4 (SCH2); 3.5 - 4.5 (CONHCH2, NCH2); 4.6 - 5.0 (OCOCH); 7.0 - 8.0 (NCHCO, ph).

[0277] Example 8-1: Synthesis of monomer NA-S-C7A

[0278]

[0279] Weigh 2-(azepan-1-yl)thiol (2.00 g, 12.6 mmol) and propiolic acid (0.88 g, 12.6 mmol), dissolve them in 10 mL of DMF, react under ultraviolet light irradiation for 3 h, and remove the solvent by rotary evaporation to obtain product C. Then dissolve product C in 100 mL of DCM, add (4S)-4-(4-aminobutyl)-2,5-oxazolidinedione (2.17 g, 12.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 4.91 g, 25.2 mmol), and N-hydroxysuccinimide (NHS, 2.91 g, 25.2 mmol), and stir and react at room temperature for 24 h. After the reaction is completed, wash twice with 1 M NaOH, wash twice with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter off the sodium sulfate solid, concentrate by rotary evaporation, and purify by silica gel column to obtain monomer NA-S-C7A.

[0280] Chemical shift of NA-S-C7A: 1 1H-NMR (CDCl3), δ (ppm): 1.2 - 1.4 (NCH2CH2); 2.0 - 2.4 (NCH2); 2.6 - 3.0 (NHCOCH2, SCH2); 3.5 - 4.5 (NHCHCO, CONHCH2, SCH).

[0281] Example 8-2: Synthesis of triblock polymer PEG-PCL-P(NA-S-C7A)

[0282]

[0283]

[0284] Accurately weigh polyethylene glycol (PEG 5k, 5.00 g, 1 mmol) was added to a Schlenk flask. After dewatering at 110 °C under vacuum for 1 h, the heating was stopped. Boc-Ser(TBDMS)-OH (4.2 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar were added. 17 mL of anhydrous DCM was drawn into the Schlenk flask under an argon atmosphere, sealed and reacted at room temperature for 48 h. Post-treatment of the reaction: The insoluble reaction products were removed by filtration. The liquid phase was collected and concentrated to a certain volume, precipitated in excess ether, filtered and dried, and then dissolved in ethanol. Recrystallization was carried out at -20 °C, and after suction filtration and drying, the white solid Boc-Ser(TBDMS)-PEG was obtained.

[0285] A dry and clean 25 mL single-necked flask was taken, 2.00 g of Boc-Ser(TBDMS)-PEG was added and dissolved in 15 mL of DCM. 2 mL of trifluoroacetic acid was added, and the reaction was stirred at room temperature for 2 h. The reaction solution was concentrated to a certain volume, dissolved in ethanol and recrystallized twice at -20 °C, and the white solid NH2-Ser(OH)-PEG was obtained by filtration.

[0286] The Schlenk flask, glass syringe and needle used in the reaction were dried in an oven at 120 °C for 2 h to remove water. The vacuum was evacuated and argon was filled three times. Under argon protection, ε-caprolactone monomer (CL, 6.15 g, 53 mmol), 0.50 g of dried NH2-Ser(TBDMS)-PEG and 5% Sn(Oct)2 solution were added. 6 mL of freshly distilled toluene was drawn with a glass syringe to dissolve them, and the mixture was placed in an oil bath preheated to 120 °C. After stirring until dissolved, the reaction was carried out for 48 h. The reaction was quenched with liquid nitrogen, a few drops of dichloromethane were added to dilute the reaction solution, and it was precipitated and centrifuged 3 times in excess methanol. The precipitate was collected and dried in a vacuum drying oven for 24 h to obtain a white solid, which was the product PEG-PCL-NH2.

[0287] PEG-PCL-NH2 (100 mg, 0.01 mmol) and NA-S-C7A monomer (1.08 g, 2 mmol) were dissolved in DMF (5 mL) in a reaction tube. After three cycles of "freezing - evacuating - purging with argon - dissolving", the reaction mixture was stirred at room temperature for 72 h, and then precipitated with cold ether or methanol. The polymer was purified by centrifugation and dialysis. The obtained purified solution was dried in vacuo to obtain the polymer PEG-PCL-P(NA-S-C7A).

[0288] The chemical shift of PEG-PCL-P(NA-S-C7A): 11H-NMR (CDCl3), δ (ppm): 1.2 - 1.4 (NCH2CH2); 1.4 - 2.0 (OCOCH2); 2.0 - 2.4 (NCH2); 2.6 - 3.0 (NHCOCH2, SCH2); 3.5 - 4.5 (OCH2CH2, OCH3, COOCH2, NHCHCO, CONHCH2, SCH); 4.6 - 5.1 (COOCH, CONHCH).

[0289] Example 9 - 1: Synthesis of monomer NOPC

[0290]

[0291]

[0292] Ethanolamine (3.66 g, 0.06 mol) and succinic anhydride (6.00 g, 0.06 mol) were added to a 250 mL round-bottom flask. 12 mL of pyridine and the catalyst DMAP were added, and the mixture was stirred in 80 mL of tetrahydrofuran solvent. The reaction was carried out at 40 °C in an oil bath for 24 h. After the reaction, the tetrahydrofuran was evaporated to dryness. The residue was redissolved in 100 mL of dichloromethane and washed with 0.1 M HCl. The organic phase was collected, dried over anhydrous Na2SO4, filtered, and the solvent was evaporated to obtain product C.

[0293] Product C (0.37 g, 2.3 mmol), D-α-tocopherol (1.00 g, 2.3 mmol), dicyclohexylcarbodiimide (DCC, 0.57 g, 2.76 mmol), a small amount of 4-dimethylaminopyridine (DMAP), and a magnetic stir bar were placed in a schlenk flask. 10 mL of anhydrous DCM was drawn in under an argon atmosphere, sealed, and the reaction was carried out at room temperature for 48 h. After the reaction, the insoluble substances were removed by filtration. The liquid phase was collected and concentrated to a certain volume. After column chromatography, the solvent was removed by rotary evaporation to obtain pure product E.

[0294] Product E (1.15 g, 2 mmol) and 0.28 mL of triethylamine (TEA, 2 mmol) were added to a round-bottom flask, and 10 mL of DCM was added as the solvent. Methyl acryloyl chloride (0.2 mL, 2 mmol) was added dropwise under ice bath conditions at a rate of 1 drop every 3 seconds. After the addition was completed, the ice bath was removed, and the reaction was carried out at room temperature for 16 h. After the reaction, it was washed twice with deionized water, then dried over anhydrous Na2SO4, filtered, and the liquid was concentrated by rotary evaporation. It was purified by silica gel column to obtain product NOPC.

[0295] Chemical shift of NOPC: 11H-NMR (CDCl3), δ (ppm): 1.0 - 1.2 (CHCH3); 1.2 - 1.5 (CH2CH2); 1.5 - 1.9 (CCH3, CH2CH); 2.3 - 2.6 (OCOCH2, phCH3, CCH2); 2.6 - 3.0 (OCOCH2); 3.1 - 3.5 (CONHCH2); 4.0 - 4.5 (COOCH2); 5.4 - 6.0 (CCH2).

[0296] Example 9 - 2: Synthesis of triblock polymer PEG - PNOPC - P(PO - C6A)

[0297]

[0298] Accurately weigh polyethylene glycol (PEG 5k , 5.00 g, 1 mmol) into a Schlenk flask, and after removing water under vacuum at 110 °C for 1 h, stop heating. Add O - [(1,1 - dimethylethyl)dimethylsilyl] - N - (tert - butoxycarbonyl) - L - serine (Boc - Ser(TBDMS) - OH, 4.2 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4 - dimethylaminopyridine (DMAP) and a magnetic stir bar. Under an argon atmosphere, pipette 17 mL of anhydrous DCM into the Schlenk flask, seal it, and react at room temperature for 48 h. Post - reaction treatment: Filter to remove insoluble reaction products, collect the liquid phase, concentrate it to a certain volume, precipitate in excess diethyl ether, filter, dry the diethyl ether, and then dissolve it in ethanol. Recrystallize at - 20 °C, filter, and dry to obtain the white solid Boc - Ser(TBDMS) - PEG.

[0299] Take a dry and clean 25 mL single - neck flask, dissolve 2.00 g of Boc - Ser(TBDMS) - PEG in 15 mL of DCM, add 2 mL of trifluoroacetic acid, and stir and react at room temperature for 2 h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol, and recrystallize twice at - 20 °C. Filter to obtain the white solid NH2 - Ser(OH) - PEG.

[0300] Accurately weigh CTA - COOH (4 g, 10 mmol) into a Schlenk flask, add NH2 - Ser(OH) - PEG (5.40 g, 1 mmol), dicyclohexylcarbodiimide (DCC, 0.25 g, 1.2 mmol), a small amount of 4 - dimethylaminopyridine (DMAP) and a magnetic stir bar. Under an argon atmosphere, pipette 10 mL of anhydrous DCM into the Schlenk flask, seal it, and react at room temperature for 48 h. Post - reaction treatment: Filter to remove insoluble reaction products, rotary evaporate and concentrate the product, recrystallize at - 20 °C, filter, and dry to obtain CTA - Ser(OH) - PEG.

[0301] CTA-Ser(OH)-PEG (50 mg, 0.01 mmol), NOPC monomer (115 mg, 0.2 mmol) and a small amount of AIBN were added to a branched reaction tube and dissolved ultrasonically in 600 μL of DMSO (HPLC grade). After complete dissolution, it was frozen and thawed (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring and dissolution at room temperature - argon filling) 3 times under argon protection to remove air, and then the reaction system was placed in a water bath at 70 °C for reaction for 24 h. After the reaction, air was introduced and the reaction was terminated by rapid cooling in liquid nitrogen. The obtained product was dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted NOPC monomer, etc. The solution after dialysis was freeze-dried to remove water to obtain the dry product PEG-PNOPC-OH.

[0302] PEG-PNOPC-OH (100 mg, 0.01 mmol), PO-C6A (80 mg, 0.2 mmol) and 2 mL of freshly distilled toluene were added to a dry polymerization tube. Under nitrogen protection, a toluene solution of Al(O i Pr)3 with a concentration of 0.425 mmol / mL (3 μL, 1.26 μmol) was added to this solution, and the reaction was carried out at 70 °C for 48 h. The obtained solution was inactivated with 1 mol / L acetic acid and precipitated into excess ether. The precipitate was dried under vacuum to obtain the product PEG-PNOPC-P(PO-C6A).

[0303] Chemical shift of PEG-PNOPC-P(PO-C6A): 1 1H-NMR (CDCl3), δ (ppm): 1.0 - 1.2 (CHCH3); 1.2 - 1.5 (CH2CH2); 1.5 - 1.9 (SCCH3, SCH2CH2, NCH2CH2, CCH3, CH2CH); 2.0 - 2.3 (NCH2); 2.3 - 2.6 (NHCOCH2, OCOCH2, phCH3, CCH2); 2.6 - 3.0 (OCOCH2); 3.1 - 3.5 (OCH2CH2, CONHCH2); 3.6 - 3.9 (PO3CH2); 4.0 - 4.5 (CONHC H, COOCH2).

[0304] Example 10-1: Synthesis of monomer SCN

[0305]

[0306] Ethylene glycol (3.72 g, 0.06 mol) and succinic anhydride (6.00 g, 0.06 mol) were added to a 250 mL round-bottom flask. 12 mL of pyridine and the catalyst DMAP were added, and the mixture was stirred in 80 mL of tetrahydrofuran solvent. The reaction was carried out at 40 °C in an oil bath for 24 h. After the reaction, tetrahydrofuran was rotary evaporated to dryness. Then, it was redissolved in 100 mL of dichloromethane and washed with 0.1 M HCl. The organic phase was collected, dried over anhydrous Na2SO4, filtered by suction, and the solvent was rotary evaporated to obtain product C.

[0307] Subsequently, product C (1.84 g, 12.6 mmol) was dissolved in 100 mL of DCM. 6-(2-Hydroxyethoxy)-2-benzothiazolecarbonitrile (2.77 g, 12.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 4.91 g, 25.2 mmol), and N-hydroxysuccinimide (NHS, 2.91 g, 25.2 mmol) were added, and the mixture was stirred at room temperature for 24 h. After the reaction, it was washed twice with 1 M NaOH and twice with deionized water. Anhydrous sodium sulfate was added to remove water, and it was magnetically stirred and dried for 12 h. Then, the sodium sulfate solid was removed by suction filtration, and after rotary evaporation and concentration, it was purified by silica gel column chromatography to obtain product E.

[0308] Product E (0.73 g, 2 mmol) and 0.28 mL of triethylamine (TEA, 2 mmol) were added to a round-bottom flask. Then, 10 mL of DCM was added as the solvent. Under ice bath conditions, 0.2 mL of methacryloyl chloride (2 mmol) was added dropwise at a rate of 1 drop every 3 seconds. After the addition was complete, the ice bath was removed, and the reaction was carried out at room temperature for 16 h. After the reaction, it was washed twice with deionized water, then dried over anhydrous Na2SO4, filtered by suction, and the liquid was rotary evaporated and concentrated. It was purified by silica gel column chromatography to obtain product SCN.

[0309] Chemical shift of SCN: 1 1H-NMR (CDCl3), δ (ppm): 1.8 - 2.0 (CCH2); 2.3 - 2.6 (COCH2); 3.5 - 4.0 (CONHCH2); 4.1 - 4.5 (phOCH2, COOCH2); 5.5 - 6.2 (CCH2); 7.0 - 8.2 (ph).

[0310] Example 10 - 2: Synthesis of monomer MP-C8A

[0311]

[0312] Dissolve 2,2-bis(hydroxymethyl)propionic acid (1.69 g, 12.6 mmol) in 100 mL of DCM, add 2-(azacyclooctan-1-yl)amine (2.00 g, 12.6 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 4.91 g, 25.2 mmol), and N-hydroxysuccinimide (NHS, 2.91 g, 25.2 mmol), and stir the reaction at room temperature for 24 h. After the reaction is completed, wash it twice with 1 M NaOH and twice with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter off the sodium sulfate solid by suction filtration. After rotary evaporation and concentration, transfer it to a round-bottom flask with a side arm. Under nitrogen protection, add methyl chloroformate (3.55 g, 37.8 mmol) and anhydrous tetrahydrofuran, stir for 0.5 h, then add triethylamine (3.82 g, 37.8 mmol), and react at 0 °C in an ice bath for 3 h. After that, remove the ice bath and react at room temperature overnight. After the reaction is completed, rotary evaporate and concentrate the reaction solution, and purify it through a silica gel column to obtain the product MP-C8A monomer.

[0313] Chemical shift of MP-C8A: 1 1H-NMR(CDCl3), δ(ppm): 1.2 - 1.4 (NCH2CH2, CCH3); 2.3 - 2.7 (NCH2); 3.3 - 3.4 (CONHCH2); 4.3 - 4.5 (COOCH2).

[0314] Example 10-3: Synthesis of three-armed polymer PEG-PSCN-P(MP-C8A)

[0315]

[0316]

[0317] Accurately weigh polyethylene glycol (PEG 5k , 5.00 g, 1 mmol) into a schlenk flask, and stop heating after removing water under vacuum at 110 °C for 1 h. Add O-[(1,1-dimethylethyl)dimethylsilyl]-N-(tert-butoxycarbonyl)-L-serine (Boc-Ser(TBDMS)-OH, 4.2 g, 10 mmol), dicyclohexylcarbodiimide (DCC, 2.48 g, 12 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar. Under an argon atmosphere, pipette 17 mL of anhydrous DCM into the schlenk flask, seal it, and react at room temperature for 48 h. Post-reaction treatment: Filter off the insoluble reaction products, collect the liquid phase and concentrate it to a certain volume, precipitate it in excess ether, filter and dry the ether, then dissolve it in ethanol, recrystallize it at -20 °C, filter and dry it to obtain a white solid Boc-Ser(TBDMS)-PEG.

[0318] Take a dry and clean 25 mL single-necked flask, add 2.00 g of Boc-Ser(TBDMS)-PEG and dissolve it in 15 mL of DCM. Add 2 mL of trifluoroacetic acid and stir the reaction at room temperature for 2 h. Concentrate the reaction solution to a certain volume, dissolve it in ethanol and recrystallize twice at -20 °C, and filter to obtain the white solid NH2-Ser(OH)-PEG.

[0319] Accurately weigh CTA-COOH (4 g, 10 mmol) into a schlenk flask, add NH2-Ser(OH)-PEG (5.40 g, 1 mmol), dicyclohexylcarbodiimide (DCC, 0.25 g, 1.2 mmol), a small amount of 4-dimethylaminopyridine (DMAP) and a magnetic stir bar. Under an argon atmosphere, pipette 10 mL of anhydrous DCM into the schlenk flask, seal it and react at room temperature for 48 h. Post-reaction treatment: Filter to remove the insoluble reaction products, concentrate the product by rotary evaporation and recrystallize at -20 °C, filter with suction and dry to obtain CTA-Ser(OH)-PEG.

[0320] Add CTA-Ser(OH)-PEG (50 mg, 0.01 mmol), SCN monomer (100 mg, 0.2 mmol) and a small amount of AIBN to a branched reaction tube, and ultrasonically dissolve them in 600 μL of DMSO (HPLC grade). After complete dissolution, freeze-thaw (liquid nitrogen freezing - vacuum pumping for 15 min - magnetic stirring and dissolution at room temperature - argon filling) 3 times under argon protection to remove air, and then place the reaction system in a 70 °C water bath and react for 24 h. After the reaction, pass air through and quickly cool in liquid nitrogen to terminate the reaction. The obtained product is dialyzed in deionized water for 1 week (changing water twice a day) to remove the reaction solvent DMSO and unreacted SCN monomer, etc. The dialyzed solution is freeze-dried to remove water to obtain the dry product PEG-PSCN-OH.

[0321] The schlenk flask, glass syringe and needles used in the reaction are dried in an oven at 120 °C for 2 h to remove water, evacuated and filled with argon three times. Under argon protection, add MP-C8A monomer (60 mg, 0.2 mmol), dried PEG-PSCN-OH (100 mg, 0.01 mmol), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 1.5 mg, 0.01 mmol), dissolve it in 1 mL of DMF, stir until dissolved and react at room temperature for 24 h. The reaction solution is precipitated and centrifuged 3 times in excess ether, and the precipitate is collected and dried in a vacuum drying oven for 24 h to obtain a white solid, which is the product PEG-PSCN-P(MP-C8A).

[0322] The chemical shift of PEG-PSCN-P(MP-C8A): 11H-NMR (CDCl3), δ (ppm): 1.2 - 2.0 (SCCH3, SCH2CH2, CCH2, NCH2CH2, CCH3); 2.3 - 2.7 (COCH2, NCH2); 3.3 - 3.4 (CONHCH2); 3.5 - 4.0 (CONHCH2); 4.3 - 4.5 (COOCH2, phOCH2); 5.5 - 6.2 (CCH2); 7.0 - 8.2 (ph).

[0323] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A polymer carrier, characterized in that, The polymer carrier has a three-arm structure, and the three arms are a hydrophilic segment, a hydrophobic segment, and a responsive segment respectively. The hydrophilic segment is a hydrophilic segment with the ability to resist protein adsorption; the responsive segment is a group that can maintain hydrophobicity and electrical neutrality in the blood and turn into hydrophilicity and positive charge at the lesion site. The responsive segment includes at least one of the following structures: Formula (17), where m is from 10 to 200, and R` is H or an alkyl group having 1 to 5 carbon atoms. ， R1 is an alkyl group with 1 to 5 carbon atoms, and x = 1 to 8; Equation (18), where m = 10 to 200, Equation (19), where m is from 10 to 200, ； Formula (20), where m is from 10 to 200, ； Formula (21), where m ranges from 10 to 200, 。 2. The polymer carrier according to claim 1, characterized in that, The hydrophilic segment is selected from at least one of a polyethylene glycol segment, a poly(N-(2-hydroxypropyl)methacrylamide) segment, a polyvinylpyrrolidone segment, a poly(2-methacryloyloxyethyl phosphorylcholine) segment, a poly(methacryloylethyl sulfobetaine) polyoxazoline segment, a polyamino acid segment, and a polyphosphate segment.

3. The polymer carrier according to claim 2, wherein The hydrophilic segment includes at least one of the following structures: In formula (1), m is from 20 to 200; Formula (2), where m ranges from 20 to 200, ； In formula (3), R` is H or an alkyl group having 1 to 5 carbon atoms, and m is from 20 to 200. ； Equation (4), where m is from 20 to 200, ； , formula (5), where m is from 20 to 200, ； , formula (6), where m is from 20 to 200, 。 4. The polymer carrier according to claim 1, wherein The hydrophobic segment is selected from at least one of a polyester segment, a polycarbonate segment, a polyphosphate segment, a polyorthoester segment, a polyanhydride segment, a poly(p-dioxanone) segment, a poly(meth)acrylate segment, a poly(meth)acrylamide segment, a polyurethane segment, and a polyamino acid and its side-chain functionalized derivative segment.

5. The polymer carrier according to claim 4, characterized in that, The hydrophobic segment includes at least one of the following structures: Formula (7), where m is from 10 to 200; In formula (8), m is from 10 to 200, and x is from 1 to 4; Formula (9), where m is from 10 to 200; Formula (10), where m ranges from 10 to 200, ； Formula (11), where m is from 10 to 200, ； Equation (12), where m is from 10 to 200, ， ， R1 and R2 can form a ring by bonding and are selected from at least one of the following structures: ； Equation (13), where m ranges from 10 to 200, ， ， R1 and R2 can form a ring by bonding and are selected from at least one of the following structures: ； Formula (14), where m ranges from 10 to 200, ； Formula (15), R` is H or an alkyl group having 1 to 5 carbon atoms, m is from 10 to 200, ，x=1~8； Equation (16), where m is from 10 to 200, 。 6. The preparation method of the polymer carrier according to any one of claims 1 to 5, characterized in that This method includes obtaining it by respectively initiating the polymerization of a hydrophilic monomer, a hydrophobic monomer, and a responsive monomer through a multifunctional initiator by utilizing the difference in the polymerization reaction types of different monomers; and / or, first synthesizing a hydrophilic segment molecule, a hydrophobic segment molecule, and a responsive segment molecule with end-group functionalization, and then bonding the hydrophilic segment molecule, the hydrophobic segment molecule, and the responsive segment molecule to the same molecular chain through a multifunctional initiator by a bioorthogonal chemical reaction or a "click" chemical reaction.

7. The preparation method of the polymer carrier according to claim 6, characterized in that, The multifunctional initiator is selected from at least one of the following compounds: ； The multifunctional initiator is selected from at least one of the following compounds: 。 8. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains a drug and the polymer carrier according to any one of claims 1 to 5, and the drug and the polymer carrier are combined in the form of a micelle or the drug is bonded to the polymer carrier.

9. The pharmaceutical composition according to claim 8, wherein, The drug is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, irinotecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, Taltobulin, maytansinol, dolastatin A, auristatin E, auristatin F, PiericidinA, ansamitocin P 3, dolastatin 10, β-Amanitin, and responsive prodrugs of the above chemotherapeutic drugs, and the responsive mode is selected from at least one of photo-responsiveness, radiation-responsiveness, reactive oxygen species-responsiveness, hypoxia-responsiveness, reduction-responsiveness, pH-responsiveness, and enzyme-responsiveness.

Images