Polymer-drug conjugate with charge reversal caused by hydrolysis of fibroblast activation protein-α response and preparation method and application thereof

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis, and a preparation method and application thereof. The polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis has a structure shown in formula (1). The polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis provided by the present invention has a high anti-tumor efficacy and can significantly reduce tumor volume.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a polymer-drug conjugate with charge reversal caused by hydrolysis of fibroblast activation protein-α (FAP-α) protein in response to hydrolysis, and a preparation method and application thereof. Background Art

[0002] According to the China Cancer Statistics Report, with the growth and aging of the population, the incidence and mortality of cancer are still increasing year by year. So far, no treatment with a high cure rate has been found for most cancers. With the advancement of medical treatment, more and more treatments are applied to clinical practice, including surgical resection, chemotherapy and radiotherapy. Among them, surgical resection adjuvant chemotherapy is the only way to cure cancer, but not all patients are suitable for surgical resection. It is necessary to consider the body's own condition, and surgical resection has the disadvantages of postoperative complications, incomplete surgical resection and trauma caused by excessive resection. For patients who cannot undergo surgical resection, chemotherapy and radiotherapy can only be chosen for treatment. Although the current clinical comprehensive chemotherapy method of multiple drugs and the continuous development of new chemotherapy drugs have prolonged the survival of patients and improved the survival rate, there are disadvantages such as toxic side effects and insignificant treatment effects, which will not only reduce the quality of life of patients but also limit the continuity of clinical drug treatment, ultimately leading to limited treatment effects and excessive side effects. Summary of the invention

[0003] The first object of the present invention is to provide a polymer-drug conjugate with good anti-tumor effect and charge reversal induced by FAP-α protein response to hydrolysis.

[0004] The second object of the present invention is to provide a method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein response to hydrolysis.

[0005] The third object of the present invention is to provide a polymer-drug conjugate prepared by the above method, wherein the FAP-α protein responds to hydrolysis to cause charge reversal.

[0006] The fourth object of the present invention is to provide the use of the polymer-drug conjugate with charge reversal caused by the FAP-α protein in response to hydrolysis in the preparation process of a drug carrier.

[0007] Specifically, the polymer-drug conjugate of the FAP-α protein provided by the present invention that responds to hydrolysis and causes charge reversal has a structure shown in formula (1);

[0008]

[0009] R 11 R is a linker; 12is an anti-tumor drug group; R 21 -NH-(CH2) x -、-O-(CH2) x -、-CO-(CH2) x -or-S-(CH2) x -, x is 2 to 6; R 22 is a zwitterionic group; R 31 is -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S- or -(CH2) z -COO-(CH2) t -S-, q is 1 to 5, z is 0 to 5, t is 1 to 5; R 32 It is a zwitterionic group; m, n, and p are the molar ratios of the three structural units respectively, and m:n:p is (5-40%):(10-95%):(0-85%), and m+n+p=100%.

[0010] In a preferred embodiment, R 11 At least one selected from the following structures;

[0011]

[0012] In formula (2), R 13 For aromatic ring, -CO-(CH2) z -、-O-(CH2) z -、-NH-(CH2) z -、-CO-NH-(CH2) z -、-S-(CH2) z -、-CO-(CH2CH2O) z -、-O-(CH2CH2O) z -、-NH-(CH2CH2O) z -or-S-(CH2CH2O) z -, z is 1 to 14;

[0013] In formula (3), R 14 For aromatic ring, -CO-(CH2) z -、-O-(CH2) z -、-NH-(CH2) z -、-CO-NH-(CH2) z -、-S-(CH2) z -、-CO-(CH2CH2O) z -、-O-(CH2CH2O) z -、-NH-(CH2CH2O) z-or-S-(CH2CH2O) z -, z is 1 to 14;

[0014] In formula (4), R 15 For aromatic ring, -CO-(CH2) z -CO-, -O-(CH2) z -CO-, H-(CH2) z -CO-, -S-(CH2) z -CO-, -CO-(CH2CH2O) z -CO-, -O-(CH2CH2O) z -CO-, -NH-(CH2CH2O) z -CO- or -S-(CH2CH2O) z -CO-, z is 1 to 14; R 16 H or

[0015] In formula (5), R 17 For aromatic ring, -CO-(CH2) z -CO-, -O-(CH2) z -CO-, H-(CH2) z -CO-, -S-(CH2) z -CO-, -CO-(CH2CH2O) z -CO-, -O-(CH2CH2O) z -CO-, -NH-(CH2CH2O) z -CO- or -S-(CH2CH2O) z -CO-, z is 1 to 14; R 18 H or

[0016] In formula (6), R 19 For aromatic ring, -O-(CH2) z -O-, -NH-(CH2) z -NH-, -S-(CH2) z -NH-, -CO-NH-(CH2) z -or-S-(CH2) z -O-, z is 1 to 14;

[0017] In formula (7), R 20 For aromatic ring, -O-(CH2) z -O-, -NH-(CH2) z -O-, -S-(CH2) z-O-, -CO-NH-(CH2) z -、-O-(CH2CH2O) z -、-NH-(CH2CH2O) z -or-S-(CH2CH2O) z -, z is 1 to 14;

[0018] In the above formulae, linkers are each independently a group having at least one of reduction, active oxygen, pH and enzyme responsiveness.

[0019] In a preferred embodiment, R 22 At least one selected from the following structures;

[0020]

[0021] In the above formulas, R 23 and R 24 Each independently represents H or a C1-C5 alkyl group, R 25 and R 27 Each is independently a C1-C5 alkylene group, R 26 It is a C1-C5 alkyl group.

[0022] In a preferred embodiment, R 32 At least one selected from the following structures;

[0023]

[0024] In the above formulas, R 33 and R 34 Each independently represents H or a C1-C5 alkyl group, R 35 and R 37 Each independently represents a C1-C5 alkylene group, R 36 It is a C1-C5 alkyl group.

[0025] In a preferred embodiment, the number average molecular weight of the polymer-drug conjugate is 10,000 to 80,000 Da.

[0026] In a preferred embodiment, m is 5 to 40%, n is 10 to 95%, and p is 0 to 85%.

[0027] In a preferred embodiment, the polymer-drug conjugate is a random copolymer or a block copolymer.

[0028] The method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein response to hydrolysis provided by the present invention comprises modifying a drug precursor onto a polymer backbone to obtain the conjugate; the polymer backbone has a structure shown in formula (8);

[0029]

[0030] R 11 ` is a linker; R 21 -NH-(CH2) x -、-O-(CH2) x -、-CO-(CH2) x -or-S-(CH2) x -, x is 2 to 6; R 22 is a zwitterionic group; R 31 is -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S- or -(CH2) z -COO-(CH2) t -S-, q is 1 to 5, z is 0 to 5, t is 1 to 5; R 32 It is a zwitterionic group; m, n, and p are the molar ratios of the three structural units respectively, and m:n:p is (5-40%):(10-95%):(0-85%), and m+n+p=100%.

[0031] In a preferred embodiment, in the preparation of the polymer-drug conjugate with charge flipping caused by FAP-α protein responsive to hydrolysis, the modification method is that R 11 `Perform a click chemistry reaction with a drug precursor, wherein the group of the click chemistry reaction is selected from at least one of azide, trans-cyclooctene, tetrazine, alkyne, dibenzocyclooctyne, bicyclo[6,1,0]nonyne, lipoic acid and maleimide.

[0032] In a preferred embodiment, during the preparation of the polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis, R 11 `Selected from at least one of the following structures;

[0033]

[0034] In formula (9), R 12 ` is an aromatic ring, -CO-(CH2) z -、-O-(CH2) z -、-NH-(CH2) z -、-CO-NH-(CH2) z -、-S-(CH2) z -、-CO-(CH2CH2O) z -、-O-(CH2CH2O) z -、-NH-(CH2CH2O) z -or-S-(CH2CH2O)z -, z is 1 to 14;

[0035] In formula (10), R 13 ` is an aromatic ring, -CO-(CH2) z -CO-, -O-(CH2) z -CO-, H-(CH2) z -CO-, -S-(CH2) z -CO-, -CO-(CH2CH2O) z -CO-, -O-(CH2CH2O) z -CO-, -NH-(CH2CH2O) z -CO- or -S-(CH2CH2O) z -CO-, z is 1 to 14; R 14 ` is H or

[0036] In formula (11), R 15 ` is an aromatic ring, -O-(CH2) z -O-, -NH-(CH2) z -NH-, -S-(CH2) z -NH- or -S-(CH2) z -O-, z is 1 to 14;

[0037] In formula (12), R 16 ` is an aromatic ring, -O-(CH2) z -O-, -NH-(CH2) z -O-, -S-(CH2) z -O-, -CO-NH-(CH2) z -、-O-(CH2CH2O) z -、-NH-(CH2CH2O) z -or-S-(CH2CH2O) z -, z is 1 to 14.

[0038] In a preferred embodiment, in the preparation of the polymer-drug conjugate with charge flipping caused by FAP-α protein in response to hydrolysis, the polymer backbone has at least one of the following structures;

[0039]

[0040]

[0041]

[0042] R 17`、R 21 ` and R 31 `Each independently is H or a C1-C5 alkyl group; R 18 `、R 22 ` and R 32 ` are each independently O or NH.

[0043] In a preferred embodiment, during the preparation of the FAP-α protein responsive hydrolysis-induced charge reversal polymer-drug conjugate, the polymer backbone is obtained from FAP-α responsive monomers by random copolymerization or block copolymerization.

[0044] In a preferred embodiment, in the preparation of the polymer-drug conjugate with charge flipping caused by FAP-α protein in response to hydrolysis, the drug precursor has at least one of the following structures;

[0045]

[0046] The linker is a group having at least one of reduction, active oxygen, pH and enzyme responsiveness; the drug is an anti-tumor drug.

[0047] In a preferred embodiment, in the preparation of the polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis, the anti-tumor drug is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, exotecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansinol, talanstatin A, auristatin E, auristatin F, piericidinA, ansamitocin P 3, aplysia 10 and β-Amanitin.

[0048] In a preferred embodiment, in the preparation of the polymer-drug conjugate with charge flipping caused by FAP-α protein in response to hydrolysis, the drug precursor has at least one of the following structures;

[0049]

[0050]

[0051]

[0052] The present invention also provides a polymer-drug conjugate prepared by the above method, wherein the FAP-α protein responds to hydrolysis to cause charge reversal.

[0053] In addition, the present invention also provides the use of the polymer-drug conjugate with charge reversal caused by the FAP-α protein in response to hydrolysis in the preparation process of a drug carrier.

[0054] One of the theoretical foundations for the development of existing anti-tumor drugs is to utilize the drug's penetrability in the tumor site. The penetration of anti-tumor drugs in tumor tissue is a key factor that hinders the drug's effectiveness. Tumor tissue has pathological characteristics such as high cell density, dense matrix, and high intratumor pressure. Therefore, the large size of anti-tumor drugs makes it very difficult for them to diffuse in the tumor. After leaking out of the blood vessels, they cannot diffuse to cells far away from the blood vessels to complete drug delivery, resulting in low efficacy.

[0055] After in-depth and extensive research, the inventors of the present invention found that tumor-associated fibroblasts (CAFs) in the tumor microenvironment express FAP-α, and some tumors also express FAP-α. Based on this, the present invention cleverly utilizes the characteristic of FAP-α protein responding to hydrolysis to cause charge reversal to synthesize a polymer-drug conjugate with a structure shown in formula (1) for tumor treatment, and utilizes fibroblast activation protein (FAP-α protein) to cut the polypeptide to induce charge reversal, so that its charge changes from amphoteric to positive, and then adsorbs and triggers endothelial cell endocytosis, intracellular transport and contralateral exocytosis (transcytosis). Since the transcytosis process is an active transport process in which cells consume energy, factors such as high osmotic pressure and high cell density in the tumor that hinder traditional passive diffusion have no effect on this active transport process. Therefore, the carrier has a strong intratumoral penetration ability and can deliver drugs to every cell, thereby having a high anti-tumor efficacy and significantly reducing tumor volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 The 4T1 cytotoxicity test results of monomethyl auristatin E (MMAE) and the two drug-coupled polymers obtained in Examples 1-5 and 1-7 are shown;

[0057] Figure 2 Graph showing the pharmacokinetic evaluation results of monomethyl auristatin E (MMAE) and the two drug-coupled polymers obtained in Examples 1-5 and 1-7;

[0058] Figure 3 Figure 1 is a graph showing the results of tumor inhibition experiments on monomethyl auristatin E (MMAE) and two drug-coupled polymers obtained in Examples 1-5 and 1-7;

[0059] Figure 4 This is the charge reversal result of (P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT)) polymer under the action of FAPα protein;

[0060] Figure 5The figure is a cytotoxicity test result diagram of camptothecin (CPT) and two drug-coupled polymers obtained in Example 2-5 and Example 2-7;

[0061] Figure 6 The pharmacokinetic evaluation results of camptothecin (CPT) and the two drug-coupled polymers obtained in Examples 2-5 and 2-7 are shown;

[0062] Figure 7 The figure is a result of the tumor inhibition experiment of camptothecin (CPT) and the two drug-coupled polymers obtained in Example 2-5 and Example 2-7;

[0063] Figure 8 The figure is a cytotoxicity test result diagram of camptothecin (CPT) and two drug-coupled polymers obtained in Examples 3-7 and 3-8;

[0064] Fig. 9 The pharmacokinetic evaluation results of camptothecin (CPT) and the two drug-coupled polymers obtained in Examples 3-7 and 3-8 are shown;

[0065] Fig.10 The graph shows the results of tumor inhibition experiments on camptothecin (CPT) and the two drug-coupled polymers obtained in Examples 3-7 and 3-8. DETAILED DESCRIPTION

[0066] The polymer-drug conjugate with charge reversal caused by FAP-α protein response hydrolysis provided by the present invention comprises a drug-carrying structural unit and a FAP-α protein response structural unit, and preferably further comprises a hydrophilic structural unit. As long as the polymer-drug conjugate comprises a drug-carrying structural unit and a FAP-α protein response structural unit, the charge reversal caused by FAP-α protein response hydrolysis can be achieved, and a good anti-tumor effect can be obtained. When the polymer-drug conjugate further comprises a hydrophilic structural unit, the polymer-drug conjugate micelles can be effectively prevented from being cleared, the action time of the drug can be increased, and the purpose of exerting a long-term effect in the blood can be achieved.

[0067] In the present invention, the polymer-drug conjugate with charge reversal caused by FAP-α protein response to hydrolysis has a structure shown in formula (1). Wherein, formula (1) is only used to represent the composition of the polymer-drug conjugate with charge reversal caused by FAP-α protein response to hydrolysis and the content of each structural unit, and is not used to represent the connection relationship between each structural unit. The polymer-drug conjugate with charge reversal caused by FAP-α protein response to hydrolysis can be a random copolymer or a block copolymer, without special limitation. In addition, the molar content of the drug-carrying structural unit is represented by m, the molar content of the FAP-α protein response structural unit is represented by n, and the molar content of the hydrophilic structural unit is represented by p. Wherein, m, n and p are determined by the feed amount. m:n:p is (5-40%):(10-95%):(0-85%) and m+n+p=100%. Specifically, m is 5-40%, such as 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, 32%, 35%, 38%, 40% or any value therebetween. n is 10-95%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or any value therebetween. p is 0-85%, such as 0, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or any value therebetween.

[0068] In the present invention, the drug-carrying structural unit has a structure shown in formula (1-1):

[0069]

[0070] In formula (1-1), R 11 R is a linker; 12 is an anti-tumor drug group. 11 As long as the anti-tumor drug can be bonded to the polymer main chain, it is preferably a structure shown in formula (2) to formula (7). In formula (2) to formula (7), z is 1 to 14, such as 1, 2, 3, 4, 5, 6, 8, 10, 12, 14 or any value therebetween; linker is a group having at least one of reduction, active oxygen, pH and enzyme responsiveness. When linker is a group having at least one of reduction, active oxygen, pH and enzyme responsiveness, the corresponding polymer-drug conjugate also has at least one of the properties of reduction, active oxygen, pH and enzyme responsiveness, and has stronger universality. R 12is an anti-tumor drug group. Specific examples of anti-tumor drugs include, but are not limited to, at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, exotecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansinol, talanstatin A, auristatin E, auristatin F, piericidin A, ansamitocin P 3, caudate 10, and β-Amanitin.

[0071] In the present invention, the FAP-α protein response structural unit has a structure shown in formula (1-2):

[0072]

[0073] In formula (1-2), R 21 -NH-(CH2) x -、-O-(CH2) x -、-CO-(CH2) x -or-S-(CH2) x -, x is 2 to 6 (e.g., 2, 3, 4, 5, 6 or any value therebetween). 22 is a zwitterionic group, that is, a group with both positive and negative charges. Specifically, R 22 Preferably, at least one of the following structures is selected:

[0074]

[0075] In the above formulas, R 23 and R 24 Each independently represents H or a C1-C5 alkyl group, R 25 and R 27 Each independently represents a C1-C5 alkylene group, R 26 The alkyl group is C1 to C5. Specific examples of the alkyl group of C1 to C5 include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl or neopentyl. Specific examples of the alkylene group of C1 to C5 include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, tert-pentylene or neopentylene.

[0076] In the present invention, the hydrophilic structural unit has a structure shown in formula (1-3):

[0077]

[0078] In formula (1-3), R 31 is -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S- or -(CH2) z -COO-(CH2) t -S-; q ​​is 1 to 5, such as 1, 2, 3, 4, 5 or any value therebetween; z is 0 to 5, such as 0, 1, 2, 3, 4, 5 or any value therebetween; t is 1 to 5, such as 1, 2, 3, 4, 5 or any value therebetween. R 32 is a zwitterionic group, that is, a group with both positive and negative charges. Specifically, R 32 Preferably, at least one of the following structures is selected:

[0079]

[0080]

[0081] In the above formulas, R 33 and R 34 Each independently represents H or a C1-C5 alkyl group, R 35 and R 37 Each independently represents a C1-C5 alkylene group, R 36 The alkyl group is C1 to C5. Specific examples of the alkyl group of C1 to C5 include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl or neopentyl. Specific examples of the alkylene group of C1 to C5 include, but are not limited to, methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, tert-butylene, n-pentylene, isopentylene, tert-pentylene or neopentylene.

[0082] In the present invention, the main chain of the FAP-α protein-responsive hydrolysis-induced charge flipping polymer-drug conjugate can be selected from at least one of poly(meth)acrylate, poly(meth)acrylamide, polyamino acid, polycarbonate, etc., without particular limitation.

[0083] The preparation method of the polymer-drug conjugate with charge reversal caused by FAP-α protein response to hydrolysis provided by the present invention comprises modifying the drug precursor to a polymer backbone. The polymer backbone is obtained by copolymerizing the monomers. This method of introducing the drug by polymerization followed by modification can avoid the problem that many drug monomers cannot be directly polymerized and thus cannot be smoothly connected to the polymer. The modification method is that R 11`Perform a click chemistry reaction with a drug precursor, wherein the group of the click chemistry reaction is selected from at least one of azide, trans-cyclooctene, tetrazine, alkyne, dibenzocyclooctyne, bicyclo[6,1,0]nonyne, lipoic acid and maleimide.

[0084] In order to further understand the present invention, the present invention is described in detail below in conjunction with embodiments, but the present invention is not limited to these embodiments or application examples. Changes, substitutions, combination simplifications, etc. made by those skilled in the art under the core guiding idea of ​​the present invention should be included in the protection scope of the present invention.

[0085] Example 1-1: Synthesis of Monomeric AzMA

[0086]

[0087] Dissolve chloropropylamine hydrochloride (1.2 g, 9.2 mmol) in 30 mL of deionized water. After it is completely dissolved, add sodium azide (2.25 g, 34.6 mmol) to the above solution and react at 100 °C in the dark for 24 h. After the reaction is completed, slowly cool to room temperature and add potassium hydroxide until solid potassium hydroxide is seen. At this time, oil droplets can also be seen on the liquid surface. Then use ether to extract 3 times, remove water with anhydrous MgSO4, and rotary evaporate to obtain the intermediate product.

[0088] The intermediate product (0.94 g, 9.1 mmol), 1.89 mL of triethylamine (1.38 g, 13.7 mmol) and 4-tert-butylcatechol (3.32 mg, 0.002 mmol) were dissolved in 20 mL of DCM, and 1.94 mL of methacryloyl chloride (1.18 g, 10 mmol) was added at 0 ° C. The reaction was allowed to proceed overnight at room temperature, and the mixture was drained at low temperature and washed with pure water three times. Then, MgSO4 was used to remove water, and the product AzMA was separated by a silica gel column (the mobile phase was dichloromethane: methanol = 100: 1 (V: V)).

[0089] Example 1-2: Synthesis of drug precursor DBCO-Val-Cit-PABC-MMAE

[0090]

[0091] MMAE (0.011 g, 0.014 mmol), DBCO-Val-Cit-PABC-PNP (0.023 g, 0.027 mmol) and N-hydroxybenzotriazole (HOBt) (0.004 g, 0.030 mmol) were dissolved in DMF (0.3 mL), diisopropylethylamine (DIEA) (0.011 mL) was added, and the mixture was stirred at room temperature for 16 hours. After that, DMF was removed under reduced pressure, and the crude reaction product was separated by silica gel column (mobile phase was dichloromethane:methanol=13:1 (V:V)) to obtain a white solid (DBCO-Val-Cit-PABC-MMAE, 9.2 mg, yield 49%).

[0092] Chemical shift of DBCO-Val-Cit-PABC-MMAE: 1 H-NMR(CDCl3), δ(ppm): 0.9-1.1(CH2CH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CCH2CH2C, COCCH2CH3, CHCH2, CHCH2CH2); 2.8-3.0(NCH3); 3.2-3.6 (NCH2CH3,OCH3); 3.7-4.2(COCH2NH,COOCH2,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).

[0093] Example 1-3: Synthesis of FAP-α protein-responsive monomeric MA-N-GP

[0094]

[0095] Under argon, Boc-glycine-L-proline (Boc-GP, 2.50 g, 4.6 mmol) and carbonyldiimidazole (CDI, 1.50 g, 4.6 mmol) were added to a flask, and 25 mL of anhydrous dichloromethane was added. The mixture was stirred thoroughly to dissolve the mixture, and the mixture was stirred at room temperature for 2 hours for activation. 2-aminoethylmethacrylamide hydrochloride (MA-N-NH2-HCl, 0.92 g, 5.5 mmol) and triethylamine (TEA, 4.7 mL, 16.6 mmol) were added to another flask, and 30 mL of anhydrous dichloromethane was added. L of anhydrous dichloromethane, stir at room temperature for 2 hours to obtain MA-N-NH2 reaction solution; add the activated reaction solution in the first flask dropwise into the MA-N-NH2 reaction solution using a constant pressure funnel, stir at room temperature for 24 hours, and after the reaction is completed, wash three times with 0.5M HCl, dry the washed product solution with anhydrous magnesium sulfate, filter, and finally concentrate by rotary evaporation, separate and purify by silica gel column chromatography using ethyl acetate as the mobile phase, collect the second point, remove the mobile phase by rotary evaporation, and obtain a white solid powder (monomer MA-N-GP, 1.6 g, yield 47%).

[0096] Example 1-4: Synthesis of FAP-α protein responsive polymer

[0097]

[0098] (1) Using the RAFT polymerization method, a clean Schlenk tube was placed in an oven at 120°C to dry. After drying, the reaction tube was taken out and cooled to room temperature. A paper funnel was rolled up with filter paper and placed at the mouth of the tube. The monomers MA-N-GP (500 mg, 1.28 mmol), AzMA (70 mg, 0.42 mmol), and initiator azobisisobutyronitrile (AIBN, 1.6 mg, 0.01 mmol) were added to the bottom of the reaction tube through the paper funnel; the dithioester chain transfer agent (CTA-OH, 6.2 mg, 0.02 mmol) was weighed, and 800 μL of ultra-dry dimethyl sulfoxide was added. (DMSO) was dissolved, and the solution was added to the bottom of the Schlenk tube and stirred to dissolve; then, a pumping device was used to cycle "liquid nitrogen freezing-vacuuming for 10 minutes-argon passing-thawing" for 3 times, and then the reaction tube filled with argon was placed in a 70°C oil bath and reacted for 48 hours; after the reaction, 15mL of ethyl acetate was added dropwise to the reaction solution to precipitate an orange-yellow solid product, and the supernatant was poured out by centrifugation, and the precipitation was repeated twice to remove the unreacted monomers and DMSO solvent, and dried for 24 hours to obtain a prepolymer.

[0099] (2) 80 mg of prepolymer was dissolved in 2 mL of dichloromethane. After complete dissolution, 400 μL of trifluoroacetic acid was added in an ice bath and stirred for 1 hour to remove the Boc protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and then dried in a vacuum oven at room temperature for 4 hours. 4 mL of ultrapure water was added to the dried flask and stirred to dissolve, and the pH was adjusted to 8.0 with saturated sodium bicarbonate solution. 50 mg of 2-ethoxy-2-oxide-1,3,2-dioxaphospholane was weighed and dissolved in 1 mL of ultrapure water, added to the reaction solution, and stirred for 36 hours in a water bath at 26°C. After the reaction was completed, the reaction solution was transferred into a dialysis bag (MWCO=1000Da) and dialyzed with ultrapure water for 24 hours. The water was changed every 6 hours. The whole process was protected from light. The polymer solution obtained by dialysis was the FAP-α protein-responsive polymer, which was directly stored in a 4°C refrigerator.

[0100] Chemical shift of FAP-α protein-responsive polymer: 1 H-NMR (MeOD), δ (ppm): 0.7-1.0 (CH2CH3); 1.6-2.1 (CCH2CH2C, CH2CH3); 2.8-3.2 (N(CH2)2, N3CH2); 3. 2-3.4(N(CH3)3); 3.6-3.8(CH2N(CH3)3); 4.0-4.4((NCH2CH2)2,COOCH2CH2O,POCH2); 6.0-7.0(NHCO).

[0101] Example 1-5: Synthesis of FAP-α protein-responsive polymer P(MA-N-GPP)-co-P(AzMA-MMAE) containing MMAE drug linker

[0102]

[0103] The FAP-α protein responsive polymer obtained in Example 1-4 (67 mg, 6.67 mmol), DBCO-Val-Cit-PABC-MMAE (110 mg, 66.7 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol) were weighed and dissolved in a mixed solvent of 0.3 mL DMF and water (DMF: water = 7:3 (V:V)). After pumping twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then pumped three times. The reaction was carried out at 45° C. for 48 h. After the reaction was completed, ice ether was used for precipitation three times. After DMSO was dialyzed, it was dialyzed with deionized water and lyophilized to obtain the product, which was the FAP-α protein responsive polymer P(MA-N-GPP)-co-P(AzMA-MMAE) containing the MMAE drug linker.

[0104] Chemical shifts of FAP-α protein-responsive polymers containing MMAE drug linkers: 1 H-NMR(CDCl3), δ(ppm): 0.9-1.1(CH2CH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CCH2C H2C,COCCH2CH3,CHCH2,CHCH2CH2); 2.8-3.0(NCH3); 3.2-3.6(NCH2CH3,OCH3); 3.7-4.2(COC H2NH,COOCH2); 4.2-4.6(OCOOCH2,NHCOCH,POCH2); 5.2-5.9(NCH2C); 6.0-7.0,7.5-8.0(NHCO); 7.0-7.7(ph).

[0105] Example 1-6: Synthesis of FAP-α protein-responsive polymers containing hydrophilic monomers

[0106]

[0107] (1) Using the RAFT polymerization method, place a clean Schlenk tube in an oven at 120°C to dry. After drying, take out the reaction tube and cool it to room temperature. Wrap a paper funnel with filter paper and place it on the tube mouth. Monomer MA-N-GP (500 mg, 1.28 mmol), AzMA (70 mg, 0.42 mmol), dimethylaminoethyl methacrylate (DMAEMA, 294 mg, 1.28 mmol), initiator azobisisobutyronitrile (AIBN, 1.6 mg, 0.01 mmol) were weighed and added to the bottom of the reaction tube through a paper funnel; chain transfer agent (CTA-OH, 6.2 mg, 0.02 mmol) was weighed, 800 μL of ultra-dry dimethyl sulfoxide (DMSO) was added to dissolve, and the dissolved solution was added to the bottom of the Schlenk tube and stirred to dissolve; then the pumping device "liquid nitrogen freezing-vacuuming for 10 minutes-argon passing-thawing" was used for 3 cycles, and then the reaction tube filled with argon was placed in a 70°C oil bath and reacted for 48 hours; after the reaction was completed, 15 mL of ethyl acetate was added dropwise to the reaction solution to precipitate an orange-yellow solid product, the supernatant was centrifuged and poured out, and the precipitation was repeated twice to remove the unreacted monomer and DMSO solvent, and the prepolymer was dried for 24 hours to obtain a prepolymer.

[0108] (2) 80 mg of prepolymer was dissolved in 2 mL of dichloromethane. After complete dissolution, 400 μL of trifluoroacetic acid was added in an ice bath and stirred for 1 hour to remove the Boc protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and then dried in a vacuum oven at room temperature for 4 hours. 4 mL of ultrapure water was added to the dried flask and stirred to dissolve, and the pH was adjusted to 8.0 with saturated sodium bicarbonate solution. 50 mg of 2-ethoxy-2-oxide-1,3,2-dioxaphospholane was weighed and dissolved in 1 mL of ultrapure water, added to the reaction solution, and stirred for 36 hours in a water bath at 26°C. After the reaction was completed, the reaction solution was transferred into a dialysis bag (MWCO=1000Da) and dialyzed with ultrapure water for 24 hours. The water was changed every 6 hours. The whole process was protected from light. The polymer solution obtained by dialysis was the amphiphilic FAP-α protein-responsive polymer, which was directly stored in a 4°C refrigerator.

[0109] Example 1-7: Synthesis of FAP-α protein responsive polymer P(DP)-co-P(MA-N-GPP)-co-P(AzMA-MMAE) containing MMAE drug linker and hydrophilic monomer

[0110]

[0111] The FAP-α protein-responsive polymer containing a hydrophilic monomer obtained in Example 1-6 (100 mg, 6.67 mmol), DBCO-Val-Cit-PABC-MMAE (110 mg, 66.7 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol) were weighed and dissolved in 0.3 mL of a mixed solvent of DMF and water (DMF: water = 7:3 (V:V)). After pumping twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then pumped three times. The reaction was carried out at 45° C. for 48 h. After the reaction was completed, ice ether was used for precipitation three times. After DMSO was dialyzed, it was dialyzed with deionized water and lyophilized to obtain the product, which was the FAP-α protein-responsive polymer P(DP)-co-P(MA-N-GP)-co-P(AzMA-MMAE) containing an MMAE drug linker and a hydrophilic monomer.

[0112] Application Example 1-1

[0113] (1) Preparation of polymer injection

[0114] P(MA-N-GPP)-co-P(AzMA-MMAE) and P(DP)-co-P(MA-N-GP)-co-P(AzMA-MMAE) were respectively dissolved in normal saline, and polymer injection was prepared at an MMAE equivalent concentration of 0.2 mg / kg body weight to obtain a homogeneous, clear and transparent polymer injection.

[0115] (2) Cytotoxicity assay

[0116] Monomethyl auristatin E (MMAE) and two polymer injections were tested for 4T1 cytotoxicity. The results are shown in Figure 1 .from Figure 1 It can be seen that the drug-conjugated polymer containing hydrophilic monomers (P(DP)-co-P(MA-N-GP)-co-P(AzMA-MMAE)) has strong cytotoxicity to 4T1 cells, but due to its anti-protein adsorption effect, it is not easy to enter the cell membrane, so it has relatively lower cytotoxicity than the polymer without hydrophilic monomers (P(MA-N-GPP)-co-P(AzMA-MMAE)) and free drug (monomethyl auristatin E). The results of cytotoxicity experiments show that both synthesized polymer drug conjugates have a killing effect on 4T1 and have obvious cytotoxicity.

[0117] (3) Pharmacokinetic evaluation

[0118] Take 2 mL of the two drug-coupled polymer solutions obtained in Examples 1-5 and 1-7, respectively, add 200 μL of 1 mg / mL Cy5.5-alykne solution, add copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) after evacuation twice under argon protection, and stir at room temperature for 24 hours. After the reaction is completed, dialyze with methanol for 3 hours, and then dialyze with ultrapure water for 24 hours to obtain a Cy5.5 fluorescently labeled polymer solution.

[0119] After the mice were well adapted to the environment, 6 mice were divided into two groups, with three mice in each group. The Cy5.5 fluorescently labeled polymer solution was injected into the tail vein of the mice at a concentration of 5 mg / kg. The serum clearance half-life t 1 / 2 Then, blood was collected from the capillaries around the mouse's eyes, about 20 μL of blood was collected, and the fluorescence value was measured. The results are as follows: Figure 2 As shown. Figure 2 It can be seen that the t 1 / 2 The t of P(DP)-co-P(MA-N-GP)-co-P(AzMA-CPT) is 3.6 h. 1 / 2 The duration of blood circulation is 5.6 hours. By polymerizing hydrophilic monomers, the micelles can be effectively prevented from being removed, thereby increasing the blood circulation time and ensuring sufficient long blood circulation.

[0120] (4) Tumor inhibition experiment

[0121] The inhibitory effect of MMAE drug-coupled polymer on the tumor of mouse breast cancer cell 4T1-bearing mice was studied. When the tumor grew to about 100 mm 3 The experiment was started. MMAE free drug and two drug-coupled polymer injection solutions were prepared, and PBS buffer was used as the blank group. The drug was administered by tail vein injection. The dosage was 0.2 mg / kg MMAE equivalent, and each mouse was injected with 100 μL each time. The drug was administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15, and the tumor volume of the mice was measured. The results are shown in Figure 3 .from Figure 3 It can be seen that compared with the blank group, free drug MMAE treatment can inhibit tumor growth to a certain extent, but its inhibitory effect is significantly weaker than that of polymer drug conjugates; due to their longer blood circulation time, drug-conjugated polymers containing hydrophilic monomers have a more advantageous tumor inhibition effect.

[0122] Example 2-1: Synthesis of Monomeric AzMA

[0123] The same method as in Example 1-1 was used to obtain monomer AzMA.

[0124] Example 2-2: Synthesis of FAP-α protein-responsive monomeric MA-N-GP

[0125] The same method as in Example 1-3 was used to obtain the monomer MA-N-GP.

[0126] Example 2-3: Synthesis of drug precursor DBCO-SS-CPT

[0127]

[0128] (1) Weigh (2-hydroxyethyl) disulfane (0.72 mL, 6 mmol) and DBCO-COOH (2 g, 6 mmol) into a round-bottom flask, add 40 mL of anhydrous dichloromethane and stir to dissolve in an ice bath, then dissolve EDC (1 g, 6 mmol) in 10 mL of anhydrous dichloromethane and add to a constant pressure funnel, add dropwise into the round-bottom flask under nitrogen protection, control the drip rate to 4 s / drop, and stir at room temperature for 24 hours. After the reaction is completed, filter the insoluble matter, concentrate the filtrate by rotary evaporation, and separate and purify by silica gel column chromatography using a mobile phase of dichloromethane: methanol = 20:1 (V:V), collect the product spots, and remove the mobile phase by rotary evaporation to obtain solid DBCO-SS-OH.

[0129] (2) Under argon, 30 mL of anhydrous dichloromethane was added to camptothecin (CPT, 1.00 g, 2.87 mmol) and 4-dimethylaminopyridine (DMAP, 1.05 g, 8.62 mmol) and stirred thoroughly to make them evenly dispersed; triphosgene (BTC, 0.28 g, 0.96 mmol) was weighed in a fume hood and dissolved in 10 mL of anhydrous dichloromethane in a round-bottom flask, and then added dropwise to the dichloromethane solution of CPT. The mixture was stirred for 30 minutes. After the solution turned yellow-green, DBCO-SS-OH (1.39 g, 3.16 mmol) was weighed and dissolved in 15 mL of anhydrous dichloromethane. The mixture was slowly added dropwise to the round-bottom flask and stirred for 48 hours at room temperature. After the reaction is completed, the solution turns orange-yellow, and the insoluble by-product salt is removed by suction filtration, and the product is concentrated by rotary evaporation. Dichloromethane / methanol 40:1 (V:V) is used as the mobile phase, and column chromatography is used for separation and purification. The second point is collected, which is the product point, and the mobile phase is removed by rotary evaporation to obtain a light yellow solid powder DBCO-SS-CPT (1.45 g, yield 73%).

[0130] Chemical shift of DBCO-SS-CPT: 1 H-NMR(CDCl3), δ(ppm): 0.9-1.1(CH2CH3,CCH3,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,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).

[0131] Example 2-4: Synthesis of FAP-α protein responsive polymer P(MA-N-GP)-co-P(AzMA)

[0132]

[0133] Using the RAFT polymerization method, MA-N-GP (300 mg, 0.78 mmol), AzMA (40.6 mg, 0.26 mmol), and initiator azobisisobutyronitrile (AIBN, 2 mg, 0.01 mmol) were weighed and added to the bottom of the Schlenk reaction tube; the dithioester chain transfer agent (CTA-OH, 3.5 mg, 0.01 mmol) was weighed and added to 800 μL of ultra-dry dimethyl sulfoxide (DMSO) to dissolve, and the dissolved solution was added to the bottom of the Schlenk tube, and all the raw materials were fully stirred to dissolve; then the pumping device "liquid nitrogen freezing-vacuuming for 15 minutes-argon passing-thawing" was used for 3 cycles, and the argon-filled reaction tube was placed in a 70°C oil bath for continuous reaction for 48 hours; after the reaction was completed, 15 mL of ethyl acetate was added dropwise to the reaction solution to precipitate a white solid product, the supernatant was centrifuged and poured out, and the precipitation was repeated twice to remove the unreacted monomers and DMSO solvent, and the polymer was obtained by drying for 24 hours.

[0134] 40 mg of polymer was dissolved in 2 mL of dichloromethane. After complete dissolution, 200 μL of trifluoroacetic acid was added under ice bath conditions, and the reaction was stirred in an ice bath for 1 hour to remove the Boc protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and dichloromethane was entrained twice, and then dried in a vacuum oven at room temperature for 4 hours, 4 mL of ultrapure water was added and stirred to dissolve, and after adjusting the pH to 8.0 with a saturated sodium bicarbonate solution, 25 mg of propane sultone and 1 mL of ultrapure water were added, and the reaction was stirred for 24 hours under 26 ° C water bath conditions. After the reaction was completed, the reaction solution was transferred into a dialysis bag (MWCO = 1000 Da), dialyzed with ultrapure water for 24 hours to remove unreacted propane sultone, and the water was changed every 6 hours, and the solid powder was obtained by freeze drying, which was the FAP-α protein responsive polymer P (MA-N-GP) -co-P (AzMA).

[0135] Chemical shift of P(MA-N-GP)-co-P(AzMA) polymer: 1 H-NMR (CDCl3), δ (ppm): 0.7-1.0 (CH2CH3); 1.6-2.1 (CCH2CH2C, CH2CH3); 2.8-3.2 (N3CH2); 3.2- 3.4(N(CH3)3); 3.6-3.8(CH2N(CH3)3); 4.0-4.6((SCH2CH2)2,NCOCH,NHCOCH2); 6.0-7.0(NHCO).

[0136] Example 2-5: Synthesis of CPT drug-coupled FAP-α protein-responsive polymer P(MA-N-GP)-co-P(AzMA-CPT)

[0137]

[0138] The FAP-α protein responsive polymer obtained in Example 2-4 (100 mg, 0.01 mmol), DBCO-SS-CPT (73 mg, 0.1 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol) were weighed and dissolved in 0.3 mL of a mixed solvent of DMF and water (DMF: water = 7:3 (V:V)). After pumping twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then pumped three times. The mixture was reacted at 45° C. for 48 h. After the reaction was completed, it was precipitated with ice ether three times, dialyzed with DMSO and then dialyzed with deionized water, and lyophilized to obtain the product, which was the CPT drug-coupled FAP-α protein responsive polymer P(MA-N-GP)-co-P(AzMA-CPT).

[0139] Chemical shift of P(MA-N-GP)-co-P(AzMA-CPT) polymer: 1 H-NMR(CDCl3), δ(ppm): 0.7-1.1(CH2CH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CCH2CH2C,COCCH2CH3,CHCH2,CHCH2CH2); 2.8-3.2((SCH2) 2, N3CH2); 3.7-4.2(COCH2NH,COOCH2); 4.2-4.6((SCH2CH2)2,OCOOCH2,NHCOCH); 5.2-5.9(NCH2,COOCH2C).

[0140] Example 2-6: Synthesis of polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA)

[0141]

[0142]

[0143] DMAEMA salt (129 mg, 0.82 mmol), MA-N-GP (209 mg, 0.54 mmol), AzMA (42 mg, 0.27 mmol), and initiator azobisisobutyronitrile (AIBN, 2 mg, 0.01 mmol) were weighed and added to the bottom of the Schlenk reaction tube; a chain transfer agent (CTA-COOH, 3.5 mg, 0.01 mmol) was weighed and added to 1000 μL of ultra-dry dimethyl sulfoxide (DMSO) to dissolve, and the dissolved solution was added to the bottom of the Schlenk tube, and all the raw materials were fully stirred to dissolve; then, a pumping device "liquid nitrogen freezing-vacuuming for 15 minutes-argon passing-thawing" was used for 3 cycles, and the argon-filled reaction tube was placed in a 70°C oil bath and reacted continuously for 48 hours; after the reaction was completed, the reaction solution was diluted with methanol and precipitated with ice ether, the supernatant was centrifuged and poured out, and the precipitation was repeated twice to remove the unreacted monomers and DMSO solvent, and the polymer was obtained after drying for 24 hours.

[0144] 40 mg of polymer was dissolved in 2 mL of dichloromethane. After complete dissolution, 200 μL of trifluoroacetic acid was added under ice bath conditions, and the reaction was stirred in an ice bath for 1 hour to remove the Boc protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and dichloromethane was entrained twice, and then dried in a vacuum oven at room temperature for 4 hours, 4 mL of ultrapure water was added and stirred to dissolve, and after adjusting the pH to 8.0 with a saturated sodium bicarbonate solution, 25 mg of propane sultone and 1 mL of ultrapure water were added, and the reaction was stirred for 24 hours under 26 ° C water bath conditions. After the reaction was completed, the reaction solution was transferred into a dialysis bag (MWCO = 1000 Da), dialyzed with ultrapure water for 24 hours to remove unreacted propane sultone, and the water was changed every 6 hours, and the solid powder was obtained by freeze drying, which was the FAP-α protein responsive polymer P (DS) -co-P (MA-N-GP) -co-P (AzMA).

[0145] Chemical shift of P(DS)-co-P(MA-N-GP)-co-P(AzMA) polymer: 1 H-NMR (CDCl3), δ (ppm): 0.6-1.2 (CH2CH 3,CCH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CHCH2,CHCH2CH2,CH2CH3,CCH2CH2CCH2C); 2.8-3.2(NH(CH3)2,N3CH2); 3.3-3.6(CONHCH2,(CH3) 2NHCH2,CHNCH2); 3.8-4.1(NCOCH2); 4.3-4.6((SCH2CH2)2,COOCH2CH2NH,NH(CH3)2,(COOCH2CH2S)2,CHN); 5.2-5.5(NCH2C); 5.6-5.8(COOCH2C).

[0146] Example 2-7: Synthesis of CPT drug-coupled FAP-α protein-responsive polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT)

[0147]

[0148] The polymer obtained in Example 2-6 (150 mg, 0.01 mmol), DBCO-SS-CPT (73 mg, 0.1 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol) were weighed and dissolved in 0.3 mL of a mixed solvent of DMF and water (DMF: water = 7:3 (V:V)). After pumping twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then pumped three times. The reaction was carried out at 45° C. for 48 h. After the reaction was completed, it was precipitated with ice ether three times, dialyzed with DMSO and then dialyzed with deionized water, and lyophilized to obtain the CPT drug-coupled FAP-α protein responsive polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT).

[0149] Chemical shift of P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) polymer: 1 H-NMR (CDCl3), δ (ppm): 0.6-1.2 (CH2CH 3, CCH3,); 1.3-1.5(C(CH3)3); 1.7-2.4(CHCH2,CHCH2CH2,CH2CH3,CCH2CH2CCH2C); 2.8-3.2(NH(CH3)2,S(CH2) 2,N3CH2); 3.3-3.6(CONHCH2,(CH3)2NHCH2,CHNCH2); 3.8-4.1(NCOCH2); 4.3-4.6((SCH2CH2)2,C OOCH2CH2NH,COOCH2CH2NH(CH3)2,(COOCH2CH2S)2,CHN); 5.2-5.5(NCH2C); 5.6-5.8(COOCH2C).

[0150] Application Example 2-1

[0151] (1) Preparation of polymer injection

[0152] P(MA-N-GP)-co-P(AzMA-CPT) and P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) were respectively dissolved in normal saline, and polymer injection was prepared at a CPT equivalent concentration of 10 mg / kg body weight to obtain a homogeneous, clear and transparent polymer injection.

[0153] (2) P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) charge reversal experiment in response to FAP-α protein

[0154] Prepare a 50 ng / mL FAP-α solution with PBS buffer, dissolve 5 mg of polymer (P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT)) in 5 mL of ultrapure water, mix the FAP-α solution and the polymer solution at a volume ratio of 4:1, and place the reaction solution on a shaker at 37°C and 100 rpm for reaction. Take 200 μL of the reaction solution at 0, 0.25, 0.5, 1, 2, 4, and 6 hours, filter through a 0.45 μm filter, and use DLS to detect the changes in Zeta potential at different time points. Figure 4 It can be seen that after the polymer is mixed with the FAP-α protein, the charge reversal is completed in a very short time, and the Zeta point is converted to positive charge.

[0155] (3) Cytotoxicity assay

[0156] Camptothecin (CPT) and the two polymer injections were tested for 4T1 cytotoxicity, and the results are shown in Figure 5 .from Figure 5It can be seen that the drug-conjugated polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) containing hydrophilic monomers has strong cytotoxicity to 4T1 cells, but due to its anti-protein adsorption effect, it is not easy to enter the cell membrane, so it has relatively lower cytotoxicity than the polymer P(MA-N-GP)-co-P(AzMA-CPT) without hydrophilic monomers and free drug (camptothecin). The results of cytotoxicity experiments show that both synthesized polymer drug conjugates have a killing effect on 4T1 and have obvious cytotoxicity.

[0157] (4) Pharmacokinetic evaluation

[0158] Take 2 mL of the P(MA-N-GP)-co-P(AzMA-CPT) solution obtained in Example 2-5 and the P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) solution obtained in Example 2-7, respectively, and add 200 μL of 1 mg / mL Cy5.5-alykne solution, respectively, and stir at room temperature for 24 hours. After the reaction is completed, dialyze with methanol for 3 hours and then dialyze with ultrapure water for 24 hours to obtain a Cy5.5 fluorescently labeled polymer solution, which is recorded as P(MA-N-GP)-co-P(AzMA-CPT) Cy5.5 ,P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) Cy5.5 .

[0159] After the mice were well adapted to the environment, 6 mice were divided into two groups, with three mice in each group. The Cy5.5 fluorescently labeled polymer solution was injected into the tail vein of the mice at a concentration of 5 mg / kg. The serum clearance half-life t 1 / 2 Then, blood was collected from the capillaries around the mouse's eyes. About 50 μL of blood was collected and placed in the living body for testing before the blood coagulated. The fluorescence value was measured. The results are as follows: Figure 6 As shown. Figure 6 It can be seen that the t 1 / 2 The t of P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) is 4.6 h. 1 / 2 The duration of the blood circulation is 7.5 hours. By polymerizing the hydrophilic monomers, the micelles can be effectively prevented from being removed, thereby increasing the blood circulation time and ensuring sufficient long blood circulation.

[0160] (5) Tumor inhibition experiment

[0161] The inhibitory effect of CPT drug-coupled polymer on the tumor of mouse breast cancer cell 4T1-bearing mice was studied. When the tumor grew to about 100 mm3 The experiment was started. CPT free drug and two drug-coupled polymer injection solutions were prepared, and PBS buffer was used as the blank group. The drug was administered by tail vein injection. The dosage was 5 mg / kg CPT equivalent, and each mouse was injected with 100 μL each time. The drug was administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15, and the tumor volume of the mice was measured. The results are shown in Figure 7 .from Figure 7 It can be seen that compared with the blank group, free drug CPT treatment can inhibit tumor growth to a certain extent, but its inhibitory effect is significantly weaker than that of polymer drug conjugates; due to their longer blood circulation time, drug-conjugated polymers containing hydrophilic monomers have a more advantageous tumor inhibition effect.

[0162] Example 3-1: Synthesis of azide monomer

[0163]

[0164] Dissolve (4S)-4-(4-chlorobutyl)-2,5-oxazolidinedione (1.59 g, 9.2 mmol) in 30 mL of deionized water. After it is completely dissolved, add sodium azide (2.25 g, 34.6 mmol) to the above solution and react at 100°C in the dark for 24 hours. After the reaction is completed, slowly cool to room temperature and add potassium hydroxide until potassium hydroxide solid is seen. At this time, oil droplets can also be seen on the liquid surface. Then use ether to extract 3 times, anhydrous MgSO4 to remove water, and rotary evaporate to obtain the product. Then separate it through a silica gel column (the mobile phase is dichloromethane: methanol = 100:1 (V:V)) to obtain the azide monomer.

[0165] Example 3-2: Synthesis of FAP-α protein-responsive monomers

[0166]

[0167] Under argon, Boc-glycine-L-proline (Boc-GP, 2.50 g, 4.6 mmol) and carbonyldiimidazole (CDI, 1.50 g, 4.6 mmol) were added to a flask, and 25 mL of anhydrous dichloromethane was added. The mixture was stirred thoroughly to dissolve the mixture, and the mixture was stirred at room temperature for 2 hours for activation. In another flask, (4S)-4-(4-aminobutyl)-2,5-oxazolidinedione (0.95 g, 5.5 mmol) and triethylamine (TEA, 4.7 mL, 16 .6mmol), 30mL of anhydrous dichloromethane was added, and after being fully stirred at room temperature for 2 hours, the activated reaction solution in the first flask was added dropwise into the reaction solution using a constant pressure funnel, and the reaction was stirred at room temperature for 24 hours. After the reaction was completed, the unreacted salt was filtered out and washed three times with 0.5M HCl. The washed product solution was dried over anhydrous magnesium sulfate, filtered, and finally concentrated by rotary evaporation. Silica gel column chromatography was performed with ethyl acetate as the mobile phase for separation and purification. The mobile phase was removed by rotary evaporation to obtain a white solid powder (1.6g, yield 47%).

[0168] Example 3-3: Synthesis of hydrophilic monomer

[0169]

[0170] N-tert-Butyloxycarbonyl-L-glutamic acid-α-tert-butyl ester (5.8 g, 0.029 mol) and allyl alcohol (30 mL, 0.442 mol) were mixed in a round-bottom flask (250 mL), and trimethylsilyl chloride (4 mL, 0.031 mol) was added to the mixture via a syringe. The resulting suspension was heated to 80° C. and stirred until it became uniform. The solvent was removed under vacuum at 60° C., and ether (300 mL) was added for precipitation to obtain a white solid, which was collected by filtration and further purified by recrystallization in ethanol / ether to obtain a white solid product.

[0171] The white solid product was dissolved in 10 mL of dichloromethane. After it was completely dissolved, 3 mL of trifluoroacetic acid was added under ice bath conditions and stirred for 12 hours to remove the tert-butyl ester protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and the product was entrained with dichloromethane twice, and then dried in a vacuum oven at room temperature for 4 hours to obtain allyl glutamate.

[0172] Under nitrogen protection, allyl-L-glutamate (2.5 g, 13 mmol), triphosgene (1.9 g, 6.4 mmol) and anhydrous tetrahydrofuran (40 mL) were added to a round-bottom flask (250 mL), and the resulting mixture was stirred at room temperature for 24 hours, during which allyl glutamate gradually dissolved, and the solvent was removed under vacuum to obtain an oily liquid, which was then dissolved in ethyl acetate (20 mL) and washed with a cold saturated NaHCO3 / H2O solution. The organic layer was separated and dried over anhydrous MgSO4 at 0°C, and after suction filtration and rotary evaporation, allyl oxazolidinedione propionate was obtained.

[0173] Allyl oxazolidinedionepropionate (3 g, 0.014 mol) and aminothiol (1.47 g, 0.014 mol) were dissolved in 20 mL of tetrahydrofuran, stirred and reacted at 50° C. for 2 hours, the solvent was removed by rotary evaporation, and the NCA-DS monomer was obtained after vacuum drying.

[0174] Example 3-4: Synthesis of drug precursor DBCO-SS-CPT

[0175] The same method as Example 2-3 was used to obtain the drug precursor DBCO-SS-CPT.

[0176] Example 3-5: Synthesis of FAP-α protein responsive polymer P(AzAA)-co-P(AA-N-GPS)

[0177]

[0178] The FAP-α protein response monomer (146 mg, 0.685 mmol) and the azide monomer (73 mg, 0.685 mmol) were dissolved in DMF (5 mL) in a reaction tube, and then n-butylamine / DMF stock solution (CI = 0.253 M, 136 mL, 34.4 mmol) was added with a syringe. The reaction mixture was stirred at room temperature for 48 hours, then concentrated by rotary evaporation, and then chloroform was added to dissolve, precipitated with cold ether or methanol, and the polymer was purified by centrifugation and dialysis. After vacuum drying, the polymer P(AzAA)-co-P(AA-N-GP) was obtained.

[0179] 40 mg of polymer was dissolved in 2 mL of dichloromethane. After complete dissolution, 200 μL of trifluoroacetic acid was added under ice bath conditions, and the reaction was stirred in an ice bath for 1 hour to remove the Boc protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and dichloromethane was entrained twice, and then dried in a vacuum oven at room temperature for 4 hours, 4 mL of ultrapure water was added and stirred to dissolve, and after adjusting the pH to 8.0 with a saturated sodium bicarbonate solution, 25 mg of propane sultone and 1 mL of ultrapure water were added, and the reaction was stirred for 24 hours under 26 ° C water bath conditions. After the reaction was completed, the reaction solution was transferred into a dialysis bag (MWCO = 1000 Da), dialyzed with ultrapure water for 24 hours to remove unreacted propane sultone, and the water was changed every 6 hours, and the solid powder was obtained by freeze drying, which was the FAP-α protein responsive polymer P (AzAA) -co-P (AA-N-GPS).

[0180] Chemical shift of FAP-α protein response polymer P(AzAA)-co-P(AA-N-GPS): 1 H-NMR (CDCl3), δ (ppm): 0.7-1.0 (CH2CH3); 1.6-2.1 (CCH2CH2C, CH2CH3); 2.8-3.2 ( , N(CH2)2,N3CH2); 3.2-3.4(N(CH3)3); 3.6-3.8(CH2N(CH3)3); 4.0-4.4((SCH2CH2) 2, NCOCH2,NHCOCH2,COOCH2CH2O,POCH2); 6.0-7.0(NHCO).

[0181] Example 3-6: Synthesis of FAP-α protein responsive polymer P(AzAA)-co-P(AA-N-GPS)-co-P(DS)

[0182]

[0183]

[0184] The hydrophilic monomer (82 mg, 0.685 mmol), the azide monomer (73 mg, 0.685 mmol), and the FAP-α protein response monomer (146 mg, 0.685 mmol) were dissolved in DMF (5 mL) in a reaction tube, and then the n-butylamine / DMF stock solution (C I=0.253M, 136mL, 34.4mmol), the reaction mixture was stirred at room temperature for 48 hours, then concentrated by rotary evaporation, the concentrated solution was dissolved in chloroform, precipitated with cold ether or methanol, the polymer was purified by centrifugation and dialysis, and the purified solution was vacuum dried to obtain the polymer P(AzAA)-co-P(AA-N-GP)-co-P(DS).

[0185] The obtained polymer was dissolved in 3 mL of dichloromethane. After it was completely dissolved, 300 μL of trifluoroacetic acid was added under ice bath conditions, and the reaction was stirred in an ice bath for 1 hour to remove the Boc protecting group. After the reaction was completed, dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and dichloromethane was entrained twice, and then dried in a vacuum oven at room temperature for 4 hours. The obtained product was added to the dried flask with 4 mL of ultrapure water and stirred to dissolve, and the pH was adjusted to 8.0 with a saturated sodium bicarbonate solution to obtain a reaction solution. 25 mg of propane sultone and 1 mL of ultrapure water were added, and the reaction was stirred for 24 hours under 26 ° C water bath conditions. After the reaction was completed, the reaction solution was transferred into a dialysis bag (MWCO = 1000 Da) and dialyzed with ultrapure water for 24 hours to remove unreacted propane sultone, and the water was changed every 6 hours. The solid powder was obtained by freeze drying, which is the FAP-α protein responsive polymer P (AzAA) -co-P (AA-N-GPS) -co-P (DS).

[0186] Chemical shift of FAP-α protein-responsive polymer P(AzAA)-co-P(AA-N-GPS)-co-P(DS): 1 H-NMR (CDCl3), δ (ppm): 0.6-1.2 (CH2CH 3, CCH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CHCH2,CHCH2CH2,CH2CH3,CCH2CH2CCH2C); 2.8-3.2(S(CH2) 2, NH(CH3)2,N3CH2); 3.3-3.6(CONHCH2,(CH3)2NHCH2,CHNCH2); 3.8-4.2(NCOCH2); 4.2-4.6((SCH2CH2) 2, COOCH2CH2NH,COOCH2CH2NH(CH3)2,(COOCH2CH2S)2,CHN); 5.2-5.5(NCH2C); 5.6-5.8(COOC H2C).

[0187] Example 3-7: Synthesis of FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS) containing CPT drug linker

[0188]

[0189] The polymer P(AzAA)-co-P(AA-N-GPS) (100 mg, 0.01 mmol), DBCO-SS-CPT (73 mg, 0.1 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol) were weighed and dissolved in 0.3 mL of a mixed solvent of DMF and water (DMF: water = 7:3 (V:V)). After pumping twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then pumped three times. The reaction was carried out at 45°C for 48 hours. After the reaction was completed, it was precipitated with ice ether three times, dialyzed with DMSO and then dialyzed with deionized water, and lyophilized to obtain the product, which is the FAP-α protein responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS) containing a CPT drug linker.

[0190] Chemical shifts of FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS) containing CPT drug linker: 1 H-NMR(CDCl3), δ(ppm): 0.7-1.1(CH2CH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CCH2CH2C,COCCH2CH3,CHCH2,CHCH2CH2); 2.8-3.2((SCH2) 2, N3CH2); 3.7-4.2(COCH2NH,COOCH2,COOCH2); 4.2-4.6((SCH2CH2) 2, OCOOCH2, NCOCH2, NHCOC H2); 5.2-5.9 (NCH2C, COOCH2C).

[0191] Example 3-8: Synthesis of FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) containing CPT drug linker

[0192]

[0193] The polymer P(AzAA)-co-P(AA-N-GPS)-co-P(DS) (150 mg, 0.01 mmol), DBCO-SS-CPT (73 mg, 0.1 mmol) and PMEDTA (1.772 μg, 20 μL, 0.001 mmol) were weighed and dissolved in 0.3 mL of a mixed solvent of DMF and water (DMF: water = 7:3 (V:V)). After evacuation twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then evacuation was repeated three times. The reaction was carried out at 45°C for 48 h. After the reaction was completed, ice ether was used for precipitation three times. After DMSO was dialyzed, it was dialyzed with deionized water and lyophilized to obtain the product, which was the FAP-α protein responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) containing a CPT drug linker.

[0194] Chemical shifts of FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) containing CPT drug linker: 1 H-NMR (CDCl3), δ (ppm): 0.6-1.2 (CH2CH 3, CCH3,CCH3,CCH3); 1.3-1.5(C(CH3)3); 1.7-2.4(CHCH2,CHCH2CH2,CH2CH3,CCH2CH2CCH2C); 2.8-3.2(S(CH2) 2, NH(CH3)2,S(CH2) 2, N3CH2); 3.3-3.6(CONHCH2,(CH3)2NHCH2,CHNCH2); 3.8-4.1(NCOCH2); 4.3-4.6((SCH2CH2) 2, COOCH2CH2NH,NHCOCH2,COOCH2CH 2, N(CH3)2,(COOCH2CH2S)2,CHN); 5.2-5.5(NCH2C); 5.6-5.8(COOCH2C).

[0195] Application Example 3-1:

[0196] (1) Preparation of polymer injection

[0197] P(AzAA-CPT)-co-P(AA-N-GPS) and P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) were respectively dissolved in normal saline, and polymer injections were prepared at a CPT equivalent concentration of 5 mg / kg body weight to obtain homogeneous, clear and transparent polymer injections.

[0198] (2) Cytotoxicity assay

[0199] Camptothecin (CPT) and two polymer injections were tested for 4T1 cytotoxicity. The results are shown in Figure 8 .from Figure 8 It can be seen that the drug-conjugated polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) containing amphiphilic monomers has strong cytotoxicity to 4T1 cells, but due to its anti-protein adsorption effect, it is not easy to enter the cell membrane, so it has relatively low cytotoxicity compared with the block polymer P(AzAA-CPT)-co-P(AA-N-GPS) without hydrophilic monomers and free drug (camptothecin). The results of cytotoxicity experiments show that both synthesized polymer drug conjugates have a killing effect on 4T1 and have obvious cytotoxicity.

[0200] (3) Pharmacokinetic evaluation

[0201] 2 mL of the polymers obtained in Examples 3-7 and 3-8 were respectively prepared into solutions, and 200 μL of 1 mg / mL Cy5.5-alykne solution was added dropwise, and the mixture was stirred at room temperature for 24 hours. After the reaction, the mixture was dialyzed with methanol for 3 hours and then with ultrapure water for 24 hours to obtain a Cy5.5 fluorescently labeled polymer solution.

[0202] After the mice were well adapted to the environment, 6 mice were divided into two groups, with three mice in each group. The Cy5.5 fluorescently labeled polymer solution was injected into the tail vein of the mice at a concentration of 5 mg / kg. The serum clearance half-life t 1 / 2 Then, blood was collected from the capillaries around the mouse's eyes. About 50 μL of blood was collected and placed in the living body for testing before the blood coagulated. The fluorescence value was measured. The results are as follows: Fig. 9 As shown. Fig. 9 It can be seen that the t of P(AzAA-CPT)-co-P(AA-N-GPS) 1 / 2 The t of P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) is 4 h. 1 / 2 The duration of blood circulation is 6.7 hours. By polymerizing hydrophilic monomers, the micelles can be effectively prevented from being removed, thereby increasing the blood circulation time and ensuring sufficient long blood circulation.

[0203] (4) Tumor inhibition experiment

[0204] The inhibitory effect of CPT drug-coupled polymer on the tumor of mouse breast cancer cell 4T1-bearing mice was studied. When the tumor grew to about 70 mm 3The experiment was started. CPT free drug and two drug-coupled polymer injection solutions were prepared, and PBS buffer was used as the blank group. The drug was administered by tail vein injection. The dosage was 5 mg / kg CPT equivalent, and each mouse was injected with 100 μL each time. The drug was administered on days 1, 4, 7, 10, and 13, for a total of five times. The mice were weighed on days 1, 3, 5, 7, 9, 11, 13, and 15, and the tumor volume of the mice was measured. The results are shown in Fig.10 .from Fig.10 It can be seen that compared with the blank group, free drug CPT treatment can inhibit tumor growth to a certain extent, but its inhibitory effect is significantly weaker than that of polymer drug conjugates; due to their longer blood circulation time, drug-conjugated polymers containing hydrophilic monomers have a more advantageous tumor inhibition effect.

[0205] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis, characterized in that: The method comprises modifying a drug precursor onto a polymer backbone; the polymer backbone has at least one of the following structures; R 17 `、R 21 ` and R 31 `Each independently is H or a C1-C5 alkyl group; R 18 `、R 22 ` and R 32 ` are each independently O or NH.

2. The method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis according to claim 1, characterized in that: The polymer backbone is obtained by random copolymerization or block copolymerization of FAP-α responsive monomers.

3. The method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis according to claim 1, characterized in that: The drug precursor has at least one of the following structures; The linker is a group having at least one of reduction, active oxygen, pH and enzyme responsiveness; the drug is an anti-tumor drug.

4. The method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis according to claim 3, characterized in that: The anti-tumor drug is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, exotecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansinol, talanstatin A, auristatin E, auristatin F, piericidin A, ansamitocin P 3, aplysia 10 and β-Amanitin.

5. The method for preparing a polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis according to claim 1, characterized in that: The drug precursor has at least one of the following structures; 6. A polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis, prepared by the method according to any one of claims 1 to 5.

7. Use of the polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis as claimed in claim 6 in anti-tumor drugs.

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