Fibroblast activation protein-α-responsive hydrolysis-induced charge-reversal polymer-drug conjugate, preparation method therefor and use thereof

Abstract

The present invention belongs to the technical field of medicine, and particularly relates to an FAP-α protein-responsive hydrolysis-induced charge-reversal polymer-drug conjugate, a preparation method therefor and the use thereof. Said conjugate has a structure shown as formula (1). Said conjugate provided by the present invention has excellent anti-tumor curative effect, and can remarkably reduce the tumor volume.

Description

Polymer-drug conjugate with charge reversal responsive to hydrolysis of fibroblast activation protein-α and preparation method and application thereof Technical Field

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

[0002] According to the China Cancer Statistics Report, cancer incidence and mortality continue to increase annually due to population growth and aging. Currently, no treatment with a high cure rate has been found for most cancers. With advances in medical care, a growing number of treatment options are being applied clinically, including surgical resection, chemotherapy, and radiotherapy. Surgical resection supplemented with chemotherapy is the only curative option for cancer, but not all patients are suitable for surgical resection, as their physical condition must be considered. Surgical resection also carries disadvantages such as postoperative complications, incomplete resection, and traumatic injury from excessive resection. For patients who cannot undergo surgical resection, chemotherapy and radiotherapy are the only treatment options. While current multi-drug combination chemotherapy approaches and the continued development of new chemotherapeutic agents have prolonged patient survival and improved survival rates, they are associated with toxic side effects and insignificant therapeutic effects. This not only reduces patient quality of life but also limits the continuity of clinical drug treatment, ultimately leading to limited therapeutic efficacy 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 responsive to FAP-α protein hydrolysis.

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

[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 undergoes charge reversal in response to hydrolysis.

[0006] A fourth object of the present invention is to provide the use of the polymer-drug conjugate having charge reversal upon hydrolysis of the FAP-α protein in the preparation 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] R11 is a linker; R 12 is an antitumor drug group; R 21 -NH-(CH2) x -、-O-(CH2) x -, -CO-(CH2) x -, -CO-NH-(CH2) x -、-(CH2) x -、-(CH2) x1 O(CH2) x2 S(CH2) x3 or -S-(CH2) x -, x is 2 to 6, x1, x2 and x3 are each independently 1 to 6; R 22 is a zwitterionic group; R 31 -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S-, -(CH2) z -O-(CH2) t -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 respectively the molar ratios of the three structural units and m:n:p is (5-40%):(10-95%):(0-85%) and m+n+p=100%.

[0009] In a preferred embodiment, R 11 At least one selected from the following structures;

[0010] 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;

[0011] 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;

[0012] 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

[0013] 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

[0014] 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;

[0015] 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;

[0016] In the above formulae, each linker is independently a group having at least one of reduction, active oxygen, pH, and enzyme responsiveness.

[0017] In a preferred embodiment, R 22 At least one selected from the following structures;

[0018] 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 It is a C1-C5 alkyl group.

[0019] In a preferred embodiment, R 32 At least one selected from the following structures;

[0020] 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.

[0021] In a preferred embodiment, the number average molecular weight of the polymer-drug conjugate is 10,000 to 80,000 Da.

[0022] In a preferred embodiment, m is 5 to 40%, n is 10 to 95%, and p is 0 to 85%.

[0023] In a preferred embodiment, the polymer-drug conjugate is a random copolymer or a block copolymer.

[0024] 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 a drug precursor onto a polymer backbone; the polymer backbone has a structure shown in formula (8);

[0025] R 11 ` is a linker; R 21 -NH-(CH2) x -、-O-(CH2) x -, -CO-(CH2) x -, -CO-NH-(CH2) x -、-(CH2) x -、-(CH2) x1 O(CH2) x2 S(CH2) x3 or -S-(CH2) x -, x is 2 to 6, x1, x2 and x3 are each independently 1 to 6; R 22 is a zwitterionic group; R 31 -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S-, -(CH2) z -O-(CH2) t -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 respectively the molar ratios of the three structural units and m:n:p is (5-40%):(10-95%):(0-85%) and m+n+p=100%.

[0026] In a preferred embodiment, during the preparation of the polymer-drug conjugate with charge reversal caused by FAP-α protein responsive to hydrolysis, the modification is carried out by 11 `Performing 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.

[0027] In a preferred embodiment, during the preparation of the polymer-drug conjugate with charge reversal responsive to FAP-α protein hydrolysis, R 11 `Selected from at least one of the following structures;

[0028] 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;

[0029] 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

[0030] 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;

[0031] 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.

[0032] In a preferred embodiment, during the preparation of the polymer-drug conjugate with charge reversal upon hydrolysis of the FAP-α protein, the polymer backbone has at least one of the following structures:

[0033] R 17 `、R 21 ` and R 31 `Each independently is H or C1~C5 alkyl; R 18 `、R 22 ` and R 32 ` are each independently O or NH.

[0034] In a preferred embodiment, during the preparation of the FAP-α protein-responsive hydrolysis-induced charge-flipping polymer-drug conjugate, the polymer backbone is obtained from FAP-α-responsive monomers by random copolymerization or block copolymerization.

[0035] In a preferred embodiment, during the preparation of the polymer-drug conjugate with charge reversal upon hydrolysis of the FAP-α protein, the drug precursor has at least one of the following structures:

[0036] The linker is a group having at least one of reduction, active oxygen, pH and enzyme responsiveness; the drug is an anti-tumor drug.

[0037] In a preferred embodiment, in the process of preparing the polymer-drug conjugate with charge reversal responsive to FAP-α protein hydrolysis, the anti-tumor drug is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, exitecan, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, calicheamicin, gambogic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansol, tylanstatin A, auristatin E, auristatin F, piericidin A, ansamitocin P 3, aplysia 10, and β-Amanitin.

[0038] In a preferred embodiment, during the preparation of the polymer-drug conjugate with charge reversal upon hydrolysis of the FAP-α protein, the drug precursor has at least one of the following structures:

[0039] The present invention also provides a polymer-drug conjugate prepared by the above method, wherein the FAP-α protein responds to hydrolysis and causes charge reversal.

[0040] 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.

[0041] One of the theoretical foundations of current anti-tumor drug development is the ability of drugs to penetrate tumors. However, the penetration of anti-tumor drugs into tumor tissue is a key factor hindering their effectiveness. Tumor tissue is characterized by high cell density, a dense matrix, and elevated intratumoral pressure. Consequently, the large size of anti-tumor drugs makes intratumoral diffusion difficult. After leaking out of blood vessels, they are unable to reach cells farther from the blood vessels to complete delivery, resulting in low efficacy.

[0042] 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 property of FAP-α protein to respond to hydrolysis and induce charge reversal to synthesize a polymer-drug conjugate with the structure shown in formula (1) for tumor treatment. The fibroblast activation protein (FAP-α protein) is used to cut the polypeptide to induce charge reversal, causing its charge to change from amphoteric to positive, thereby adsorbing and triggering endothelial cell endocytosis, intracellular transport and contralateral exocytosis (transcytosis). Since the transcytosis process is an active transport process that consumes 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 the drug to every cell, thereby having a high anti-tumor efficacy and significantly reducing tumor volume. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 is a graph showing the 4T1 cytotoxicity test results of monomethyl auristatin E (MMAE) and two drug-coupled polymers obtained in Examples 1-5 and 1-7;

[0044] FIG2 is a 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;

[0045] FIG3 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;

[0046] FIG4 is a graph showing the charge reversal of the (P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT)) polymer under the action of FAPα protein;

[0047] FIG5 is a graph showing the cytotoxicity test results of camptothecin (CPT) and two drug-coupled polymers obtained in Examples 2-5 and 2-7;

[0048] FIG6 is a graph showing the pharmacokinetic evaluation results of camptothecin (CPT) and the two drug-coupled polymers obtained in Examples 2-5 and 2-7;

[0049] FIG6 is a graph showing the results of tumor inhibition experiments on camptothecin (CPT) and two drug-coupled polymers obtained in Examples 2-5 and 2-7;

[0050] FIG8 is a graph showing the cytotoxicity test results of camptothecin (CPT) and two drug-coupled polymers obtained in Examples 3-7 and 3-8;

[0051] FIG9 is a graph showing the pharmacokinetic evaluation results of camptothecin (CPT) and the two drug-coupled polymers obtained in Examples 3-7 and 3-8;

[0052] FIG10 is a graph showing 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

[0053] The polymer-drug conjugates provided herein that undergo charge reversal in response to FAP-α protein hydrolysis comprise a drug-carrying structural unit and a FAP-α protein-responsive structural unit, preferably also comprising a hydrophilic structural unit. The polymer-drug conjugates, provided only with the drug-carrying structural unit and the FAP-α protein-responsive structural unit, can achieve charge reversal in response to FAP-α protein hydrolysis, resulting in a favorable anti-tumor effect. When the polymer-drug conjugates also include a hydrophilic structural unit, they can effectively prevent clearance of polymer-drug conjugate micelles, prolonging the drug's duration of action and achieving prolonged action in the blood.

[0054] In the present invention, the FAP-α protein-responsive hydrolysis-induced charge-flipping polymer-drug conjugate has a structure as shown in formula (1). Formula (1) is only used to represent the composition of the FAP-α protein-responsive hydrolysis-induced charge-flipping polymer-drug conjugate and the content of each structural unit, and is not used to represent the connection relationship between the structural units. The FAP-α protein-responsive hydrolysis-induced charge-flipping polymer-drug conjugate can be a random copolymer or a block copolymer, without particular limitation. In addition, the molar content of the drug-carrying structural unit is represented by m, the molar content of the FAP-α protein-responsive structural unit is represented by n, and the molar content of the hydrophilic structural unit is represented by p. Here, m, n, and p are determined by the feed amount. The ratio of 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.

[0055] In the present invention, the drug-carrying structural unit has a structure shown in formula (1-1):

[0056] In formula (1-1), R 11 is a linker; R 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 preferred to have 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, exitecan, 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, aplysialine 10, and β-Amanitin.

[0057] In the present invention, the FAP-α protein response structural unit has a structure shown in formula (1-2):

[0058] In formula (1-2), R 21 -NH-(CH2) x -、-O-(CH2) x -, -CO-(CH2) x -, -CO-NH-(CH2) x -、-(CH2) x -、-(CH2) x1 O(CH2) x2 S(CH2) x3 or -S-(CH2) x -, x is 2 to 6 (such as 2, 3, 4, 5, 6 or any value therebetween), x1, x2 and x3 are each independently 1 to 6 (such as 1, 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:

[0059] 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 is a C1-C5 alkyl group. Specific examples of the C1-C5 alkyl group 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 C1-C5 alkylene group 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.

[0060] In the present invention, the hydrophilic structural unit has a structure shown in formula (1-3):

[0061] In formula (1-3), R 31 -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S-, -(CH2) z -O-(CH2) t -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:

[0062] 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 is a C1-C5 alkyl group. Specific examples of the C1-C5 alkyl group 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 C1-C5 alkylene group 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.

[0063] 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.

[0064] The method for preparing a polymer-drug conjugate with charge reversal in response to hydrolysis of FAP-α protein provided by the present invention comprises modifying a drug precursor onto a polymer backbone. The polymer backbone is obtained by copolymerizing 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 incorporated into the polymer. The modification method is to modify the R11 `Performing 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.

[0065] In order to further understand the present invention, the present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments or application examples. Changes, substitutions, combinations and simplifications made by those skilled in the art under the core guiding principles of the present invention should all be included in the scope of protection of the present invention.

[0066] Example 1-1: Synthesis of Monomeric AzMA

[0067] 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 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, remove water with anhydrous MgSO4, and rotary evaporation to obtain the intermediate product.

[0068] 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 pumped out at low temperature and washed three times with pure water. MgSO4 was then used to remove water, and the product AzMA was separated by silica gel column (mobile phase: dichloromethane: methanol = 100:1 (V:V)).

[0069] Example 1-2: Synthesis of prodrug DBCO-Val-Cit-PABC-MMAE

[0070] 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), and diisopropylethylamine (DIEA) (0.011 mL) was added. The mixture was stirred at room temperature for 16 hours, and then DMF was removed under reduced pressure. The crude reaction product was separated by silica gel column (mobile phase: dichloromethane: methanol = 13:1 (V:V)) to obtain a white solid (DBCO-Val-Cit-PABC-MMAE, 9.2 mg, yield 49%).

[0071] 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).

[0072] Example 1-3: Synthesis of FAP-α protein-responsive monomeric MA-N-GP

[0073] 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, 2-aminoethylmethacrylamide hydrochloride (MA-N-NH2-HCl, 0.92 g, 5.5 mmol) and triethylamine (TEA, 4.7 mL, 16.6 mmol) were added, and 30 mL of anhydrous dichloromethane was added. L anhydrous dichloromethane, stirred at room temperature for 2 hours to obtain a MA-N-NH2 reaction solution; the activated reaction solution in the first flask was added dropwise into the MA-N-NH2 reaction solution using a constant pressure funnel, and the reaction was stirred at room temperature for 24 hours. After the reaction, it was washed three times with 0.5M HCl, and 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 second point was collected, and the mobile phase was removed by rotary evaporation to obtain a white solid powder (monomer MA-N-GP, 1.6 g, yield 47%).

[0074] Example 1-4: Synthesis of FAP-α protein-responsive polymers

[0075] (1) Using the RAFT polymerization method, a clean Schlenk tube was placed in an oven at 120°C for drying. After drying, the reaction tube was taken out and cooled to room temperature. A paper funnel was rolled up with filter paper and placed on the tube mouth. The monomers MA-N-GP (500 mg, 1.28 mmol), AzMA (70 mg, 0.42 mmol), and the initiator azobisisobutyronitrile (AIBN, 1.6 mg, 0.01 mmol) were added to the bottom of the reaction tube through the paper funnel; the disulfide 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 gas-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, 15 mL 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. The precipitation was repeated twice to remove unreacted monomers and DMSO solvent, and the prepolymer was dried for 24 hours to obtain a prepolymer.

[0076] (2) Dissolve 80 mg of prepolymer in 2 mL of dichloromethane. After complete dissolution, add 400 μL of trifluoroacetic acid in an ice bath and stir the mixture for 1 hour to remove the Boc protecting group. After the reaction is complete, remove the dichloromethane and trifluoroacetic acid by rotary evaporation, and then dry in a vacuum oven at room temperature for 4 hours. Add 4 mL of ultrapure water to the dried flask and stir to dissolve. Adjust the pH to 8.0 with saturated sodium bicarbonate solution. Weigh 50 mg of 2-ethoxy-2-oxide-1,3,2-dioxaphosphorane and dissolve it in 1 mL of ultrapure water. Add it to the reaction solution and stir the reaction in a water bath at 26°C for 36 hours. After the reaction is complete, transfer the reaction solution into a dialysis bag (MWCO = 1000 Da) and dialyze it with ultrapure water for 24 hours. Change the water every 6 hours. Keep it away from light throughout the process. The polymer solution obtained by dialysis is the FAP-α protein-responsive polymer, which is directly stored in a refrigerator at 4°C.

[0077] 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).

[0078] Example 1-5: Synthesis of FAP-α Protein Responsive Polymer P(MA-N-GPP)-co-P(AzMA-MMAE) Containing MMAE Drug Linker

[0079] 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 0.3 mL of a mixed solvent of DMF and water (DMF:water = 7:3 (v:v)). The mixture was pumped twice under argon protection, and copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5 (w:w)) were added. The mixture was then pumped three more times and reacted at 45° C. for 48 h. After completion of the reaction, the mixture was precipitated three times with glacial ether, dialyzed against DMSO, and then dialyzed against deionized water. The product was lyophilized to obtain the FAP-α protein-responsive polymer P(MA-N-GPP)-co-P(AzMA-MMAE) containing an MMAE drug linker.

[0080] 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(CCH2CH2C, COCCH2CH3, CHCH2, CHCH2CH2); 2.8-3.0(NCH3); 3. 2-3.6(NCH2CH3,OCH3); 3.7-4.2(COCH2NH,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).

[0081] Example 1-6: Synthesis of FAP-α protein-responsive polymers containing hydrophilic monomers

[0082] (1) Using the RAFT polymerization method, dry a clean Schlenk tube in an oven at 120°C. After drying, remove the tube and cool it to room temperature. Wrap a paper funnel with filter paper and place it on the tube opening. The monomers MA-N-GP (500 mg, 1.28 mmol), AzMA (70 mg, 0.42 mmol), dimethylaminoethyl methacrylate (DMAEMA, 294 mg, 1.28 mmol), and initiator azobisisobutyronitrile (AIBN, 1.6 mg, 0.01 mmol) were weighed and added to the bottom of the reaction tube through a paper funnel; the chain transfer agent (CTA-OH, 6.2 mg, 0.02 mmol) was weighed and dissolved in 800 μL of ultra-dry dimethyl sulfoxide (DMSO), and the solution was added to the bottom of the Schlenk tube and stirred to dissolve; then, the pumping device was used to cycle "liquid nitrogen freezing-vacuuming for 10 minutes-argon passing-thawing" for 3 times, and then the argon-filled reaction tube was placed in a 70°C oil bath and reacted for 48 hours; after the reaction, 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.

[0083] (2) 80 mg of prepolymer was dissolved in 2 mL of dichloromethane. After complete dissolution, 400 μL of trifluoroacetic acid was added under ice bath conditions and the mixture 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 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-dioxaphosphorane was weighed and dissolved in 1 mL of ultrapure water, added to the reaction solution, and stirred in a water bath at 26°C for 36 hours. After the reaction was completed, the reaction solution was transferred to a dialysis bag (MWCO = 1000 Da) 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 refrigerator at 4°C.

[0084] Example 1-7: Synthesis of FAP-α Protein Responsive Polymer P(DP)-co-P(MA-N-GPP)-co-P(AzMA-MMAE) Comprising MMAE Drug Linker and Hydrophilic Monomer

[0085] 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)). The mixture was pumped twice under argon protection, and copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5 (w:w)) were added. The mixture was then pumped three more times and reacted at 45° C. for 48 h. After completion of the reaction, the mixture was precipitated three times with glacial ether, dialyzed against DMSO, and then dialyzed against deionized water. The product was lyophilized to obtain the FAP-α protein-responsive polymer P(DP)-co-P(MA-N-GPP)-co-P(AzMA-MMAE) containing an MMAE drug linker and a hydrophilic monomer.

[0086] Application Example 1-1

[0087] (1) Preparation of polymer injection

[0088] P(MA-N-GPP)-co-P(AzMA-MMAE) and P(DP)-co-P(MA-N-GPP)-co-P(AzMA-MMAE) were respectively dissolved in normal saline, and polymer injection was prepared at an equivalent concentration of 0.2 mg / kg body weight MMAE to obtain a homogeneous, clear and transparent polymer injection.

[0089] (2) Cytotoxicity assay

[0090] Monomethyl auristatin E (MMAE) and two polymer injections were tested for 4T1 cell toxicity, and the results are shown in Figure 1. As can be seen from Figure 1, the drug-conjugated polymer containing a hydrophilic monomer (P(DP)-co-P(MA-N-GPP)-co-P(AzMA-MMAE)) has strong cytotoxicity against 4T1 cells. However, due to its anti-protein adsorption effect, it is difficult to enter the cell membrane. Therefore, compared with the polymer without a hydrophilic monomer (P(MA-N-GPP)-co-P(AzMA-MMAE)) and the free drug (monomethyl auristatin E), it has relatively low cytotoxicity. The results of the cytotoxicity experiment show that both synthesized polymer drug conjugates have a killing effect on 4T1 cells and have significant cytotoxicity.

[0091] (3) Pharmacokinetic evaluation

[0092] 2 mL of each of the two drug-conjugated polymer solutions obtained in Examples 1-5 and 1-7 was added to 200 μL of a 1 mg / mL Cy5.5-alykne solution. After evacuation twice under argon, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added and stirred at room temperature for 24 hours. After completion of the reaction, the solution was dialyzed against methanol for 3 hours and then against ultrapure water for 24 hours to obtain a Cy5.5 fluorescently labeled polymer solution.

[0093] After the mice had adapted to the environment, 6 mice were divided into two groups, with three mice in each group. 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 eyes of the mice. About 20 μL of blood was collected and its fluorescence value was measured. The results are shown in Figure 2. As can be seen from Figure 2, the t 1 / 2 The t of P(DP)-co-P(MA-N-GPP)-co-P(AzMA-CPT) was 3.6 h. 1 / 2 The duration of the micelles was 5.6 hours. By polymerizing the hydrophilic monomers, the micelles could be effectively prevented from being removed, thereby increasing the blood circulation time and ensuring sufficient long-term blood circulation.

[0094] (4) Tumor inhibition experiment

[0095] The inhibitory effect of MMAE drug-coupled polymer on 4T1 breast cancer cell-bearing mice was studied. When the tumor grew to about 100 mm 3The experiment began. Injectable solutions of free MMAE and two drug-conjugated polymers were prepared, with PBS buffer used as a blank control. Drugs were administered via tail vein injection at a dose of 0.2 mg / kg MMAE equivalent, with 100 μL injected per mouse. Administration was repeated five times on days 1, 4, 7, 10, and 13. Mice were weighed and tumor volumes were measured on days 1, 3, 5, 7, 9, 11, 13, and 15. The results are shown in Figure 3. As can be seen from Figure 3, compared to the blank control, treatment with free MMAE inhibited tumor growth to some extent, but its inhibitory effect was significantly weaker than that of the polymer-drug conjugate. Due to its longer blood circulation time, the drug-conjugated polymer containing a hydrophilic monomer exhibited a more dominant tumor-inhibiting effect.

[0096] Example 2-1: Synthesis of Monomeric AzMA

[0097] The same method as Example 1-1 was used to obtain monomer AzMA.

[0098] Example 2-2: Synthesis of FAP-α protein-responsive monomeric MA-N-GP

[0099] The same method as in Example 1-3 was used to obtain the monomer MA-N-GP.

[0100] Example 2-3: Synthesis of prodrug DBCO-SS-CPT

[0101] (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 in an ice bath to dissolve. Then, dissolve EDC (1 g, 6 mmol) in 10 mL of anhydrous dichloromethane and add it to a constant pressure funnel. Under nitrogen protection, add it dropwise into the round-bottom flask at a rate of 4 s / drop. Stir and react at room temperature for 24 hours. After the reaction is completed, the insoluble matter is filtered off, and the filtrate is concentrated by rotary evaporation. 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.

[0102] (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 disperse them evenly. In a fume hood, triphosgene (BTC, 0.28 g, 0.96 mmol) was weighed and dissolved in 10 mL of anhydrous dichloromethane in a round-bottom flask. The solution was then added dropwise to the CPT dichloromethane solution and stirred for 30 minutes. After the solution turned yellow-green, the product DBCO-SS-OH (1.39 g, 3.16 mmol) from the previous step was weighed and dissolved in 15 mL of anhydrous dichloromethane. The solution was slowly added dropwise to the round-bottom flask and stirred at room temperature for 48 hours. After the reaction was completed, the solution turned orange-yellow, and the insoluble by-product salt was removed by filtration. The product was concentrated by rotary evaporation and separated and purified by column chromatography using dichloromethane / methanol 40:1 (v:v) as the mobile phase. The second spot was collected, which was the product spot. The mobile phase was removed by rotary evaporation to obtain a light yellow solid powder DBCO-SS-CPT (1.45 g, yield 73%).

[0103] 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).

[0104] Example 2-4: Synthesis of FAP-α protein responsive polymer P(MA-N-GP)-co-P(AzMA)

[0105] 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 disulfide 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 solution was added to the bottom of the Schlenk tube and stirred thoroughly to dissolve all the raw materials; then, the pumping device was used to "liquid nitrogen freeze-vacuum 15 minutes-argon gas-thaw" cycle 3 times, and the argon-filled reaction tube was placed in a 70°C oil bath for continuous reaction for 48 hours; after the reaction, 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 monomer and DMSO solvent, and the polymer was dried for 24 hours.

[0106] 40 mg of the polymer was dissolved in 2 mL of dichloromethane. Once completely dissolved, 200 μL of trifluoroacetic acid was added in an ice bath and stirred for 1 hour to remove the Boc protecting group. After completion of the reaction, the dichloromethane and trifluoroacetic acid were removed by rotary evaporation. The polymer was then entrained twice with dichloromethane and dried in a vacuum oven at room temperature for 4 hours. The polymer was then dissolved by stirring in 4 mL of ultrapure water. The pH was adjusted to 8.0 with saturated sodium bicarbonate solution. 25 mg of propane sultone and 1 mL of ultrapure water were added and stirred in a 26°C water bath for 24 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed against ultrapure water for 24 hours to remove unreacted propane sultone. The water was changed every 6 hours and the polymer was lyophilized to obtain a solid powder, the FAP-α protein-responsive polymer P(MA-N-GP)-co-P(AzMA).

[0107] 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).

[0108] Example 2-5: Synthesis of CPT Drug-Conjugated FAP-α Protein Responsive Polymer P(MA-N-GP)-co-P(AzMA-CPT)

[0109] 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 evacuation twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5 (w:w)) were added, and then evacuated three times. The reaction was carried out at 45° C. for 48 h. After completion of the reaction, the product was precipitated three times with glacial ether, dialyzed against DMSO and then dialyzed against deionized water, and lyophilized to obtain the product, which is the CPT drug-conjugated FAP-α protein-responsive polymer P(MA-N-GP)-co-P(AzMA-CPT).

[0110] 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).

[0111] Example 2-6: Synthesis of polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA)

[0112] 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; the 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 stirred thoroughly to dissolve all the raw materials; then, the pumping device was used to "freeze with liquid nitrogen-vacuum for 15 minutes-pass argon-thaw" 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, 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 unreacted monomers and DMSO solvent, and the polymer was obtained after drying for 24 hours.

[0113] 40 mg of the polymer was dissolved in 2 mL of dichloromethane. Once completely dissolved, 200 μL of trifluoroacetic acid was added in an ice bath and stirred for 1 hour to remove the Boc protecting group. After completion of the reaction, the dichloromethane and trifluoroacetic acid were removed by rotary evaporation. The polymer was then entrained twice with dichloromethane and dried in a vacuum oven at room temperature for 4 hours. The polymer was then dissolved by stirring in 4 mL of ultrapure water. The pH was adjusted to 8.0 with saturated sodium bicarbonate solution. 25 mg of propane sultone and 1 mL of ultrapure water were added and stirred in a 26°C water bath for 24 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed against ultrapure water for 24 hours to remove unreacted propane sultone. The water was changed every 6 hours and the polymer was lyophilized to obtain a solid powder, which is the FAP-α protein-responsive polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA).

[0114] 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).

[0115] Example 2-7: Synthesis of CPT Drug-Conjugated FAP-α Protein Responsive Polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT)

[0116] 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 evacuation twice under argon protection, copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate: vitamin C = 1:5 (w:w)) were added, and then evacuated three times. The reaction was carried out at 45° C. for 48 h. After completion of the reaction, the mixture was precipitated three times with glacial ether, dialyzed against DMSO and then dialyzed against 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).

[0117] 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).

[0118] Application Example 2-1

[0119] (1) Preparation of polymer injection

[0120] 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.

[0121] (2) P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) charge reversal experiment in response to FAP-α protein

[0122] A 50 ng / mL FAP-α solution was prepared in PBS buffer. 5 mg of the polymer (P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT)) was dissolved in 5 mL of ultrapure water. The FAP-α solution and polymer solution were mixed at a volume ratio of 4:1. The reaction solution was shaken at 37°C and 100 rpm for reaction. 200 μL of the reaction solution was sampled at 0, 0.25, 0.5, 1, 2, 4, and 6 hours, filtered through a 0.45 μm filter, and the zeta potential changes at different time points were measured using DLS. As shown in Figure 4, after mixing with the FAP-α protein, the polymer undergoes a rapid charge reversal, with the zeta potential shifting to a positive state.

[0123] (3) Cytotoxicity assay

[0124] Camptothecin (CPT) and the two polymer injections were subjected to 4T1 cell cytotoxicity tests, and the results are shown in Figure 5. As can be seen from Figure 5, the drug-conjugated polymer P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) containing a hydrophilic monomer has strong cytotoxicity to 4T1 cells. However, due to its anti-protein adsorption effect, it is difficult to enter the cell membrane. Therefore, compared with the polymer P(MA-N-GP)-co-P(AzMA-CPT) without a hydrophilic monomer and the free drug (camptothecin), it has relatively lower cytotoxicity. The results of the cytotoxicity experiment show that both synthesized polymer drug conjugates have a killing effect on 4T1 cells and have significant cytotoxicity.

[0125] (4) Pharmacokinetic evaluation

[0126] 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 were respectively added dropwise with 200 μL of a 1 mg / mL Cy5.5-alykne solution and stirred at room temperature for 24 hours. After the reaction, the solution was dialyzed with methanol for 3 hours and then with ultrapure water for 24 hours to obtain a Cy5.5 fluorescently labeled polymer solution, which was respectively designated as P(MA-N-GP)-co-P(AzMA-CPT). Cy5.5 ,P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) Cy5.5 .

[0127] After the mice had adapted to the environment, 6 mice were divided into two groups, with three mice in each group. 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 eyes. About 50 μL of blood was put into the living body for detection before the blood coagulation. The fluorescence value was measured. The results are shown in Figure 6. As can be seen from Figure 6, the t 1 / 2 The t of P(DS)-co-P(MA-N-GP)-co-P(AzMA-CPT) is 4.6h. 1 / 2 The duration of the micelles 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-term blood circulation.

[0128] (5) Tumor inhibition experiment

[0129] The inhibitory effect of CPT drug-coupled polymer on 4T1 breast cancer cell-bearing mice was studied. When the tumor grew to about 100 mm 3 The experiment began. Free CPT and two drug-conjugated polymer injections were prepared, with PBS buffer used as a blank control. Each mouse was administered via tail vein injection at a CPT equivalent of 5 mg / kg, with 100 μL injected per dose. Dosing was performed five times on days 1, 4, 7, 10, and 13. Mice were weighed and tumor volumes were measured on days 1, 3, 5, 7, 9, 11, 13, and 15. The results are shown in Figure 7. As shown in Figure 7, free CPT treatment inhibited tumor growth to some extent compared to the blank control, but its inhibitory effect was significantly weaker than that of the polymer-drug conjugate. Due to its longer blood circulation time, the drug-conjugated polymer containing a hydrophilic monomer exhibited a superior tumor-inhibiting effect.

[0130] Example 3-1: Synthesis of azide monomer

[0131] (4S)-4-(4-chlorobutyl)-2,5-oxazolidinedione (1.59 g, 9.2 mmol) was dissolved in 30 mL of deionized water. After it was completely dissolved, sodium azide (2.25 g, 34.6 mmol) was added to the above solution and reacted at 100°C in the dark for 24 hours. After the reaction was completed, it was slowly cooled to room temperature and potassium hydroxide was added until potassium hydroxide solid was seen. At this time, oil droplets could also be seen on the liquid surface. Then, it was extracted with ether three times, and water was removed by anhydrous MgSO4. The product was rotary evaporated to obtain the product, which was then separated by silica gel column (mobile phase: dichloromethane: methanol = 100:1 (V:V)) to obtain the azide monomer.

[0132] Example 3-2: Synthesis of FAP-α protein-responsive monomers

[0133] 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 the mixture was stirred at room temperature for 2 hours. The activated reaction solution in the first flask was added dropwise to the reaction solution using a constant pressure funnel, and the mixture was stirred at room temperature for 24 hours. After the reaction, the unreacted salt was filtered 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. The product was separated and purified by silica gel column chromatography using ethyl acetate as the mobile phase. The mobile phase was removed by rotary evaporation to obtain a white solid powder (1.6g, yield 47%).

[0134] Example 3-3: Synthesis of hydrophilic monomer

[0135] 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 homogeneous. 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 from ethanol / ether to obtain a white solid product.

[0136] The above white solid product was dissolved in 10 mL of dichloromethane. After complete dissolution, 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. After that, it was dried in a vacuum oven at room temperature for 4 hours to obtain allyl glutamate.

[0137] 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). The resulting mixture was stirred at room temperature for 24 hours, during which time allyl glutamate gradually dissolved. 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. After suction filtration and rotary evaporation, allyl oxazolidinedione propionate was obtained.

[0138] Allyl oxazolidinedione propionate (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, and the solvent was removed by rotary evaporation. The NCA-DS monomer was obtained after vacuum drying.

[0139] Example 3-4: Synthesis of prodrug DBCO-SS-CPT

[0140] The same method as in Example 2-3 was used to obtain the drug precursor DBCO-SS-CPT.

[0141] Example 3-5: Synthesis of FAP-α protein responsive polymer P(AzAA)-co-P(AA-N-GPS)

[0142] 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 dissolved in chloroform. The mixture was 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.

[0143] 40 mg of the polymer was dissolved in 2 mL of dichloromethane. Once completely dissolved, 200 μL of trifluoroacetic acid was added in an ice bath and stirred for 1 hour to remove the Boc protecting group. After completion of the reaction, the dichloromethane and trifluoroacetic acid were removed by rotary evaporation. The polymer was then entrained twice with dichloromethane and dried in a vacuum oven at room temperature for 4 hours. The polymer was then dissolved by stirring in 4 mL of ultrapure water. The pH was adjusted to 8.0 with saturated sodium bicarbonate solution. 25 mg of propane sultone and 1 mL of ultrapure water were added and stirred in a 26°C water bath for 24 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed against ultrapure water for 24 hours to remove unreacted propane sultone. The water was changed every 6 hours and the polymer was lyophilized to obtain a solid powder, which is the FAP-α protein-responsive polymer P(AzAA)-co-P(AA-N-GPS).

[0144] Chemical shift of FAP-α protein-responsive 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).

[0145] Example 3-6: Synthesis of FAP-α protein responsive polymer P(AzAA)-co-P(AA-N-GPS)-co-P(DS)

[0146] The hydrophilic monomer (82 mg, 0.685 mmol), the azide monomer (73 mg, 0.685 mmol), and the FAP-α protein-responsive 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 =0.253 M, 136 mL, 34.4 mmol), the reaction mixture was stirred at room temperature for 48 hours, then concentrated by rotary evaporation, the concentrated solution was dissolved in chloroform, and 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).

[0147] The resulting polymer was dissolved in 3 mL of dichloromethane. Once completely dissolved, 300 μL of trifluoroacetic acid was added in an ice bath and the mixture was stirred for 1 hour to remove the Boc protecting group. After completion of the reaction, the dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and the mixture was entrained twice with dichloromethane. The mixture was then dried in a vacuum oven at room temperature for 4 hours. The resulting product was dissolved in 4 mL of ultrapure water in a dried flask and stirred. The pH was adjusted to 8.0 with saturated sodium bicarbonate solution to obtain a reaction solution. 25 mg of propane sultone and 1 mL of ultrapure water were added, and the mixture was stirred in a 26°C water bath for 24 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag (MWCO = 1000 Da) and dialyzed against ultrapure water for 24 hours to remove unreacted propane sultone, with the water changed every 6 hours. The resulting solid powder was lyophilized to obtain the FAP-α protein-responsive polymer P(AzAA)-co-P(AA-N-GPS)-co-P(DS).

[0148] 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(COOCH2C).

[0149] Example 3-7: Synthesis of FAP-α Protein Responsive Polymer P(AzAA-CPT)-co-P(AA-N-GPS) Containing CPT Drug Linker

[0150] 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 h. After completion of the reaction, it was precipitated with glacial ether three times, dialyzed against DMSO and then dialyzed against 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.

[0151] Chemical shifts of the FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS) containing a 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, NHCOCH2); 5.2-5.9 (NCH2C, COOCH2C).

[0152] 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

[0153] 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)). The mixture was pumped twice under argon protection, and copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5 (w:w)) were added. The mixture was then pumped three times and reacted at 45°C for 48 h. After completion of the reaction, the mixture was precipitated three times with glacial ether, dialyzed against DMSO and then dialyzed against deionized water, and lyophilized to obtain the product, which is the FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) containing a CPT drug linker.

[0154] Chemical shifts of the FAP-α protein-responsive polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) containing a 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).

[0155] Application Example 3-1:

[0156] (1) Preparation of polymer injection

[0157] 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.

[0158] (2) Cytotoxicity assay

[0159] Camptothecin (CPT) and two polymer injections were tested for 4T1 cytotoxicity, and the results are shown in Figure 8. As can be seen from Figure 8, the drug-conjugated polymer P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS), which contains amphiphilic monomers, has strong cytotoxicity against 4T1 cells. However, due to its anti-protein adsorption effect, it is difficult to enter the cell membrane. Therefore, compared with the block polymer P(AzAA-CPT)-co-P(AA-N-GPS) without hydrophilic monomers and the free drug (camptothecin), it has relatively lower cytotoxicity. The results of the cytotoxicity experiment show that both synthesized polymer drug conjugates have a killing effect on 4T1 cells and have significant cytotoxicity.

[0160] (3) Pharmacokinetic evaluation

[0161] 2 mL of each polymer obtained in Examples 3-7 and 3-8 was prepared into a solution. 200 μL of a 1 mg / mL Cy5.5-alykne solution was added dropwise to each solution. The mixture was stirred at room temperature for 24 hours. After the reaction, the solution was dialyzed against methanol for 3 hours and then against ultrapure water for 24 hours to obtain a Cy5.5 fluorescently labeled polymer solution.

[0162] After the mice had adapted to the environment, 6 mice were divided into two groups, with three mice in each group. 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 eyes. About 50 μL of blood was put into the living body for detection before the blood coagulation. The fluorescence value was measured. The results are shown in Figure 9. As can be seen from Figure 9, the t 1 / 2 The t of P(AzAA-CPT)-co-P(AA-N-GPS)-co-P(DS) is 4h. 1 / 2 The duration of the micelles is 6.7 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-term blood circulation.

[0163] (4) Tumor inhibition experiment

[0164] The inhibitory effect of CPT drug-coupled polymer on 4T1 breast cancer cell-bearing mice was studied. When the tumor grew to about 70 mm 3The experiment began. Free CPT and two drug-conjugated polymer injections were prepared, with PBS buffer used as a blank control. Each mouse was administered via tail vein injection at a CPT equivalent of 5 mg / kg, with 100 μL injected per dose. Dosing was performed on days 1, 4, 7, 10, and 13, for a total of five doses. Mice were weighed and tumor volumes were measured on days 1, 3, 5, 7, 9, 11, 13, and 15. The results are shown in Figure 10. As shown in Figure 10, free CPT treatment inhibited tumor growth to some extent compared to the blank control, but its inhibitory effect was significantly weaker than that of the polymer-drug conjugate. Due to its longer blood circulation time, the drug-conjugated polymer containing a hydrophilic monomer exhibited a superior tumor-inhibiting effect.

[0165] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative 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 purpose of the present invention.

Claims

1. A polymer-drug conjugate that responds to FAP-α hydrolysis to cause charge inversion, characterized in that The polymer-drug conjugate that responds to hydrolysis of FAP-α protein to cause charge inversion has the structure shown in formula (1); R 11 is a linker; R 12 is an anti-tumor drug group; R 21 is -NH-(CH2) x -, -O-(CH2) x -, -CO-(CH2) x -, -CO-NH-(CH2) x -, -(CH2) x -, -(CH2) x1 O(CH2) x2 S(CH2) x3 or -S-(CH2) x -, x is 2 to 6, x1, x2 and x3 are each independently 1 to 6; R 22 is a zwitterionic group; R 31 is -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S-, -(CH2) z -O-(CH2) t -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 is a zwitterionic group; m, n, p are the molar ratios of the three structural units and m:n:p is (5 to 40%):(10 to 95%):(0 to 85%) and m + n + p = 100%.

2. The polymer-drug conjugate that responds to hydrolysis of the FAP-α protein to cause charge inversion according to claim 1, wherein R 11 selected from at least one of the following structures; In formula (2), R 13 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 -, and z is from 1 to 14; In formula (3), R 14 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 -, and z is from 1 to 14; In formula (4), R 15 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 16 is H or In formula (5), R 17 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 18 is H or In formula (6), R 19 is an 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 from 1 to 14; In formula (7), R 20 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 -, and z is from 1 to 14; In the above formulas, each linker is independently a group having at least one of reducibility, reactive oxygen species, pH, and enzyme responsiveness.

3. The polymer-drug conjugate that responds to FAP-α protein hydrolysis to cause charge inversion according to claim 1, wherein R 22 selected from at least one of the following structures; In the above formulas, R 23 and R 24 are each independently H or an alkyl group having 1 to 5 carbon atoms, R 25 and R 27 are each independently an alkylene group having 1 to 5 carbon atoms, and R 26 is an alkyl group having 1 to 5 carbon atoms.

4. The polymer-drug conjugate that responds to hydrolysis of FAP-α protein to cause charge inversion according to claim 1, characterized in that, R 32 selected from at least one of the following structures; In the above formulas, R 33 and R 34 are each independently H or an alkyl group having 1 to 5 carbon atoms, R 35 and R 37 are each independently an alkylene group having 1 to 5 carbon atoms, and R 36 is an alkyl group having 1 to 5 carbon atoms.

5. The polymer-drug conjugate that responds to FAP-α protein hydrolysis to cause charge inversion according to claim 1, wherein The number average molecular weight of the polymer-drug conjugate is 10,000 to 80,000 Da; m is 5 to 40%, n is 10 to 95%, and p is 0 to 85%; the polymer-drug conjugate is a random copolymer or a block copolymer.

6. A preparation method of a polymer-drug conjugate that responds to the hydrolysis of FAP-α protein to cause charge inversion, characterized in that, The method includes obtaining by modifying a prodrug to a polymer backbone; the polymer backbone has a structure shown in formula (8); R 11 ` is a linker; R 21 is -NH-(CH2) x -, -O-(CH2) x -, -CO-(CH2) x -, -CO-NH-(CH2) x -, -(CH2) x -, -(CH2) x1 O(CH2) x2 S(CH2) x3 or -S-(CH2) x -, x is 2 to 6, x1, x2 and x3 are each independently 1 to 6; R 22 is an zwitterionic group; R 31 is -NH-, -O-, -OCO-, -NHCO-, -COO-, -CONH-, -S-, -(CH2) z -O-(CH2) t -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 is an zwitterionic group; m, n, p are the molar ratios of the three structural units and m:n:p is (5 to 40%):(10 to 95%):(0 to 85%) and m + n + p = 100%.

7. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 6, characterized in that, The modification method is R in the polymer backbone 11 `to carry out a click chemical reaction with the drug precursor, and the groups for the click chemical reaction are selected from at least one of azide, trans-cyclooctene, tetrazine, alkyne, dibenzocyclooctyne, bicyclo[6,1,0]nonyne, lipoic acid and maleimide.

8. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 6, characterized in that, R 11 selected from at least one of the following structures; 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 from 1 to 14; 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 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 from 1 to 14; 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 -, and z is from 1 to 14.

9. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 6, characterized in that, The polymer backbone has at least one of the following structures; R 17 `, R 21 `, and R 31 ` are each independently H or an alkyl group having 1 to 5 carbon atoms; R 18 `, R 22 `, and R 32 ` are each independently O or NH.

10. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 6, characterized in that, The polymer backbone is obtained by random copolymerization or block copolymerization of FAP-α-responsive monomers.

11. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 6, characterized in that, The drug precursor has at least one of the following structures; The linker is a group having at least one of reducibility, reactive oxygen species, pH, and enzyme responsiveness; Drug is an anti-tumor drug.

12. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 11, characterized in that, The anti-tumor 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, Piericidin A, ansamitocin P3, dolastatin 10, and β-Amanitin.

13. The preparation method of the polymer-drug conjugate with charge inversion caused by hydrolysis in response to FAP-α protein according to claim 6, characterized in that, The drug precursor has at least one of the following structures; 14. The FAP-α protein-responsive hydrolytic charge-reversal polymer-drug conjugate prepared by the method according to claim 6.

15. Use of the FAP-α protein-responsive hydrolytic charge-reversal polymer-drug conjugate according to claim 1 in anti-tumor treatment.

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