A tumor microenvironment-responsive polymer-drug conjugate, and a preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a tumor microenvironment responsive polymer-drug conjugate as well as a preparation method and application thereof. The polymer-drug conjugate has a structure as shown in formula (1), wherein R1 is a hydrophilic group with anti-protein adsorption capacity; R2 is a microenvironment responsive group; R3 is a linker which is connected with R1 and R2 through a carbon-carbon bond, a phosphorus ester bond, an ester bond, an amide bond, an urethane bond and an ether bond respectively, and is connected with R4 through various chemical bonds formed through bio-orthogonal chemical reaction; R4 is an anti-tumor drug group with a-linker-Drug structure, and linker is a group with at least one of reduction, active oxygen, pH and enzyme responsiveness. The polymer-drug conjugate obtained in this way can be enriched and penetrated in a large amount at a tumor tissue site, and through the breaking of the responsive linker bond, the drug is released at the tumor tissue site, thereby producing a good tumor treatment effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a tumor microenvironment-responsive polymer-drug conjugate, its preparation method, and its application. Background Technology

[0002] With population growth and aging, cancer incidence and mortality rates continue to rise annually, and no highly curative treatment has been found for most cancers. Advances in medical technology have led to the application of more and more cancer therapies in clinical practice, including surgical resection, chemotherapy, and radiotherapy. Among these, surgical resection combined with chemotherapy is the only method that can cure cancer. However, not all patients are suitable for surgical resection; the patient's individual physical condition must be considered. Furthermore, surgical resection has drawbacks such as postoperative complications, incomplete resection, and trauma caused by excessive resection. For patients who cannot undergo surgical resection, chemotherapy and radiotherapy are the only treatment options.

[0003] However, most anticancer drugs have poor water solubility and can cause adverse reactions in healthy tissues. Nanoparticle drug delivery systems combining polymers and targeted drugs offer a more effective and safer option for cancer treatment. Polymer-drug conjugates can increase the water solubility of anticancer drugs and embed the drug within the polymer, resulting in a low risk of drug burst release during circulation. The advantages of therapeutic polymer-drug conjugates over other delivery systems have been recognized in preclinical trials using various in vivo / in vitro cancer models. However, current polymer-drug conjugates lack specific recognition of tumor tissues. Improving drug accumulation and targeted release at tumor tissues remains a key technical challenge for enhancing the therapeutic efficacy of polymer-drug conjugates. Summary of the Invention

[0004] The primary objective of this invention is to address the problem that existing polymer-drug conjugates lack specific recognition of tumor tissue, resulting in poor therapeutic efficacy due to drug accumulation and targeted release at tumor tissue. The invention provides a tumor microenvironment-responsive polymer-drug conjugate that improves specific recognition of tumor tissue, thereby enabling drug accumulation and targeted release at tumor tissue and enhancing therapeutic efficacy.

[0005] The second objective of this invention is to provide a method for preparing the above-mentioned tumor microenvironment-responsive polymer-drug conjugate.

[0006] A third objective of this invention is to provide the application of the aforementioned tumor microenvironment-responsive polymer-drug conjugates in antitumor drug delivery and tumor therapy.

[0007] In traditional nanomedicine delivery, when nanomedicines enter the body, they lack specific recognition of tumor tissues. During delivery, the nanomedicines are easily engulfed by the MPS system due to their large size or filtered by the renal tubules and glomeruli due to their small size. At the same time, due to the complex biochemical environment in the body, nanomedicines are prone to leakage from the delivery system, causing toxic side effects on other healthy tissues. This results in anti-tumor drugs not being accurately delivered to the tumor tissue and released to produce a therapeutic effect, leading to a very low cure rate for tumors.

[0008] After in-depth and extensive research, the inventors of this invention discovered that the acidic pH, hypoxia, dense tissue matrix, and upregulation of certain enzymes in the tumor microenvironment provide conditions for targeted drug release in responsive drug delivery systems. Based on this, this invention ingeniously designed and synthesized a polymer-drug conjugate with the structure shown in formula (1). Utilizing hydrophilic groups with anti-protein adsorption capabilities, it evades the phagocytosis of the MPS system, resulting in a long circulating half-life in vivo. Simultaneously, by utilizing microenvironment-responsive groups, the polymer-drug conjugate can accumulate and penetrate extensively in tumor tissue through the EPR effect. Finally, through the cleavage of responsive linker bonds, the drug is released at the tumor site, producing a good therapeutic effect. The polymer-drug conjugate provided by this invention can efficiently deliver drugs to each cancer cell and release them to produce a therapeutic effect, causing rapid apoptosis of cancer cells and significantly reducing tumor volume in a short period.

[0009] Specifically, the polymer-drug conjugate has the structure shown in formula (1);

[0010]

[0011] In formula (1), R1 is a hydrophilic group with anti-protein adsorption ability; R2 is a group with microenvironment responsiveness; R3 is a linker, which is connected to R1 and R2 through carbon-carbon bonds, phosphoester bonds, ester bonds, amide bonds, urethane bonds, and ether bonds respectively, and is connected to R4 through various chemical bonds formed by bioorthogonal chemical reactions; R4 is an antitumor drug group with a -linker-Drug structure, and linker is a group with at least one of reducing, reactive oxygen species, pH and enzyme responsiveness.

[0012] In a preferred embodiment, R1 is derived from at least one of polydiols, polyvinyl alcohols, poly(meth)acrylates, poly(meth)acrylamides, polyamino acids, and polyphosphates.

[0013] In a preferred embodiment, R1 has at least one of the following structures:

[0014] m = 10 ~ 200; m = 10 ~ 200;

[0015] m = 10 ~ 200, R 11 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0016] m = 10 ~ 200, R 11 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0017] m = 10 ~ 200, R 11 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0018] m = 10 ~ 200; m = 10 ~ 200;

[0019] m = 10 ~ 200, R 11 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0020] m = 10 ~ 200, R 11 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0021] m = 10 ~ 200, R 11 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0022] In equations (2) and (3), R 11 `and R 11 Each is an alkyl group that is independently C1-C4;

[0023] In equation (4), R 12 `is H, F, Cl, Br, I or CH3, R 12 `` is a C1-C4 alkylene group, R 12 for

[0024] In equations (5) and (6), R 13 `and R 14 Each of the following can be independently identified as H, F, Cl, Br, I, CH3. R 13 ``and R14 Each is independently a C1-C4 alkylene group. R 13 and R 14 Each independently

[0025] In equations (7) and (8), R 15 and R 16 Each independently n = 1 - 2;

[0026] In equations (9) and (10), R 17 and R 18 Each can be independently -CH3, -CH2CH3, -(CH2)2OH or -CH2CHOHCH2OH, R 17 `and R 18 Each is independently a C1-C4 alkylene group or -CH2CH2-O-;

[0027] In equation (11), R 19 `is H, F, Cl, Br, I or CH3, R 19 `` is a C1-C4 alkylene group, R 19 It can be -CH3, -CH2CH3, -CH2CH(CH3)2, or -(CH2)3CH3;

[0028] In equations (2)-(11), * indicates the position where the R1 group is bonded to the R3 group.

[0029] In a preferred embodiment, R2 is derived from at least one of poly(meth)acrylate, poly(meth)acrylamide, polyurethane, polyβ-amino ester, polycarbonate, polyamino acid, polypeptide, and nucleic acid.

[0030] In a preferred embodiment, R2 has at least one of the following structures:

[0031] m = 10 ~ 200, R 20 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0032] m = 10 ~ 200, R 20 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0033] m = 10 ~ 200; m = 10 ~ 200;

[0034] m = 10 ~ 200, R 20 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0035] m = 10 ~ 200, R 20 It can be -O-, -NH-, -CO-, -OCO-, or -NHCO-;

[0036] m = 10 ~ 200; m = 10 ~ 200;

[0037] m = 10 ~ 200;

[0038] m = 10 ~ 200;

[0039] m = 10 ~ 200;

[0040] In equations (12) and (13), R 21 `and R 22 Each of the following can be independently identified as H, F, Cl, Br, I, CH3. R 21 ``and R 22 Each is independently a C1-C4 alkylene group. R 21 and R 22 Each independently x = 1 - 8;

[0041] In equations (14) and (15), R 23 and R 24 Each independently x1 = 1 - 4, x2 = 2 - 6;

[0042] In equations (16) and (17), R 27 `and R 28 Each is independently a C1-C4 alkylene group, R 25 `and R 26 Each can be independently -H, -CH3, or -CH2CH3, R 25 and R 26 Each independently Among them, R 25” is -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, -CHCH3-CH2CH3, -CHCH3-(CH2)2CH3, -CH(CH3)2 or -CHCH2CH3-CH2CH3, x1=1-4, x2=2-8;

[0043] In equations (18) and (19), R 27 and R 28 Each independently Among them, R 26 ' is -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, -CHCH3-CH2CH3, -CHCH3-(CH2)2CH3, -CH(CH3)2 or -CHCH2CH3-CH2CH3, x1=1-2, x2=1-4, x3=2-6;

[0044] In equations (20), (21), and (22), R 29 For -CH3, -CH2CH3, -(CH2)2CH3, -CHCH3-CH2CH3, -CHCH2CH3-CH2CH3, -(CH2)3CH3, -(CH2)4CH3, -(CH2)2-N(CH2)4, -(CH2)2-N(CH2)5, -(CH2)2-N(CH2)6 or -(CH2)2-N(CH2)7, R 29 ' is an alkylene group with no or no C1-C4 atoms, R 30 -(CH2) x1 -, -CH2-C(CH)4C-CH2- or -(CH2CH2O) x2 - where x1 = 1 - 6, x2 = 1 - 16;

[0045] In formulas (12)-(22), * indicates the position or H where the R2 group is bonded to the R3 group.

[0046] In a preferred embodiment, R3, which is bonded to R4, has at least one of the following structures:

[0047]

[0048] n = 1 - 4;

[0049] n = 1 - 4,

[0050]

[0051] In this context, * indicates the position where the R3 group is bonded to the R1 and R2 groups.

[0052] In a preferred embodiment, the linker in the R4 group is at least one of the group structures that are responsive to glutathione, reactive oxygen species, hypoxia, pH, ATP, esterase, cathepsin, matrix metalloproteinase, nitroreductase, aspartate protease, gamma-glutamyl transferase, or fibroblast activating protein.

[0053] In a preferred embodiment, the antitumor drug in the R4 group is selected from at least one of doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, eczema, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, chachiin, gambogeylic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansine, tyransatine A, auristatin E, auristatin F, Piericidin A, anserine P3, salicylate 10 and β-Amanitin, BTZ, fluorouracil, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, glucose oxidase, lactate oxidase, catalase, and therapeutic nucleic acids.

[0054] The method for preparing tumor microenvironment-responsive polymer-drug conjugates provided by the present invention includes: obtaining polymer-drug conjugates by reacting a polymer carrier with a drug precursor through a click chemical reaction.

[0055] In a preferred embodiment, the click chemistry reaction is selected from at least one of azide, trans-cyclooctene, tetrazine, alkyne, dibenzocyclooctene, bicyclo[6,1,0]nonyne, thioctic acid and maleimide.

[0056] In a preferred embodiment, the polymer carrier has one or more of the following structures:

[0057]

[0058]

[0059] In equations (33)-(46), R1 has at least one of the structures shown in equations (2)-(11), and R2 has at least one of the structures shown in equations (12)-(22). This indicates that the bonds are connected to R1 and R2 via carbon-carbon bonds, phosphoester bonds, ester bonds, amide bonds, urethane bonds, and ether bonds, respectively.

[0060] In a preferred embodiment, the drug precursor has at least one of the following structures:

[0061]

[0062] In a preferred embodiment, the drug precursor has at least one of the following structures:

[0063]

[0064] Attached Figure Description

[0065] Figure 1 The particle size distribution of the polymer-drug conjugate (PC7A-MMAE-PEG) obtained in Example 1 at different pH values ​​is shown below.

[0066] Figure 2 Potential diagrams of the polymer-drug conjugate (PC7A-MMAE-PEG) obtained in Example 1 at different pH values;

[0067] Figure 3 The graphs show the 3T3 and 4T1 cytotoxicity of methylaurestatin E (MMAE) and the polymer-drug conjugate (PC7A-MMAE-PEG) obtained in Example 1.

[0068] Figure 4 Cytotoxicity graphs of PBS, methylaurestatin E (MMAE), PC7A-PEG (PC7A-Lys(N)-PEG) obtained in Example 1, and the polymer-drug conjugate PC7A-MMAE-PEG at different pH values;

[0069] Figure 5 The figure shows the tumor inhibition experiment results of PBS, methylaurestatin E (MMAE), and the polymer-drug conjugate (PC7A-MMAE-PEG) obtained in Example 1;

[0070] Figure 6 Cytotoxicity diagrams for 7-ethyl-10-hydroxycamptothecin (SN38) and the polymer-drug conjugate (PC7A-SN38-PEG) obtained in Example 7.

[0071] Figure 7 Cytotoxicity of PBS, 7-ethyl-10-hydroxycamptothecin (SN38), and the polymer-drug conjugate (PC7A-SN38-PEG) obtained in Example 7 at different pH values;

[0072] Figure 8 The pharmacokinetic evaluation results of the polymer-drug conjugate (PC7A-SN38-PEG) obtained in Example 7 are shown in the figure.

[0073] Figure 9The figure shows the tumor inhibition experiment results of PBS, 7-ethyl-10-hydroxycamptothecin (SN38) and the polymer-drug conjugate (PC7A-SN38-PEG) obtained in Example 7;

[0074] Figure 10 The image shows the gel electrophoresis diagram of the polymer-protein conjugate (PC7A-GOD-PEG) obtained in Examples 3-2.

[0075] Figure 11 Particle size diagrams of glucose oxidase (GOD) and the polymer-protein conjugate (PC7A-GOD-PEG) obtained in Example 9 at different pH values;

[0076] Figure 12 Potential diagrams of glucose oxidase (GOD) and the polymer-protein conjugate (PC7A-GOD-PEG) obtained in Example 9 at different pH values;

[0077] Figure 13 Activity graphs of glucose oxidase (GOD) and the polymer-protein conjugate (PC7A-GOD-PEG) obtained in Example 9 at different pH values;

[0078] Figure 14 This is a graph showing the in vitro glucose-consuming ability of the polymer-protein conjugate (PC7A-GOD-PEG) obtained in Example 9. Detailed Implementation

[0079] The tumor microenvironment-responsive polymer-drug conjugate provided by the present invention has the structure shown in formula (1);

[0080]

[0081] Wherein, R1 is a hydrophilic group with anti-protein adsorption capability. R1 is preferably derived from at least one of polydiols, polyvinyl alcohols, poly(meth)acrylates, poly(meth)acrylamides, polyamino acids, and polyphosphates. Specifically, examples include, but are not limited to, at least one of polyethylene glycol, poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone, poly(2-methacryloyloxyethylphosphorylcholine), poly(methacryloylethylsulfobetaine), and poly(methacryloylethylcarboxybetaine). More preferably, R1 has at least one of the structures shown in formulas (2)-(11). In formulas (2)-(11), specific examples of C1-C4 alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl; specific examples of C1-C4 alkylene groups include, but are not limited to: methylene, ethylene, n-propylene, isopropylene, n-butylene, sec-butylene, isobutylene, or tert-butylene. m is preferably an integer from 10 to 200, such as 10, 20, 50, 80, 100, 120, 150, 180, 200, or any integer between them. n is preferably an integer from 1 to 2, such as 1 or 2.

[0082] R2 is a microenvironment-responsive group. R2 is preferably derived from at least one of poly(meth)acrylate, poly(meth)acrylamide, polyurethane, polyβ-amino ester, polycarbonate, polyamino acid, polypeptide, and nucleic acid. More preferably, R2 has at least one of the structures shown in formulas (12)-(22). In formulas (12)-(12), specific examples of C1-C4 alkylene groups include, but are not limited to: methylene, ethylene, n-propylene, isopropylene, n-butylene, secondary butylene, isobutylene, or tert-butylene. m is preferably an integer from 10 to 200, such as 10, 20, 50, 80, 100, 120, 150, 180, 200, or any integer between them. In formulas (12) and (13), x is preferably an integer from 1 to 8, such as 1, 2, 3, 4, 5, 6, 7, or 8. In equations (14) and (15), x1 is preferably an integer from 1 to 4, such as 1, 2, 3, or 4; x2 is preferably an integer from 2 to 6, such as 2, 3, 4, 5, or 6. In equations (16) and (17), x1 is preferably an integer from 1 to 4, such as 1, 2, 3, or 4; x2 is preferably an integer from 2 to 8, such as 2, 3, 4, 5, 6, 7, or 8. In equations (18) and (19), x1 is preferably an integer from 1 to 2, such as 1 or 2; x2 is preferably an integer from 1 to 4, such as 1, 2, 3, or 4; x3 is preferably an integer from 2 to 6, such as 2, 3, 4, 5, or 6. In equations (20), (21) and (22), x1 is preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5 or 6; x2 is preferably an integer from 1 to 16, such as 1, 2, 5, 8, 10, 12, 14, 16 or any integer between them.

[0083] R3 is a linker that is bonded to R1 and R2 via carbon-carbon bonds, phosphoester bonds, ester bonds, amide bonds, urethane bonds, and ether bonds, respectively, and is bonded to R4 via various chemical bonds formed through bioorthogonal chemical reactions. Specifically, R3 bonded to R4 preferably has at least one of the structures shown in formulas (23)-(32). Wherein, n is preferably an integer from 1 to 4, such as 1, 2, 3, or 4.

[0084] R4 is an antitumor drug group with a -linker-Drug structure, and the linker is a group having at least one of the following: reducing, reactive oxygen species, pH, and enzyme responsiveness. When the linker is a group having at least one of the following: reducing, reactive oxygen species, pH, and enzyme responsiveness, the polymer-drug conjugate is correspondingly endowed with at least one of the following: reducing, reactive oxygen species, pH, and enzyme responsiveness. This enables the polymer-drug conjugate to respond to the tumor microenvironment, improve its specific recognition of tumor tissue, and release the drug at a specific site in the tumor tissue, thereby producing a therapeutic effect. The linker is preferably a group structure having at least one of the following: glutathione, reactive oxygen species, hypoxia, pH, ATP, esterase, cathepsin, matrix metalloproteinase, nitroreductase, aspartate protease, gamma-glutamyl transferase, or fibroblast activating protein responsiveness. Specific examples of antitumor drugs in R4 include, but are not limited to: at least one of the following: doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, eczema, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, chachiin, gambogeylic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansine, tyransine A, auristatin E, auristatin F, Piericidin A, anserine P3, sulphurin 10 and β-Amanitin, BTZ, fluorouracil, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, glucose oxidase, lactate oxidase, catalase, and therapeutic nucleic acid.

[0085] In this invention, the preparation method of the tumor microenvironment-responsive polymer-drug conjugate includes: obtaining the polymer-drug conjugate by reacting a polymer carrier with a drug precursor via a click chemical reaction. This method of first synthesizing the polymer carrier and then attaching the drug precursor to the polymer carrier via a click chemical reaction not only allows for better drug access to the polymer carrier, avoiding the problem of drug failure due to direct polymerization of drug monomers and polymer monomers, but also allows for greater flexibility by designing drug access points on the polymer carrier to adjust the amount of drug in the polymer-drug conjugate, thus meeting different drug delivery requirements. The polymer carrier can be obtained by reacting compound I with an R3 linker group, compound II with an R1 hydrophilic group, and compound III with an R2 responsive group. Preferably, the polymer carrier has at least one of the structures shown in formulas (33)-(46). The drug precursor has at least one of the structures shown in formulas (47)-(53). More preferably, the drug precursor has at least one of the structures shown in formulas (54)-(60).

[0086] The present invention will be described in detail below through specific embodiments.

[0087] Example 1: Preparation of PC7A-VCMMAE-PEG conjugate

[0088] (1) Synthesis of polymer carrier BIBB-Lys(DL-α-LA)-PEG

[0089]

[0090] (1-1) Weigh polyethylene glycol (3.03 g, 0.606 mmol, PEG5k) and remove water under vacuum at 110 °C for 2 h. Then cool to room temperature. Add 10 mL of anhydrous dichloromethane solution (DCM), A:Fmoc-Lys(Boc)-OH (2.84 g, 6.06 mmol), dicyclohexylcarbodiimide (1.50 g, 7.27 mmol, DCC), and 4-dimethylaminopyridine (74 mg, 0.606 mmol, DMAP). React at 25 °C in the dark for 48 h. Place the reaction flask in an ice bath for 5 h. Filter to remove solid insoluble matter. Concentrate the filtrate to a certain volume and add 10 times the volume of ethanol. After dissolving completely, recrystallize at -20 °C for 6 h. After filtration and drying, obtain white solid B:Fmoc-Lys(Boc)-PEG.

[0091] (1-2) Weigh B:Fmoc-Lys(Boc)-PEG (1g, 0.19mmol) into 10mL DMF solution and stir to dissolve. Add 3mL Piperidine to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20℃. After filtration and drying, obtain white solid C:N-Lys(Boc)-PEG.

[0092] (1-3) Synthesis of BIBB-Lys(Boc)-PEG:

[0093] Weigh C:N-Lys(Boc)-PEG (2.04 g, 0.39 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add 2-bromoisobutyric acid active ester (2.09 g, 3.12 mmol, BIBS) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20 °C. After filtration and drying, obtain white solid D:BIBB-Lys(Boc)-PEG.

[0094] (1-4) After drying the polymerization tube in an oven at 120℃ for 2 hours, weigh out D:BIBB-Lys(Boc)-PEG (100 mg, 0.015 mmol), C7A-HCl (200 mg, 0.81 mmol), and pentamethyldiethylenetriamine (10 μL, 0.09 mmol, PMDETA) and add them to the polymerization tube under nitrogen protection, followed by 0.6 mL of anhydrous methanol solution. After two cycles of "freezing-vacuuming-dissolving-nitrogen purging", CuBr (6 mg, 0.045 mmol) was added under nitrogen protection. After three more cycles of "freezing and thawing", the reaction was carried out at 45℃ in the dark for 24 hours. After the reaction was completed, the polymer was transferred to a 5000 Da dialysis bag and dialyzed with DMF for 40 h, then with acidic PBS (phosphate buffer) aqueous solution for 48 h, then with deionized water for 48 h. Finally, it was transferred to a 50 mL centrifuge tube and lyophilized to obtain a white solid E:PC7A-Lys(Boc)-PEG.

[0095] (1-5) Weigh E:PC7A-Lys(Boc)-PEG (1g, 0.18mmol) into 20mL of DCM solution and stir to dissolve. Slowly add trifluoroacetic acid (3mL, 39mmol, TFA) to the solution and react at 25℃ for 4h. After concentrating the reaction solution, add 15 times the volume of ethanol solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid F:PC7A-Lys(N)-PEG.

[0096] (1-6) Weigh F:PC7A-Lys(N)-PEG (3g, 0.56mmol) into 50mL of DCM solution and stir to dissolve. Slowly add DL-α-LA-NHS (1.7g, 5.6mmol, DL-α-lipoic acid-NHS) to the solution and react in the dark at 25℃ for 24h. After concentrating the reaction solution, add 10 times the volume of tetrahydrofuran solution (THF). Recrystallize twice at -20℃. After filtration and drying, obtain white solid G:BIBB-Lys(DL-α-LA)-PEG.

[0097] NMR data for BIBB-Lys(DL-α-LA)-PEG: 1H NMR (400MHz, CDCl3) δ 6.79 (s, 1H), 6.17 (d, J = 7.7Hz, 1H), 4.65-4.58 (m, 1H), 4.35-3.92 (m, 38H), 3.85 (s, 3H), 3.66-3.54 (s, 456H), 3.48 (dd, J = 5.8, 4.1Hz, 4H), 3.40-3.19 (m, J = 6.7Hz, 4H), 3.10-2.72 (m, J = 6.7Hz, 71H), 2.30-2.07 (m, 5H), 1.94-1.40 (m, 110H), 1.06-0.64 (m, 52H).

[0098] (2) Synthesis of the drug precursor VCMMAE

[0099]

[0100] (2-1) Weigh 43 mg of Mc-Val-Cit-PAB (0.075 mmol) and dissolve it in 1 mL of anhydrous DMF solution. Then, add 45.6 mg of bis(p-nitrophenyl) carbonate (0.15 mmol) and 9.5 mg of DIPEA (9.5 mg, 0.075 mmol, N-ethyldiisopropylamine) to the reaction flask and react at 25 °C for 16 h. First, obtain the crude product by vacuum distillation of the reaction solution, then treat it with 3 mL of methanol to obtain the precipitate. Centrifuge at 10,000 rpm for 10 min to remove the supernatant. The obtained precipitate is further washed twice with methanol to obtain a pale yellow solid, which is Mc-Val-Cit-PAB-PNP.

[0101] (2-2) Weigh Mc-Val-Cit-PAB-PNP (45 mg, 0.061 mmol), MMAE (49 mg, 0.0732 mmol, methylaurestatin E) and 1-hydroxybenzotriazole (7.2 mg, 0.061 mmol, HOBT) and dissolve them in 1.5 mL of anhydrous DMF solution and stir at 25 °C for 2 min. Then add pyridine (12 mg, 0.15 mmol) dropwise and stir at 25 °C in the dark for 24 h. The crude product obtained by vacuum distillation is then slurried with a mixture of ethyl acetate and methanol for 15 min to obtain red solid VCMMAE.

[0102] VCMMAE NMR data: 1H NMR (400MHz, DMSO) δ 10.00 (d, J = 14.0Hz, 1H), 8.31 (s, 1H), 8.08 (d, J = 7.6Hz, 2H), 7.95–7.74 (m, 2H), 7.68–7.49 (m, 4H), 7.40–7.14 (m, 7H), 7.01 (s, 2H), 5.96 (d, J = 5.4Hz, 2H), 5.52–5.30 (m, 4H), 5.20–4.9 2(m,3H),4.54-4.13(m,6H),4.04(q,J=7.1Hz,4H),3.28-3.10(m,8H),3.08-2.81(m,8H),2.14(tq,J=14.2,6. 9Hz, 5H), 1.99 (s, 3H), 1.87-1.29 (m, 18H), 1.18 (td, J = 7.3, 2.6Hz, 5H), 1.08-0.94 (m, 6H), 0.92-0.72 (m, 24H).

[0103] (3) Synthesis of PC7A-MMAE-PEG conjugate

[0104] G:BIBB-Lys(DL-α-LA)-PEG (0.5 g, 0.09 mmol) was weighed and dissolved in 2 mL of anhydrous MeOH solution. Then, under nitrogen protection, TCEP (50 μL, 0.27 mmol) was slowly added dropwise. The color of the reaction solution changed from pale yellow to colorless. The reaction was then carried out in the dark at 25 °C under nitrogen protection for 4 h.

[0105] Under nitrogen protection, 0.5 mL of methanol solution containing VCMMAE (355 mg, 0.27 mmol) was added to the reaction solution from the previous step. After stirring for 2 min, TEA (38 μL, 0.27 mmol, triethylamine) was added, and the reaction was carried out at 25 °C in the dark for 24 h. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed in DMSO solution for 40 h, with the solution changed every 10 h on average. Then, the solution was transferred to water and dialyzed for 48 h, with the solution changed every 10 h on average. Finally, the solution was transferred to a 50 mL centrifuge tube and lyophilized to obtain a pink solid PC7A-MMAE-PEG conjugate with the structure shown below.

[0106]

[0107] NMR data for PC7A-MMAE-PEG: 1H NMR (400MHz, DMSO) δ 8.12 (s, 4H), 7.91-7.78 (m, 3H), 7.58 (s, 4H), 7.29 (dd, J = 15.5, 7.7Hz, 7H), 6.00 (s, 3H), 5.43 (s, 4H), 5.05 (s, 4H), 4.55-4.33 (m, 10H), 4.31-3.91 (m, 39H), 3.66-3.44 (s, 461H), 3.38 (d, J = 5.0Hz, 5H), 3.23-2.72 (m, 75H), 2.62-2.07 (m, 17H), 1.94-1.40 (m, 130H), 1.06-0.64 (m, 72H).

[0108] Example 2: Preparation of PC5A-VCMMAE-PVP Couplings

[0109] (1) Synthesis of polymer carrier PVP-lys(DL-α-LA)-PC5A

[0110] (1-1) Weigh B:CTA-OH (3.22 g, 12.12 mmol) into a round-bottom flask with a side arm. Add 100 mL of anhydrous DCM solution, A:Fmoc-lys(Boc)-OH (2.84 g, 6.06 mmol), DCC (1.56 g, 7.27 mmol), and DMAP (74 mg, 0.606 mmol) to the flask. After reacting at 25 °C in the dark for 48 h, place the flask in an ice bath for 5 h. Filter to remove the solid insoluble matter. Concentrate the filtrate to a certain volume and purify it by silica gel column chromatography with DCM:MeOH (40:1). Collect the second spot and remove the mobile phase by rotary evaporation to obtain the red solid product C:Fmoc-lys(Boc)-CTA.

[0111] (1-2) Weigh C (1g, 1.39mmol) into 10mL DMF solution and stir to dissolve. Add 2.5mL Piperidine and react at 25℃ for 8h. After concentrating the reaction solution, perform silica gel column chromatography separation and purification using DCM:MeOH (20:1). Collect the third spot and remove the mobile phase by rotary evaporation to obtain the red solid product D:N-lys(Boc)-CTA.

[0112] (1-3) Weigh D (0.77 g, 1.56 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add BIBS (2.09 g, 3.12 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of diethyl ether solution and recrystallize twice at -20 °C. After filtration and drying, obtain red solid E:BIBB-lys(Boc)-CTA.

[0113] (1-4) After drying the polymerization tubes in an oven at 120℃ for 2 hours, E (100 mg, 0.15 mmol), F: 3-Vinyl-2-pyrrolidinone (91 μL, 5.1 mmol), and Bpy (46 mg, 0.3 mmol) were weighed and added to the polymerization tubes under nitrogen protection, followed by 0.6 mL of anhydrous DMF solution. After two cycles of "freezing-vacuuming-dissolving-nitrogen purging", CuBr (40 mg, 0.3 mmol) was added under nitrogen protection, followed by three more "freeze-thaw" cycles, and then the reaction was carried out at 110℃ in the dark for 48 hours. After the reaction was completed, the polymer was transferred to a 5000 Da dialysis bag, dialyzed with DMF for 40 hours, then dialyzed with acidic PBS aqueous solution for 48 hours, and then dialyzed with deionized water for 48 hours. Finally, it was transferred to a 50 mL centrifuge tube and freeze-dried to obtain the pink solid product G: PVP-lys(Boc)-CTA.

[0114] (1-5) After drying the polymerization tube in an oven at 120℃ for 2 hours, weigh G (100 mg, 0.035 mmol), C5AMMA (186 mg, 1.05 mmol), and AIBN (5.74 mg, 0.035 mmol) and add them to the polymerization tube under nitrogen protection, followed by 0.6 mL of anhydrous 1-4 dioxane solution. After three cycles of "freezing-vacuuming-dissolving-nitrogen purging", the reaction was carried out at 70℃ in the dark for 36 hours. After the reaction was completed, the polymer was transferred to a 5000 Da dialysis bag, dialyzed with DMF for 40 hours, then dialyzed with acidic PBS aqueous solution for 48 hours, and then dialyzed with deionized water for 48 hours. Finally, it was transferred to a 50 mL centrifuge tube and freeze-dried to obtain a powdery white solid product H:PVP-lys(Boc)-PC5A.

[0115] (1-6) Weigh H (1g, 0.182mmol) into 20mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of diethyl ether solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid I:PVP-lys-PC5A.

[0116] (1-7) Weigh I (2.92 g, 0.56 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add DL-α-LA-NHS (3 g, 5.6 mmol) to the solution and react in the dark at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of THF solution and recrystallize twice at -20 °C. After filtration and drying, obtain white solid J:PVP-lys(DL-α-LA)-PC5A.

[0117]

[0118] (2) Synthesis of the drug precursor VCMMAE

[0119] It was prepared according to the method in Example 1(2).

[0120] (3) Synthesis of PC5A-VCMMAE-PVP conjugate

[0121] The PC5A-VCMMAE-PVP conjugate was prepared according to the method in Example 1 (3), except that the same molar amount of J:PVP-lys(DL-α-LA)-PC5A (0.47g, 0.09mmol) was used instead of G:BIBB-Lys(DL-α-LA)-PEG (0.5g, 0.09mmol), the amount of TCEP added was 66μL, 0.36mmol, the amount of TEA added was 25μL, 0.18mmol, and all other conditions were the same, so that a pink solid PC5A-VCMMAE-PVP conjugate with the following structure could be prepared.

[0122]

[0123] NMR data for PC5A-MMAE2-PEG: 1H NMR (400MHz, DMSO) δ 8.12 (s, 8H), 7.91-7.78 (m, 6H), 7.58 (s, 8H), 7.42-7.35 (m, 5H) 7.29 (dd, J = 15.5, 7.7Hz, 14H), 6.00 (s, 6H), 5.43 (s, 8H), 5.05 (s, 6H), 4.55-4.33 (m, 14H). 5H),4.31-3.91(m,39H),3.66-3.44(m,25H),3.38(d,J=5.0Hz,5H),3.23-2.72(m,75 H),2.62-2.07(m,47H),1.94-1.40(m,162H),1.69-1.54(m,41H)1.06-0.64(m,107H).

[0124] Example 3: Preparation of SN38-PMNZ-PMPC Couplings

[0125] (1) Synthesis of polymer carrier N3-PMNZ-PMPC

[0126] (1-1) Weigh B:ONP (16.35 g, 0.054 mol) into a round-bottom flask with a side arm. Add 300 mL of anhydrous DCM solution, A (1.67 g, 0.027 mol), DCC (6.23 g, 0.032 mol), and DMAP (0.6 g, 0.0054 mol). First, react under nitrogen protection at 0 °C for 2 h. Then, remove the ice bath and react under nitrogen protection at 25 °C for 24 h. Then, place the reaction flask in an ice bath for 5 h. Filter to remove solid insoluble matter. Concentrate the filtrate to a certain volume and then purify it by silica gel column chromatography with DCM:MeOH (40:1). Collect the first spot and remove the mobile phase by rotary evaporation to obtain the white solid product C:OH-ONP.

[0127] (1-2) Weigh C (1.92 g, 5.4 mmol) into a round-bottom flask with a side arm. Add 50 mL of anhydrous DCM solution, D:Boc-Lys(Fmoc)-OH (1.27 g, 2.7 mmol), DCC (0.63 g, 3.2 mmol), and DMAP (0.06 g, 0.54 mmol). After reacting at 25 °C for 48 h, place the reaction flask in an ice bath for 5 h. Filter to remove the solid insoluble matter. Concentrate the filtrate to a certain volume and purify it by silica gel column chromatography with DCM:MeOH (30:1). Remove the mobile phase by rotary evaporation to obtain the white solid product E:Boc-Lys(Fmoc)-ONP.

[0128] (1-3) Weigh E (1.02 g, 1.39 mmol) into 10 mL of DMF solution and stir to dissolve. Add 2.5 mL of Piperidine and react at 25 °C for 8 h. After concentrating the reaction solution, perform silica gel column chromatography separation and purification using DCM:MeOH (20:1). Collect the third spot and remove the mobile phase by rotary evaporation to obtain the solid product F:Boc-Lys-ONP.

[0129] (1-4) Weigh F (0.81 g, 1.56 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add BIBS (2.09 g, 3.12 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of diethyl ether solution and recrystallize twice at -20 °C. After filtration and drying, obtain solid product G:Boc-BIBB-ONP.

[0130] (1-5) After drying the polymerization tubes in an oven at 120℃ for 2 hours, weigh G (110 mg, 0.15 mmol), MPC (320 mg, 2.6 mmol, 2-methacryloyloxyethylphosphocholine), and Bpy (46 mg, 0.3 mmol, bipyridine) and add them to the polymerization tubes under nitrogen protection, followed by 0.6 mL of anhydrous 1-4 dioxane solution. After two cycles of "freezing-vacuuming-dissolving-nitrogen purging", CuBr (40 mg, 0.3 mmol) was added under nitrogen protection, followed by three more "freeze-thaw" cycles, and then the reaction was carried out at 110℃ in the dark for 48 hours. After the reaction was completed, the polymer was transferred to a 5000 Da dialysis bag, dialyzed with DMF for 40 hours, then dialyzed with acidic PBS aqueous solution for 48 hours, and finally dialyzed with deionized water for 48 hours. The product was then transferred to a 50 mL centrifuge tube and freeze-dried to obtain a white solid product H:Boc-ONP-PMPC.

[0131] (1-6) After drying the polymerization tubes in an oven at 120°C for 2 hours, H (170 mg, 0.035 mmol), I (430 mg, 1.17 mmol), and BPO (9.74 mg, 0.07 mmol) were weighed and added to the polymerization tubes under nitrogen protection, followed by 0.6 mL of anhydrous DMSO solution. After three cycles of "freezing-vacuuming-dissolving-nitrogen purging", the reaction was carried out at 70°C in the dark for 36 hours. After the reaction was completed, the polymer was transferred to a 5000 Da dialysis bag and dialyzed with DMF for 40 hours, then with acidic PBS aqueous solution for 48 hours, and then with deionized water for 48 hours. The polymer was then transferred to a 50 mL centrifuge tube and freeze-dried to obtain a white solid product J:Boc-Lys-PMNZ-PMPC.

[0132] (1-7) Weigh J (1g, 0.11mmol) into 20mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of ether solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid K:Lys-PMNZ-PMPC.

[0133] (1-8) Weigh K (2.58 g, 0.38 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add N3-NHS (1.64 g, 7.6 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of diethyl ether solution and recrystallize twice at -20 °C. After filtration and drying, obtain a white solid L:N3-PMNZ-PMPC.

[0134]

[0135] (2) Synthesis of the drug precursor ALK-SS-SN38

[0136]

[0137] (2-1) Weigh B (1.135 g, 5.4 mmol) into a round-bottom flask with a side arm. Add 10 mL of anhydrous DCM solution, A (0.15 g, 2.7 mmol), EDC (0.623 g, 3.2 mmol), and DMAP (0.06 g, 0.54 mmol). React at 0 °C for 3 h, remove the ice bath, and then react at 25 °C for 24 h. Then place the reaction flask in an ice bath for 5 h. Filter to remove the solid insoluble matter. Concentrate the filtrate to a certain volume and then purify it by silica gel column chromatography with DCM:MeOH (15:1). Collect the second spot and remove the mobile phase by rotary evaporation to obtain the white solid product C:ALK-SS-COOH.

[0138] (2-2) Under nitrogen purging, C (1.00 g, 2.54 mmol) and DMAP (78 mg, 0.508 mmol) were added to 30 mL of anhydrous DMF solution and stirred thoroughly to disperse them evenly, yielding mixed solution I. In a fume hood, BTC (0.889 g, 3.048 mmol) and SN38-O-Boc (1.24 g, 2.54 mmol) were weighed and dissolved in 3 mL of anhydrous DMF solution in a round-bottom flask, yielding mixed solution II. Mixed solution II was added dropwise to mixed solution I, and the mixture was stirred at 25 °C for 48 h. After the reaction was complete, the solution turned orange-yellow. Insoluble byproduct salts were removed by filtration, and the solution was concentrated by rotary evaporation. Using dichloromethane / methanol (30:1) as the mobile phase, the solution was purified by column chromatography. The second spot was collected, which is the product spot. The mobile phase was removed by rotary evaporation, yielding a pale yellow solid product D:ALK-SS-SN38-O-Boc.

[0139] (2-3) Weigh D (1 g, 0.13 mmol) into 20 mL of DCM solution and stir to dissolve. Slowly add TFA (3 mL, 39 mmol) to the solution. React at 0 °C for 8 h, remove the ice-water bath, and then react at 20 °C for 3 h. After the reaction is complete, use dichloromethane / methanol 20:1 (V:V) as the mobile phase for column chromatography to separate and purify the product. Collect the third spot, which is the product spot. Remove the mobile phase by rotary evaporation to obtain the pale yellow solid product E:ALK-SS-SN38.

[0140] NMR data of ALK-SS-SN38: 1H NMR (400MHz, DMSO) δ 10.07 (s, 1H), 7.65-7.46 (m, 3H), 6.74 (s, 1H), 4.76-4.72 (m, 2H), 4.22 (s, 2H), 3.49-3.39 (m, 4H), 3.08-2.93 (m, 5H), 2.83-2.75 (m, 6H), 2.55-2.49 (m, 4H), 2.02 (m, J=4.9Hz, 2H), 1.18 (m, 3H), 0.89 (m, 3H).

[0141] (3) Synthesis of SN38-PMNZ-PMPC conjugate

[0142] Weigh out L (100 mg, 0.01 mmol) and ALK-SS-SN38 (73 mg, 0.4 mmol) and dissolve them in 0.3 mL of mixed solvent (DMF:water = 7:3). After two evacuations under nitrogen protection, add copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5), and then evacuate three more times. React at 25 °C for 24 h. After the reaction is complete, precipitate three times with ice-cold diethyl ether, dialyze against DMSO, and then dialyze against deionized water. Freeze-dry to obtain a yellow solid product SN38-PMNZ-PMPC, which has the structure shown below.

[0143]

[0144] NMR data of SN38-PMNZ-PMPC: 1H NMR (400MHz, DMSO) δ 8.05-7.91 (m, 18H), 7.65-7.46 (m, 3H), 6.97-6.74 (m, 18H), 4.76-4.58 (m, 41H), 4.47-4.31 (m, 82H), 3.61-3.45 (m, 68H), 3.49-3.34 (m, 142H), 1.72-1.61 (m, 38H), 1.53-1.42 (m, 14H), 1.39-1.27 (m, 77H).

[0145] Example 4: Preparation of PLCN-MMAE-PHPMA Conjugate

[0146] (1) Synthesis of polymer support PCLN-lys(N3)-PHPMA

[0147] (1-1) Synthesis of MA-Z-AZO in the hypoxic segment:

[0148] Weigh A (3.00 g, 15 mmol) into a Schlenk flask, add B (6.2 g, 30 mmol), DCC (3.72 g, 18 mmol), DMAP (74 mg, 0.606 mmol), and a magnetic stir bar. Add 40 mL of anhydrous dichloromethane to the Schlenk flask under nitrogen atmosphere, seal, and react at room temperature for 48 h. After the reaction is complete, filter to remove insoluble matter, collect the liquid phase, concentrate to a certain volume, separate by column chromatography, remove the mobile phase by rotary evaporation, and dry in a vacuum oven overnight to obtain product C.

[0149] C (1g, 2.85mmol) was dissolved in 10mL of dichloromethane, and 3mL of trifluoroacetic acid was added. The mixture was reacted at room temperature for 12h. Dichloromethane and trifluoroacetic acid were removed by rotary evaporation, and the mixture was dried in a vacuum oven overnight to obtain D.

[0150] Weigh D (1.00 g, 3.7 mmol) and TEA (1.87 g, 12.5 mmol) into a round-bottom flask, add 15 mL of dichloromethane, and stir magnetically until fully dissolved. Continue stirring in an ice-water bath. Then weigh methacryloyl chloride (1.93 g, 18.5 mmol), dissolve it in 5 mL of dichloromethane, place it in a dropping funnel, and slowly add it dropwise to the round-bottom flask at a rate of 3 drops per second. After the addition is complete, continue the reaction overnight. After the reaction is complete, wash both sides with deionized water, add anhydrous sodium sulfate to remove water, stir magnetically and dry for 12 h, then filter to remove sodium sulfate solid, remove part of the solvent by rotary evaporation, concentrate the product, separate by column chromatography, remove the mobile phase by rotary evaporation, and dry in a vacuum oven overnight to obtain MA-N-AZO.

[0151] NMR data of MA-N-AZO: 1H NMR (400MHz, CDCl3) δ 7.91-7.87 (m, 4H), 7.56-7.32 (m, 5H), 5.79-5.72 (s, 2H), 3.55 (t, 2H), 2.85 (t, 2H), 1.98 (s, 3H).

[0152]

[0153] (1-2) Synthesis of PCLN-lys(N3)-PHPMA:

[0154] First, E:BIBB-lys(Boc)-CTA is prepared according to the methods (1-1)-(1-3) in Embodiment 2, except that the following steps are used:

[0155] E (100 mg, 0.4 mmol), HPMA (2.00 g, 15.5 mmol), and AIBN (32 mg, 0.2 mmol) were added to a side-ended reaction tube and sonicated in 600 μL of DMSO (HPLC grade). After complete dissolution, the mixture was subjected to three freeze-thaw cycles in liquid nitrogen under nitrogen protection to remove air. The reaction system was then placed in a 70°C water bath for 24 h. After the reaction was completed, air was introduced and the reaction was rapidly cooled in liquid nitrogen to terminate the reaction. The obtained product was dialyzed against deionized water for one week (with water changed twice daily) to remove the reaction solvent DMSO and unreacted HPMA monomer. The dialyzed solution was lyophilized to obtain a pink solid product F:BIBB-lys(Boc)-PHPMA.

[0156] The Schlenk flask was dried in an oven at 120°C. F (0.1 g, 0.023 mmol), MA-N-AZO (0.1 g, 0.28 mmol), and PMEDTA (10 μL, 0.09 mmol) were weighed into a reaction tube and dissolved in 0.6 mL of DMSO. After two cycles of "freezing-vacuuming-nitrogen purging-dissolving," CuBr (6 mg, 0.045 mmol) was added under nitrogen protection. This was followed by another cycle of "freezing-vacuuming-nitrogen purging-dissolving," and the mixture was reacted in a 70°C water bath in the dark for 24 h. After the reaction was complete, a small amount of DMSO was added to dissolve the solid, which was then dialyzed against deionized water and dried to obtain a pink solid product G:PCLN-lys(Boc)-PHPMA.

[0157] Weigh G (1 g, 0.12 mmol) into 20 mL of DCM solution and stir to dissolve. Slowly add TFA (3 mL, 39 mmol) to the solution and react at 25 °C for 12 h. After concentrating the reaction solution, add 15 times the volume of diethyl ether solution and recrystallize three times at -20 °C. After filtration and drying, obtain pink solid H:PCLN-lys-PHPMA.

[0158] Weigh H (2.85 g, 0.56 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add N3-NHS (1.18 g, 5.6 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of THF solution and recrystallize twice at -20 °C. After filtration and drying, obtain a powdery white solid I:PCLN-lys(N3)-PHPMA.

[0159]

[0160] (2) Synthesis of the drug precursor DBCO-PEG3-VCMMAE

[0161] Purchased from MCE, brand name HY-111012

[0162] (3) Synthesis of PLCN-MMAE-PHPMA conjugate

[0163] Under nitrogen protection, I:PCLN-lys(N3)-PHPMA (150 mg, 0.014 mmol) was dissolved in 1 mL of DMSO solution, followed by the addition of 0.5 mL of DMSO solution containing DBCO-PEG3-MMAE (534 mg, 0.27 mmol). The reaction was carried out at 25 °C in the dark for 24 h. The reaction solution was first transferred to a 3500 Da dialysis bag and dialyzed in DMSO solution for 40 h, with the solution being changed every 10 h on average. Then, it was transferred to water and dialyzed for 48 h, with the solution being changed every 10 h on average. Finally, it was transferred to a 50 mL centrifuge tube and lyophilized to obtain a pale yellow solid product, PLCN-MMAE-PHPMA, which has the structure shown below.

[0164]

[0165] PLCN-MMAE-PHPMA NMR data: 1H NMR (400MHz, DMSO) δ 8.01 (m, 8H), 7.90-7.74 (m, 65H), 7.62-7.39 (m, 94H), 7.40-7.14 (m, 7H), 5.37-5.29 (m, 29H), 4.54-4.13 (m, 6H), 3.59-3.51 (m, 34H), 3.42-3.27 (m, 30H), 2.83-2.75 (m, 38H), 1.87-1.29 (m, 18H), 1.27-1.05 (m, 216H), 0.94-0.74 (m, 25H).

[0166] Example 5: Preparation of PC8A-SN38-PGLU Coupling

[0167] (1) Synthesis of polymer precursor PC8A-N3-PGLU

[0168] (1-1) Synthesis of Lys-NCA-Glu:

[0169] Under nitrogen purging, Fmoc-Glu (0.532 g, 1.212 mmol) and CDI (0.395 g, 1.212 mmol) and 25 mL of anhydrous dichloromethane were added to a flask and stirred thoroughly to dissolve. The mixture was then activated at room temperature for 2 h. In another flask, Lys-NCA (0.21 g, 1.212 mmol) and TEA (1.12 mL, 3.6 mmol) and 30 mL of anhydrous dichloromethane were added and stirred thoroughly at room temperature for 2 h. The activated reaction solution from the first flask was then added dropwise to the second flask using a constant pressure funnel, and the mixture was stirred at room temperature for 72 h. After the reaction was complete, the unreacted salt was filtered out, washed three times with 0.5 M HCl, and the washed product solution was dried over anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and purified by silica gel column chromatography. The mobile phase was removed by rotary evaporation to obtain a white solid powder, Lys-NCA-Glu.

[0170] NMR data for Lys-NCA-Glu: 1H NMR (400MHz, DMSO): δ 8.11 (d, J = 8.1Hz, 1H), 7.90–7.55 (m, 5H), 7.38–7.27 (m, 5H), 6.38 (m, J = 7.3Hz, 1H), 4.70 (m, 2H), 4.46–4.33 (m, 3H), 3.05 (m, 2H), 2.05–1.76 (m, 6H), 1.55–1.35 (m, 15H).

[0171]

[0172] (1-2) Synthesis of PC8A-N3-PGLU:

[0173] First, D:N-lys(Boc)-CTA was prepared according to the method in (1-1)-(1-2) of Example 2, except that the following steps were used:

[0174] Under nitrogen protection, E:Lys-NCA-Glu (200 mg, 0.337 mmol) was dissolved in 0.5 mL of DMF to obtain a colorless solution. D (75 mg, 0.012 mmol) was dissolved in 200 μL of DMF and added to the above colorless solution using a micropipette. A small amount of LiHMDS catalyst was added, and the reaction was carried out at 25 °C for 72 h. The solution was then concentrated by rotary evaporation, dissolved in chloroform, precipitated with cold diethyl ether or methanol, and centrifuged, dialyzed, and lyophilized to obtain the pale red product F:CTA-Lys(Boc)-PGLU.

[0175] The Schlenk flask was dried in an oven at 120°C. F (0.1 g, 0.016 mmol), C8AMMA (0.1 g, 0.29 mmol), and AIBN (2.78 mg, 0.2 mmol) were weighed into a reaction tube and dissolved in 0.6 mL of chloroform. After three cycles of "freezing-vacuuming-nitrogen purging-dissolving", the mixture was reacted in a water bath at 70°C in the dark for 24 h. After the reaction was completed, a small amount of DMSO was added to dissolve the mixture. After dialysis with deionized water, the solution was dried to obtain a pink solid product G:PC8A-Lys(Boc)-PGLU.

[0176] Weigh G (1g, 0.12mmol) into 20mL of DCM solution and stir to dissolve. Slowly add TFA (5mL, 65mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of diethyl ether solution and recrystallize three times at -20℃. After filtration and drying, obtain pink solid H:PC8A-Ly(N)-PGLU.

[0177] H (1.638 g, 0.2 mmol) was weighed and dissolved in 30 mL of DCM solution. L-Lys(Boc)2-NHS (0.24 g, 0.6 mmol) was slowly added to the solution. The mixture was reacted at 25 °C for 24 h. After concentrating the reaction solution, 10 times the volume of ethanol solution was added. The mixture was recrystallized twice at -20 °C. After filtration and drying, a pink solid I:PC8A-Lys(Lys)-PGLU was obtained.

[0178] Weigh I (1 g, 0.11 mmol) into 20 mL of DCM solution and stir to dissolve. Slowly add TFA (3 mL, 39 mmol) to the solution and react at 25 °C for 12 h. After concentrating the reaction solution, add 15 times the volume of ethanol solution and recrystallize three times at -20 °C. After filtration and drying, obtain a white solid J:PC8A-Lys(Lys-N)-PGLU.

[0179] Weigh J (1.1 g, 0.12 mmol) into 30 mL of DCM solution and stir to dissolve. Slowly add L-Lys(Boc)2-NHS (0.62 g, 0.96 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20 °C. After filtration and drying, obtain pink solid K:PC8A-Boc-PGLU.

[0180] Weigh K (1g, 0.11mmol) into 10mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of ethanol solution and recrystallize three times at -20℃. After filtration and drying, obtain a white solid L:PC8A-N-PGLU.

[0181] Weigh L (1g, 0.11mmol) into 10mL of DCM solution and stir to dissolve. Slowly add N3-NHS (1.27g, 1.1mmol) to the solution and react at 25℃ for 24h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20℃. Filter and dry to obtain a white solid M:PC8A-N3-PGLU.

[0182]

[0183] (2) Synthesis of the drug precursor DBCO-SS-SN38

[0184] (2-1) Weigh SN38 (2 g, 5 mmol, 7-ethyl-10-hydroxycamptothecin) and Boc2O (0.56 g, 2.5 mmol, di-tert-butyl dicarbonate) into a round-bottom flask, add 30 mL of DCM solution, and react at 25 °C for 5 h in the dark. Detect by TLC, purify by column chromatography, collect the second spot (the product), and dry to obtain a yellow solid SN38-O-Boc.

[0185] (2-2) Weigh DBCO-SS-OH (1.39 g, 3.16 mmol, dibenzocyclooctyn-SS-acid) and add 15 mL of LDM solution, SN38-O-Boc (1.43 g, 2.87 mmol), triphosgene BTC (0.71 g, 3.5 mmol), and a small amount of DMAP. React at room temperature in the dark for 48 h. Post-treatment: Place the reaction flask in an ice bath for 5 h, and filter to remove the solid insoluble matter. Purify by column chromatography, collecting the second spot, which is the product spot. Remove the mobile phase by rotary evaporation to obtain a yellow solid powder, DBCO-SS-SN38-O-Boc.

[0186] (2-3) Weigh DBCO-SS-SN38-O-Boc (1 g, 2.27 mmol) into 10 mL of DCM solution and stir to dissolve. Slowly add TFA (2 mL, 65 mmol) and react in an ice-water bath in the dark for 6 h. After extraction with DCM and water, separate and purify by column chromatography, and dry to obtain a pale yellow solid DBCO-SS-SN38-O-Boc.

[0187] NMR data from DBCO-SS-SN38: 1H NMR (400MHz, DMSO) δ 10.07 (s, 1H), 7.77–7.08 (m, 11H), 4.74–4.32 (m, 4H), 3.52–3.49 (m, 4H), 2.92–2.75 (m, 4H), 2.65–2.52 (m, 6H), 2.02 (t, J = 4.9Hz, 2H), 1.2–0.89 (m, 6H).

[0188]

[0189] (3) Synthesis of PC8A-SN38-PGLU conjugate

[0190] Under nitrogen protection, M:PC8A-N3-PGLU (150 mg, 0.016 mmol) was dissolved in 1 mL of DMF solution, followed by the addition of 0.5 mL of DMSO solution containing DBCO-SS-SN38 (224 mg, 0.24 mmol). The reaction was carried out at room temperature in the dark for 24 h. Then, 10 mL of DMF solution was added, and the mixture was stirred for 10 min. Finally, 2.5 mL of piperidine solution was added, and the reaction was continued for 8 h under nitrogen protection. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed in DMSO solution for 40 h, with the solution changed every 10 h on average. The solution was then transferred to water and dialyzed for 48 h, with the solution changed every 10 h on average. Finally, the solution was transferred to a 50 mL centrifuge tube and lyophilized to obtain a pale yellow solid product, PC8A-SN38-PGLU, which has the structure shown below.

[0191]

[0192] NMR data of PC8A-SN38-PGLU: 1H NMR (400MHz, DMSO) δ 12.21 (m, 18H), 9.01-8.76 (m, 41H), 8.32-8.09 (m, 22H), 7.68-7.28 (m, 62H), 6.74 (s, 5H), 4.74-4.48 (m, 96H), 4.35-4.22 (m, 38H), 3.55-3.49 (m, 20H), 2.95-2.82 (m, 46H), 2.55-2.05 (m, 165H), 2.02 (s, 45H), 1.96-1.76 (m, 52H), 1.63-1.18 (m, 298H), 0.89 (m, 18H).

[0193] Example 6: Preparation of (PC7A)2-MMAE-PPE

[0194] (1) Synthesis of polymer precursor PC7A-ALK-PPE

[0195]

[0196] (1-1) Synthesis of Lys-NCA-PC7A:

[0197] Under nitrogen protection, A (596.25 mg, 3.75 mmol) was dissolved in 3 mL of DMF solution, followed by the addition of B (87.5 mg, 1.25 mmol) and DMAP (57 mg, 0.5 mmol). After stirring at room temperature for 2 min, TEA (40 μL, 1 mmol) was added. The reaction was then carried out under ultraviolet light (UV = 365 nm) for 4 h. After the reaction was completed, the reaction solution was concentrated, and 10 times its volume of diethyl ether solution was added. The solution was recrystallized twice at -20 °C, filtered, and dried to obtain a pale yellow solid C.

[0198] Under nitrogen purging, C (0.47 g, 1.212 mmol), CDI (0.395 g, 1.212 mmol), and 25 mL of anhydrous dichloromethane were added to a flask and stirred thoroughly to dissolve them. The mixture was then activated at room temperature for 2 h. In another flask, A (0.21 g, 1.212 mmol) and TEA (1.12 mL, 3.6 mmol) were added, along with 30 mL of anhydrous dichloromethane. After stirring thoroughly at room temperature for 2 h, the activated reaction solution from the first flask was added dropwise to the reaction solution using a constant pressure funnel. The mixture was stirred at room temperature for 72 h. After the reaction was complete, the unreacted salt was filtered out, washed three times with 0.5 M HCl, and the washed product solution was dried over anhydrous magnesium sulfate, filtered, concentrated by rotary evaporation, and purified by silica gel column chromatography. The mobile phase was removed by rotary evaporation to obtain a white solid powder, Lys-NCA-C7A.

[0199] NMR data of Lys-NCA-PC7A: 1H NMR (400MHz, DMSO) δ 4.33 (t, 1H), 3.88 (t, 1H), 3.09–2.88 (m, 12H), 2.62–2.54 (m, 8H), 1.78–1.66 (m, 18H), 1.53 (m, 2H), 1.25 (m, 2H).

[0200] (1-2) Synthesis of PC7A-ALK-PPE:

[0201]

[0202] Weigh A (254 mg, 0.54 mmol) into a round-bottom flask with a side arm. Add 300 mL of anhydrous DCM solution, ethylene glycol (38 mg, 0.54 mmol), DCC (0.623 g, 0.32 mmol), and DMAP (18 mg, 0.054 mmol). React under nitrogen protection at 0°C for 2 h. After removing the ice bath, react under nitrogen protection at 25°C for 24 h. Place the reaction flask in an ice bath for 5 h. Filter to remove insoluble solids. Concentrate the filtrate to a certain volume and purify by silica gel column chromatography with DCM:MeOH (30:1). Collect the first spot, remove the mobile phase by rotary evaporation, and obtain the white solid product B:Fmoc-Lys(Boc)-OH.

[0203] In a dry polymerization tube, B (102 mg, 0.2 mmol), C (1.00 g, 6 mmol), and 8 mL of freshly distilled toluene were added. Under nitrogen protection, a toluene solution of Sn(Oct)₂ at a concentration of 0.425 mmol / mL (3 μL, 1.26 μmol) was added, and the reaction was carried out at 70 °C for 48 h. The resulting solution was deactivated with 1 mol / L acetic acid, and the precipitate was placed in excess diethyl ether. The precipitate was dried under vacuum to obtain product D:Fmoc-Lys(Boc)-PPE.

[0204] Weigh D (1g, 0.35mmol) into 10mL of DMF solution and stir to dissolve. Add 2.5mL of Piperidine and react at 25℃ for 8h. After concentrating the reaction solution, precipitate into excess diethyl ether. Dry the precipitate under vacuum to obtain product E:N-Lys(Boc)-PPE.

[0205] Under nitrogen protection, E (89 mg, 0.0337 mmol) was dissolved in 0.3 mL of DMF to obtain a colorless solution; Lys-NCA-C7A (385 mg, 0.6 mmol) was dissolved in DMF (200 μL) and added to the above colorless solution using a micropipette. A small amount of DBU catalyst was added, and the mixture was reacted at 25 °C for 72 h. The mixture was then concentrated by rotary evaporation, dissolved in chloroform, precipitated with cold ether or methanol, and centrifuged, dialyzed, and lyophilized to obtain product F:PC7A-Lys(Boc)-PPE.

[0206] Weigh F (1g, 0.09mmol) into 20mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of diethyl ether solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid G:PC7A-Lys-PPE.

[0207] G (373 mg, 0.0337 mmol) was dissolved in 1 mL of DCM to obtain a colorless solution. Propylori (8 mg, 0.51 mmol) was dissolved in DMF (200 μL) and added to the above colorless solution using a micropipette. DCC (130 mg, 0.61 mmol) and a small amount of DMAP were added, and the mixture was reacted at 25 °C for 24 h. After that, the mixture was concentrated by rotary evaporation, precipitated with diethyl ether, centrifuged, and dried to obtain the solid product H:PC7A-ALK-PPE.

[0208] (2) Synthesis of the drug precursor Da-LA-MMAE

[0209]

[0210] LA-MMAE (119 mg, 0.09 mmol) was weighed and dissolved in 2 mL of anhydrous MeOH solution. Under nitrogen protection, TCEP (50 μL, 0.34 mmol) was slowly added dropwise. The color of the reaction solution changed from pale yellow to colorless. The reaction was then carried out in the dark at room temperature for 4 h. The filtrate was concentrated to a certain volume, and 10 times the volume of ethyl acetate solution was added. The solution was recrystallized twice at -20 °C. After filtration and drying, a pale yellow solid, Da-LA-MMAE, was obtained.

[0211] Da-LA-MMAE NMR data: 1H NMR (400MHz, DMSO) δ 12.00 (d, J = 14.0Hz, 1H), 8.31 (s, 1H), 8.08 (d, J = 7.6Hz, 2H), 7.95–7.74 (m, 2H), 7.68–7.49 (m, 4H), 7.40–7.14 (m, 7H), 5.96 (d, J = 5.4Hz, 2H), 5.52–5.30 (m, 4H), 5.20–4.92 (m, 3H), 4.54– 4.13(m,6H),4.04(q,J=7.1Hz,4H),3.28-3.10(m,8H),3.08-2.81(m,8H),2.14(tq,J=14.2,6.9Hz,5H) ,1.99(s,3H),1.87-1.29(m,18H),1.18(td,J=7.3,2.6Hz,5H),1.08-0.94(m,6H),0.92-0.72(m,24H).

[0212] (3) Synthesis of (PC7A)2-MAE-PPE conjugate

[0213] Under nitrogen protection, H:PC7A-ALK-PPE (210 mg, 0.018 mmol) was dissolved in 1 mL of methanol, followed by the addition of 1 mL of methanol solution containing Da-LA-MMAE (47 mg, 0.054 mmol). After stirring for 2 min, TEA (4 μL, 0.027 mmol) was added, and the mixture was reacted at 25 °C in the dark for 24 h. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed in DMSO solution for 40 h, with the solution being changed every 10 h on average. The solution was then transferred to water and dialyzed for 48 h, with the solution being changed every 10 h on average. Finally, the solution was transferred to a 50 mL centrifuge tube and lyophilized to obtain a white solid product (PC7A)2-MAE-PPE conjugate with the following structure.

[0214]

[0215] NMR data for (PC7A)2-MMAE-PPE: 1H NMR (400MHz, DMSO) 8.21-8.08 (m, 23H), 7.95-7.45 (m, 12H), 7.40-7.14 (m, 14H), 5.96-5.52 (m, 12H), 5.20-4.92 (m, 23H), 4.54-4.13 (m, 88H), 4.04-3.92 (m, 46H), 3.28- 2.81(m,118H),2.62-2.54(m,71H),2.14(m,10H),1.99(s,6H),1.87-1.80(m,36H), 1.78-1.53(m,198H),1.49-1.19(m,220H),1.08-0.94(m,12H),0.92-0.72(m,48H).

[0216] Example 7 Synthesis of PC7A-SN38-PEG

[0217] (1) Synthesis of polymer precursor PC7A-(N3)-PEG

[0218] First, F:PC7A-Lys(N)-PEG was prepared according to steps (1-1)-(1-5) in Example 1, with the difference being:

[0219]

[0220] (1-6) Weigh F (12.04 g, 0.2 mmol) into 10 mL of DCM solution and stir to dissolve. Slowly add L-Lys(Boc)2-NHS (0.86 g, 2 mmol) to the solution and react at room temperature for 24 h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20 °C. After filtration and drying, obtain white solid G:PC7A-Lys(Lys-Boc)-PEG.

[0221] (1-7) Weigh G (1g, 0.1mmol) into 20mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at room temperature for 12h. After concentrating the reaction solution, add 15 times the volume of ethanol solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid H:PC7A-Lys(Lys)-PEG.

[0222] (1-8) Weigh H (110 mg, 0.01 mmol) into 10 mL of DMF solution and stir to dissolve. Slowly add N3-NHS (0.24 g, 0.1 mmol) to the solution and react at room temperature for 24 h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20 °C. After filtration and drying, obtain white solid I:PC7A-(N3)-PEG.

[0223] (2) Synthesis of the drug precursor DBCO-SS-SN38

[0224] DBCO-SS-SN38 was prepared according to the steps in Example 5 (2).

[0225] (3) Synthesis of PC7A-SN38-PEG conjugate

[0226] Under nitrogen protection, I:PC7A-(N3)-PEG (150 mg, 0.014 mmol) was dissolved in 1 mL of DMSO solution, followed by the addition of 0.5 mL of DBCO-SS-SN38 DMSO solution (234 mg, 0.27 mmol). The reaction was carried out at room temperature in the dark for 36 h. The reaction solution was then transferred to a 3500 Da dialysis bag and dialyzed in DMSO solution for 40 h, with the solution being changed every 10 h on average. The solution was then transferred to water and dialyzed for 48 h, with the solution being changed every 10 h on average. Finally, the solution was transferred to a 50 mL centrifuge tube and lyophilized to obtain a pale yellow solid PC7A-SN38-PEG with the structure shown below.

[0227]

[0228] NMR data of PC7A-SN38-PEG: 1H NMR (400MHz, DMSO) δ 10.17 (s, 2H), 7.77-7.08 (m, 22H), 4.74-4.42 (m, 12H), 4.31-3.91 (m, 41H), 3.72-3.63 (s, 454H), 3.52-3.18 (m, 72H), 2.02 (t, J=4.9Hz, 4H), 1.94-1.40 (m, 96H), 1.06-0.84 (m, 68H).

[0229] Example 8: Preparation of PNMA-VCMMAE-PEG conjugate

[0230] (1) Synthesis of polymer precursor PNMA-Lys-PEG

[0231] First, N-Lys(Boc)-PEG was prepared following the steps (1-1) and (1-2) in Example 1, with the difference being:

[0232]

[0233] (1-3) In a round-bottom flask, C (1.00 g, 2.1 mmol) and triethylamine (0.42 g, 4.2 mmol) were dissolved in 10 mL of dichloromethane. After thorough dissolution by magnetic stirring, the mixture was stirred continuously in an ice-water bath. Then, D (0.19 g, 2.1 mmol) was weighed and dissolved in 2 mL of dichloromethane. The solution was placed in a dropping funnel and slowly added dropwise to the round-bottom flask at a rate of 3 drops per second. After the addition was complete, the reaction was continued overnight. After the reaction was completed, the mixture was washed twice with deionized water, and anhydrous sodium sulfate was added to remove water. The mixture was dried by magnetic stirring for 16 h. Then, the sodium sulfate solid was removed by suction filtration, the solvent was removed by rotary evaporation, and the concentrated product was dissolved in ethanol and recrystallized twice at -20 °C. The product was filtered to obtain a white solid E:NMA-Lys(Boc)-PEG.

[0234] (1-4) Add E (70 mg, 0.007 mmol), G (20 mg, 0.118 mmol), F (30 mg, 0.176 mmol) and 1 mL DMSO to the reaction tube. After three cycles of "freezing-vacuuming-nitrogen purging-dissolving", react at 60 °C in the dark for 24 h. Then add N-methylimidazole (2.3 mg, 0.03 mmol) and imidazole (28 mg, 0.0004 mmol) to the flask and heat to 70 °C. React in the dark for 24 h. After dialyzing the reaction solution, freeze dry to obtain the solid product H:PNMA-Lys(Boc)-PEG.

[0235] (1-5) Weigh H (1g, 0.11mmol) into 20mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of ethanol solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid PNMA-Lys-PEG.

[0236] Weigh PNMA-Lys-PEG (2.85 g, 0.56 mmol) into 50 mL of DCM solution and stir to dissolve. Slowly add DL-α-LA-NHS (1.58 g, 2.8 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of THF solution and recrystallize twice at -20 °C. After filtration and drying, obtain a white solid I:PNMA-Lys(DHLA)-PEG.

[0237] (2) Synthesis of the drug precursor VCMMAE

[0238] VCMMAE was prepared according to the steps in Example 1 (2).

[0239] (3) Synthesis of PNMA-VCMMAE-PEG

[0240] PNMA-VCMMAE-PEG was prepared according to the steps in Example 1 (3), except that the same molar amount of I:PNMA-Lys(DHLA)-PEG was used instead of G, the amount of TCEP added was 35 μL (0.18 mmol), and the other conditions were the same. PNMA-VCMMAE-PEG was prepared with the structure shown below.

[0241]

[0242] NMR data of PNMA-MMAE-PEG: 1H NMR(400MHz,DMSO)δ8.12(s,4H),7.91-7.78(m,3H),7.58(s,4H),7.29(dd, J=15.5,7.7Hz,7H),6.00(s,3H),5.43(s,4H),5.05(s,4H),4.55-4.33(m,10 H),4.31-3.91(m,107H),3.66-3.44(m,529H),3.38(d,J=5.0Hz,5H),3.23-2 .72(m,75H),2.62-2.07(m,153H),1.94-1.40(m,130H),1.06-0.64(m,72H).

[0243] Example 9: Preparation of PC7A-GOD-PEG conjugate

[0244] (1) Synthesis of polymer precursors

[0245] First, F:PC7A-Lys(N)-PEG was prepared according to steps (1-1)-(1-5) in Example 1, with the difference being:

[0246]

[0247] (1-6) Weigh F (100 mg, 0.01 mmol) into 10 mL of DCM solution and stir to dissolve. Slowly add N3-NHS (0.21 g, 0.1 mmol) to the solution and react at room temperature for 24 h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20 °C. After filtration and drying, obtain white solid G:PC7A-Lys(N3)-PEG.

[0248] (2) Synthesis of the drug precursor DBCO-PEG4-GOD

[0249]

[0250] DBCO-PEG4-NHS ester (5 mg, 7.7 μmol) was dissolved in DMSO (50 μL) for later use. GOD powder (15 mg, 250 U / mg) was dissolved in 1.2 mL of PBS solution. The prepared DMSO solution was added dropwise to the PBS mixture. The mixture was stirred slowly at room temperature in the dark for 36 h. Then, it was transferred to a 50000 Da dialysis bag and dialyzed for 24 h to remove unreacted DBCO-PEG4-NHS ester. Finally, the dialysate was collected into an ultrafiltration tube of about 50000 rpm and centrifuged at 5000 rpm until 1 mL of residual liquid remained. The solution was then filtered through a membrane to obtain DBCO-PEG4-GOD concentrate.

[0251] (3) Synthesis of PC7A-GOD-PEG

[0252] Dissolve 50 mg of G in 0.5 mL of deionized water and pass it through a membrane. Under sterile conditions, slowly add G dropwise to the concentrated solution of DBCO-PEG4-GOD. Stir slowly at room temperature in the dark for 12 h. Then transfer it to a 50000 Da dialysis bag and dialyze for 24 h to remove unreacted PC7A-Lys(N3)-PEG. Finally, concentrate the dialysate into an ultrafiltration tube of about 50000 rpm and centrifuge at 5000 rpm until about 1-1.5 mL of liquid remains. The resulting yellow product is PC7A-GOD-PEG, which has the structure shown below.

[0253]

[0254] NMR data for PC7A-N3-PEG: 1H NMR (400MHz, DMSO) δ 4.59-4.02 (m, 35H), 3.72-3.43 (s, 454H), 3.38-2.68 (m, 42H), 1.81-1.68 (m, 133H), 1.25-0.87 (m, 54H).

[0255] Example 10: Preparation of PNNM-SN38-PMeOX conjugate

[0256] (1) Synthesis of polymer precursor PNNM-N3-PMeOX

[0257] E:CTA-Lys(Boc)-BIBB was prepared according to the methods (1-1)-(1-3) in Example 2, with the difference being:

[0258] (2-4) Add E (100 mg, 0.4 mmol) and F (1.32 g, 15.5 mmol, structure: ) to the polymerization tube. The 18 μL of acetonitrile (HPLC grade) and TEA (18 μL, 0.2 mmol) were ultrasonically dissolved in 600 μL of acetonitrile. After complete dissolution, the mixture was subjected to three freeze-thaw cycles in liquid nitrogen under nitrogen protection to remove air. The reaction system was then placed at 25 °C for 18 h. After the reaction was completed, air was introduced and the reaction was rapidly cooled in liquid nitrogen to terminate the reaction. The resulting product was dialyzed against deionized water for one week (with water changed twice daily) to remove the reaction solvent DMSO and unreacted F monomers. The dialyzed solution was lyophilized to give a pale red solid product G:CTA-Lys(Boc)-PMeOX.

[0259] (2-5) After drying the Schlenk flask in an oven at 120℃, weigh G (112 mg, 0.048 mmol), NNM (0.12 g, 0.64 mmol), and AIBN (8 mg, 0.048 mmol) into a polymerization tube and dissolve them in 0.6 mL of chloroform. After three cycles of "freezing-vacuuming-nitrogen purging-dissolving", the mixture was reacted in an oil bath at 70℃ in the dark for 24 h under nitrogen protection. After the reaction was completed, a small amount of DMSO was added to dissolve the product. After dialysis with deionized water and drying, a pink solid product H:PNNM-(Boc)-PMeOX was obtained.

[0260] (2-6) Weigh H (1g, 0.21mmol) into 10mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of ether solution and recrystallize three times at -20℃. After filtration and drying, obtain pink solid I:PNNM-Lys-PMeOX.

[0261] (2-7) Weigh I (90 mg, 0.02 mmol) into 10 mL of DCM solution and stir to dissolve. Slowly add L-Lys(Boc)2-NHS (29 mg, 0.06 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of diethyl ether solution and recrystallize twice at -20 °C. After filtration and drying, obtain a pink solid J:PNNM-Lys(Lys-Boc)-PMeOX.

[0262] (2-8) Weigh J (1g, 0.21mmol) into 10mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of ethanol solution and recrystallize three times at -20℃. After filtration and drying, obtain white solid K:PNNM-Lys(Lys)-PMeOX.

[0263] (2-9) Weigh K (46 mg, 0.01 mmol) into 10 mL of DCM solution and stir to dissolve. Slowly add N3-NHS (0.24 g, 0.1 mmol) to the solution and react at 25 °C for 24 h. After concentrating the reaction solution, add 10 times the volume of diethyl ether solution and recrystallize twice at -20 °C. After filtration and drying, obtain a white solid L:PNNM-N3-PMeOX, which has the structure shown in formula (10-1).

[0264] (2) Synthesis of the drug precursor ALK-SS-SN38

[0265] ALK-SS-SN38 was prepared according to the method in Example 3 (2).

[0266] (3) Synthesis of PNNM-SN38-PMeOX conjugate

[0267] Weigh out L:PNNM-N3-PMeOX (48 mg, 0.01 mmol) and ALK-SS-SN38 (21 mg, 0.1 mmol) and dissolve them in a 0.3 mL mixture of DMF and water (DMF:water = 7:3). After two evacuations under nitrogen protection, add copper sulfate pentahydrate and vitamin C (copper sulfate pentahydrate:vitamin C = 1:5), and then evacuate three more times. React at 25 °C for 24 h. After the reaction is complete, precipitate three times with ice-cold ether, dialyze with DMSO, and then dialyze with deionized water. Freeze dry to obtain a pale yellow solid product M, which is PNNM-SN38-PMeOX, and has the structure shown in formula (11-2).

[0268] NMR data of PNNM-SN38-PMeOX: 1H NMR (400MHz, DMSO) δ 10.07 (s, 2H), 7.92-7.85 (m, 5H), 7.65-7.19 (m, 28H), 6.79-6.74 (m, 18H), 4.76-4.56 (m, 34H), 4.22 (s, 5H), 3.68-3.59 (m, 114H), 3.49-3.39 (m, 8H), 3.08-2.93 (m, 10H), 2.83-2.75 (m, 12H), 2.55-2.49 (m, 8H), 2.10-2.02 (m, 62H), 1.27-1.18 (m, 36H), 0.89 (m, 6H).

[0269]

[0270] Example 11: Preparation of PMPC-MMAE-PC7A Coupling

[0271] (1) Synthesis of polymer carrier PMPC-PSC7A

[0272] (1-1) Synthesis of C7A-S: Under nitrogen protection, A (250 mg, 1.25 mmol) was dissolved in 3 ml of DMF solution, followed by the addition of B (87.5 mg, 1.25 mmol) and DMAP (57 mg, 0.5 mmol). After stirring at room temperature for 2 min, TEA (40 μL, 1 mmol) was added, and the reaction was carried out under ultraviolet light (UV = 365 nm) for 4 h. After the reaction was completed, the reaction solution was concentrated and 10 times the volume of diethyl ether solution was added. The solution was recrystallized twice at -20 °C, filtered and dried to obtain a pale yellow solid C7A-S.

[0273]

[0274] NMR data for C7A-S: 1H NMR (400MHz, CDCl3) δ 4.55 (d, 2H), 4.30 (d, 2H), 4.13 (t, 2H), 3.09 (m, 4H), 2.62–2.42 (m, 6H), 1.94 (m, 2H), 1.78–1.66 (m, 8H), 1.27 (s, 3H).

[0275] (1-2) Weigh A:Fmoc-Lys(Boc) (254 mg, 0.54 mmol) into a round-bottom flask with a side arm. Add 300 mL of anhydrous DCM solution, ethylene glycol (38 mg, 0.54 mmol), ECC (0.623 g, 0.32 mmol), and DMAP (18 mg, 0.054 mmol). First, react under nitrogen protection and at 0 °C for 2 h. Then, remove the ice bath and continue the reaction under nitrogen protection and at 25 °C for 48 h. Then, place the reaction flask in an ice bath for 5 h. Filter to remove the solid insoluble matter. Concentrate the filtrate to a certain volume and then perform silica gel column chromatography separation and purification using DCM:MeOH (30:1). Collect the first spot and remove the mobile phase by rotary evaporation to obtain the white solid product B:Fmoc-Lys(Boc)-OH.

[0276] (1-3) Under nitrogen protection, B (245 mg, 0.48 mmol), C:C7A-S (516 mg, 1.44 mmol), and DBU (18 mg, 0.192 mmol) were weighed into a polymerization tube and dissolved in 1.5 mL of anhydrous DMF. After three cycles of "freezing-vacuuming-nitrogen purging-dissolving", the reaction was carried out in an oil bath at 25 °C in the dark for 24 h under nitrogen protection. After the reaction was completed, a small amount of DMSO was added to dissolve the product, which was then dialyzed against deionized water and dried to obtain a white solid product D:Fmoc-Lys(Boc)-PSC7A.

[0277] (1-4) Weigh D (1g, 0.12mmol) into 10mL DMF solution and stir to dissolve. Add 2.5mL Piperidine and react at 25℃ for 8h. After concentrating the reaction solution, add 10 times the volume of diethyl ether solution and recrystallize twice at -20℃. Filter and dry to obtain white solid E:Lys(Boc)-PSC7A.

[0278] (1-5) Weigh E (2.8g, 0.39mmol) into 50mL of DCM solution and stir to dissolve. Slowly add BIBS (2.09g, 3.12mmol) to the solution and react at 25℃ for 24h. After concentrating the reaction solution, add 10 times the volume of ethanol solution and recrystallize twice at -20℃. Filter and dry to obtain white solid F:BIBBLys(Boc)-PSC7A.

[0279] (1-6) After drying the polymerization tubes in an oven at 120℃ for 2 hours, F (113 mg, 0.015 mmol), MPC (202 mg, 0.6 mmol), and Bpy (5 mg, 0.03 mmol) were weighed and added to the polymerization tubes under nitrogen protection. Then, 0.6 mL of a mixture of 1,4-dioxane and water (2:3) was added. After two cycles of "freezing-vacuuming-dissolving-nitrogen purging," CuBr (4.5 mg, 0.03 mmol) was added under nitrogen protection. Following three more "freeze-thaw" cycles, the reaction was carried out at 110℃ in the dark for 24 hours. After the reaction, the polymer was transferred to a 5000 Da dialysis bag and dialyzed with DMF for 40 hours, then with acidic PBS aqueous solution for 48 hours, and finally with deionized water for 48 hours. The polymer was then transferred to a 50 mL centrifuge tube and freeze-dried to obtain a white solid, G:PMPC-Lys(Boc)-PSC7A.

[0280] (1-7) Weigh G (1g, 0.084mmol) into 10mL of DCM solution and stir to dissolve. Slowly add TFA (3mL, 39mmol) to the solution and react at 25℃ for 12h. After concentrating the reaction solution, add 15 times the volume of diethyl ether solution and recrystallize three times at -20℃. Filter and dry to obtain white solid H:PMPC-Lys-PSC7A.

[0281] (1-8) H (396 mg, 0.0337 mmol) was dissolved in 1 mL of DCM to obtain a colorless solution; propargyl acid (8 mg, 0.51 mmol) was dissolved in DMF (200 μL), and then added to the above colorless solution using a micropipette, along with DCC (130 mg, 0.61 mmol) and a small amount of NHS. The reaction was carried out at 25 °C for 24 h. Afterwards, the solution was concentrated by rotary evaporation, precipitated with diethyl ether, centrifuged, and dried to obtain solid product I: PMPC-PSC7A, which has the structure shown in formula (11-1).

[0282] (2) Synthesis of the drug precursor Da-LA-MMAE

[0283] Da-LA-MMAE was prepared according to the method in Example 6(2).

[0284] (3) Synthesis of PMPC-MMAE-PSC7A

[0285] PMPC-MMAE-PSC7A was prepared according to the method in Example 6 (3), except that the same molar amount of I:PMPC-PSC7A (212 mg, 0.018 mmol) was used instead of H:PC7A-ALK-PPE (210 mg, 0.018 mmol), and all other conditions were the same, and a white solid product PMPC-MMAE-PSC7A with the structure shown in formula (11-2) was obtained.

[0286] PMPC-MMAE-PMP NMR data: 1H NMR (400MHz, DMSO) δ 12.00 (d, J = 14.0Hz, 2H), 8.31 (s, 2H), 8.08 (d, J = 7.6Hz, 4H), 7.95-7.74 (m, 4H), 7.68-7.49 (m, 8H), 7.40-7.14 (m, 14H), 5.96 (d, J = 5.4Hz, 4H), 5.52-5.30 (m, 8H), 5.20-4.92 (m, 6H), 4. 54-4.13(m,96H),4.08-3.61(m,64H),3.28-3.10(m,8H),3.08-2.81(m,211H),2.54-2.42(m,68H),1. 99-1.83(m,82H),1.87-1.59(m,138H),1.38-1.27(m,105H),1.08-0.94(m,12H),0.92-0.72(m,48H).

[0287]

[0288] The following provides examples of the application of three typical commonly used conjugates (PC7A-VCMMAE-PEG, PC7A-SN38-PEG, and PC7A-GOD-PEG).

[0289] Application Example 1 (PC7A-VCMMAE-PEG)

[0290] (1) Preparation of micelles:

[0291] Preparation of micelles (pH=7.4): 10 mg of PC7A-VCMMAE-PEG polymer was dissolved in 1 mL of DMF solution and slowly injected into 9 mL of pH=7.4 phosphate buffer solution under high-speed stirring at a rate of 12 μL / s using a microinjection pump. After completion, the solution was stirred at high speed at room temperature for 12 h. Then, it was transferred to a 7000 Da dialysis bag and stirred at medium speed for 48 h. The pH=7.4 phosphate buffer solution was changed every 8 h to ensure complete dialysis of the DMF. Finally, the micelles were filtered through a 0.45 μm membrane and stored in a light-protected refrigerator at 4 °C.

[0292] Preparation of micelles (pH=6.5): The preparation of these micelles is basically the same as the preparation process described above, except that the phosphate buffer solution with pH=7.4 is replaced with a phosphate buffer solution with pH=6.5.

[0293] (2) Determination of micelle size and potential:

[0294] When determining the particle size and potential of micelles at different pH values, it is first necessary to prepare phosphate buffer solutions with the same pH as the micelles to be tested (pH=7.4 and pH=6.5). Then, the instrument is rinsed multiple times with this buffer solution, followed by rinsing the instrument three times with the micelles to be tested. Afterward, the hydrated particle size and Zeta potential of the micelles can be determined according to the user manual. The results are as follows: Figure 1 and Figure 2 As shown. From Figure 1 and Figure 2 It was confirmed that the PC7A-MMAE-PEG conjugate exhibits significant acid-responsive behavior and undergoes charge reversal and size transition. This is beneficial for both the accumulation of the drug conjugate in tumor tissue and for better drug penetration.

[0295] (3) Cytotoxicity test:

[0296] The cytotoxicity of 4T1 and 3T3 cells to drug-loaded micelles (MMAE, PC7A-VCMMAE-PEG) was tested. Eight drug concentrations were set up: 100, 50, 20, 10, 5, 2.5, 1, and 0 nmol, with a culture time of 24 h. Cell morphology changes were initially observed under a microscope. Then, the old mixture was removed, and 0.1 mL of freshly prepared CCK-8 mixture was added to each well. The cells were incubated for another 1 h, and the absorbance of each well was measured using a microplate reader to calculate cell viability. The cytotoxicity test for 3T3 cells was basically the same as above, except that the number of seeded cells was increased, and the cells needed to be incubated in a constant temperature incubator for 24-48 h. The number of cells used in the cytotoxicity test was kept consistent with that of 4T1 cells. All other procedures remained the same. The results are shown below. Figure 3 As shown. By Figure 3 It can be seen that MMAE has a strong killing effect on 3T3 cells and 4T1 cells, while PC7A-MMAE-PEG also has a strong killing effect on 4T1 cells. This indicates that PC7A-MMAE-PEG can release the drug through the acid response of the tumor environment, and the released MMAE has a killing effect on 4T1 cells.

[0297] (4) Toxicity test of drug-loaded micelles at different pH values:

[0298] Five specific pH values ​​were set for this experiment: pH = 8.23, pH = 7.85, pH = 7.24, pH = 6.71, and pH = 6.23. The drug-loaded micelles were PBS, PC7A-VCMMAE-PEG, PC7A-PEG (PC7A-Lys(N)-PEG), and MMAE, respectively. The preparation method of the drug-loaded micelles is as described in (1). Since the pH of the culture medium will change to different degrees over time, according to the preliminary experiment, a very small amount of standard acid solution needs to be added every 4-5 hours to ensure the pH of the mixture. Then, the mixture is cultured under the same conditions for 24 hours. The cell morphology changes are observed under a microscope. Then, the old mixture is removed, and 0.1 mL of freshly prepared CCK-8 mixture is added to each well. After culturing in a constant temperature incubator for 1 hour, the absorbance of each well is measured with an enzyme-linked immunosorbent assay (ELISA) reader, and the cell viability is calculated. The results are as follows. Figure 4 As shown. From Figure 4 It can be seen that PC7A-VCMMAE-PEG exhibits strong acid-responsive cytotoxicity. Under moderately alkaline conditions, its cytotoxicity is relatively low, while when it reaches an acidic environment, PC7A-VCMMAE-PEG micelles exhibit a strong killing effect. This is closely related to the charge reversal and size inversion of the drug micelles under acidic conditions.

[0299] (5) Tumor suppression experiment:

[0300] Twenty-five mice with tumor volumes ranging from 90 to 100 mm were selected. 3 Mice were randomly divided into five groups, with five mice in each group. One group served as the control group, injected with PBS. The other groups were injected with free MMAE (0.1 mg / kg) and PC7A-VCMMAE-PEG micelles (MMAE was used as an indicator, at 0.2 mg / kg, 0.5 mg / kg, and 1.5 mg / kg). The body weight and tumor volume of each group of mice were measured every two days. The drugs were administered via tail vein every two days for five administrations. The experiment was terminated based on the actual results. Curves were plotted based on the data, and the results are shown below. Figure 5 As shown. According to Figure 5 As can be seen, tumor growth in the PBS group was extremely rapid, while the tumor-suppressing effect of PC7A-MMAE-PEG was dose-dependent; the higher the dose, the slower the tumor growth. When the dose reached 1.5 mg / kg, tumor growth was almost completely inhibited.

[0301] Application Example 2 (PC7A-SN38-PEG)

[0302] (1) Preparation of micelles:

[0303] As described in (1) of the preparation method and application example 1-1, simply replace PC7A-VCMMAE-PEG with PC7A-SN38-PEG.

[0304] (2) Cytotoxicity assays of SN38 and PC7A-SN38-PEG:

[0305] Eight drug concentrations were set up in this experiment: 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 μg / mL, with a culture time of 24 h. Cell morphology changes were initially observed under a microscope. Then, the old mixture was removed, and 0.1 mL of freshly prepared CCK-8 mixture was added to each well. The cells were then incubated in a constant temperature incubator for another 1 h. The absorbance of each well was measured using a microplate reader, and cell viability was calculated. The results are shown below. Figure 6 As shown. From Figure 6 It can be seen that PC7A-SN38-PEG is more cytotoxic than SN38 at high concentrations. This is because SN38 is extremely hydrophobic, and some of the drug is precipitated out. However, after polymer modification, the solubility of SN38 is improved, thus enhancing the therapeutic effect.

[0306] (3) Toxicity test of drug-loaded micelles at different pH values:

[0307] Five specific pH values ​​were set for this experiment: pH = 8.23, pH = 7.85, pH = 7.24, pH = 6.71, and pH = 6.23. The drug-loaded micelles were PBS, SN38, and PC7A-SN38-PEG, respectively. The preparation method of the drug-loaded micelles is as described in (1). Since the pH of the culture medium will change to different degrees over time, according to the preliminary experiment, a very small amount of standard acid solution needs to be added every 4-5 hours to ensure the pH of the mixture. Continue the same culture conditions as above and culture for 24 hours. Preliminary observation of cell morphology changes was performed under a microscope. Then, the old mixture was removed, and 0.1 mL of freshly prepared CCK-8 mixture was added to each well. After culturing in a constant temperature incubator for another 1 hour, the absorbance of each well was measured with an ELISA reader, and the cell viability was calculated. The results are as follows. Figure 7 As shown. Figure 7 The cytotoxicity of PC7A-SN38-PEG exhibits a strong acid-responsive behavior. Under moderately alkaline conditions, the cytotoxicity is relatively low, while when it reaches an acidic environment, the PC7A-SN38-PEG micelles exhibit a strong killing effect. This is closely related to the charge reversal and size inversion of the drug micelles under acidic conditions.

[0308] (3) Pharmacokinetic evaluation

[0309] Preparation of Cy7.5-loaded fluorescein micelles: 5 mg of PC7A-SN38-PEG polymer was dissolved in 0.5 mL of DMF solution, and 0.5 mg of Cy7.5 fluorescein was dissolved in 0.5 mL of DMF solution. After filtration through a 0.22 μm membrane, the mixture was thoroughly mixed with the drug-loaded solution and then slowly injected into 4 mL of neutral phosphate buffer at a rate of 15 μL / s using a microinjection pump. The mixture was then stirred at high speed at room temperature for 12 h. The mixture was then transferred to a 7000 Da dialysis bag and stirred at medium speed for 48 h, with the phosphate buffer changed every 8 h to ensure complete dialysis of the DMF. Finally, the micelles were filtered through a 0.45 μm membrane and stored in a light-protected refrigerator at 4 °C.

[0310] After the mice had adapted to their environment, six mice were divided into two groups of three. Each group received a Cy7.5 fluorescently labeled polymer micelle injected into the tail vein at a concentration of 5 mg / kg. After the serum clearance half-life (t1 / 2), approximately 50 μL of blood was collected from the capillaries around the eyes of the mice and placed in vivo before coagulation for fluorescence detection. The results are shown below. Figure 8 As shown. (Through) Figure 8 It can be seen that when SN38 is made into a polymer-drug conjugate, it can effectively avoid the removal of micelles, increase blood circulation time, ensure sufficient long blood circulation, and facilitate accumulation at the tumor site through the EPR effect.

[0311] (4) Tumor suppression experiment

[0312] Twenty mice were selected, with a weight of approximately 18-20g and a tumor volume of 90-100mm. 3 Mice were randomly divided into four groups of five each. One group served as the control group, injected with PBS. The other groups were injected with free SN38 (5 mg / kg) or PC7A-SN38-PEG micelles (SN38 was the indicator, 5 mg / kg and 10 mg / kg). The body weight and tumor volume of each mouse were measured every two days. The drug was administered via tail vein every two days for five administrations. The experiment was terminated based on the observed results. Curves were plotted based on the data. The results are shown below. Figure 9 As shown. According to Figure 9 As can be seen, PC7A-SN38-PEG has excellent therapeutic effects, inhibiting tumor growth in a short period of time, exhibiting excellent biocompatibility, and enhancing the therapeutic effect.

[0313] Application Example 3 (PC7A-GOD-PEG)

[0314] (1) Electrophoresis experiment of PC7A-GOD-PEG:

[0315] The grafting rate of GOD on the surface was determined using SDS-PAGE denaturing gel electrophoresis. By comparing with the marker, the molecular weight of the modified protein could be obtained, thus inferring the grafting rate. The specific procedure was as follows: 80 μL of 0.5 mg / mL PC7A-GOD-PEG solution was mixed with 20 μL of loading buffer 5*, and incubated at 100 °C for 15 min. The sample (20 μL) was then added to the gel, and electrophoresis was performed at 130 V for 1 h. After electrophoresis, staining was performed at room temperature for 1 h, followed by destaining in destaining solution for 16 h. The results are shown below. Figure 10 As shown. According to Figure 10 It can be seen that when polymer:GOD = 30:1, the integrity of the modification is the best, with about 14 polymer chains grafted. This is somewhat different from the theoretical value. This may be because there is steric hindrance between the polymer chains, making it difficult to connect all the polymers to the surface of GOD.

[0316] (2) Determination of PC7A-GOD-PEG particle size and potential:

[0317] For the determination of particle size and potential of unmodified GOD under neutral conditions, a phosphate buffer with the same pH as unmodified GOD needs to be prepared first. The instrument should then be rinsed multiple times with this buffer, followed by rinsing the instrument three times with the unmodified GOD sample to be tested. Afterward, the particle size and potential of the micelles can be determined according to the usage specifications.

[0318] When determining the particle size and potential of PC7A-GOD-PEG at different pH values, it is first necessary to prepare phosphate buffer solutions with the same pH as the test samples (pH=7.4 and pH=6.7). Then, rinse the instrument several times with this buffer solution, followed by rinsing the instrument three times with the test samples. After this, the particle size and potential of the micelles can be determined according to the usage specifications. The results are as follows: Figure 11 and Figure 12 As shown.

[0319] from Figure 11 and 12It can be seen that the PC7A-GOD-PEG conjugate has a particle size of approximately 110-130 nm under neutral conditions, exhibiting a relatively long cycle time and half-life. It can also accumulate at the tumor site through the EPR effect. Under acidic conditions, the particle size is approximately 15-25 nm; the smaller particle size allows for better drug penetration, delivering more GOD into cancer cells to exert its effect. Under neutral conditions, the PC7A-GOD-PEG nanoparticle drug delivery system exhibits a slightly increased negative potential due to the PEGylated protective shell. This helps prevent adsorption by plasma proteins during blood circulation, and PEG also helps prevent the drug delivery system from being cleared by RES. Upon reaching an acidic environment, PC7A undergoes protonation, giving the nanoparticle drug delivery system a positive charge, which attracts the negatively charged cell membranes of cancer cells, enhancing tumor uptake.

[0320] (3) Determination of pH-responsive PC7A-GOD-PEG enzyme activity:

[0321] Preparation of H2O2-Methylaniline standard curve: First, prepare standard H2O2 solutions with concentrations of 24 μg / mL, 27.8 μg / mL, 38.4 μg / mL, 48 μg / mL, 60 μg / mL, 67.2 μg / mL, and 72 μg / mL for later use. Prepare solution A by mixing N-Methylaniline solution (2.5 mL), glucose solution (0.3 mL), and horseradish peroxidase solution (0.1 mL). Add 0.1 mL of the prepared solution to each solution. After mixing again, add H2SO4 solution (2 mL, 2 mol / L). Measure the OD value at 540 nm. Plot a standard curve with OD as the x-axis and H2O2 concentration as the y-axis.

[0322] The PC7A-GOD-PEG enzyme solution to be tested was diluted with PBS solution to a GOD activity of 0.8–2.8 U / mL.

[0323] Add the diluted test sample solution (0.1 mL) to the prepared solution A. After reacting for 3 min, add 2 mL of H2SO4 solution (2 mol / L). After cooling to room temperature, measure the OD value at 510 nm. The inactivated enzyme solution serves as the negative control, and the unmodified enzyme solution serves as the positive control. Calculate the enzyme activity at different pH values ​​based on the standard curve.

[0324] according to Figure 13It can be seen that the PC7A-GOD-PEG conjugate exhibits significant differences in enzyme activity under neutral and acidic conditions. This is because, under neutral conditions, the active site of GOD is encapsulated by PC7A and cannot function. Under acidic conditions, PC7A changes from hydrophobic to hydrophilic, releasing the active site of GOD, which rapidly decomposes glucose and produces hydrogen peroxide. Therefore, PC7A-GOD-PEG exhibits a distinct acid-responsive release of GOD characteristic.

[0325] (4) Determination of glucose consumption by the drug delivery system:

[0326] Plotting the standard curve: Prepare 1 mL of each of the isogradient Glu standard solutions using a method similar to that described above. Mix with 2 mL of DNS reagent and measure the OD value of the solution at 540 nm. Measure five times at each point and take the average value. Plot the standard curve with Glu concentration on the x-axis and OD value on the y-axis. Glucose consumption experiment: Add a certain amount of active PC7A-GOD-PEG solution to a Glu (5.0 mM) solution. Collect 0.5 mL of sample solution in 1.5 mL of DNS reagent at different time points, heat at 100℃ for 4 min, then quickly transfer to ice water for 15 min, and measure the OD value at 540 nm. Combine the standard curve to evaluate the glucose consumption of the system, using inactivated enzyme solution as a negative control and unmodified enzyme solution as a positive control. Figure 14 It can be seen that heat-inactivated GOD is basically unable to consume glucose in the system. Unmodified GOD can rapidly catalyze the reaction of glucose with oxygen, and the glucose content decreases rapidly in a short period of time. The PC7A-GOD-PEG conjugate has a relatively weak ability to catalyze the reaction of glucose with oxygen under neutral conditions. Most of the active sites are wrapped by the hydrophobic response segment PC7A. As the reaction continues, the product gluconic acid changes the pH environment of the system. When a certain pH is reached, PC7A becomes positively charged and becomes the hydrophilic part, and most of the GOD active sites are released, enhancing the ability to consume glucose. The glucose content in the system continues to decrease and eventually tends to stabilize.

[0327] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A tumor microenvironment-responsive polymer-drug conjugate, characterized in that, The polymer-drug conjugate has the structure shown in formula (1); Equation (1), In formula (1), R2 is a microenvironment-responsive group; R4 is an antitumor drug group with a -linker-Drug structure; R2 has the structure shown in the following formula: ，m=10~200， In the above structure, R 21 `is F, Cl, Br, or I, R 21 `` represents a C1-C4 alkylene group, x=1-8, and * indicates the position where the R2 group is bonded to the R3 group; The R1 has the structure shown in equation (2) or equation (3): Equation (2), m = 10~200; Equation (3), m = 10~200; In equations (2) and (3), R 11 `and R 11 Each is an alkyl group that is independently C1-C4; The linker has at least one of the following structures: ， ， ， In the above structure, z1=0~5, z2=1~5, z3=4~10, and * indicates the position where the linker segment is connected to the R3 group or Drug bond; R3 is a connector and has at least one of the following structures: ， ，n=1-4， ， In the above structure, * indicates the position where the R3 group is bonded to the R1 and R2 groups.

2. The tumor microenvironment-responsive polymer-drug conjugate according to claim 1, characterized in that, The antitumor drug in the R4 group is selected from at least one of the following: doxorubicin, epirubicin, gemcitabine, cisplatin, carboplatin, paclitaxel, camptothecin, eczema, mitomycin C, methotrexate, 7-ethyl-10-hydroxycamptothecin, maytansine, alpha-amanitin, MMAE, MMAF, DM4, chachiin, gambogeylic acid, rhein, vincristine, colchicine, eribulin, taltobulin, maytansine, tyransine A, auristatin E, auristatin F, Piericidin A, anserine P3, salicylate 10 and β-Amanitin, BTZ, fluorouracil, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, glucose oxidase, lactate oxidase, and catalase.

3. The method for preparing the tumor microenvironment-responsive polymer-drug conjugate according to claim 1 or 2, characterized in that, The method includes: obtaining a polymer-drug conjugate by reacting a polymer carrier with a drug precursor via a click chemistry reaction.

4. The method for preparing the tumor microenvironment-responsive polymer-drug conjugate according to claim 3, characterized in that, The reagents used in the click chemical reaction are selected from at least one of azide, dibenzocyclooctylene, lipoic acid, and maleimide.

5. The method for preparing the tumor microenvironment-responsive polymer-drug conjugate according to claim 3, characterized in that, The polymer carrier has one or more of the following structures: ; ,n=4; ,n=1-4; In the above structure, R1 has the structure shown in equation (2) or (3). This indicates that the structures R1 and R2 are bonded to the * position in the aforementioned structures by forming ester or amide bonds, respectively.

6. The method for preparing the tumor microenvironment-responsive polymer-drug conjugate according to claim 3, characterized in that, The drug precursor has at least one of the following structures: Equation (48), Equation (51).

7. The method for preparing the tumor microenvironment-responsive polymer-drug conjugate according to claim 3, characterized in that, The drug precursor has at least one of the following structures: Equation (54), Equation (55), Equation (56).

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