A modularly constructed targeted-tumor-killing polypeptide-drug conjugate, preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a modularly constructed targeted-tumor-killing polypeptide-drug conjugate as well as a preparation method and application. The conjugate has a dendritic six-branch scaffold structure, and the scaffold can realize replaceable combination of a targeting polypeptide and a chemotherapeutic drug through chemical modification, so that precise drug delivery can be realized for different tumor types. Among them, a blood vessel binding peptide and an albumin binding peptide are selected as model polypeptides, and a common clinical drug doxorubicin is selected as a model drug. The polypeptide-drug conjugate has high drug loading and stability, can improve the pharmacokinetic characteristics of poorly soluble drugs, prolong in-vivo circulation, has small molecular weight and strong tissue permeability, can be precisely enriched in tumors, has good structural adjustability and functional expansion potential, and exhibits significant anti-tumor effect and safety.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a modularly constructed targeted-tumor-killing polypeptide-drug conjugate, its preparation method, and its application. Background Technology

[0002] The key to cancer treatment lies in achieving precise drug delivery to the lesion site. However, traditional chemotherapy drugs, lacking molecular-level targeting, often exhibit non-specific distribution in the body, easily causing significant systemic toxicity, thus limiting dose escalation and further improvement in treatment efficacy. In recent years, antibody-drug conjugates (ADCs) have improved tumor selectivity to some extent by introducing antibody-mediated targeting mechanisms. However, their molecular weight is typically around 150 kDa, their large size and limited tissue penetration significantly restrict their diffusion and uniform distribution within solid tumors, making it difficult to achieve sufficient coverage of tumor tissue. Furthermore, the complex manufacturing process and high cost of ADCs also limit their widespread clinical application.

[0003] On the other hand, the high heterogeneity of tumors further complicates precision medicine. Significant differences exist in the expression levels of molecular markers among different patients and within the same tumor, while sensitivity and resistance to different chemotherapeutic drugs also exhibit marked individual variations. Therefore, delivery strategies relying on single targets or fixed drug combinations are insufficient to meet the needs of personalized treatment. There is an urgent need to develop a drug delivery system that combines high efficiency with structural tunability to achieve flexible combination and optimized configuration of targeting and therapeutic units.

[0004] Peptide-drug conjugates (PDCs), as an emerging targeted therapy strategy, typically consist of a homing peptide, a linker, and a cytotoxic drug payload. Compared to ADCs, PDCs offer advantages such as small molecular weight (usually between 1 and 5 kDa), high tissue permeability, and low immunogenicity, showing broad application prospects in the field of precision tumor delivery. Through rational design, peptide ligands can specifically recognize and bind to tumor-associated receptors, such as integrins and growth factor receptors, and leverage their small size to penetrate physiological barriers and enter tumor tissues, thereby achieving selective targeted delivery to tumor cells. However, existing PDC systems mostly focus on the design of single-target peptides or fixed drug payloads, lacking a modular construction strategy that allows for flexible substitution of target peptides and chemotherapeutic drugs, making it difficult to adapt to different tumor types and individualized treatment needs.

[0005] Therefore, constructing a modular and adjustable delivery system based on PDC to achieve interchangeable combinations of targeting and therapeutic units is of great significance for improving the adaptability and scalability of precision cancer treatment. Summary of the Invention

[0006] Breast cancer is one of the most common malignant tumors among women worldwide, with a continuously rising incidence and persistently high mortality rate, making it a significant disease burden that seriously threatens women's health and public health. Triple-negative breast cancer (TNBC), lacking expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), lacks effective molecularly targeted therapies, and clinical treatment primarily relies on cytotoxic chemotherapy drugs. However, existing chemotherapy drugs such as paclitaxel, docetaxel, and doxorubicin are mostly hydrophobic small molecules, exhibiting significant limitations in their pharmacokinetic behavior, including short half-lives, insufficient tumor targeting, limited tissue penetration, and lack of selective distribution in the body. These limitations result in limited anti-tumor efficacy and significant systemic toxicity.

[0007] From the perspective of delivery mechanisms, the complex tissue structure, abnormal vascular system, and dense extracellular matrix of solid tumors constitute multiple physiological barriers, significantly limiting the effective penetration and uniform distribution of drugs within tumor tissue. Furthermore, the molecular heterogeneity and metabolic reprogramming characteristics of tumors further exacerbate the uncertainty of drug delivery and treatment response, making the traditional "single-target-single-drug" treatment model insufficient to meet the needs of precision medicine. Therefore, constructing a drug delivery system that combines efficient delivery capabilities, adjustable structure, and multiple targeting mechanisms has become a key scientific challenge urgently needing breakthroughs in the field of cancer treatment.

[0008] Against this backdrop, this invention proposes a modularly constructed peptide-drug conjugate system. By introducing a molecular scaffold with a six-branched dendritic structure, programmable combination and dynamic regulation of targeting peptides and chemotherapeutic drugs are achieved, thereby constructing a targeted delivery platform with high structural tunability and functional extensibility. Using tubule-binding peptides, albumin-binding peptides, and doxorubicin as model units, this invention constructed a six-branched dendritic peptide-drug conjugate and systematically evaluated its potential application value in breast cancer treatment.

[0009] Furthermore, this invention designs polypeptide units with complementary targeting mechanisms to address the pathophysiological characteristics and metabolic microenvironment of breast cancer. On one hand, tumor tissues generally exhibit enhanced nutrient uptake and energy metabolism. Albumin, as an important endogenous nutrient and transport carrier, accumulates significantly more in tumor tissues than in normal tissues. This invention introduces the albumin-binding domain variant ABD035 as a targeting module, enabling the coupling system to form a stable complex with endogenous albumin, thereby prolonging in vivo circulation time and enhancing the passive and active co-accumulation of tumor tissues.

[0010] On the other hand, platelets play a crucial regulatory role in tumorigenesis, progression, and metastasis. P-selectin, an adhesion molecule expressed on the cell membrane surface after platelet activation, can specifically interact with P-selectin glycoprotein ligand 1 (PSGL-1) or CD44, which are highly expressed on the surface of tumor cells. This invention introduces a P-selectin ligand peptide as a targeting module, enabling the coupling system to selectively bind to activated platelets. Utilizing endogenous platelets as a "biodelivery carrier," it achieves targeted transport and efficient accumulation at tumor sites, thereby further improving drug delivery efficiency and therapeutic precision.

[0011] In this invention, a six-branched dendritic PDC backbone is formed by coupling 7-(N-tert-butoxycarbonylamino)heptanoic acid with tris(hydroxymethyl)aminomethane and serine. The six activated PNP groups provide conditions for subsequent high coupling with the cytotoxic drug doxorubicin, while simultaneously increasing the drug loading capacity of DOX. Furthermore, to achieve active targeting of tumor sites, this invention uses the albumin-targeting peptide ABD035 and the platelet-targeting peptide PSN for modification. After reaching the tumor cells through the active targeting action of ABD035 or PSN, the PDC releases the drug under the action of intracellular esterases, precisely killing tumor cells. Through functional modification and multi-branched design of the carrier backbone, this invention obtains a drug delivery system with ideal biocompatibility, high drug loading capacity, and tumor targeting, which is expected to enhance the therapeutic efficacy against triple-negative breast cancer.

[0012] Through the above design, this invention constructs a peptide-drug conjugate system that integrates multiple targeting mechanisms, modular structural design, and efficient delivery functions, providing a universal and scalable strategy for precision treatment of tumors, such as breast cancer.

[0013] To achieve the above objectives, the present invention provides the following technical solution:

[0014] This invention provides a modularly constructed targeted-tumor-killing peptide-drug conjugate, characterized in that: the conjugate has a tree-like six-branch scaffold structure, and the scaffold can be chemically modified to achieve interchangeable combinations of targeted peptides and chemotherapeutic drugs, enabling precise drug delivery for different tumor types.

[0015] Among them, the target peptides were blood vessel-binding peptide (PSN) and albumin-binding peptide (ABD035) as model peptides, and the chemotherapy drug was the common clinical drug doxorubicin (DOX) as the model drug.

[0016] This invention provides a method for modularly constructing high-drug-load drug-targeting peptide conjugates with tumor-killing properties. The method for preparing high-drug-loaded doxorubicin peptide conjugates using platelet or albumin peptides as targeting peptides and doxorubicin as a model drug is characterized by comprising the following steps: (1) 7-(N-tert-butoxycarbonylamino)heptanoic acid, tris(hydroxymethyl)aminomethane, and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline were dissolved in anhydrous ethanol and stirred under argon protection. After the reaction was completed, most of the solvent was removed by rotary evaporation under reduced pressure. Anhydrous diethyl ether pre-cooled to -20 °C was added to precipitate a solid precipitate, which was filtered to obtain a white solid. The precipitate was dried under vacuum to obtain compound Boc-C7-OH3. (2) Dissolve Boc-C7-OH3 in anhydrous THF (tetrahydrofuran), add PNP-Cl (p-nitrophenyl chloroformate) first, then slowly add pyridine dropwise under ice bath and stir under argon protection; after the reaction is completed, remove the solvent by rotary evaporation under reduced pressure, redissolve with anhydrous DCM (dichloromethane), extract to remove impurities, and dry; obtain the PNP-activated compound Boc-C7-PNP3 by silica gel column chromatography. (3) Dissolve Boc-C7-PNP3 in anhydrous THF, add serine alcohol and DMAP (4-dimethylaminopyridine), and stir the reaction under argon protection at room temperature. After the reaction is completed, remove the solvent by rotary evaporation under reduced pressure, add anhydrous diethyl ether pre-cooled at -20 °C to precipitate a solid precipitate, filter to obtain a yellow solid, and dry the precipitate under vacuum to obtain compound Boc-C7-OH6. (4) Pyridine and PNP-Cl were dissolved in anhydrous THF and mixed. Boc-C7-OH6 was dissolved in anhydrous THF and slowly added to the above system. The reaction was stirred under argon protection at 45 °C. After the reaction was completed, the compound Boc-C7-PNP6 was obtained by silica gel column chromatography. (5) Dissolve Boc-C7-PNP6 in anhydrous DMSO (dimethyl sulfoxide), add DOX (doxorubicin) and DIPEA (N,N-diisopropylethylamine), and stir the reaction under argon protection at room temperature; after the reaction is completed, dialyze the resulting solution with DMF (N,N-dimethylformamide) and deionized water for 24 h, and freeze dry to obtain the compound Boc-C7-DOX6 with six doxorubicins attached; (6) Dissolve Boc-C7-DOX6 in DCM solution containing 33% trifluoroacetic acid. Stir the solution at room temperature after dissolution. After the reaction is complete, remove most of the solvent by rotary evaporation under reduced pressure. Add anhydrous diethyl ether pre-cooled at -20 °C to precipitate a solid precipitate. Filter the precipitate to obtain a red solid. Dry the precipitate under vacuum to obtain compound NH2-C7-DOX6. (7) Dissolve NH2-C7-DOX6 in anhydrous DMSO, add DIPEA and 6-(maleimide)hexanoic acid succinimide ester, and stir the reaction under argon protection at room temperature. After the reaction is completed, add a small amount of glacial acetic acid to the above system to adjust the pH to 6, and then add PSN modified with C-terminal cysteine ​​of platelet-targeting peptide or ABD035 modified with C-terminal cysteine ​​of albumin-targeting peptide. Continue to stir the reaction under argon protection at room temperature. After the reaction is completed, dialyze the resulting solution with DMF and DCM in sequence, evaporate under reduced pressure, and dry the product under vacuum to obtain platelet-targeting doxorubicin peptide conjugate PSN-C7-DOX6 and albumin-targeting doxorubicin peptide conjugate ABD035-C7-DOX6, respectively.

[0017] Preferably, in step (1), the molar ratio of 7-(N-tert-butoxycarbonylamino)heptanoic acid, tris(hydroxymethyl)aminomethane and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline is 1:0.8:1.2; more preferably, the reaction temperature is 60 °C and the reaction time is 16 h.

[0018] Preferably, in step (2), the molar ratio of Boc-C7-OH3, PNP-Cl and pyridine is 1:4:5; the reaction time at room temperature is 16 h; the extraction reagent is 1.33 mol / L potassium hydrogen sulfate solution, and the drying agent is anhydrous sodium sulfate; more preferably, the eluent of the chromatographic column is dichloromethane:methanol 20:1, v:v.

[0019] Preferably, in step (3), the molar ratio of Boc-C7-PNP3, serine alcohol and DMAP is 1:4:0.4; and the reaction time at room temperature is 16 h.

[0020] Preferably, in step (4), the molar ratio of Boc-C7-OH6, pyridine and PNP-Cl is 1:10:12; the reaction time at room temperature is 16 h; more preferably, the eluent for the chromatographic column is petroleum ether: ethyl acetate 2:3, v: v.

[0021] Preferably, in step (5), the molar ratio of Boc-C7-PNP6, DOX and DIPEA is 1:12:24; the reaction time at room temperature is 16 h; and more preferably, the molecular weight cutoff of the dialysis bag is 2 kDa.

[0022] Preferably, the room temperature reaction time in step (6) is 2 h.

[0023] Preferably, in step (7), the molar ratio of NH2-C7-DOX6, DIPEA, 6-(maleimide)hexanoic acid succinimide ester to C-terminal cysteine-modified PSN is 1:1.2:1.1:1.2; the reaction time at room temperature is 24 h; the molecular weight cutoff of the dialysis bag is 2 kJ; the molar ratio of NH2-C7-DOX6, DIPEA, 6-(maleimide)hexanoic acid succinimide ester to C-terminal cysteine-modified ABD035 is 1.2:1.2:1.1:1; the reaction time at room temperature is 24 h; and the molecular weight cutoff of the dialysis bag is 7 kJ.

[0024] The above-mentioned modularly constructed targeted-tumor-killing peptide-drug conjugate is used in the preparation of drugs for treating breast cancer.

[0025] This invention discloses a modularly constructed targeted-tumor-killing peptide-drug conjugate, comprising a hexabranched dendritic backbone, the cytotoxic drug doxorubicin, and either peptide ABD035 or PSN, which actively targets tumor lesions. The peptide-drug conjugate enters tumor tissue and cells via ABD035 targeting endogenous albumin or PSN targeting CD44, and then releases the drug DOX under the action of intracellular esterases, precisely killing tumor cells.

[0026] In this invention, an amidation reaction is performed between the carboxyl group of 7-(N-tert-butoxycarbonylamino)heptanoic acid and tris(hydroxymethyl)aminomethane having three hydroxyl groups to obtain a three-branched dendritic polymer. These three hydroxyl groups are then coupled to serine alcohol having two hydroxyl groups via urethane bonds to obtain the hexa-branched dendritic framework with improved drug loading. Subsequently, by removing the protecting group, maleimide active groups are added via an amidation reaction. Modification with ABD035 or PSN is achieved through Michael addition of maleimide and thiol groups.

[0027] The room temperature mentioned in this invention refers to 25°C.

[0028] Compared with the prior art, the present invention has the following advantages: (1) The polypeptide-drug conjugate of the present invention has high drug loading capacity and good stability, which can significantly improve the pharmacokinetic properties of poorly soluble small molecule drugs and prolong the in vivo circulation time.

[0029] (2) The conjugates described in this invention have small molecular weight and strong tissue permeability, exhibiting good penetration and retention capabilities in tumor tissues, which is conducive to achieving efficient delivery and precise enrichment.

[0030] (3) The present invention adopts a modular construction strategy and, based on a tree-like six-branch scaffold structure, realizes the interchangeable combination of targeted peptides and chemotherapeutic drugs, with good structural tunability and functional scalability.

[0031] (4) The conjugates constructed using blood vessel-binding peptide, albumin-binding peptide and doxorubicin as models in this invention showed significant anti-tumor effects and good in vivo safety, verifying the universality and application potential of this strategy. Attached Figure Description

[0032] Figure 1 This is the synthetic route for Boc-C7-PNP6 in this invention; Figure 2 The synthesis route of PSN-C7-DOX6 and ABD035-C7-DOX6 in this invention is shown below; Figure 3 The 1H NMR spectrum of Boc-C7-OH3; Figure 4 Mass spectra of Boc-C7-OH3; Figure 5 The 1H NMR spectrum of Boc-C7-PNP3; Figure 6 Mass spectra of Boc-C7-PNP3; Figure 7 The 1H NMR spectrum of Boc-C7-OH6; Figure 8 Mass spectra of Boc-C7-OH6; Figure 9 The 1H NMR spectrum of Boc-C7-PNP6; Figure 10 Mass spectrometry for Boc-C7-PNP6; Figure 11 The 1H NMR spectrum of Boc-C7-DOX6; Figure 12 The 1H NMR spectrum of NH2-C7-DOX6; Figure 13 The 1H NMR spectrum of PSN-C7-DOX6; Figure 14The 1H NMR spectrum of ABD035-C7-DOX6.

[0033] Figure 15 Tumor volume monitoring graphs in breast cancer model animals under different treatment groups; Figure 16 Figures showing the survival monitoring of breast cancer model animals in different treatment groups; Figure 17 Figures showing weight monitoring in breast cancer model animals under different treatment groups; Figure 18 Images of hematoxylin and eosin staining of organs from breast cancer model animals in different treatment groups. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0035] English definition: THF: Tetrahydrofuran; PNP-Cl: p-Nitrophenylchloroformate; DCM: Dichloromethane; DMAP: 4-Dimethylaminopyridine; DMSO: Dimethyl sulfoxide; DOX: Doxorubicin; DIPEA: N,N-Diisopropylethylamine; DMF: N,N-Dimethylformamide.

[0036] Example 1 A method for preparing highly drug-targeting peptide conjugates with modular tumor-killing properties includes the following steps: (1) The method for synthesizing Boc-C7-OH3 includes the following steps: 1 g of 7-(N-tert-butoxycarbonylamino)heptanoic acid, 0.39 g of tris(hydroxymethyl)aminomethane, and 1.21 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline were dissolved in 50 mL of anhydrous ethanol and reacted at 60 °C under argon protection with stirring for 16 h. The reaction process is shown in Formula I. After the reaction, most of the solvent was removed by rotary evaporation under reduced pressure. The remaining solution was added to anhydrous diethyl ether pre-cooled at -20 °C to precipitate a solid precipitate, which was filtered to obtain a white solid. The precipitate was dried under vacuum to obtain 0.79 g of compound Boc-C7-OH3. Its specific structure and 1H NMR spectrum are shown in Formula I. Figure 3 As shown, the mass spectrometry is as follows Figure 4 As shown.

[0037]

[0038] Formula I (2) 250 mg of Boc-C7-OH3 and 578.2 mg of PNP-Cl were dissolved in 15 mL of anhydrous THF. 280 μL of pyridine was slowly added dropwise under ice bath conditions, and the mixture was stirred for 16 h under argon protection at room temperature. The reaction process is as shown in Formula II. After the reaction, the solvent was removed by rotary evaporation under reduced pressure. The solution was reconstituted with anhydrous DCM and extracted with 1.33 mol / L potassium hydrogen sulfate solution to remove excess pyridine. The remaining DCM solution was dried with anhydrous sodium sulfate. The solution was purified by silica gel column chromatography (dichloromethane:methanol = 20:1) to obtain a total of 346.9 mg of compound Boc-C7-PNP3. Its specific structure and 1H NMR spectrum are shown in Formula II. Figure 5 As shown, the mass spectrometry is as follows Figure 6 As shown.

[0039]

[0040] Formula II (3) 346.9 mg Boc-C7-PNP3, 149.9 mg serine, and 20.8 mg DMAP were dissolved in 10 mL of anhydrous THF and stirred for 16 h under argon protection at room temperature. The reaction process is as shown in Formula III. After the reaction, most of the solvent was removed by rotary evaporation under reduced pressure. Anhydrous diethyl ether pre-cooled to -20 °C was added to precipitate a solid. The solid was filtered to obtain a yellow solid. The precipitate was dried under vacuum to obtain 230.8 mg of compound Boc-C7-OH6. Its specific structure and 1H NMR spectrum are shown in Formula III. Figure 7 As shown, the mass spectrometry is as follows Figure 8 As shown.

[0041]

[0042] Formula III (4) Dissolve 86 μL of pyridine and 259.2 mg of PNP-Cl in 15 mL of anhydrous THF, and dissolve 70 mg of Boc-C7-OH6 in 2 mL of anhydrous THF. Add the solutions dropwise to the above system and stir for 16 h under argon protection at 45 °C. The reaction process is as shown in Formula IV. After the reaction, the solution was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 2:3) to obtain 31.8 mg of compound Boc-C7-PNP6. The synthesis process of Boc-C7-PNP6 is as follows: Figure 1 As shown, its specific structure and proton nuclear magnetic resonance spectrum are as follows: Figure 9 As shown, the mass spectrometry is as follows Figure 10 As shown.

[0043]

[0044] Formula IV (5) 20.0 mg Boc-C7-PNP6, 82.0 mg DOX, and 48 μL DIPEA were dissolved in 5 mL of anhydrous DMSO and stirred for 16 h under argon protection at room temperature. The reaction process is shown in Formula V. After the reaction, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 2 kDa, dialyzed with DMF for 8 h and deionized water for 16 h, and then lyophilized to form a red powder, yielding 42.1 mg of compound Boc-C7-DOX6. Its specific structure and 1H NMR spectrum are shown in Formula V. Figure 11 As shown.

[0045]

[0046] Formula V (6) 32.6 mg of Boc-C7-DOX6 was dissolved in 7.2 mL of DCM solution containing 33% trifluoroacetic acid, and the mixture was stirred at room temperature for 2 h. The reaction process is as shown in Formula VI. After the reaction, most of the solvent was removed by rotary evaporation under reduced pressure. The solution was then added to anhydrous diethyl ether pre-cooled to -20 °C to precipitate a solid precipitate. The precipitate was filtered to obtain a red powder, which was then dried under vacuum to obtain 10.3 mg of NH2-C7-DOX6. Its specific structure and 1H NMR spectrum are shown in Formula VI. Figure 12 As shown.

[0047]

[0048] Formula VI (7) 14.0 mg NH2-C7-DOX6, 0.6 μL DIPEA, and 1.12 mg succinimide 6-(maleimide)hexanoate were dissolved in 5 mL anhydrous DMSO and stirred for 24 h under argon protection at room temperature. After the reaction, a small amount of glacial acetic acid was added to the above system to adjust the pH to 6, and then 4.0 mg of platelet-targeting peptide C-terminal cysteine-modified PSN was added. The reaction was continued to be stirred for 24 h under argon protection at room temperature. After the reaction, the resulting solution was placed in a dialysis bag with a molecular weight cutoff of 2 kDa and dialyzed with DMF for 8 h and DCM for 16 h, respectively. The product was then dried under vacuum by rotary evaporation under reduced pressure to obtain 9.8 mg PSN-C7-DOX6. The synthesis process of PSN-C7-DOX6 is as follows. Figure 2 As shown, its specific structure and proton nuclear magnetic resonance spectrum are as follows: Figure 13 As shown.

[0049] The platelet-targeting peptide C-terminal cysteine-modified PSN in this step was replaced with albumin-targeting peptide C-terminal cysteine-modified ABD035, and the molecular weight cutoff of the dialysis bag was changed to 7 kDa. Other reactants and reaction conditions remained unchanged, yielding ABD035-C7-DOX6. The synthesis process of ABD035-C7-DOX6 is as follows: Figure 2 As shown, its specific structure and proton nuclear magnetic resonance spectrum are as follows: Figure 14 As shown. The reaction process is as described in equation VII.

[0050]

[0051] Formula VII After digestion and centrifugation, GL261-luci cells in good growth condition were subjected to a 6×10⁻⁶ ppm solution. 7 The cell suspension was resuspended at a concentration of / mL in HBSS without FBS and penicillin antibodies and placed on ice. Mice were anesthetized and fixed in a stereotaxic apparatus. After shaving the head and preparing the skin, the skin was incised with a scalpel, and the external skull mucosa was wiped with a cotton swab soaked in 3% H2O2 to expose the anterior fontanelle. 5 μL of the cell suspension was injected intracranially 2 mm to the right of the anterior fontanelle to a depth of 3 mm.

[0052] On the tenth day after the GBM model was established, the model mice were randomly divided into 5 groups of 6 mice each, and treated with different PDC and control preparations. The administration frequency was once every 5 days for a total of 3 times. Tumor volume, mouse weight and survival rate were recorded at the same time.

[0053] The formulations for the different dosing groups are as follows: G1: normal saline; G2: Dox 5 mg / kg; G3: BOC-C7-DOX6; G4: ABD035-C7-DOX6; G5: PSN-C7-DOX6.

[0054] On day 22 after administration, the model mice were euthanized by cervical dislocation, and the heart, liver, spleen, lungs, and kidneys were quickly removed, paraffin sections were prepared, and H&E staining was performed to examine the safety of each PDC formulation.

[0055] Results of the tumor chemotherapy efficacy of peptide-drug conjugates as follows: Figure 15 , 16 As shown: ABD035-C7-DOX6 (G4) and PSN-C7-DOX6 (G5) can significantly slow down tumor growth and effectively prolong the survival of animals.

[0056] The therapeutic safety profile of peptide-drug conjugates, with results as follows: Figure 17 , 18 As shown, none of the drugs affected the body weight of the animal models, and no significant organ toxicity was observed.

Claims

1. A method for preparing a modularly constructed targeted-tumor-killing peptide-drug conjugate, characterized in that, Includes the following steps: (1) 7-(N-tert-butoxycarbonylamino)heptanoic acid, tris(hydroxymethyl)aminomethane, and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline were dissolved in anhydrous ethanol and stirred under argon protection. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure. Then, anhydrous diethyl ether pre-cooled to -20 °C was added to precipitate a solid precipitate. The precipitate was filtered to obtain a white solid. The precipitate was dried under vacuum to obtain compound Boc-C7-OH3. (2) Dissolve Boc-C7-OH3 in anhydrous tetrahydrofuran, add p-nitrophenyl chloroformate first, then slowly add pyridine dropwise under ice bath, and stir the reaction under argon protection; after the reaction is completed, remove the solvent by rotary evaporation under reduced pressure, redissolve with anhydrous dichloromethane, extract and remove impurities, and dry. The PNP-activated compound Boc-C7-PNP3 was obtained by silica gel column chromatography separation and purification. (3) Dissolve Boc-C7-PNP3 in anhydrous THF, add serine alcohol and 4-dimethylaminopyridine and stir under argon protection at room temperature; after the reaction is completed, remove the solvent by rotary evaporation under reduced pressure, add anhydrous diethyl ether pre-cooled at -20 °C to precipitate a solid precipitate, filter to obtain a yellow solid; dry the precipitate under vacuum to obtain compound Boc-C7-OH6; (4) Pyridine and PNP-Cl were dissolved in anhydrous THF and mixed. Boc-C7-OH6 was dissolved in anhydrous THF and slowly added to the above system. The reaction was stirred under argon protection at 45 °C. After the reaction was completed, the compound Boc-C7-PNP6 was obtained by silica gel column chromatography. (5) Dissolve Boc-C7-PNP6 in anhydrous dimethyl sulfoxide, add doxorubicin and N,N-diisopropylethylamine, and stir the reaction under argon protection at room temperature; after the reaction is completed, dialyze the resulting solution with N,N-dimethylformamide and deionized water for 24 h, freeze dry and dehydrate to obtain the compound Boc-C7-DOX6 with six doxorubicins attached. (6) Dissolve Boc-C7-DOX6 in DCM solution containing 33% trifluoroacetic acid. Stir the solution at room temperature after dissolution. After the reaction is complete, remove most of the solvent by rotary evaporation under reduced pressure. Add anhydrous diethyl ether pre-cooled at -20 °C to precipitate a solid precipitate. Filter the precipitate to obtain a red solid. Dry the precipitate under vacuum to obtain compound NH2-C7-DOX6. (7) Dissolve NH2-C7-DOX6 in anhydrous DMSO, add DIPEA and 6-(maleimide)hexanoic acid succinimide ester, and stir the reaction under argon protection at room temperature. After the reaction is completed, add a small amount of glacial acetic acid to the above system to adjust the pH to 6, and then add platelet-targeting peptide PSN modified with C-terminal cysteine ​​or albumin-targeting peptide ABD035 modified with C-terminal cysteine. Continue to stir the reaction under argon protection at room temperature. After the reaction is completed, dialyze the resulting solution with DMF and DCM in sequence, evaporate under reduced pressure, and dry the product under vacuum to obtain platelet-targeting doxorubicin peptide conjugate PSN-C7-DOX6 and albumin-targeting doxorubicin peptide conjugate ABD035-C7-DOX6, respectively.

2. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, In step (1), the molar ratio of 7-(N-tert-butoxycarbonylamino)heptanoic acid, tris(hydroxymethyl)aminomethane and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline is 1:0.8:1.2; more preferably, the reaction temperature is 60 °C and the reaction time is 16 h.

3. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, In step (2), the molar ratio of Boc-C7-OH3, PNP-Cl and pyridine is 1:4:5; the reaction time at room temperature is 16 h; the extraction reagent is 1.33 mol / L potassium hydrogen sulfate solution, and the drying agent is anhydrous sodium sulfate; preferably, the eluent for the chromatographic column is dichloromethane:methanol 20:1, v:v.

4. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, In step (3), the molar ratio of Boc-C7-PNP3, serine alcohol and DMAP is 1:4:0.4; the reaction time at room temperature is 16 h.

5. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, In step (4), the molar ratio of Boc-C7-OH6, pyridine and PNP-Cl is 1:10:12; the reaction time at room temperature is 16 h; and more preferably, the eluent for the chromatographic column is petroleum ether: ethyl acetate 2:3, v: v.

6. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, In step (5), the molar ratio of Boc-C7-PNP6, DOX and DIPEA is 1:12:24; the reaction time at room temperature is 16 h; preferably, the molecular weight cutoff of the dialysis bag is 2 kDa.

7. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, The room temperature reaction time in step (6) is 2 h.

8. The method for preparing the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 1, characterized in that, In step (7), the molar ratio of NH2-C7-DOX6, DIPEA, 6-(maleimide)hexanoic acid succinimide ester to C-terminal cysteine-modified PSN is 1:1.2:1.1:1.2; the reaction time at room temperature is 24 h; the molecular weight cutoff of the dialysis bag is 2 kJ; the molar ratio of NH2-C7-DOX6, DIPEA, 6-(maleimide)hexanoic acid succinimide ester to C-terminal cysteine-modified ABD035 is 1.2:1.2:1.1:1; the reaction time at room temperature is 24 h; the molecular weight cutoff of the dialysis bag is 7 kJ.

9. Modularly constructed targeted-tumor-killing peptide-drug conjugates prepared by the preparation method according to any one of claims 1-8.

10. The use of the modularly constructed targeted-tumor-killing peptide-drug conjugate according to claim 9 in the preparation of a medicament for treating breast cancer.

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