A polypeptide delivery system based on cationic nanocarriers and its preparation method and use

Abstract

The present invention discloses a polypeptide delivery system based on cationic nanocarriers, comprising a cationic nanocarrier and a stable polypeptide inhibitor; the sequence of the cationic nanocarrier is Fmoc-RRMEHRMEW, and the two Met are connected by 1,2-di(bromomethyl)benzene to form a cyclic peptide with sulfonium salts at both ends; the amino acid sequence of the stable polypeptide inhibitor is: Cyclo(isoD-NS-Dap)-D-W-N-E-P-A-E-E-W-G-N-W-V-D-E. The present invention also provides a preparation method of the above-mentioned polypeptide delivery system and its application in the preparation of drugs for interfering with the formation of MTDH / SND1 complex and treating triple-negative breast cancer. The polypeptide delivery system of the present invention has good cell permeability and low toxicity, and after entering the cell, it can effectively target SND1 and interfere with the formation of MTDH / SND1 complex in tumor cells, and exhibits good anti-tumor activity in triple-negative breast cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of medicinal chemistry and relates to a polypeptide delivery system, specifically a cationic nanocarrier-based stable polypeptide delivery system targeting MTDH / SND1, and a preparation method and use thereof. Background Art

[0002] As a class of endogenous molecules in organisms, peptides possess high biocompatibility and low cytotoxicity. Furthermore, the application of various peptide stabilization methods to modify peptide molecules can enhance the biophysical and chemical properties of linear peptides, and they can be used as bioactive materials in a variety of applications, such as cell culture, tissue engineering, and drug delivery. Cationic peptide-based delivery systems have emerged as a new class of non-viral vectors, offering advantages over polymer and lipid-based carriers, such as improved biocompatibility, design versatility, ease of synthesis, and a lower risk of immune response.

[0003] The previous research team rationally designed a 9-amino acid sulfonium salt peptide wpc carrier. The positive charge brought by the modification of the peptide Met side chain with sulfonium salt can enhance the electrostatic interaction between the peptide and the nucleic acid molecule, and the elimination of the positive charge under high GSH concentration promotes the release of nucleic acid molecules, playing a role in nucleic acid delivery.

[0004] A series of studies have demonstrated that nanoparticles formed by the copolymerization of wpc peptide vectors and siRNA have a modest inhibitory effect on the growth and proliferation of HeLa cells, arresting them in the G2 phase. The transfection efficiency is comparable to that of commercial transfection reagents Lipo-2000 and Oligo. Furthermore, wpc peptide vector-siRNA copolymerized nanoparticles have a modest inhibitory effect on HeLa tumor xenografts in nude mice, with no significant biotoxicity to various organs and tissues during administration. Beyond nucleic acid delivery, wpc vectors also hold great potential for delivering peptides and proteins.

[0005] MTDH and SND1 can interact to form a functional complex that mediates cancer development. Tryptophans at positions 394 and 401 of MTDH can penetrate into two hydrophobic pockets of the SND1 protein, and the binding of these two pockets plays a crucial role in the binding of MTDH and SND1.

[0006] Based on these two pockets, the present invention mutated the original sequence (Ala at position 396 was mutated to Glu to further enhance binding to the SND1 protein), and a terminal aspartic acid-based cross-linking ring closure method was used to cyclize the original sequence to maintain the binding conformation of the original sequence in a complex system, thereby improving the physicochemical properties of the polypeptide, such as binding, cellular uptake, and serum stability.

[0007] Given that the developed stable peptide contains a high number of negatively charged amino acids, the team sought to utilize a cationic carrier to assist the stable peptide in entering cells and exerting its effect. Therefore, they introduced a 9-amino acid sulfonium salt peptide carrier, WPC, developed by the research team. The WPC carrier is positively charged overall and has the potential to enable negatively charged nucleic acids, proteins, or peptides to enter cells and exert their effect. Based on the previous successful efficacy of WPC in siRNA delivery, the present invention utilizes the positively charged WPC carrier to enhance the cellular uptake of the peptide, thereby reducing the peptide's dosage concentration and studying its anti-tumor activity in triple-negative breast cancer cells. Summary of the Invention

[0008] The present invention provides a cationic nanocarrier-based polypeptide delivery system, a preparation method, and uses thereof. The cationic nanocarrier-based polypeptide delivery system, a preparation method, and uses thereof are intended to solve the technical problem of poor efficacy of drugs for treating triple-negative breast cancer in the prior art.

[0009] The present invention provides a cationic nanocarrier-based peptide delivery system (NS-E-cyc / wpc), comprising a cationic nanocarrier (named as the wpc carrier) and a stable peptide inhibitor (named as the NS-E-cyc peptide); the sequence of the cationic nanocarrier is Fmoc-RRMEHRMEW, and the two Met groups of the cationic nanocarrier are connected by 1,2-di(bromomethyl)benzene to form a cyclic peptide with sulfonium salts at both ends; the amino acid sequence of the stable peptide inhibitor is: Cyclo(isoD-NS-Dap)-DWNEPAEEWGNWVDE.

[0010] Furthermore, in the polypeptide delivery system, the concentration of the cationic nanocarrier is

[0011] 200-240 μM, and the concentration of the stable polypeptide inhibitor is 20-40 μM.

[0012] Furthermore, the structural formula of the cationic nanocarrier is:

[0013]

[0014] Furthermore, the structural formula of the stable polypeptide inhibitor is:

[0015]

[0016] R is Ac, H or βA-FITC.

[0017] The present invention also provides a method for preparing the above-mentioned cationic nanocarrier-based polypeptide delivery system, comprising the following steps:

[0018] 1) A step for preparing cationic nanocarriers:

[0019] a) Preparation of oligopeptide solid phase resin: loading the target oligopeptide on MBHA resin by solid phase synthesis method;

[0020] b) Preparation of sulfonium salt cyclic peptide WPC carrier: After the linear peptide containing methionine is synthesized on the solid phase, an appropriate amount of resin is placed in an EP tube and 1-2 mL of TFA / TIPS / H2O / EDT shearing buffer is added and shaken for 1-2 hours. The volume ratio of TFA, TIPS, H2O, and EDT is 94:1:2.5:2.5. The shearing buffer is blown dry with nitrogen gas, and then ether is added to precipitate for 2 minutes. The supernatant is discarded after centrifugation, and the precipitated peptide is allowed to evaporate to dryness with air.

[0021] c) The polypeptide was dissolved in a 50% by volume acetonitrile / water solution to form a 10 mM polypeptide solution, which was acidified by adding 1% by volume of formic acid to a final pH of 3. 1.2 polypeptide equivalents of 1,2-bis(bromomethyl)benzene were weighed and dissolved in one-tenth the solvent volume of DMF. The solution was then added to the polypeptide solution, the reaction was allowed to proceed on a shaker, and the wpc carrier was purified by high performance liquid chromatography.

[0022] The process of the above reaction is as follows:

[0023]

[0024] 2) A step for preparing a stable polypeptide inhibitor:

[0025] a) Preparation of oligopeptide solid phase resin: loading the target oligopeptide on MBHA resin by solid phase synthesis method;

[0026] b) Preparation of Compound I: This step is carried out on a resin, and the Alloc and Allyl protecting groups of the target oligopeptide are removed in the presence of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to obtain Compound I;

[0027] c) Preparation of Compound II: Compound I undergoes a ring-closure reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole, and N-methylmorpholine to produce Compound II;

[0028] d) Preparation of modified polypeptide: The Fmoc protecting group of compound II is removed by 50% by volume morpholine, which can then be directly acetylated or modified with FITC fluorescent dye;

[0029] The process of the above reaction is as follows:

[0030]

[0031] 3) Mixing the cationic nanocarrier and the stable polypeptide inhibitor in a cell culture medium, wherein the concentration of the cationic nanocarrier is 200-240 μM, and the concentration of the stable polypeptide inhibitor is 20-40 μM.

[0032] The present invention also provides a cationic nanocarrier, the amino acid sequence of which is Fmoc-RRMEHRMEW, wherein two Met groups are connected via 1,2-di(bromomethyl)benzene to form a cyclic peptide with sulfonium salts at both ends.

[0033] The present invention also provides the use of the cationic nanocarrier in the preparation of a negatively charged polypeptide drug targeting MTDH / SND1.

[0034] The present invention also provides the use of the above polypeptide delivery system in the preparation of a drug that interferes with the formation of the MTDH / SND1 complex.

[0035] The present invention also provides the use of the above-mentioned polypeptide delivery system in the preparation of a drug for treating triple-negative breast cancer.

[0036] Based on previous studies, this invention starts from the MTDH / SND1 crystal structure and selects the SND1-binding sequence (DWNAPAEEWGNWVDE) in the MTDH protein as a starting point. By combining molecular docking technology with experimental methods such as mutation and cyclization, a stable peptide inhibitor based on terminal aspartic acid cyclization was obtained.

[0037] The present invention uses the Autodock molecular docking method to construct a model for the primary binding between a peptide derived from the MTDH sequence and the SND1 protein, attempting to explain the peptide-protein binding interface. In the original MTDH sequence, two tryptophan residues, Trp394 and Trp403, can penetrate into two hydrophobic pockets in the SND1 protein, playing a crucial role in binding to the protein. The acidic amino acid at the C-terminus also plays a significant role. Therefore, based on Trp394 and Trp403, the present invention mutated Ala at position 396 to Glu to enhance electrostatic interactions with the SND1 protein, thereby strengthening binding. Furthermore, a terminal aspartic acid was cross-linked and cyclized to maintain the secondary conformation of the peptide. Given that the stable peptide developed by the present invention contains a high number of negatively charged amino acids, a nine-amino acid sulfonium salt peptide (WPC) carrier developed by the research team was introduced. The WPC carrier is positively charged overall, allowing it to carry negatively charged nucleic acids, proteins, or peptides into cells for their effects. Previous studies have also found that wpc has achieved good therapeutic effects in the delivery of siRNA. Therefore, the present invention uses the positively charged carrier of wpc to improve the cellular uptake ability of stable polypeptides, thereby reducing the dosage concentration of the polypeptide and studying the anti-tumor activity of the polypeptide in triple-negative breast cancer cells. Experiments have shown that the polypeptide delivery system of the present invention can effectively penetrate cells and effectively interfere with the interaction of MTDH / SND1 proteins. The polypeptide delivery system of the present invention has low cytotoxicity, can effectively inhibit the growth and migration of triple-negative breast cancer, and induce G0 / G1 cycle arrest of triple-negative breast cancer cells, bringing new possibilities for breast cancer treatment.

[0038] The present invention provides a stable peptide delivery system targeting MTDH / SND1 based on cationic nanocarriers, discloses a peptide (NS-E-cyc) with a stable conformation through specific chemical modification (TD strategy), and assists the peptide to enter cells through positively charged WPC nanocarriers, which also broadens the application range of WPC cationic peptide carriers ( Figure 1 A series of biological experiments demonstrated that the stabilized peptide NS-E-cyc, when mixed with a carrier, significantly enhanced tumor cell uptake. It also effectively targeted the SND1 protein within cells and interfered with the formation of the MTDH / SND1 protein complex, thereby inhibiting the growth and migration of triple-negative breast cancer cells and inducing cell cycle arrest. The TD strategy preserves the free amino group at the peptide's N-terminus, allowing for further chemical modification and structural optimization. This type of peptide delivery system may serve as a potential treatment for triple-negative breast cancer.

[0039] Compared with existing technologies, the present invention represents a significant technological advancement. Currently, cationic peptide carriers that can be used to deliver peptides or proteins are very limited. WPC cationic peptide carriers have a relatively simple sequence and are easy to synthesize and modify. Furthermore, after modification with sulfonium salts, their positive charge is enhanced, enabling better assembly with negatively charged macromolecules, thereby improving cell penetration. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 .It is the principle diagram of the present invention.

[0041] Figure 2 .Comparison of fluorescence polarization results and binding constants between peptides and SND1 protein.

[0042] Figure 3 .Simulation diagram of the peptide and SND1 crystal structure (PDB:4QMG).

[0043] Figure 4 .The cellular uptake ability of the polypeptide after the wpc carrier is mixed with the polypeptide.

[0044] Figure 5 .Interference effect of polypeptide complex on intracellular MTDH / SND1 protein.

[0045] Figure 6 .Effects of peptide complex on proliferation, migration and cell cycle of triple-negative breast cancer cells. DETAILED DESCRIPTION

[0046] Example 1 Design and Optimization of Stable Peptide Inhibitors

[0047] Based on previous studies, the present invention, starting from the MTDH / SND1 crystal structure (PDB: 4QMG), selected the sequence in the MTDH protein that binds to SND1 (DWNAPAEEWGNWVDE, designated MS2D, with a Kd of approximately 60 nM) as a starting point. Key amino acid mutations and terminal aspartic acid cyclization modifications were performed on this original sequence. The effects of side chains of varying lengths on binding were explored, as well as the effect of ring closure on the conformation of the original linear sequence. To test the binding strength of stable peptide inhibitors to the SND1 protein, the present invention synthesized FITC-labeled peptides (MS2D-E and MS2D-D) using peptide solid-phase synthesis and expressed and purified the SND1 protein (16-339).

[0048] Fluorescence polarization (FP) was then used to test the binding ability of the peptides to the SND1 protein (16-339). It can be seen that compared with MS2D, the binding ability of the two mutant peptides was improved, and the binding ability of MS2D-E with a longer side chain was relatively better. ( Figure 2 A, 2B) MS2D-E was then cross-linked and cyclized at the terminal aspartic acid. To enhance hydrogen bonding between the cyclic peptide and the wpc carrier, the original -Ala-Ala residue within the amide ring of the cyclic peptide was mutated to -Asn-Ser. This modification was named MS2D-E-NS-E-cyc, or NS-E-cyc for short. Experimental results showed that fixing the ring closure position at the N-terminus of MS2D-E and omitting this sequence (Cyclo(isoD NS Dap)DWNEPAEEWGNWVDE)) better maintained the binding ability of the original sequence (Kd approximately 20 nM). Figure 2 A, 2B).

[0049] Further molecular docking technology revealed that NS-E-cyc can maintain the basic backbone of linear polypeptides. The two key tryptophans can extend into the hydrophobic pocket of the SND1 protein, and Glu396 can extend into the basic pocket of the SND1 protein, thereby enhancing the electrostatic interaction with the SND1 protein. Moreover, the longer the side chain, the closer the distance between the side chain and the basic amino acid, which is more conducive to binding to the protein. This may also be the reason why MS2D-E has a stronger binding ability than MS2D-D ( Figure 3 ).

[0050] Example 2 Preparation Method of Polypeptide Delivery System

[0051] The present invention also provides a method for preparing the above-mentioned polypeptide delivery system, comprising the following steps:

[0052] 1. Preparation of cationic nanocarriers (wpc carriers):

[0053] 1.1 Preparation of oligopeptide solid phase resin: The target oligopeptide is loaded on MBHA resin by standard solid phase synthesis method;

[0054] 1.2 Preparation of Sulfonium Cyclic Peptide WPC Support: After solid-phase synthesis of a linear peptide containing methionine, place an appropriate amount of resin in an EP tube and add 1-2 mL of a shearing buffer of TFA / TIPS / H2O / EDT (94:1:2.5:2.5). Shake the mixture for 1-2 hours. Drain the shearing buffer with nitrogen and then add 1 mL of cold ether to precipitate for two minutes. Centrifuge and discard the supernatant. The precipitated peptide is then air-evaporated to remove the ether. The peptide is then dissolved in a 50% acetonitrile / water solution to a concentration of approximately 10 mM. The solution is acidified (pH approximately 3) with 1% by volume formic acid. Simultaneously, 1.2 peptide equivalents of 1,2-bis(bromomethyl)benzene are weighed and dissolved in approximately one-tenth the solvent volume of DMF. The mixture is then added to the peptide solution and shaken for 24 hours. The WPC support is then purified by high-performance liquid chromatography (HPLC).

[0055] The process of the above reaction is as follows:

[0056]

[0057] 2. Preparation of stable polypeptide inhibitor (NS-E-cyc polypeptide):

[0058] 2.1 Preparation of oligopeptide solid phase resin: The target oligopeptide is loaded on MBHA resin by standard solid phase synthesis method;

[0059] 2.2 Preparation of Compound I: This step is carried out on a resin. The target oligopeptide is deprotected by removing the Alloc and Allyl protecting groups in the presence of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to obtain Compound I.

[0060] 2.3 Preparation of Compound II: Compound I undergoes a ring-closure reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole and N-methylmorpholine to produce Compound II;

[0061] 2.4 Preparation of modified polypeptides: The Fmoc protecting group of compound II is removed by 50% morpholine by volume, and then the compound can be directly acetylated or modified with FITC fluorescent dye;

[0062] The process of the above reaction is as follows:

[0063]

[0064] 3. Mix the cationic nanocarrier and the stable polypeptide inhibitor in cell culture medium at a concentration of 200-240 μM and 20-40 μM, respectively. The polypeptide delivery systems in the following examples were prepared using the above method. The cationic nanocarrier and the stable polypeptide inhibitor were mixed in cell culture medium before use.

[0065] Example 3 Introduction of amphiphilic membrane-penetrating peptide carrier and cellular uptake of polypeptide complex

[0066] Since the NS-E-cyc sequence contains many negatively charged amino acids, in order to further improve its cellular uptake ability, this paper uses a 9-amino acid sulfonium salt peptide (wpc) developed by the previous research group as a carrier to deliver the NS-E-cyc peptide into cells to exert its effect. The wpc peptide sequence contains an Fmoc protecting group and a hydrophobic part of tryptophan, as well as basic amino acids such as Arg and His and acidic amino acids such as Glu, and 1,2-di(bromomethyl)benzene connects the two Met residues to form a cyclic peptide with sulfonium salts at both ends. The wpc is positively charged as a whole and can carry negatively charged nucleic acids such as siRNA into cells. After entering the cell, the wpc-encapsulated complex is exposed to the high concentration of GSH in the tumor microenvironment, and the sulfonium salts at both ends are reduced and open the ring, releasing nucleic acids or other negatively charged drugs to exert their effects. First, the cytotoxicity of the wpc vector was evaluated. Different concentrations of wpc peptide were incubated with MDA-MB-231 cells for 72 hours. The CCK8 experimental results showed that the cell survival rate of the wpc peptide was approximately 75% at 320 μM, while the cell survival rate was still greater than 85% at 240 μM. In order to avoid cytotoxicity caused by excessively high vector concentrations, the vector concentration should not exceed 240 μM (Figure 4A).

[0067] The peptide NS-E-cyc was then mixed with the wpc carrier and incubated with MDA-MB-231 cells for 6 hours. Flow cytometry was used to examine the cellular uptake of the mixed peptide complex. The experimental results showed that compared with the peptide NS-E-cyc itself, the cellular uptake ability of the mixed NS-E-cyc / wpc peptide complex was significantly improved ( Figure 4 B). Laser confocal microscopy also demonstrated that the NS-E-cyc / wpc polypeptide complex can effectively enter cells and co-localize with lysosomes ( Figure 4 C), to some extent, it indicates that the NS-E-cyc / wpc polypeptide complex enters the cell through endocytosis.

[0068] Further experiments revealed that the peptide and SND1 had good co-localization at 24 h, suggesting that the peptide complex could release the peptide NS-E-cyc within 24 hours and then bind to the target (Figure 4D).

[0069] Example 4 Interference of the polypeptide complex on intracellular MTDH / SND1

[0070] In order to further explore whether the wpc vector carrying peptides into cells can interfere with the formation of MTDH / SND1 complex, this paper used co-immunoprecipitation (Co-IP) experiments to study the binding of MTDH to SND1 protein before and after drug addition. Figure 4As shown, compared with the group treated with the peptide NS-E-cyc alone, the group treated with the NS-E-cyc / wpc peptide complex had a more significant interference effect on MTDH / SND1, and the inhibitory effect was seen with only 20μM of the NS-E-cyc peptide in the complex. Without affecting the normal expression of the MTDH protein, the binding of MTDH to SND1 was significantly reduced, indicating that the wpc vector can carry the peptide to effectively enter the cell to target the SND1 protein, thereby inhibiting the formation of the MTDH / SND1 complex ( Figure 5 ).

[0071] Example 5 Effects of the polypeptide complex on the proliferation, migration and cell cycle of triple-negative breast cancer cells

[0072] This study used two triple-negative breast cancer cell lines (human MDA-MB-231 cells and mouse 4T1 cells) to evaluate the effects of peptide complexes on tumor cell proliferation and migration. Figure 6 As shown in A, the inhibition rate of MDA-MB-231 cell proliferation and 4T1 cell proliferation after treatment with NS-E-cyc / wpc peptide complex (20μM: 240μM) was approximately 50% and 40% respectively. The scratch test also showed that NS-E-cyc and NS-E-cyc / wpc peptide complex (20μM: 240μM) could effectively inhibit the migration of tumor cells ( Figure 6 B), and the effect of the peptide complex was more obvious, which further demonstrated that the wpc vector can effectively deliver the peptide NS-E-cyc to cells, interfere with the formation of the MTDH / SND1 complex, and thus inhibit the proliferation and migration of triple-negative breast cancer cells. Finally, the flow cytometry results showed that the peptide complex can induce G0 / G1 cell cycle arrest in triple-negative breast cancer cells, which is similar to the mechanism of action of the previously developed stable peptide inhibitor ( Figure 6 C).

Claims

1. A cationic nanocarrier-based polypeptide delivery system, characterized in that: The invention comprises a cationic nanocarrier and a stable polypeptide inhibitor; the sequence of the cationic nanocarrier is Fmoc-RRMEHRMEW, and the two Met groups of the cationic nanocarrier are connected by 1,2-di(bromomethyl)benzene to form a cyclic peptide with sulfonium salts at both ends; the structural formula of the stable polypeptide inhibitor is, ； R is Ac, H or FITC.

2. A cationic nanocarrier-based polypeptide delivery system according to claim 1, characterized in that: In the polypeptide delivery system, the concentration of the cationic nanocarrier is 200-240 μM, and the concentration of the stable polypeptide inhibitor is 20-40 μM.

3. A cationic nanocarrier-based polypeptide delivery system according to claim 1, characterized in that: The structural formula of the cationic nanocarrier is: 。 4. The method for preparing a cationic nanocarrier-based polypeptide delivery system according to claim 1, characterized in that The steps include: 1) A step for preparing cationic nanocarriers: a) Preparation of oligopeptide solid phase resin: loading the target oligopeptide onto MBHA resin by solid phase synthesis method; b) Preparation of sulfonium salt cyclic peptide WPC carrier: After the linear peptide containing methionine is synthesized on the solid phase, an appropriate amount of resin is placed in an EP tube. 1-2 mL of TFA / TIPS / H2O / EDT shearing buffer is added and shaken for 1-2 h. The volume ratio of TFA, TIPS, H2O, and EDT is 94:1:2.5:2.

5. The shearing buffer is blown dry with nitrogen gas, and then ether is added to precipitate for two minutes. Centrifuge and discard the supernatant. The precipitated peptide is allowed to evaporate to dryness with air. c) The peptide was dissolved in a 50% by volume acetonitrile / water solution to form a 10 mM peptide solution. 1% by volume of formic acid was added to acidify the solution to a final pH of 3. 1.2 peptide equivalents of 1,2-di(bromomethyl)benzene were weighed and dissolved in one-tenth the solvent volume of DMF. The solution was then added to the peptide solution and allowed to react on a shaker. The WPC carrier was purified by high performance liquid chromatography. The process of the above reaction is as follows: ； 2) A step for preparing a stable peptide inhibitor: a) Preparation of oligopeptide solid phase resin: loading the target oligopeptide onto MBHA resin by solid phase synthesis method; b) Preparation of Compound I: This step is carried out on a resin, and the Alloc and Allyl protecting groups of the target oligopeptide are removed in the presence of tetrakis(triphenylphosphine)palladium and N,N-dimethylbarbituric acid to obtain Compound I; c) Preparation of Compound II: Compound I is subjected to a ring-closure reaction in a solution of benzotriazol-1-yl-oxytripyrrolidinophosphine hexafluorophosphate, 1-hydroxybenzotriazole, and N-methylmorpholine to produce Compound II; d) Preparation of modified polypeptides: The Fmoc protecting group of compound II is removed by 50% by volume morpholine, which can then be directly acetylated or modified with FITC fluorescent dye; The process of the above reaction is as follows: ； 3) mixing the cationic nanocarrier and the stable polypeptide inhibitor in a cell culture medium to obtain a cationic nanocarrier-based polypeptide delivery system, wherein the concentration of the cationic nanocarrier is 200-240 μM, and the concentration of the stable polypeptide inhibitor is 20-40 μM.

5. Use of the polypeptide delivery system according to claim 1 in the preparation of a drug for treating triple-negative breast cancer.

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