A HER2-targeted antitumor polypeptide nanomedicine and a preparation method and application thereof

Abstract

This invention relates to a HER2-targeted antitumor peptide nanomedicine, its preparation method, and its applications. The antitumor peptide nanomedicine comprises an antitumor functional unit, a self-assembly unit, and a targeting unit connected in sequence; the targeting unit includes a peptide sequence targeting the HER2 protein. Through a specially designed peptide sequence, the peptide nanomedicine can be delivered to the lesion site via an active targeting mechanism based on molecular recognition. It then specifically binds to the HER2 target, achieving in-situ self-assembly at the tumor site. The resulting assembly exhibits a significant tumor-killing effect, and its preparation cost is low, demonstrating advantages and significant application prospects in targeted diagnosis and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a HER2-targeted antitumor polypeptide nanomedicine, its preparation method, and its application. Background Technology

[0002] In recent years, cancer has become a leading cause of death worldwide, with high morbidity and mortality rates due to its poor prognosis, high metastasis rate, and high incidence. Traditional cancer treatments include surgery, chemotherapy, and radiotherapy (RT). Among these, chemotherapy remains one of the most classic cancer treatment methods in clinical practice. However, chemotherapy drugs lack targeting specific lesions, their non-specific distribution, induction of multidrug resistance (MDR) in tumor cells, and severe systemic toxicity greatly limit the long-term use of chemotherapy. Radiotherapy, due to its poor specificity, inevitably damages normal tissues while treating cancer, leading to severe side effects. Therefore, there is an urgent need to develop a more precise and effective strategy to combat cancer.

[0003] Human epidermal growth factor receptor-2 (HER2) is a prognostic indicator for clinical treatment monitoring and an important target for tumor targeted therapy. The tumorigenic mechanism of HER2 oncoprotein involves inhibiting apoptosis, promoting proliferation, increasing tumor cell invasiveness, and promoting tumor angiogenesis and lymphangiogenesis. HER2 protein is typically expressed only during fetal development, and only at low levels in a very small number of tissues after adulthood. However, studies have shown that HER2 protein amplification or overexpression exists in more than 30% of human tumors (such as breast cancer, ovarian cancer, endometrial cancer, fallopian tube cancer, gastric cancer, and prostate cancer). Currently, the main targeted therapies for HER2 protein overexpression are Herceptin and lapatinib. Research has confirmed that HER2 protein overexpression is associated with tumor development and invasion, and can increase the risk of metastasis; transfected cell and animal models have demonstrated that it can alter tumor sensitivity to hormones and chemotherapy drugs.

[0004] With the increasing prevalence of nanobiotechnology applications in recent years, peptide self-assembly technology has attracted widespread attention from researchers. Peptides are biomolecules, thus possessing excellent biocompatibility and being easily synthesized and chemically modified, endowing peptide materials with unique biological functions. Therefore, self-assembled peptides have broad application potential in biomedical fields such as gene therapy and tumor treatment, and have received widespread attention from researchers. The application of nanotechnology in medicine has improved the targeting, accumulation, and stability of original drugs, achieving good therapeutic effects. However, changes in stability and surface properties, such as the formation of protein halos, dissolution of nanomedicines during transport, and poor penetration depth of these nanomedicines, lead to unsatisfactory therapeutic effects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a HER2-targeted antitumor polypeptide nanomedicine, its preparation method, and its application.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a HER2-targeted antitumor peptide nanomedicine, the antitumor peptide nanomedicine comprising an antitumor functional unit, a self-assembly unit and a targeting unit connected in sequence; the targeting unit comprises a peptide sequence targeting the HER2 protein.

[0008] The antitumor polypeptide nanomedicine involved in this invention comprises three functional structures: an antitumor functional unit, a self-assembly unit, and a targeting unit, as shown in the schematic diagram below. Figure 1 As shown, the three components complement and work together to exert the therapeutic effect. Through modular design, it achieves highly efficient targeting of the HER2 protein in tumor cells and excellent cell penetration. Furthermore, it can self-assemble in situ into a β-sheet conformation within tumor cells, inducing HER2 protein aggregation after assembly, thus enhancing the inhibitory effect on HER2 protein. Simultaneously, the assemblies remain at the lesion site for a long period, allowing the anti-tumor functional units to exert their therapeutic effect more effectively. This peptide nanomedicine provides a new targeted therapy strategy for tumor treatment and has broad application prospects.

[0009] Preferably, the targeting unit is derived from any one of the polypeptide sequences KCCSYL, SRMARDPQRFVVIQNE, CAHYKDPPFCVARCPS, YGLGMEHLREVRAVTS, or AVENPEYLTPQGGAAP.

[0010] All of the above-mentioned targeting units have the function of targeting the HER2 protein highly expressed in tumor cells, and can achieve excellent tumor targeting function and trigger endocytosis mechanism to improve their penetration into tumor cells.

[0011] Preferably, the self-assembly unit is derived from any one or a combination of at least two of the polypeptide sequences KHYYQNYQ, GNQQNY, KLVFF, FF, ITSVV, GFLG, DEVD, or RVRR.

[0012] The aforementioned self-assembly units all have the effect of promoting the deformation and self-assembly of peptide nanomedicines to form β-sheet structures.

[0013] Preferably, the antitumor functional unit is an antitumor drug; the antitumor drug includes any one or a combination of at least two of the following: chemical drugs, biological drugs, nanomedicines, radiopharmaceuticals, photothermal therapy drugs, or photodynamic therapy drugs.

[0014] Preferably, the chemical drug includes any one or a combination of at least two of cisplatin, doxorubicin, or paclitaxel.

[0015] For example, if the chemical drug is cisplatin, cisplatin needs to be modified by grouping, such as by oxidizing dichlorodiammineplatin by hydrogen peroxide and then reacting it with adipic anhydride to obtain a product that can be linked to a polypeptide through ester bonds.

[0016] In a second aspect, the present invention provides a method for preparing a HER2-targeted antitumor polypeptide nanomedicine according to the first aspect, the preparation method comprising:

[0017] Using resin as a carrier and amino acids that form the self-assembly unit and the targeting unit as raw materials, the amino acids are sequentially linked using a solid-phase synthesis method, and finally linked with the anti-tumor functional unit to obtain the HER2-targeted anti-tumor polypeptide nanomedicine.

[0018] The polypeptide nanomedicines involved in this invention can be prepared using conventional techniques in the field, among which the most commonly used method is solid-phase synthesis. Using Wang resin as a carrier material, the polypeptides are coupled according to their amino acid linkage sequence using a coupling agent, and finally prepared through steps such as trifluoroacetic acid pyrolysis, nitrogen drying, and ether purification.

[0019] Preferably, the preparation method includes:

[0020] (1) The C-terminus of the first amino acid in the targeting unit is fixed on the resin, the N-terminus is protected by Fmoc, the side chain is protected by Boc, and then swelling treatment is performed.

[0021] (2) The Fmoc protection of the N-terminus of the first amino acid is removed by the deprotecting agent, and after washing, the next amino acid is added to react. The amino acids are linked sequentially to obtain a polypeptide sequence fixed in the resin.

[0022] (3) The obtained peptide sequence immobilized in the resin is mixed with the antitumor functional unit modified by the group, and then the resin is shrunken, pyrolyzed, and dried with nitrogen to obtain the HER2-targeted antitumor peptide nanomedicine.

[0023] Preferably, when the antitumor functional unit is cisplatin, the preparation method of the group-modified antitumor functional unit includes: oxidizing dichlorodiammineplatinum ([Pt(Ⅱ)Cl2(NH3)2]) with peroxide to obtain the oxidation product c,c,t-[Pt(Ⅳ)(OH)2Cl2(NH3)2], and then reacting it with adipic anhydride to obtain the product.

[0024] Preferably, the reaction time is 4-12 hours, such as 4 hours, 6 hours, 8 hours, 10 hours, or 12 hours.

[0025] Preferably, the reaction temperature is 40-80℃, such as 40℃, 50℃, 55℃, 60℃, 70℃, 75℃, 80℃, etc.

[0026] Preferably, the resin is Wang resin.

[0027] Preferably, the reagent for removing the Fmoc protecting group of the first amino acid is a mixed solution of N,N-dimethylformamide (DMF), anhydrous piperazine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0028] Preferably, the detection reagent for removing the Fmoc protecting group of the first amino acid is a mixed solution of ninhydrin, phenol, ascorbic acid, and anhydrous ethanol.

[0029] Preferably, after removing the Fmoc protecting group of the last amino acid, it undergoes a condensation reaction with the cisplatin chemotherapy drug obtained above.

[0030] Preferably, the condensation reaction method is as follows: the carboxyl group of the amino acid to be linked is activated with a DMF solution of N-methylmorpholine (NMM) and benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and then added to a resin with the Fmoc protecting group removed for reaction.

[0031] Preferably, the reaction time is 0.75 to 3 hours, for example, 0.8 hours, 1 hour, 1.2 hours, 1.5 hours, 1.7 hours, 2 hours, 2.3 hours, 2.5 hours or 3 hours.

[0032] Preferably, the reagent for removing the resin and side-chain protecting groups is a mixed solution of water, ethylenedithiol, triisopropylsilane, and trifluoroacetic acid.

[0033] Thirdly, the present invention provides the use of the HER2-targeted antitumor peptide nanomedicine according to the first aspect in the preparation of medicaments for the diagnosis, prevention or treatment of tumors.

[0034] Preferably, the tumor is a HER2-overexpressing tumor.

[0035] Preferably, the tumor includes any one of breast cancer, gastric cancer, leukemia, bladder cancer, cervical cancer, or nasopharyngeal carcinoma.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The antitumor peptide nanomedicine of this invention comprises three functional components: an antitumor functional unit, a self-assembly unit, and a targeting unit. These three components work synergistically to exert their therapeutic effect. Through modular design, it achieves highly efficient targeting of the HER2 protein in tumor cells and excellent cell penetration. Furthermore, it can self-assemble in situ into a β-sheet conformation within tumor cells, inducing HER2 protein aggregation after assembly, thus enhancing the inhibitory effect on HER2 protein. Simultaneously, the assemblies remain at the lesion site for a long period, allowing the antitumor functional unit to exert its therapeutic effect more effectively. This peptide nanomedicine provides a novel targeted therapy strategy for tumor treatment and has broad application prospects. Attached Figure Description

[0038] Figure 1 This is a schematic diagram of the structure of the HER2-targeted antitumor polypeptide nanomedicine of the present invention;

[0039] Figure 2 This is the molecular structural formula of the HER2-targeted antitumor polypeptide nanomedicine of this invention;

[0040] Figure 3 This is the mass spectrum of the HER2-targeted antitumor polypeptide nanomedicine of this invention;

[0041] Figure 4 This is a liquid phase characterization diagram of the HER2-targeted antitumor peptide nanomedicine of this invention;

[0042] Figure 5 This is the circular dichroism spectrum of the HER2-targeted antitumor polypeptide nanomedicine of this invention.

[0043] Figure 6 This is the Fourier transform infrared spectrum of the HER2-targeted antitumor polypeptide nanomedicine of this invention;

[0044] Figure 7 This is a transmission electron microscope image of the HER2-targeted antitumor polypeptide nanomedicine of this invention.

[0045] Figure 8 This is a confocal imaging image at the cellular level of the HER2-targeted antitumor peptide nanomedicine of this invention;

[0046] Figure 9 This is a verification diagram of the cytotoxicity results of the HER2-targeted antitumor peptide nanomedicine of this invention;

[0047] Figure 10 This is a statistical graph of the tumor inhibition curve of the in vivo antitumor experiment of the HER2-targeted antitumor peptide nanomedicine of the present invention.

[0048] Figure 11This is a statistical graph of the survival curves of the in vivo antitumor experiments of the HER2-targeted antitumor polypeptide nanomedicine of this invention. Figure 12 This is a standard curve of liquid chromatography-mass spectrometry (LC-MS) quantification of the HER2-targeted anti-tumor peptide nanomedicine of this invention.

[0049] Figure 13 This is a graph showing the concentration change of the HER2-targeted anti-tumor polypeptide nanomedicine of this invention in vivo over time. Detailed Implementation

[0050] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof. Various improvements and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the present invention, which is obvious to those skilled in the art. Other embodiments derived from this specification are also obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0051] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0052] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0053] The mice used in the following test cases were female BALB / c nude mice (6-8 weeks old, 18-20g), purchased from Beijing Vital River Company.

[0054] Example 1

[0055] This embodiment prepares a cisplatin chemotherapy drug, and the preparation method includes the following steps:

[0056] (1) Weigh 200 mg of dichlorodiammineplatinum and add it to a 50 mL round-bottom flask. Add 20 mL of 30% H2O2. Stir the mixture at 60 °C for 4 h to obtain a pale yellow powder. Recrystallize to obtain the oxidation product. Filter the product and wash it with cold water, anhydrous ethanol, and diethyl ether. Dry the product to obtain the final product.

[0057] (2) Weigh 100 mg of the oxidation product into a 10 mL round-bottom flask, add 1.5 mL of DMF, sonicate for 30 min to dissolve, add 30 mg of adipic anhydride, stir at 25 °C for 24 h, add the mixture dropwise to a stirred ice bath of diethyl ether, and add dichloromethane. After high-speed centrifugation, remove the dichloromethane to obtain a product with a small amount of liquid. Concentrate the product for 4 h using a concentrator to obtain the final product.

[0058] Example 2

[0059] This embodiment provides a HER2-targeted antitumor peptide nanomedicine, denoted as X-KHYYQNYQKCCSYL, where X represents the cisplatin chemotherapy drug prepared in Example 1. The preparation method includes the following steps:

[0060] (1) Weigh 300 mg of Fmoc-Leu-Wang with a modified density of 0.35 mM. Resin (with the C-terminus of leucine fixed on the resin and the N-terminus protected by Fmoc) was added to a 10 mL peptide solid-phase synthesis tube. DMF activated by molecular sieves was added to swell the resin, and the DMF was filtered off using a vacuum pump. A deprotecting agent was added, and the N-terminal Fmoc protecting group was removed using a mixed solution of DBU, anhydrous piperazine, and N,N-dimethylformamide (DMF), where DBU was 2% and anhydrous piperazine was 5%. The mixture was placed on a shaker for 15 min. The tube was then washed three times alternately with DMF and DCM (dichloromethane). A small amount was taken from the peptide solid-phase synthesis tube and placed in a 1.5 mL centrifuge tube. The resin was tested using a detection reagent (A: 0.5 g ninhydrin dissolved in 10 mL anhydrous ethanol; B: 20 ​​g phenol dissolved in 5 mL anhydrous ethanol; C: 0.1 g ascorbic acid dissolved in 5 mL anhydrous ethanol, one drop each). A purple color indicated successful deprotection of the resin. The amino acids (Y) were then linked sequentially to initiate the amino acid condensation reaction.

[0061] (2) Take 10 equivalents of amino acids and HBTU according to the above amino acid sequence, dissolve them in 10 mL of coupling agent (N-methylmorpholine and DMF volume ratio of 5:95), and put them into the polypeptide solid phase synthesis tube. After shaking on a shaker for 1 hour, take a small amount from the polypeptide solid phase synthesis tube into a 1.5 mL centrifuge tube. The test reagent is colorless, which is a negative result, which proves that the condensation reaction is successful. Remove the liquid in the polypeptide solid phase synthesis tube, wash it 3 times with DMF and DCM, and obtain the polypeptide resin after the second amino acid condensation.

[0062] (3) Repeat the above reaction steps until the last amino acid (K) has reacted completely, and wash three times each with DMF and DCM. Add the cisplatin chemotherapy drug obtained in Example 1 and react for 12 hours. After the reaction is complete, wash three times each with DMF and DCM. Rinse three times with methanol and then dry.

[0063] (4) The shrunken peptide containing resin was placed in a vial containing a prepared lysis buffer (a trifluoroacetic acid solution containing 2.5% water, 2.5% ethylenedithiol, and 2.5% triisopropylsilane) and lysed in an ice-water bath for 3 hours. After drying with nitrogen to remove the trifluoroacetic acid, the resulting peptide was washed three times with anhydrous methyl tert-butyl ether and left overnight. The peptide was purified using preparative reversed-phase HPLC to obtain the HER2-targeted antitumor peptide nanodrug X-KHYYQNYQKCCSYL, whose molecular structure is shown below. Figure 2 As shown.

[0064] Test Example 1

[0065] Mass spectrometry characterization of HER2-targeted antitumor peptide nanomedicines:

[0066] The product obtained in Example 2 was dissolved in an aqueous solution (500 μg / mL), and a small amount of the solution was analyzed by liquid chromatography-mass spectrometry (LC-MS 8050). The mass spectrum is shown below. Figure 3 As shown, according to the mass spectrometry detection results, the molecular formula of the polypeptide nanomedicine is C0. 89 H 131 Cl2N 23 O 27 PtS2; [M+H] + :2284.1; [M+2H] 2+ :1142.6; [M+3H] 3+ The molecular weights are 762.0; m / z: 1142.5 and 762.0. The molecular weights are consistent with the theoretical molecular weights, indicating the successful synthesis of the peptide nanomedicine.

[0067] Test Example 2

[0068] Liquid phase characterization of HER2-targeted antitumor peptide nanomedicines:

[0069] The product obtained in Example 2 was dissolved in an aqueous solution (500 μg / mL), and a small amount of the solution was analyzed by high-performance liquid chromatography (HPLC). The HPLC chromatogram is shown below. Figure 4 As shown, according to the high-performance liquid chromatography (HPLC) results, the purity of the polypeptide nanomedicine is 98.2%.

[0070] Test Example 3

[0071] CD validation of HER2-targeted anti-tumor peptide nanomedicines:

[0072] The product obtained in Example 2 was prepared into a 20 μL stock solution of 10 mmol / mL DMSO, which was then diluted with water to a concentration of 100 μmol / mL, with a volume of 1 mL. The CD spectrum was detected using a J-1500 circular dichroism spectrometer. The circular dichroism spectroscopy method was as follows: a quartz cuvette with a path length of 1 cm was used; the scanning speed was set to 1 nm / s; the scanning range was 180 nm–265 nm; and the number of repeated scans was 3. The N2 flow rate was 15–20 L / min. The background was first subtracted using a 20% acetonitrile solution, and then the sample solutions were scanned separately. The results are as follows: Figure 5 As shown: The polypeptide nanomedicine with HER2 protein targeting function in this invention forms a β-sheet conformation at 100 μmol / mL.

[0073] Test Example 4

[0074] FTIR validation of HER2-targeted anti-tumor peptide nanomedicines:

[0075] For infrared spectroscopy determination, the sample solution was dropped onto a CaF2 slide. Specifically, the product obtained in Example 2 was prepared as a 20 μL stock solution of 10 mmol / mL DMSO, diluted with water to 100 μmol / mL (1 mL volume), and the micro-infrared structure was measured. 500 μL of the sample was placed in a 2 mL centrifuge tube and incubated at 20°C for 24 h. A small amount of the liquid was pipetted onto a CaF2 slide, dried, and then measured using an infrared spectrometer. The parameters were set as follows: scan range 1000 cm⁻¹. -1 -4000cm -1 The number of scans was 8. The results are as follows: Figure 6 As shown: The polypeptide nanomedicine with HER2 protein targeting function in this invention forms a β-sheet conformation at 100 μmol / mL.

[0076] Test Example 5

[0077] TEM validation of HER2-targeted anti-tumor peptide nanomedicine:

[0078] The product obtained in Example 2 was dissolved in a pre-prepared mixed solution of 20% acetonitrile and water to a concentration of 200 μmol / mL, and allowed to stand for 24 h to allow for complete assembly. 10 μL was dropped onto a copper mesh, and the sample was deposited for 10 min. Unadsorbed sample at the edge of the copper mesh was blotted dry along the edge using filter paper. The sample was stained with uranium acetate-hydrogen peroxide solution for 2 min, washed three times with deionized water, blotted dry along the edge with filter paper, allowed to stand, and then dried. Real-time CCD imaging was performed using a transmission electron microscope (T20). Results are as follows: Figure 7 As shown: In this invention, a polypeptide nanomedicine with HER2 protein targeting function is formed into a polypeptide nanofiber structure at 200 μmol / mL.

[0079] Test Example 6

[0080] Validation of the targeting ability of HER2-targeted anti-tumor peptide nanomedicines:

[0081] First, the peptide nanomedicines were fluorescently labeled. The fluorescent dye fluorescein isothiocyanate (FITC) was used to react with the peptide nanomedicines for 12 hours to obtain FITC-labeled peptide nanomedicines. SKOV-3 and MDA-MB-231 cells were cultured separately. When the cell density reached 80%, cell counting was performed, and 5 × 10⁶ cells of each cell type were collected. 4 Cells were seeded in different confocal cell culture dishes. After the cells were fully adhered, 50 μM of FITC-labeled peptide nanomedicine was added to stimulate the different cells for 4 hours. After washing three times with PBS, cell culture medium was added, and the uptake of nanomaterials by the cells was observed immediately under a confocal microscope with a 60× oil objective and a 488 nm excitation channel.

[0082] The results are as follows Figure 8 As shown in the figure, the peptide nanomedicine has a good targeting effect on HER2 positive cells, but no targeting effect on HER2 negative cells, proving that the designed peptide nanomedicine has excellent HER2 protein targeting performance.

[0083] Test Example 7

[0084] Validation of tumor cell toxicity of HER2-targeted anti-tumor peptide nanomedicines:

[0085] The toxicity of the peptide material to tumor cells was determined using the Cell Counting Kit-8 (CCK-8). SKOV-3 cells were cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin-dextrose antibody. When the cells reached 80% confluence, they were trypsinized, centrifuged at 1000 rpm for 5 min, and cell counts were performed. The cell concentration was then adjusted to 1 × 10⁻⁶ cells / mL. 5Cells were seeded at a density of 100 μL / mL in 96-well plates, with 100 μL of cell suspension added to each well. 100 μL of PBS was added to the outermost ring of the 96-well plate. The plates were incubated at 37°C, 5% CO2, and saturated humidity for 24 hours. The culture medium was then aspirated and replaced with complete culture medium containing the desired concentration of peptide nanomedicine. After another 24 hours of incubation, the culture medium was aspirated, and the plates were washed three times with PBS. The cells were then added to McCoy's 5A medium containing 10% CCK8 and incubated at 37°C, 5% CO2, and saturated humidity for 2 hours. The cells were wrapped in aluminum foil and transferred to an ELISA reader. The absorbance was set to A=450nm, and A=650nm was set as the reference wavelength for measurement. The data were recorded, and the cell activity (%) was calculated as (experimental group measurement value - blank value) / (control group measurement value - blank value) × 100%. The concentration gradients were set to 960, 480, 240, 120, 60, 30, 15, and 0 μM. The results were statistically analyzed.

[0086] The results are as follows Figure 9 As shown, the IC50 value of the polypeptide nanomedicine with HER2 protein targeting function involved in this invention is about 60 μM, indicating that it has a significant anti-tumor effect.

[0087] Test Example 8

[0088] Validation of the therapeutic efficacy of HER2-targeted anti-tumor peptide nanomedicines at the tumor site:

[0089] Eight-week-old female SPF-grade BALB / c nude mice were randomly divided into two groups of five each: one group received PBS and the other received peptide nanomedicine. The mice, weighing 18-20g, were housed in specialized cages in the animal facility. After seven days of acclimatization, and once normal physiological activity was observed, the mice were used to establish an animal model.

[0090] Thaw the matrix gel in advance, and place pipette tips, syringes, and centrifuge tubes at 4°C 12 hours in advance. Perform hair removal on the experimental mice 12 hours in advance. Culture SKOV-3 cells. When the cells reach 80% confluence, digest with trypsin, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, resuspend in PBS, centrifuge at 1000 rpm for 5 minutes, count the cells, and adjust the cell concentration to 5 × 10⁶ cells / mL with a cell suspension:Matrix gel ratio of 1:1. 7 After achieving a cell density of [number] cells / mL, the cells were placed on ice and immediately transferred to the animal room. Each mouse was injected with 100 μL of the cell suspension via the tail vein. The animals were observed for 30 minutes post-injection; no abnormalities were observed. The mice were observed the following day, and the tumor size was measured using calipers. When the transplanted tumor grew to approximately 100 mm... 3The experimental treatment began. Drugs were administered via tail vein, once every two days, at a dose of 5 mg / kg in 100 μL PBS, for 22 consecutive days. Tumor volume changes and survival curve changes were measured and recorded every other day for different mice.

[0091] The results are as follows Figure 10 and Figure 11 As shown in the results, the polypeptide nanomedicine involved in this invention has a significant inhibitory effect on tumors.

[0092] Test Example 9

[0093] Validation of the long-term retention effect of HER2-targeted anti-tumor peptide nanomedicines on tumor cells:

[0094] The polypeptide nanomedicine obtained in Example 2 was prepared into a 1 mg / mL drug solution and diluted to 100, 80, 20, 10, 5, and 1 μg / mL. After setting the method file using LC-MS 8050, a standard curve was prepared. The LC-MS conditions included: binary high-pressure gradient elution mode, total flow rate of 0.3 mL / min, maximum column pressure of 50 MPa, nebulizer gas flow rate of 3.0 L / min, drying gas flow rate of 10.0 L / min, heating gas flow rate of 10.0 L / min, interface temperature of 300 °C, DL temperature of 250 °C, heating block temperature of 400 °C, and injection volume of 1 μL. The time-programmed method is as follows: Solvent A is 100% mass-grade water containing 0.1% mass-grade formic acid; Solvent B is 100% mass-grade acetonitrile containing 0.1% mass-grade formic acid; at 0.01 min, 20% solvent B is used, and at 6 min, 90% solvent B is used. The resulting standard curve is shown below. Figure 12 As shown.

[0095] The polypeptide nanomedicine prepared in Example 2 was formulated to a concentration of 80 μg / mL. SKOV-3 cells were cultured, and when the growth density reached 80%, cell counting was performed, and 5 × 10⁶ cells were collected. 4Cells were seeded in confocal cell culture dishes. After complete cell adhesion, the cells were co-incubated with 80 μg / mL of peptide nanomedicine, and 200 μL samples were taken at different time points for quantitative analysis. The concentration of the peptide nanomedicine was determined using LC-MS 8050 after setting the method file. The LC-MS conditions included: binary high-pressure gradient elution mode, total flow rate of 0.3 mL / min, maximum column pressure of 50 MPa, nebulizer gas flow rate of 3.0 L / min, drying gas flow rate of 10.0 L / min, heating gas flow rate of 10.0 L / min, interface temperature of 300 °C, DL temperature of 250 °C, heating block temperature of 400 °C, and injection volume of 1 μL. The time program method is as follows: Solvent A is 100% mass spectrometry grade water containing 0.1% mass spectrometry grade formic acid; Solvent B is 100% mass spectrometry grade acetonitrile containing 0.1% mass spectrometry grade formic acid; at 0.01 min, 20% solvent B, and at 6 min, 90% solvent B.

[0096] The results are as follows Figure 13 As shown in the figure, after 12 hours of co-incubation with SKOV-3 cells, the polypeptide nanomedicine was completely consumed, indicating that the polypeptide nanomedicine involved in this invention has a long retention effect on tumor cells.

[0097] The applicant declares that this invention illustrates a HER2-targeted anti-tumor polypeptide nanomedicine, its preparation method, and its application through the above embodiments. However, this invention is not limited to the above embodiments, meaning that this invention does not necessarily rely on the above embodiments for implementation. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection and disclosure scope of this invention.

[0098] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0099] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A HER2-targeted antitumor polypeptide nanodrug, characterized in that, The anti-tumor polypeptide nanodrug comprises an anti-tumor functional unit, a self-assembly unit KHYYQNYQ and a targeting unit KCCSYL connected in sequence; the targeting unit is a polypeptide sequence targeting HER2 protein; The anti-tumor functional unit is an anti-tumor drug cisplatin.

2. The method for preparing the HER2-targeted antitumor polypeptide nanodrug according to claim 1, characterized in that, The preparation method comprises: The preparation method comprises:

3. The method for preparing the HER2-targeted antitumor polypeptide nanodrug according to claim 2, characterized in that, The preparation method comprises: (1) fixing the C-terminal of the first amino acid in the targeting unit on a resin, performing Fmoc protection on the N-terminal, performing Boc protection on the side chain, and then performing swelling treatment; (2) removing the Fmoc protection on the N-terminal of the first amino acid by a deprotection agent, adding the next amino acid for reaction after washing, and sequentially connecting the amino acids to obtain a polypeptide sequence fixed on the resin; (3) mixing the obtained polypeptide sequence fixed on the resin with an anti-tumor functional unit modified by a group, then shrinking the resin, cleaving, and blowing dry with nitrogen to obtain the HER2-targeted anti-tumor polypeptide nanodrug.

4. Application of the HER2-targeted anti-tumor polypeptide nanodrug in claim 1 in the preparation of a drug for diagnosing, preventing or treating tumors.

5. Use according to claim 4, characterized in that, The tumor is a tumor overexpressing HER2.

6. Use according to claim 4, characterized in that, The tumor comprises any one of breast cancer, gastric cancer, leukemia, bladder cancer, cervical cancer or nasopharyngeal cancer.

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