Enzymatic polypeptide self-assembly targeting degradation of her2 protein and applications thereof

Abstract

The application provides an enzymatic polypeptide self-assembly for targeting HER2 protein degradation and an application thereof, and relates to the technical field of biological medicines.The enzymatic polypeptide self-assembly for targeting HER2 protein degradation has the functions of enzyme catalytic cascade activation and enhancement of imaging and targeting HER2 protein degradation.The enzymatic polypeptide self-assembly material organically integrates the imaging unit and the treatment unit into the same carrier, avoids bringing additional physical burden to organisms, and has a good synergistic effect on intuitively and accurately revealing the targeting efficiency of polypeptide compounds targeting tumor cell membrane proteins and improving the accuracy of tumor targeted treatment, solves the problems of low membrane protein degradation efficiency and insufficient targeting in the prior art, provides a new strategy for precise tumor treatment, and has important clinical conversion value and application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an enzymatic polypeptide self-assembly that targets the degradation of HER2 protein and its applications. Background Technology

[0002] The human epidermal growth factor receptor (EGFR / ERBB) family comprises four members: HER1 (EGFR, ERBB 1), HER2 (NEU, ERBB 2), HER3 (ERBB 3), and HER4 (ERBB 4), all associated with numerous cancers. Among them, HER2 is a unique receptor molecule because it lacks a ligand but acts as a co-receptor, forming homodimers and heterodimers with the other three HER family proteins (1, 3, and 4). Furthermore, HER2 is a transmembrane glycoprotein that activates downstream signaling pathways through dimerization, promoting the proliferation and survival of malignant cancer phenotypes. HER2 is overexpressed in various tumors, including breast cancer, gastric cancer, ovarian cancer, prostate cancer, and lung cancer; therefore, HER2 is considered an important therapeutic target for these diseases. Trastuzumab, a monoclonal antibody targeting HER2, has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of HER2-positive cancers. However, acquired resistance develops after a period of use with trastuzumab. Therefore, developing effective drugs targeting HER2 protein degradation is a crucial and highly challenging task.

[0003] Drugs targeting membrane proteins typically bind to the binding sites of the target proteins, leading to problems such as acquired mutational resistance or compensatory pathways that confer resistance in vivo. Targeted protein degradation (TPD) technology, as an emerging drug development strategy, selectively degrades specific proteins, effectively overcoming the limited efficacy and resistance of traditional inhibitors and showing significant therapeutic potential. Currently, methods for degrading cell membrane proteins include lysosomal-targeting chimeras, cytokine receptor-targeting chimeras, dendritic DNA chimeras, and integrin-promoted lysosomal degradation. While these protein degradation strategies represent significant progress, many methods rely on macromolecules, such as bispecific antibodies and aptamers, which are structurally complex, difficult to synthesize, and often lack stability. Therefore, protein degradation strategies still face significant challenges.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an enzymatically catalyzed polypeptide self-assembly targeting HER2 protein degradation and its applications. This HER2-targeting polypeptide can serve as a HER2-targeting protein degrader or drug delivery carrier for the preparation of drugs to treat HER2-positive tumors. It solves the problems of low membrane protein degradation efficiency and insufficient targeting in existing technologies, providing a new strategy for precision tumor treatment and possessing significant clinical translational value and application prospects.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted: This invention provides an enzymatic peptide self-assembly targeting HER2 protein degradation, wherein the structure of the enzymatic peptide self-assembly for HER2 protein degradation is as follows: p-dye-(X)-RTYGKRPKIR; Among them, RTYGKRPKIR is the sequence that targets the polypeptide chain of the HER2 protein; p represents a phosphate group; (X) represents a self-assembly unit.

[0007] Furthermore, the self-assembly unit is a dipeptide phenylalanine-phenylalanine (FF).

[0008] Furthermore, the dye is a hydrophobic dye.

[0009] Furthermore, the dye is Cy5, and the structure of the enzymatic polypeptide self-assembly targeting HER2 protein degradation is as follows: p-Cy5-FF-RTYGKRPKIR.

[0010] This invention also provides a method for preparing the above-mentioned enzymatic polypeptide self-assemblies targeting HER2 protein degradation, comprising the following steps: S1. A polypeptide with DBCO-(X)-RTYGKRPKIR was synthesized using a solid-phase synthesis method. S2. The polypeptide obtained in S1 is linked with the azide-modified dye through a copper-free click chemical reaction to obtain the dye -(X)-RTYGKRPKIR. S3. The product obtained in S2 is phosphorylated to obtain an enzymatically catalyzed polypeptide self-assembly that targets the degradation of HER2 protein.

[0011] Furthermore, the specific steps of S1 are as follows: S101. Weigh the resin and add DCM. After swelling, remove the DCM. S102. Dissolve Fmoc-arginine in DCM, then add DIEPA and mix well. React with the resin at room temperature by shaking. After the reaction is complete, wash the resin thoroughly with DCM. S103. Then, the unreacted sites on the resin are sealed with a sealing solution, followed by washing the sealed resin with DCM and DMF. S104. Add 20% piperidine DMF solution to the resin obtained in S103, shake the reaction at room temperature to remove the Fmoc protecting group at the N-terminus of the amino acid, exposing the free amino group, and then wash thoroughly with DMF to obtain product 1. S105. In another container, Fmoc-Ile(I)-OH and HATU are dissolved in DMF, DIEPA is added and mixed evenly, and then the mixture is allowed to stand for activation to form an activated ester. S106. Add the activated ester obtained in S105 to product 1 and react it with the deprotected amino groups on the resin. After the reaction is complete, wash the resin thoroughly with DMF. S107, repeat S103 and S104, and then couple the remaining amino acids in sequence: R, K, P, K, I, R, G, Y, T, R, F, F. S108. After all amino acids are coupled, perform the final Fmoc removal to expose the N-terminal amino group of the complete polypeptide chain. Then, dissolve the polypeptide and HATU in DMF, add DIEPA and react. After the reaction, wash with DMF and DCM. S109. The polypeptide derivative obtained in S108 is removed from the resin to obtain the crude product; S110. The crude product is purified and freeze-dried to obtain a polypeptide with the DBCO-(X)-RTYGKRPKIR structure.

[0012] Furthermore, the capping solution was prepared by mixing DCM:DIEA:methanol = 17:1:2.

[0013] Furthermore, the specific steps of S2 are as follows: DBCO-(X)-RTYGKRPKIR prepared by S1 was dissolved in methanol, and then fluorescent dye molecules containing azide groups were added. Through a copper-free click chemistry reaction, a product combining the dye and the polypeptide derivative was constructed.

[0014] Furthermore, the specific steps of S3 include: Phosphorus oxychloride was added to the product obtained in S2, and the pH was adjusted to alkaline to obtain the phosphorylated product.

[0015] Furthermore, the pH is 8-10.

[0016] Furthermore, the solid-phase synthesis method of S1 employs an Fmoc protection strategy, which uses 2-Cl-Trt resin or Rink resin for protection.

[0017] This invention also provides the application of enzyme-based self-assembly targeting HER2 protein degradation peptides in the preparation of drugs for treating tumors.

[0018] Furthermore, the tumor is a tumor that highly expresses HER2.

[0019] Furthermore, the tumors that highly express HER2 include: breast cancer, gastric cancer, ovarian cancer, prostate cancer, or lung cancer.

[0020] The present invention also provides a pharmaceutical composition comprising the above-described HER2 protein-targeting degradation peptide.

[0021] This invention also provides a method for preparing enzymatically catalyzed polypeptide self-assemblies, wherein S3-obtained HER2 protein-targeting degradation polypeptides are co-incubated with alkaline phosphatase in phosphate buffer, resulting in dephosphorylation and self-assembly into nanofibers.

[0022] Furthermore, the reaction conditions for alkaline phosphatase are 36℃-38℃.

[0023] Furthermore, the pH for co-incubation with alkaline phosphatase in phosphate buffer is 8-10.

[0024] The present invention also provides an enzyme-catalyzed polypeptide self-assembly prepared by the above-mentioned method.

[0025] The present invention also provides the application of the above-mentioned enzymatic polypeptide self-assembly in the preparation of drugs for treating tumors.

[0026] Furthermore, the tumor is a tumor that highly expresses HER2.

[0027] The present invention has the following technical effects: (1) This invention prepares a polypeptide derivative containing an ALP restriction site that can be self-assembled by enzyme catalysis, enabling it to form nanofibers through enzyme catalysis at specific sites with high enzyme specificity. The nanofibers obtained by enzyme-catalyzed treatment are processed under physiological conditions without the need for high temperature or ultraviolet radiation, thus avoiding damage to bioactive substances caused by high temperature or radiation, and are more suitable for the temperature-sensitive in vivo environment.

[0028] (2) The p-dye-FF-RTYGKRPKIR prepared in this invention has a hydrophilic phosphate group (-PO3H). It is initially a water-soluble small molecule probe with pre-quenched near-infrared fluorescence and selective targeting of HER2 protein. After systemic administration, due to its small molecule diffusivity, hydrophilicity and HER2 protein targeting, p-dye-FF-RTYGKRPKIR can easily be actively targeted to tumor tissue.

[0029] (3) The p-dye-FF-RTYGKRPKIR described in this invention, when activated by endogenous ALP overexpressed on the tumor cell membrane in ALP-positive tumor tissue, emits near-infrared fluorescence at a wavelength of 720 nm. Simultaneously, p-dye-FF-RTYGKRPKIR is dephosphorylated, releasing the dephosphorylated product: dye-FF-RTYGKRPKIR. Since dye-FF-RTYGKRPKIR contains FF dipeptide, it can provide effective intermolecular interactions (e.g., hydrophobic interactions, π-π stacking) and promote molecular self-assembly. Subsequently, dye-FF-RTYGKRPKIR undergoes in-situ self-assembly at the tumor site, forming supramolecular nanofibers that can efficiently bind to the tumor cell membrane, and thus tightly bind to the HER2 protein surface on the tumor cell membrane. This process is equivalent to attaching a hydrophobic tag to the surface of the target protein. According to cellular mechanisms, it can be recognized by the cell as a partially denatured state of the protein (exposed hydrophobic core), thereby endocytizing it into the cell and degrading it through the proteasome pathway.

[0030] (4) The p-dye-FF-RTYGKRPKIR described in this invention forms supramolecular nanofibers in ALP-positive tumor tissues after enzymatic catalysis. This prolongs circulation, enhances tumor retention and retention time, and achieves the purpose of continuously activating HER2 protein degradation, thereby promoting a strong anti-tumor inhibitory effect. This method not only utilizes the high selectivity of targeting peptide derivatives and the long-term tumor retention characteristics of enzyme-catalyzed self-assembled peptides to form nanomaterials, but also shows promise for application in the efficient targeting of tumor cell membrane protein degradation.

[0031] (5) The p-dye-FF-RTYGKRPKIR described in this invention can achieve better tumor inhibition effect in HER2 positive tumor tissues compared with the control group. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 It is the structural formula of p-Cy5-FF-RTYGKRPKIR; Figure 2 It is the structural formula of p-Cy5-RTYGKRPKIR; Figure 3 This is the mass spectrum of p-Cy5-FF-RTYGKRPKIR; Figure 4 This is the mass spectrum of p-Cy5-RTYGKRPKIR; Figure 5 These are transmission electron microscope (TEM) images of p-Cy5-FF-RTYGKRPKIR before and after ALP activation; Figure 6 These are transmission electron microscope (TEM) images of p-Cy5-RTYGKRPKIR before and after ALP activation; Figure 7 These are fluorescence spectra of p-Cy5-FF-RTYGKRPKIR incubated with ALP for different times; Figure 8 It is the cytotoxicity of p-Cy5-FF-RTYGKRPKIR; Figure 9 These are the results of the immunoblotting experiment of p-Cy5-FF-RTYGKRPKIR; Figure 10 It is in vivo tumor-targeted fluorescence imaging of p-Cy5-FF-RTYGKRPKIR; Figure 11 These are in vitro near-infrared fluorescence images of major organs and tumors in tumor-bearing mice 48 h after tail vein injection of a polypeptide derivative. Figure 12 This refers to the in vivo antitumor inhibitory effect of p-Cy5-FF-RTYGKRPKIR. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0035] The reagents used in the embodiments of this invention are as follows: Various F-moc amino acids: Jier Biochemical (Shanghai) Co., Ltd., 98% purity; Dichloropolymer resin: Jier Biochemical (Shanghai) Co., Ltd., substitution rate 1.158 mmol / g; N,N-Diisopropylethylamine (DIEPA): Shanghai Aladdin Biochemical Technology Co., Ltd., purity 99%; 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU): Tianjin Xiens Biochemical Technology Co., Ltd., purity 98%; Trifluoroacetic acid (TFA): Shanghai Maclean Biochemical Technology Co., Ltd., purity 99%; Triisopropylsilane (TIS): Shanghai Aladdin Biochemical Technology Co., Ltd., purity 99%; Anhydrous dichloromethane (DCM): Tianjin Chemical Reagent Company; N,N-Dimethylformamide (DMF): Tianjin Chemical Reagent Company; Piperidine: Tianjin Chemical Reagent Company; Alkaline phosphatase (30 U / μL): Yisheng Biotechnology Co., Ltd.; Enhanced CCK-8 Reagent Kit: Tianjin Mederma Biotechnology Co., Ltd.; HER2(1C4) mouse monoclonal antibody: Tianjin Mederma Biotechnology Co., Ltd.; Balb / c mice, 6-8 weeks old, female: Henan Skebest Biotechnology Co., Ltd.

[0036] The following is a detailed explanation using specific embodiments: Example 1: Preparation of p-Cy5-RTYGKRPKIR and p-Cy5-FF-RTYGKRPKIR S101. Weigh 2-Cl-Trt resin and place it in a solid-phase reactor. Add anhydrous dichloromethane to swell it for 10 minutes, and then remove DCM. S102. Dissolve Fmoc-arginine in DCM, add DIEPA and mix well. React with the resin at room temperature with shaking for 1-2 hours (preferably 1.5 hours in this example). After the reaction is complete, wash the resin thoroughly with DCM 3-5 times (preferably 5 times in this example). S103. Add a sealing solution consisting of DCM:DIEA:methanol = 17:1:2 and react at room temperature for 30 minutes to seal unreacted sites on the resin. Then wash the sealed resin with DCM and DMF. S104. Add 20% piperidine DMF solution to the resin obtained in S103, and shake the reaction at room temperature for 20-30 minutes (preferably 25 minutes in this embodiment) to remove the Fmoc protecting group at the N-terminus of the first amino acid and expose the free amino group. Then wash thoroughly with DMF to obtain product 1. S105. In another container, dissolve Fmoc-Ile(I)-OH and HATU in DMF, add DIEPA and vortex mix evenly, let stand at room temperature for 1-2 minutes to activate, and form activated ester. S106. Add the activated ester obtained in S105 to product 1 and react with the deprotected amino groups on the resin for 1 hour. After the reaction is completed, wash the resin thoroughly with DMF 3-5 times (preferably 5 times in this example). S107, repeat S103 and S104, and then couple the remaining amino acids in sequence: R, K, P, K, I, R, G, Y, T, R, F, F. S108. After all amino acids are coupled, perform the final Fmoc removal to expose the N-terminal amino group of the complete polypeptide chain. Then, dissolve the polypeptide and HATU in DMF, add DIEPA and react. After the reaction, wash with DMF and DCM. S109. The polypeptide derivative obtained in S108 is removed from the resin to obtain the crude product; S110. The crude product is purified and freeze-dried to obtain a polypeptide with the DBCO-FF-RTYGKRPKIR structure.

[0037] S2. Construct the phosphorylated peptide derivative p-Cy5-FF-RTYGKRPKIR via a copper-free click chemistry reaction. The specific steps are as follows: The DBCO-FF-RTYGKRPKIR polypeptide derivative prepared in step 1 was dissolved in methanol, and then fluorescent dye molecules (Cy5) containing azide groups were added at a molar ratio of 1:1. The mixture was stirred overnight at room temperature.

[0038] S3. Next, excess phosphorus oxychloride (POCl3) was added to the product of S2, and the pH was adjusted to alkaline (pH=9 in this example) with pyridine. The mixture was stirred overnight at room temperature to obtain the final phosphorylated product p-Cy5-FF-RTYGKRPKIR. Finally, the molecular weight and purity were determined by high-resolution mass spectrometry. The results are shown below. Figure 2 , Figure 2 This is the high-resolution mass spectrum of p-Cy5-FF-RTYGKRPKIR, showing a quintet [M+5H] for p-Cy5-FF-HER2. 5+ The molecular weight was 478.25, which is the target molecular weight, indicating that the prepared p-Cy5-FF-RTYGKRPKIR was successful; the structural formula is shown below. Figure 1 , Figure 1 The structure is p-Cy5-FF-RTYGKRPKIR.

[0039] The preparation of the phosphorylated product p-Cy5-RTYGKRPKIR was identical to that of p-Cy5-FF-RTYGKRPKIR, except that the two phenylalanines (F) were not added during the synthesis. Results are shown below. Figure 4 The results showed a septet [M+7H] for p-Cy5-RTYGKRPKIR. 7+ The molecular weights were 300.80, which are the target molecular weights, indicating that the prepared p-Cy5-RTYGKRPKIR was successful; the structural formula is shown below. Figure 3 .

[0040] Example 2: Microstructure characterization of p-Cy5-FF-RTYGKRPKIR and p-Cy5-RTYGKRPKIR The polypeptide prepared in Example 1 was weighed and dissolved in phosphate-buffered saline (PBS, pH 7.4, 1 mL) (0.5 mM). Then, ALP (2 U / mL, 100 µL) was added and the mixture was placed in a 37°C incubator for 12 hours. After enzyme catalysis, transmission electron microscopy (TEM) samples were prepared from the samples before and after enzyme addition to observe their microstructure. The results are shown in the figure. Figure 5 and Figure 6 .

[0041] Figure 5 These are transmission electron microscopy (TEM) images of p-Cy5-FF-RTYGKRPKIR before and after ALP activation. The results show that p-Cy5-FF-RTYGKRPKIR is in a molecular state before ALP dephosphorylation and cannot assemble into nanostructures. The absence of a Tyndall effect when the solution is irradiated with a laser further confirms that p-Cy5-FF-RTYGKRPKIR is in a molecular state before ALP dephosphorylation. However, after ALP-catalyzed dephosphorylation, self-assembly occurs to form nanofibers. The presence of a Tyndall effect when the solution is irradiated with a laser further demonstrates that p-Cy5-FF-RTYGKRPKIR self-assembles into nanomicelles after ALP dephosphorylation.

[0042] Figure 6 These are transmission electron microscopy (TEM) images of p-Cy5-RTYGKRPKIR before and after ALP activation. The results show that p-Cy5-RTYGKRPKIR is in a molecular state before ALP dephosphorylation and cannot assemble into nanostructures. The absence of a Tyndall effect when the solution is irradiated with a laser further confirms that p-Cy5-RTYGKRPKIR is in a molecular state before ALP dephosphorylation. Furthermore, no colloidal formation of nanostructures was observed after ALP-catalyzed dephosphorylation; the absence of a Tyndall effect when the solution is irradiated with a laser further confirms that p-Cy5-RTYGKRPKIR cannot self-assemble into nanomicelles after ALP dephosphorylation.

[0043] Figure 5 and Figure 6 Comparing the transmission electron microscopy (TEM) results with those of the other samples, it is shown that the introduction of the self-assembly unit FF can induce the self-assembly of polypeptide derivatives to form nanostructures.

[0044] Example 3: Fluorescence spectrum of p-Cy5-FF-RTYGKRPKIR Weigh the polypeptide prepared in Example 1 and dissolve it in phosphate-buffered saline (PBS, pH=7.4, 1 mL) (0.5 mM). Take 100 µL, dilute it with PBS to 0.05 mM, then add ALP (2 U / mL, 100 µL) and place it in a 37 ℃ incubator. Incubate for different times and take samples for fluorescence spectroscopy scanning. The results are shown in the figure. Figure 7 .

[0045] Figure 7 The images show the fluorescence spectra of the p-Cy5-FF-RTYGKRPKIR peptide derivative after ALP activation at different times. The results indicate that the fluorescence intensity of p-Cy5-FF-RTYGKRPKIR changes with the duration of ALP addition, exhibiting a trend of quenching followed by activation and gradual enhancement. This is because p-Cy5-FF-RTYGKRPKIR undergoes dephosphorylation after ALP catalysis, forming hydrophobic Cy5-FF-RTYGKRPKIR, which then self-assembles to form a nanofiber structure.

[0046] Example 4: Cytotoxicity of p-Cy5-FF-RTYGKRPKIR The cytotoxicity of the p-Cy5-FF-RTYGKRPKIR peptide derivative was detected using the CCK-8 assay. The specific procedures were as follows: Mouse TUBO breast cancer cells were seeded at a density of 1000 cells / well in 96-well plates and cultured overnight at 37°C in a cell culture incubator containing 5% CO2. After cell attachment, the culture medium was removed. Then, 100 μL / well of DMEM medium containing different concentrations (0, 2, 4, 8, 16, 32, 64, 128 µM) of peptide derivatives was added, and the plates were cultured for another 24 h. The supernatant was then discarded, and 100 µL of DMEM medium containing 10% CCK-8 was added to each well, and the plates were incubated at 37°C for 30 min. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. The results are shown below. Figure 8 .

[0047] Figure 8The results show the cell viability of TUBO and 4T1 cells after incubation with different concentrations of peptide derivatives for 24 hours. The results indicated that after treatment with different concentrations of peptide derivatives, p-Cy5-FF-RTYGKRPKIR exhibited significant cytotoxicity with increasing peptide derivative concentration. Cy5 showed no significant cytotoxicity below 128 µM, while p-Cy5-RTYGKRPKIR, even at a concentration as high as 128 µM, still maintained a cell viability of 36.62%. Similarly, after treatment with different concentrations of peptide derivatives, neither Cy5 nor p-Cy5-RTYGKRPKIR showed significant cytotoxicity below 128 µM, while p-Cy5-FF-RTYGKRPKIR, at a concentration as high as 128 µM, still maintained a cell viability of 48.2%, indicating that it did not exhibit significant cytotoxicity below 128 µM. This is because TUBO cells are cells that highly express HER2 protein and contain highly expressed ALP, while 4T1 cells, although highly expressing ALP, are HER2 protein negative.

[0048] Example 5: Immunoblot assay of p-Cy5-FF-RTYGKRPKIR Western blotting (WB) was used to characterize the degradation of HER2 protein on the TUBO cell membrane by peptide derivatives. The specific procedures were as follows: TUBO cells were seeded in 6-well plates and cultured at 37°C in a cell culture incubator containing 5% CO2. Once the cell density reached approximately 80%, a 2 μM peptide solution was added, and the plates were incubated at 37°C in a 5% CO2 incubator for 24 h. The original solution was then discarded, and the cells were washed three times with PBS buffer. Cells were collected from the plates using a cell scraper and lysed using enhanced RIPA lysis buffer to obtain cell membrane proteins. After quantification using BCA, an equal volume (20 µg) of the sample was subjected to sodium dodecyl sulfate-separated polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with rapid blocking buffer for 10 min to eliminate nonspecific interference. The membrane was then washed three times with TBST for 10 min each time. Next, HER2 and GADPH antibodies (as internal control proteins) were prepared and incubated at room temperature for 2 h. The membrane was then washed three times with TBST for 10 min each time. Finally, the membrane was incubated with HRP-labeled secondary antibody at room temperature for 1 h. The strip was cleaned three times with TBST, 10 minutes each time. Next, the strip was soaked in developer for 10 seconds, and then exposed and inspected using a Tanon 5500 exposure unit. The results are shown below. Figure 9 .

[0049] Figure 9The images show Western blotting (WB) bands of HER2 protein in TUBO cells after incubation with different peptide derivatives. The results indicate that, while maintaining a relatively consistent grayscale value for the internal control protein band, the HER2 protein band on the TUBO cell membrane treated with p-Cy5-FF-RTYGKRPKIR exhibited the lowest grayscale value compared to the other three control groups. This suggests that the expression level of HER2 protein on the TUBO cell membrane treated with p-Cy5-FF-RTYGKRPKIR was significantly lower than in the other three groups. In other words, p-Cy5-FF-RTYGKRPKIR can induce the degradation of HER2 protein on the cell membrane, thereby leading to a decrease in the grayscale value of the HER2 protein band on the cell membrane. Subsequently, WB was used to confirm the degradation potential of p-Cy5-FF-RTYGKRPKIR for HER2 protein. TUBO cells treated with different concentrations of p-Cy5-FF-RTYGKRPKIR were analyzed for HER2 protein degradation. The results showed that, while maintaining a relatively consistent gray level of the internal control protein band, the degradation of HER2 protein on the TUBO cell membrane gradually increased with the gradual increase of p-Cy5-FF-RTYGKRPKIR concentration.

[0050] Example 6: In vivo tumor-targeted fluorescence imaging using p-Cy5-FF-RTYGKRPKIR After thoroughly mixing the prepared TUBO cells by pipetting, transfer them to a 1 mL syringe, ensuring no air bubbles are present. Then, pump the cells at approximately 1 × 10⁻⁶. 7 The drug was subcutaneously injected into the right back of mice at a specific density. Tumor growth was observed every other day until the tumor reached a suitable size. After anesthetizing the mice with isoflurane, the hair at the tumor site was removed with depilatory cream. The polypeptide derivative (16 µM, 200 µL) was then injected into the mice via the tail vein. The mice were then placed on a small animal in vivo imaging system for imaging. The results are shown in [Figure number missing]. Figure 10 and Figure 11 .

[0051] Figure 10 This study presents in vivo near-infrared fluorescence imaging of tumors at different time points following tail vein injection of a polypeptide derivative. The results show that the near-infrared fluorescence signal at the tumor site initially increases and then decreases over time, reaching its maximum value 4 hours after injection, indicating that the polypeptide derivative reaches maximum aggregation at the tumor site 4 hours after injection.

[0052] Figure 11These are in vitro near-infrared fluorescence images of major organs and tumors in tumor-bearing mice 48 hours after tail vein injection of the peptide derivative. Tumor-bearing mice were euthanized by cervical dislocation 48 hours after tail vein injection, and their major organs and tumors were dissected and collected for in vitro near-infrared fluorescence imaging. The near-infrared fluorescence imaging results show that, 48 hours after tail vein injection, the peptide derivative was mainly distributed in the liver and spleen. Furthermore, the enrichment level of p-Cy5-FF-RTYGKRPKIR in the tumor site was significantly higher than that of p-Cy5-RTYGKRPKIR after 48 hours. This further demonstrates that p-Cy5-FF-RTYGKRPKIR, activated by endogenous ALP, can self-assemble into nanostructures, prolonging circulation and leading to the aggregation of peptide nanomaterials in the tumor site of tumor-bearing mice. This not only enhances near-infrared fluorescence imaging but also enhances the degradation of HER2 protein in tumor cell membranes, thereby inhibiting tumor growth.

[0053] Example 7: In vivo antitumor inhibitory effect of p-Cy5-FF-RTYGKRPKIR After thoroughly mixing the prepared TUBO cells by pipetting, transfer them to a 1 mL syringe, ensuring no air bubbles are present. Then, pump the cells at approximately 1 × 10⁻⁶. 7 The tumor was subcutaneously injected into the right back of mice at a specific density. Tumor growth was observed every other day until the tumor reached approximately 100 mm. 3 At that time, tumor-bearing mice were randomly divided into 4 groups of 5 mice each, and then started peptide drug treatment. The 4 groups of tumor-bearing mice were treated as follows: (1) control group - PBS buffer injected into the tail vein; (2) Cy5 solution (16 µM) injected into the tail vein; (3) p-Cy5-HER2 solution (16 µM) injected into the tail vein; (4) p-Cy5-FF-RTYGKRPKIR solution (16 µM) injected into the tail vein. Note that the volume of each injection solution was 200 μL, and it was injected once every two days for a total of 8 injections. During the treatment, the weight and tumor size of each group of mice, i.e., length a (mm) and width b (mm), were recorded every other day, and V was calculated according to the formula V=a*b. 2 / 2. Calculate tumor volume. After the treatment experiment, all mice were euthanized by cervical dislocation. Then, the tumors of each treated mouse group were dissected and collected. Bright-field images of the tumors after treatment were captured using a camera. The results are shown in [Figure 2]. Figure 12 .

[0054] Figure 12The in vivo antitumor inhibitory effect of p-Cy5-FF-RTYGKRPKIR was observed. The experimental results showed that, compared with the control group, p-Cy5-FF-RTYGKRPKIR had the most significant tumor inhibitory effect during the 15-day treatment period. This further indicates that p-Cy5-FF-RTYGKRPKIR has the strongest degradation effect on HER2 protein on the cell membrane of HER2 protein-positive tumor cells, thereby inhibiting the growth of HER2 protein-positive tumors.

[0055] This peptide self-assembled assembly targeting HER2 protein degradation possesses the function of enzymatic catalytic cascade activation, enhancing near-infrared fluorescence imaging and targeting HER2 protein degradation. This enzymatic peptide self-assembly material, which organically integrates imaging and therapeutic units onto the same carrier, avoids imposing additional physical burden on the organism, and the combination of the two has a good synergistic effect on intuitively and accurately revealing the targeting efficiency of peptide compounds against tumor cell membrane proteins and improving the precision of targeted tumor therapy. Furthermore, the peptide self-assembled assembly targeting HER2 protein degradation described in this invention mainly achieves a cascade activation mechanism through an enzyme-triggered fluorescence quenching recovery, fluorescence reaction, and in-situ peptide self-assembly strategy, which can simultaneously promote tumor near-infrared fluorescence imaging and target HER2 protein degradation. By integrating diagnostic and therapeutic functions, this therapeutic diagnostic strategy improves the precision of targeted protein degradation, providing a promising approach for cancer treatment.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. An enzymatic polypeptide self-assembly targeting HER2 protein degradation, characterized in that, The structure of the enzymatic polypeptide self-assembly targeting HER2 protein degradation is as follows: p-dye-(X)-RTYGKRPKIR; Among them, RTYGKRPKIR is the sequence that targets the polypeptide chain of the HER2 protein; p represents a phosphate group; (X) represents a self-assembly unit.

2. The enzymatic polypeptide self-assembly targeting HER2 protein degradation according to claim 1, characterized in that, The self-assembly unit is a dipeptide phenylalanine-phenylalanine.

3. The enzymatic polypeptide self-assembly targeting HER2 protein degradation according to claim 1, characterized in that, The dye is a hydrophobic dye.

4. The enzymatic polypeptide self-assembly targeting HER2 protein degradation according to any one of claims 2-3, characterized in that, The dye is Cy5, and the structure of the enzymatic polypeptide self-assembly targeting HER2 protein degradation is as follows: p-Cy5-FF-RTYGKRPKIR.

5. A method for preparing an enzymatically catalyzed polypeptide self-assembly targeting HER2 protein degradation as described in any one of claims 1-4, characterized in that, Includes the following steps: S1. A polypeptide with DBCO-(X)-RTYGKRPKIR was synthesized using a solid-phase synthesis method. S2. The polypeptide obtained in S1 is linked with the azide-modified dye through a copper-free click chemical reaction to obtain the dye -(X)-RTYGKRPKIR. S3. The product obtained in S2 is phosphorylated to obtain an enzymatically catalyzed polypeptide self-assembly that targets the degradation of HER2 protein.

6. The method for preparing enzymatically catalyzed polypeptide self-assemblies targeting HER2 protein degradation according to claim 5, characterized in that, The specific steps of S3 include: adding phosphorus oxychloride to the product obtained in S2, adjusting the pH to alkaline, and obtaining a phosphorylated product; The alkalinity specifically refers to a pH of 8-10.

7. A pharmaceutical composition, characterized in that, Includes the enzymatic peptide self-assembly targeting HER2 protein degradation as described in any one of claims 1-4, and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is a lyophilized powder for injection or an injection solution.

9. The use of an enzymatic polypeptide self-assembly targeting HER2 protein degradation as described in any one of claims 1-4 and the pharmaceutical composition as described in any one of claims 5-7 in the preparation of a medicament for treating tumors.

10. The application according to claim 9, characterized in that, The tumor is a tumor that highly expresses HER2.

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