A polypeptide nanodrug capable of inducing necroptosis and autophagy-related synergistic apoptosis, and a preparation method and application thereof

Abstract

The present application belongs to the technical field of biomedical materials and nano drugs, and discloses a polypeptide nano drug capable of inducing necrosis and autophagy related synergistic apoptosis and a preparation method and application thereof. X-DDP1 and DDP1 are self-assembled to form a responsive polypeptide nano drug, which provides a delivery platform for broad-spectrum chemotherapy drugs such as buforin RBG, irinotecan CPT11 or camptothecin CPT, realizes a Caspase-3 / GSH double-response mechanism, improves drug release specificity and spatial controllability, can promote RIPK3 aggregation and activate necrotic apoptosis in cells, can effectively inhibit the growth of cancer, and has potential clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and nanomedicines, and in particular to a polypeptide nanomedicine that can induce necrosis and autophagy-related synergistic apoptosis, its preparation method, and its application. Background Technology

[0002] The occurrence and progression of tumors are closely related to abnormal cell death signals. Traditional anticancer therapies often rely on inducing apoptosis, but due to the presence of anti-apoptotic mechanisms in tumor cells, such as caspase inactivation or Bcl-2 overexpression, the efficacy is limited. Necrotizing apoptosis is a programmed necrosis pathway that depends on the aggregation of receptor-interacting protein kinases RIPK1 and RIPK3 to form a complex. Its activation can bypass the apoptosis pathway, induce tumor cell death, and promote immune responses, thus possessing good anti-tumor potential.

[0003] In recent years, peptide self-assembly materials have been widely used for drug delivery and cell death regulation due to their excellent biocompatibility, molecular designability, and responsive modulation capabilities. However, most reported peptide drug systems focus on apoptosis or chemosynergistic mechanisms, and few materials can achieve specific activation of necroptotic signals through precise molecular design. Therefore, developing peptide nanomedicines that can responsively release and activate necrosis and autophagy-related apoptosis pathways in the characteristic environment of tumor cells has significant scientific and clinical application value. Summary of the Invention

[0004] The purpose of this invention is to address the technical deficiencies in existing technologies by providing a polypeptide nanomedicine that can induce necrosis and autophagy-related synergistic apoptosis, along with its preparation method and applications. This drug is a polypeptide nanomedicine (RDP) with dual Caspase-3 and GSH responsiveness. It can achieve drug release within tumor cells and induce RIPK3 aggregation, thereby activating necrosis and autophagy-related apoptosis pathways and achieving highly efficient inhibition of colorectal cancer.

[0005] The technical solution adopted to achieve the purpose of this invention is: A peptide nanomedicine capable of inducing necrosis and autophagy-related synergistic apoptosis is co-assembled from peptides DDP1 and X-DDP1, wherein: The structural formula of DDP1 is: ; The chemotherapy drug X is bufoginine RBG, irinotecan CPT11, or camptothecin CPT; When chemotherapy drug X is bufoginine RBG, X-DDP1 is RBG-DDP1, and the structural formula of RBG-DDP1 is: ; When chemotherapy drug X is irinotecan CPT11, X-DDP1 is CPT11-DDP1, and the structural formula of CPT11-DDP1 is: ; When chemotherapy drug X is camptothecin (CPT), X-DDP1 is CPT-DDP1, and the structural formula of CPT-DDP1 is: .

[0006] In the above technical solution, the polypeptide nanomedicine is a spherical nanoparticle with a diameter of 30~70nm.

[0007] Another aspect of the present invention includes a method for preparing the responsive peptide nanomedicine comprising the following steps: X-DDP1 and DDP1 were dissolved in PBS solution to prepare X-DDP1 mother liquor and DDP1 mother liquor respectively. The X-DDP1 mother liquor and DDP1 mother liquor were mixed and annealed. X-DDP1 and DDP1 self-assembled to form nanoparticles, and the mother liquor of the responsive peptide nanomedicine was obtained. The responsive peptide nanomedicine was obtained by freeze drying.

[0008] In the above technical solution, the volume ratio of X-DDP1 mother liquor to DDP1 mother liquor is 1:(5~15), preferably 1:9, the annealing reaction temperature is 60~80℃, preferably 80℃, and the annealing reaction time is 30~60 min, preferably 30 min.

[0009] In the above technical solution, DDP1 is synthesized by Fmoc solid-phase synthesis: dichloro resin is weighed and placed in a reactor, solvent is added to swell the resin, the terminal amino acids protected by Fmoc, HBTU and DIEA are dissolved in the solvent, after the reaction with the resin is completed, the Fmoc protecting group is removed; the above steps are repeated, and after all amino acids are linked, the protecting group of the last amino acid is removed, the peptide DDP1 is cleaved from the resin using a cleavage agent, and then DDP1 is obtained by acetyl capping.

[0010] In the above technical solution, X-DDP1 is prepared through the following steps: CDDP1 peptide is dissolved in a solvent, a chemotherapeutic drug X derivative is added, and the reaction is carried out at room temperature in the dark under a nitrogen atmosphere. After the reaction is completed, the reaction solution is freeze-dried to obtain X-DDP1, wherein the structural formula of CDDP1 is: ; When chemotherapy drug X is bufoginine RBG, the derivative of chemotherapy drug X is an RBG derivative, and the structural formula of the RBG derivative is: ; When the chemotherapy drug is irinotecan CPT11, the chemotherapy drug X derivative is a CPT11 derivative, and the structural formula of the CPT11 derivative is: ; When the chemotherapy drug is camptothecin (CPT), the chemotherapy drug X derivative is a CPT derivative, and the structural formula of the CPT derivative is: .

[0011] Another aspect of the present invention includes the application of the responsive peptide nanomedicine in the preparation of antitumor pharmaceutical products.

[0012] Another aspect of the present invention includes an antitumor drug formulation comprising the responsive peptide nanomedicine and pharmaceutically acceptable excipients.

[0013] In the above technical solution, the pharmaceutically acceptable excipients are one or more of the following: ethanol, propylene glycol, polyethylene glycol, diethylene glycol, triacetin, glycerol, dextrin, povidone, stearyl alcohol, stearic acid, microcrystalline cellulose, starch, lactose, mannitol, sodium bicarbonate, calcium carbonate, low-substituted hydroxypropyl methylcellulose, magnesium stearate, and talc.

[0014] In the above technical solution, the dosage form of the antitumor drug preparation is injection, tablet, pill, capsule, suspension or emulsion.

[0015] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention achieves a dual-response mechanism (Caspase-3 / GSH), improving drug release specificity and spatial control.

[0016] 2. The polypeptide sequence designed in this invention mimics the core aggregation fragment of RIPK1, which can promote RIPK3 aggregation and activate necrotic apoptosis in cells.

[0017] 3. The system structure of this invention is stable and the preparation method is simple, providing a delivery platform for broad-spectrum chemotherapy drugs such as bufoginine RBG, irinotecan CPT11, or camptothecin CPT.

[0018] 4. Both in vivo and in vitro experiments of this invention have demonstrated that RDP can effectively inhibit the growth of colorectal cancer and has potential clinical application value. Attached Figure Description

[0019] Figure 1 The chemical structural formulas and characterization of each component of the peptide nanodrug RDP, which can induce necrosis and autophagy-related synergistic apoptosis according to the present invention, include the chemical structural formula of the control molecule DP1.

[0020] Figure 2The UPLC curves of the peptide nanodrug RDP, which can induce necrosis and autophagy-related synergistic apoptosis according to the present invention, are shown in the presence of GSH and Caspase-3.

[0021] Figure 3 AFM and TEM images of the RDP-formed assembly prepared by co-assembly of DDP1 and RBG-DDP1.

[0022] Figure 4 The binding constants of the peptide nanodrug RDP, which can induce necrosis and autophagy-related co-apoptosis, and the control molecule DP1 to RIPK3 protein are shown in this invention.

[0023] Figure 5 A shows the quantitative analysis of cell necrosis in HT-29 cells cultured for 24 hours using flow cytometry under different sample treatments; B shows the viability of HT-29 cells cultured for 24 hours under different sample treatments; C shows the immunoblotting detection of RIPK3, pRIPK3, and pMLKL levels in HCT116 cells cultured for 24 hours under different sample treatments; D shows the LDH release from HT-29 cells cultured for 24 hours under different sample treatments.

[0024] Figure 6 Confocal microscopy images of HT-29 cells cultured with DP1, DDP1, and RDP in the presence of cell uptake inhibitors.

[0025] Figure 7 This paper presents a transcriptome sequencing analysis of HT-29 cells cultured for 24 hours after treatment with RDP, a polypeptide nanodrug that induces necrosis and autophagy-related synergistic apoptosis, according to the present invention. A shows a volcano plot of differentially expressed genes; B shows KEGG analysis of differentially expressed genes; C shows a heatmap of upregulation and downregulation of major related genes; and D shows GO enrichment analysis of differentially expressed genes.

[0026] Figure 8 In the table, A represents the ATP release amount after treating HT-29 cells with different samples; B represents the ULK1 content after treating HT-29 cells with different samples; C represents the LC3B content after treating HT-29 cells with different samples; and D represents the p62 content after treating HT-29 cells with different samples.

[0027] Figure 9 In the figure, A represents the tumor growth curve of mice in each treatment group; B represents the image of the isolated tumor of mice in each group after 21 days of treatment; C represents the weight of the isolated tumor of mice in each group after 21 days of treatment; and D represents the body weight change curve of mice in each treatment group.

[0028] Figure 10 This diagram illustrates the response, release, and induction of necrosis and apoptosis, as well as the regulation of autophagy, of the GSH and Caspase-3 dual-responsive peptide nanodrug RDP in tumor cells, for efficient drug delivery and tumor therapy. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Example

[0030] Example 1.1 The preparation of responsive peptide nanomedicines (RDPs) includes the following steps: Step 1: Synthesize peptides RBG-DDP1 and DDP1 (structural formula as shown in Figure 1). Figure 1 (as shown) (1) The peptide DDP1 was synthesized according to the structural formula using the conventional Fmoc solid-phase synthesis method. Specifically, dichloromethane was added to swell the resin, and four equivalents of Fmoc-protected amino acids, four equivalents of HBTU, and six equivalents of DIEA were dissolved in DMF and reacted with the resin for 1.5 to 2 hours. The Fmoc protecting groups were removed with 25% piperidine. The above steps were repeated until the ligation was complete. The protecting group of the last amino acid was removed, and the peptide was cleaved from the resin using a cleavage agent to obtain the intermediate peptide. ; The intermediate peptide was capped using a capping solution prepared with glacial acetic acid and pyridine in a 1:1 volume ratio. The reaction was carried out for 30 min until the ninhydrin test showed no color change, indicating successful capping. The resulting product was DDP1, with the following structural formula: .

[0031] (2) For peptide RBG-DDP1: The intermediate peptide is linked to cysteine ​​to synthesize CDDP1. After the linkage is complete, the protecting group of the last amino acid is removed, and the peptide is cleaved from the resin using a cleavage agent to obtain the peptide CDDP1.

[0032] The structural formula for CDDP1 is:

[0033] The dried peptide CDDP1 was dissolved in DMSO, and the drug bufojinin derivative was added. The reaction was carried out at room temperature in the dark for one day under a nitrogen atmosphere. After the reaction was completed, the reaction solution was freeze-dried to obtain the solid RBG-DDP1. The structural formula of the bufoginine derivative is: ; The structural formula of RBG-DDP1 is: .

[0034] Step 2: Co-assemble peptide DDP1 and RBG-DDP1 to prepare RDP: Lyophilized peptide DDP1 and lyophilized RBG-DDP1 were dissolved in PBS solution at pH 7.4 to obtain peptide stock solutions of the same concentration. The peptide stock solutions of DDP1 and RBG-DDP1 were mixed at a volume ratio of 9:1 and annealed at 80°C for 30 minutes to prepare RDP stock solution, which was then freeze-dried to obtain RDP.

[0035] Example 1.2 Replace the chemotherapy drug in Example 1.1 with irinotecan CPT11, and the preparation principle is the same as in Example 1.1.

[0036] Step 1: Synthesize peptides CPT11-DDP1 and DDP1; The synthesis methods for DDP1 and CDDP1 are the same as in Example 1.1; The synthesis method of CPT11-DDP1 involves dissolving the dried peptide CDDP1 in DMSO, adding a CPT11 derivative, the structural formula of which is as follows:

[0037] The reaction was carried out at room temperature in the dark under a nitrogen atmosphere for one day. After the reaction was completed, the reaction solution was freeze-dried to obtain CPT11-DDP1. The structural formula of CPT11-DDP1 is as follows: ; Step 2, same as in Example 1.1, involves co-assembling peptide DDP1 and CPT11-DDP1 to prepare a responsive peptide nanomedicine that delivers CPT11.

[0038] Example 1.3 Replace the chemotherapy drug in Example 1.1 with camptothecin CPT, and the preparation principle is the same as in Example 1.1.

[0039] Step 1: Synthesize peptides CPT-DDP1 and DDP1; The synthesis methods for DDP1 and CDDP1 are the same as in Example 1.1; The synthesis method of CPT-DDP1 involves dissolving the dried peptide CDDP1 in DMSO, adding a CPT derivative, the structural formula of which is:

[0040] The reaction was carried out at room temperature in the dark under a nitrogen atmosphere for one day. After the reaction was completed, the reaction solution was freeze-dried to obtain CPT-DDP1. The structural formula of CPT-DDP1 is as follows: ; Step 2, same as in Example 1.1, involves co-assembling peptide DDP1 and CPT-DDP1 to prepare a responsive peptide nanomedicine for delivering CPT.

[0041] The following examples use the RDP prepared in Example 1.1 as an example to verify its performance. Example

[0042] RDP was diluted in HEPES buffer, and GSH and Caspase-3 were added. UPLC was then used to test its responsiveness. The test method is as follows: (1) Dilute the RDP stock solution to 100M concentration with HEPES buffer, and add Caspase-3 enzyme and GSH, where the enzyme concentration is 0.2 u / mL and the GSH concentration is 5 mM.

[0043] (2) Set the incubation time (0, 20 min, 30 min) under constant temperature of 37℃. (3) Samples taken at different times were tested by UPLC-MS.

[0044] like Figure 2 The UPLC curves show that after adding GSH and Caspase-3, the free drug peak (RBG), peptide DDP1, CDDP1 peaks, and the DP1 peak of the fragment cleaved by Caspase-3 enzyme can be observed. Furthermore, as time increases, only the free drug peak RBG and peptide DP1 peak are observed at 30 minutes, indicating that it has a good ability to release RBG and DP1 in response to Caspase-3 enzyme and GSH within 30 minutes.

[0045] The structural formula of DP1 is: .

[0046] Its morphology was observed using transmission electron microscopy (TEM) and atomic force microscopy (AFM). Observations using AFM and TEM showed, for example... Figure 3 As shown, spherical nanoparticle structures with diameters of approximately 30–70 nm are visible. This result demonstrates that RDPs can form stable nanostructures through molecular self-assembly. Example

[0047] The binding affinity of RDP to RIPK3 protein was tested using the MST method: RIPK3 was labeled according to the manufacturer's standard instructions. The labeled RIPK3 solution was diluted to 100 nM with phosphate buffer containing 0.05% Tween 20. RDP and control molecule DP1 were added to the solution, with the peptide concentration ranging from 7.6 nM to 500 mM. The peptide and protein mixture was incubated at room temperature for 30 minutes, and the Kd values ​​of the peptide and RIPK3 were measured using a Monolith NT.115 instrument.

[0048] like Figure 4 The results showed that the binding constant between RDP and RIPK3 protein was approximately 26 mM, which was much higher than that of the functional molecule DP1 after release, indicating that it requires the release of the functional fragment DP1 to enhance its binding ability with RIPK3 protein. Example

[0049] The toxic effects of RDP on HT-29 cells: The cytotoxic effects of RDP on HT-29 cells were assessed using the CCK-8 assay and flow cytometry. The specific methods are as follows: (1) Cell preparation: Human colorectal cancer cell line HT-29 was selected and cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin and streptomycin, and maintained in logarithmic growth phase at 37℃ and 5% CO2.

[0050] (2) Experimental grouping and treatment: HT-29 cells were seeded in 96-well plates at a density of 5 × 10⁶ cells / well. 3 After cells / wells adhered, PBS was added to the control group, RBG group, DDP1 group and RDP treatment group respectively, with the drug concentration range set at 0-100 mM.

[0051] (3) CCK-8 detection: After incubation for 24 h, 10 μL of CCK-8 working solution was added to each well and incubated for another 2 h. The absorbance (OD450) was measured at a wavelength of 450 nm.

[0052] HT-29 cells were seeded in 96-well plates (5 × 10⁻⁶ cells per well). 3 Cells / well were incubated with PBS, RDP, and other control groups at concentrations ranging from 0 to 100 mM. After 24 h of incubation, CCK-8 reagent was added and the OD450 value was measured.

[0053] like Figure 5 Results A and B showed that cell viability in the RDP-treated group decreased significantly with increasing concentration, with an IC50 of approximately 35 mM, which was significantly lower than that of other components. Example

[0054] Studies on the regulation of intracellular protein expression by RDP: Western blot was used to analyze the ability of RDP to regulate key proteins involved in necrosis and apoptosis.

[0055] HT-29 cells were treated with RDP (50M, 24h), and proteins were extracted by lysis. Proteins were separated by SDS-PAGE and transferred to a PVDF membrane for the detection of RIPK3, p-RIPK3, and p-MLKL. The specific steps are as follows: Western blot analysis was performed on the expression of RIPK3, pRIPK3, and pMLKL in HT-29 human colorectal cancer cells after exposure to the peptide nanodrug RDP and control molecules (PBS, DP1, DDP1, RBG, RBG-DDP1). Cells were seeded at a density of 2 × 10⁵ in six-well plates and incubated overnight at 37°C. Then, 50 mM of the peptide nanodrug RDP and other control molecules were added and cultured for 24 hours. Cells were then treated with RIPA lysis buffer, and proteins were collected. Protein concentration was measured using a Bradford protein quantification kit. Proteins were boiled with loading buffer at 100°C for 10 minutes. Subsequently, 20 mg of protein from each sample was loaded onto an SDS-PAGE gel and electrophoresed onto a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, then incubated overnight at 4°C with the corresponding specific primary antibody, and finally incubated at room temperature with a horseradish peroxidase-conjugated secondary antibody for 1 hour. h later, the protein bands were imaged using ECL chemiluminescence detection on a Tanon-5200Multi instrument.

[0056] like Figure 5 Results C and D showed that RDP treatment significantly upregulated the expression of pRIPK3 and p-MLKL, indicating activation of the necroptosis pathway; while the changes in other control groups were not significant. These results validate that RDP specifically regulates necroptosis signaling by inducing RIPK3 aggregation. Example

[0057] Characterization of RDP cellular uptake: The uptake of RDP cells was determined using confocal microscopy, with the following specific steps: HT-29 human colorectal cancer cells were analyzed at 1 × 10⁻⁶ cells per well. 5Cells were seeded at a density of [number] cells per confocal dish and incubated overnight at 37°C. After washing with PBS, the cells were pretreated for 1 hour with endocytosis inhibitors 50 mM chlorpromazine (CPZ), 1 mM methyl-β-cyclodextrin (MBCD), or 50 mM amiloride. The cells were then added to a 50 mM peptide nanodrug RDP containing 2% TAMRA-DDP1, along with control molecules DP1 and DDP1. After incubation for 2 hours, the cells were washed with PBS, and cell uptake was observed using a confocal microscope. Figure 6 As shown.

[0058] like Figure 6 As shown, incubation with CPZ (clathrin-mediated endocytosis inhibitor) significantly reduced the intracellular fluorescence intensity of DP1, DDP1, and RDP cells, indicating a significant decrease in cellular uptake. This suggests that the clathrin-mediated endocytosis pathway plays an important role in the uptake of these peptides. Furthermore, the red fluorescence intensity in cells treated with amiloride also decreased to some extent, indicating that amiloride can also inhibit peptide uptake to some extent, suggesting that some peptides can also enter cells via macropinocytosis. MBCD, however, had almost no effect on peptide uptake, indicating that peptide uptake mainly occurs through other pathways. Example

[0059] To elucidate the molecular mechanism of RDP in programmed cell death, we validated its regulation of autophagy at the cellular omics and ultrastructural levels. The specific methods are as follows: (1) Transcriptomics analysis HT-29 human colorectal cancer cells were selected, seeded in six-well plates, and cultured to 70% confluence. A PBS control group and an RDP treatment group (RDP concentration 50 mM) were established. After 24 hours of treatment, total RNA was extracted using the Trizol method. RNA samples were sequenced using an Illumina NovaSeq 6000 platform after library construction.

[0060] The obtained data underwent quality control, comparison, and differential gene analysis, such as... Figure 7 The results showed that genes related to the necroptosis pathway (RIPK3, MLKL) and autophagy pathway (BECN1, ATG5, MAP1LC3B) were significantly upregulated in the RDP-treated group. GO and KEGG enrichment analyses indicated that the differentially expressed genes were mainly enriched in the "Autophagy-animal," "Necroptosis," and "Lysosome" signaling pathways. These results suggest that RDP can simultaneously activate necroptosis and autophagy, forming a synergistic programmed cell death mechanism within the cell.

[0061] (2) ELISA detection of changes in ULK1, LC3B and p62 levels like Figure 8 The results showed a significant increase in the levels of ULK1 and LC3B, indicating the initiation of autophagic flux and exhibiting characteristics of autophagic activation. However, the level of p62 protein did not decrease, suggesting that p62 accumulates in cells, blocking autophagic flux and potentially triggering a more intense intracellular stress response, especially evident in the RDP-treated group. Example

[0062] RDP's in vivo anti-tumor effects: The in vivo efficacy of RDP was evaluated using the HT-29 tumor-bearing nude mouse model.

[0063] HT-29 human colorectal cancer cells were administered at a rate of 1×10⁻⁹ / mL. 7 A tumor-bearing mouse model was established by subcutaneous injection of 100 cells into the right hind limb of female BALB / c nude mice. After 14 days of tumor growth, the mice were randomly divided into four groups: PBS, RBG, DP1, DDP1, RBG-DDP1, and RDP (n=4). The dosage was 500 mM, 200 mL, administered via tail vein injection every two days for a total of five times. The specific steps are as follows: The results showed that, Figure 9 As shown, tumor volume growth was significantly inhibited in the RDP group, showing a significant difference compared to the control group. Subjective comparison of tumor images and weight after treatment demonstrated the excellent tumor-suppressing effect of RDP. No significant abnormalities were observed in weight monitoring, indicating that RDP possesses good biocompatibility and significant antitumor activity.

[0064] like Figure 10 As shown, RDP nanoparticles were formed in vitro by co-assembling two peptides. Firstly, RDP released the drug RBG in tumor cells via a GSH response. RBG upregulated RIPK3 and activated Caspase-3 (…). Figure 10 The CASP3 in the enzyme induces cleavage of the necroptosis response sequence, thereby releasing the necroptosis functional peptide fragment DP1, which binds to RIPK3, promoting the aggregation of RIPK3 to form necrosomes and initiating the necroptosis pathway. Furthermore, the necrosomes disrupt the cell membrane, leading to ATP release and lysosomal damage, which in turn activates upstream autophagy and blocks downstream autophagy-lysosomal fusion, ultimately resulting in cell death. In summary, RDP exhibits a multimodal antitumor effect, with necroptosis as its core while simultaneously interfering with autophagy.

[0065] Similarly, when bufoginine RBG is replaced with irinotecan CPT11 or camptothecin CPT, it can achieve a multimodal antitumor effect with necrosis and apoptosis as the core while interfering with autophagy, relying on the same principle.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide nanomedicine capable of inducing necrosis and autophagy-related synergistic apoptosis, characterized in that, It was co-assembled by annealing of peptides DDP1 and X-DDP1, wherein: The structural formula of DDP1 is: ； X represents a chemotherapy drug, specifically bufogenin RBG, irinotecan CPT11, or camptothecin CPT. When chemotherapy drug X is bufoginine RBG, X-DDP1 is RBG-DDP1, and the structural formula of RBG-DDP1 is: ； When chemotherapy drug X is irinotecan CPT11, X-DDP1 is CPT11-DDP1, and the structural formula of CPT11-DDP1 is: ； When chemotherapy drug X is camptothecin (CPT), X-DDP1 is CPT-DDP1, and the structural formula of CPT-DDP1 is: 。 2. The polypeptide nanomedicine that can induce necrosis and autophagy-related synergistic apoptosis as described in claim 1, characterized in that, The polypeptide nanomedicine is a spherical nanoparticle with a diameter of 30-70 nm.

3. The method for preparing the polypeptide nanomedicine capable of inducing necrosis and autophagy-related synergistic apoptosis as described in claim 1, characterized in that, Includes the following steps: X-DDP1 and DDP1 were dissolved in PBS solution to prepare X-DDP1 mother liquor and DDP1 mother liquor respectively. The X-DDP1 mother liquor and DDP1 mother liquor were mixed and annealed. X-DDP1 and DDP1 self-assembled to form nanoparticles, and the mother liquor of responsive peptide nanomedicine was obtained. The responsive peptide nanomedicine was obtained by freeze drying.

4. The preparation method according to claim 3, characterized in that, The volume ratio of X-DDP1 mother liquor to DDP1 mother liquor is 1:(5~15), the annealing temperature is 60~80℃, and the annealing time is 30~60 min.

5. The preparation method according to claim 4, characterized in that, The volume ratio of X-DDP1 mother liquor to DDP1 mother liquor was 1:9, the annealing temperature was 80℃, and the annealing time was 30 min.

6. The preparation method according to claim 3, characterized in that, DDP1 was synthesized via Fmoc solid-phase synthesis: Dichloro resin was weighed and placed in a reactor, and solvent was added to swell the resin. The Fmoc-protected terminal amino acids, HBTU, and DIEA were dissolved in the solvent and reacted with the resin. After the reaction was completed, the Fmoc protecting groups were removed. The above steps were repeated until all amino acids were linked. The protecting group of the last amino acid was removed, and the peptide DDP1 was cleaved from the resin using a cleavage agent. Then, DDP1 was obtained by acetyl capping.

7. The preparation method according to claim 3, characterized in that, X-DDP1 was prepared by the following steps: The CDDP1 peptide was dissolved in a solvent, and a chemotherapeutic drug derivative X was added. The reaction was carried out at room temperature in the dark under a nitrogen atmosphere. After the reaction was completed, the reaction solution was freeze-dried to obtain X-DDP1, wherein the structural formula of CDDP1 is: ； When chemotherapy drug X is bufoginine RBG, the derivative of chemotherapy drug X is an RBG derivative, and the structural formula of the RBG derivative is: ； When the chemotherapy drug is irinotecan CPT11, the chemotherapy drug X derivative is a CPT11 derivative, and the structural formula of the CPT11 derivative is: ； When the chemotherapy drug is camptothecin (CPT), the chemotherapy drug X derivative is a CPT derivative, and the structural formula of the CPT derivative is: 。 8. The application of the polypeptide nanomedicine that can induce necrosis and autophagy-related synergistic apoptosis as described in claim 1 in the preparation of anti-colorectal cancer pharmaceutical products.

9. An antitumor drug formulation, characterized in that, This includes the polypeptide nanomedicine of claim 1 that can induce necrosis and autophagy-related synergistic apoptosis, as well as pharmaceutically acceptable excipients.

10. The antitumor drug formulation according to claim 9, characterized in that, The pharmaceutically acceptable excipients are one or more of the following: ethanol, propylene glycol, polyethylene glycol, triacetin, glycerin, dextrin, povidone, stearyl alcohol, stearic acid, microcrystalline cellulose, starch, lactose, mannitol, sodium bicarbonate, calcium carbonate, low-substituted hydroxypropyl methylcellulose, magnesium stearate, and talc.

11. The antitumor drug formulation according to claim 9, characterized in that, The dosage form of the antitumor drug preparation is injection, tablet, pill, capsule, suspension or emulsion.

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