Nanometer polypeptide material capable of doubly inhibiting tumor Wnt signal channel as well as preparation method and application of nanometer polypeptide material

Abstract

The invention relates to the technical field of nano biological materials, in particular to a nano polypeptide material for dual inhibition of tumor Wnt signal pathways as well as a preparation method and application of the nano polypeptide material for dual inhibition of tumor Wnt signal pathways, and the nano polypeptide material for dual inhibition of tumor Wnt signal pathways comprises polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, the hydrophobic fluorophore is connected with the last lysine of the polypeptide through an amido bond. The nano polypeptide material deforms into a nano fiber network structure in situ after targeting FZD7 protein highly expressed on the surface of a tumor cell membrane, is retained for a long time and directly blocks a binding site of a Wnt ligand and the FZD7 protein, and inhibits signal activation from the upstream of a pathway. Meanwhile, the dense fiber network indirectly limits autosecretion and paracrine processes mediated by vesicles or exosomes of mature modified Wnt protein and inhibits signal cascade amplification conduction between adjacent cells, so that key protein expression and downstream target protein transcription level in a Wnt signal channel are doubly down-regulated in internal and external aspects, and finally, the tumor treatment effect is enhanced.

Description

Technical Field

[0001] This invention relates to the field of nanobiomaterials technology, and in particular to a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, its preparation method, and its application. Background Technology

[0002] The classic Wnt signaling pathway (Wnt / β-catenin pathway) is highly conserved throughout species evolution, precisely regulating cell proliferation, differentiation, migration, and fate determination, and participating in key physiological processes such as embryonic development, adult stem cell maintenance, tissue homeostasis, and damage repair. Abnormal activation of the Wnt pathway in tumor cells promotes the development and progression of cancer cells and significantly affects tumor cell stemness.

[0003] After undergoing a series of acetylation and lipidation modifications in cells, Wnt protein ligands bind to Frizzled (FZD) receptors on the cell membrane surface via paracrine or autocrine pathways to activate downstream signaling pathways. Studies have shown that Frizzled7 (FZD7) is a subclass of the ten members of the Frizzled family that is significantly upregulated in various malignant tumors, including breast cancer and liver cancer, making it an important molecular target in tumor research. However, to date, selective inhibitors of FZD7 and Wnt protein secretion inhibitors are still in the early stages of development. The complex mechanisms of the Wnt pathway and the exocrine transport of Wnt protein remain unclear, posing significant challenges to the research and application process.

[0004] Furthermore, current small molecule inhibitors and monoclonal antibody preparations targeting the FZD receptor, or exocrine inhibitors, decoy receptors, and competitive binding inhibitors targeting the Wnt ligand, are all limited to drug intervention targeting a single link in the Wnt pathway. Often, due to the complexity of the Wnt pathway itself, which involves multiple branches, molecules, and interactions, as well as tumor heterogeneity, significant toxic side effects, branch compensatory activation, and limited applicability, it is difficult to achieve the ideal goal of "precisely blocking abnormal signals without affecting normal function."

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, its preparation method and application, in order to solve the problem that existing inhibitors of the tumor Wnt signaling pathway are difficult to accurately block abnormal signals and affect normal function.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, comprising a peptide with an amino acid sequence as shown in SEQ ID NO:1, and a hydrophobic fluorescent group connected to the terminal lysine of the peptide via an amide bond.

[0008] Secondly, the present invention provides a method for preparing a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, comprising the following steps: The amine resin was subjected to swelling treatment, and the Fmoc protecting group of the amino group of the amine resin was removed to obtain the first product; Y[Fmoc-Tyr(tBu)-OH], additives, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide were mixed and reacted with the first product to obtain the second product; The second product was reacted with a mixture of acetic anhydride, N,N-diisopropylethylamine, and N,N-dimethylformamide to obtain the third product; The fourth product is obtained by coupling amino acids onto the main chain of the third product in the following order: methionine M, valine V, histidine H, cysteine ​​C, tryptophan W, phenylalanine F, glutamic acid E, leucine L, aspartic acid D, serine S, proline P, leucine L, lysine K (Boc), leucine L, valine V, phenylalanine F, phenylalanine F, and lysine K (Dde). The Fmoc protecting group of the main chain amino group in the fourth product is removed, and the exposed amino group of the main chain is capped with acetic anhydride to obtain the fifth product. The protecting group of the side chain amino group Dde of lysine K (Dde) in the fifth product was removed by using an N,N-dimethylformamide solution containing hydrazine hydrate to obtain the sixth product; The hydrophobic fluorescent group, additives, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide and the sixth product were mixed and subjected to an amidation reaction to obtain nanopeptide materials.

[0009] Thirdly, the present invention provides the application of a nanopeptide material that dually inhibits the tumor Wnt signaling pathway in the preparation of drugs for treating breast cancer or liver cancer.

[0010] Beneficial Effects: This invention provides a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, its preparation method, and its application. The nanopeptide material that dually inhibits the tumor Wnt signaling pathway comprises a peptide with the amino acid sequence shown in SEQ ID NO:1, and a hydrophobic fluorescent group linked to the terminal lysine residue of the peptide via an amide bond. After targeting the highly expressed FZD7 protein on the surface of tumor cells, this nanopeptide material transforms in situ into a nanofiber network structure, permanently retaining and directly blocking the binding site of the Wnt ligand to the FZD7 protein, thus inhibiting signal activation upstream of the pathway. Simultaneously, the dense fiber network indirectly restricts the autocrine and paracrine processes of the mature and modified Wnt protein mediated by vesicles or exosomes, inhibiting the signal cascade amplification and transduction between neighboring cells. This dual downregulates the expression of key proteins in the Wnt signaling pathway and the transcription level of downstream target proteins from both internal and external perspectives, ultimately reducing cell invasion, migration, and overall physiological activity, and enhancing the therapeutic effect on tumors. Attached Figure Description

[0011] Figure 1 A schematic diagram of the structure of a nanopeptide material that dually inhibits the tumor Wnt signaling pathway; Figure 2 This is the mass spectrum of the TP-Fz nanopeptide material prepared in Example 1; Figure 3 The fluorescence intensity spectra of TP-Fz nanoparticles in Example 2 at different water contents are shown. Figure 4 This is a graph showing the change in particle size over time after TP-Fz nanoparticles were incubated with FZD7 protein, as detected by a particle size potentiometer in Example 3. Figure 5 This is a TEM image showing the fiber formation and changes over time after TP-Fz nanopeptide material was incubated with FZD7 protein. Figure 6 This is a graph showing the difference in FZD7 expression levels between liver cancer cells and breast cancer cells screened by Western blot analysis in Example 5. Figure 7 This is a CLSM colocalization map of TP-Fz material and FZD7 protein in HepG2 liver cancer cells and MDA-MB-231 breast cancer cells observed in Example 6. Figure 8 This is a diagram showing the fiber formation of TP-Fz nanopeptide material on the cell membrane surface as observed by TEM in Example 7. Figure 9 This is a diagram showing the fiber formation of TP-Fz nanopeptide material on the cell membrane surface as observed by SEM in Example 8. Figure 10This is a diagram from Example 9 showing the inhibition of β-catenin expression and nuclear translocation of the Wnt pathway key protein by TP-Fz nanopeptide material using protein immunoblotting. Figure 11 This is a graph showing the level of inhibition of downstream target gene transcription in the Wnt pathway by TP-Fz nanopeptide material by qPCR in Example 10. Figure 12 This is a graph showing the inhibitory effect of TP-Fz nanopeptide material on the proliferation of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells in Example 11. Figure 13 This is a graph showing the inhibition of the migration ability of TP-Fz nanopeptide material on HepG2 liver cancer cells and MDA-MB-231 breast cancer cells in Example 12. Figure 14 This is a graph showing the inhibition of the invasive ability of TP-Fz nanopeptide material on HepG2 liver cancer cells and MDA-MB-231 breast cancer cells in Example 13. Figure 15 Figure showing how TP-Fz nanopeptide materials alter the morphology, number of exosomes, and particle size of HepG2 liver cancer cells. Figure 16 Figure showing the effect of TP-Fz nanopeptide material on the overall exosome content of HepG2 liver cancer cells by exosome staining; Figure 17 The image shows the direct targeting of TP-Fz nanopeptides to HepG2 exosomes of liver cancer cells by TEM. Figure 18 To analyze the effect of exosomes secreted by cells after intervention with TP-Fz nanopeptides on the activation capacity of the Wnt pathway using Western blotting. Detailed Implementation

[0012] This invention provides a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0013] This invention provides a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, comprising a peptide with an amino acid sequence as shown in SEQ ID NO:1, and a hydrophobic fluorescent group connected to the terminal lysine of the peptide via an amide bond.

[0014] In this embodiment, after targeting the highly expressed FZD7 protein on the surface of tumor cell membranes, the nanopeptide material transforms in situ into a nanofiber network structure, which retains and directly blocks the binding site of Wnt ligands to FZD7 protein, inhibiting signal activation upstream of the pathway. Simultaneously, the dense fiber network indirectly restricts the autocrine and paracrine processes of mature and modified Wnt protein via vesicles or exosomes, inhibiting signal cascade amplification and transduction between neighboring cells. This dual downregulation of key protein expression and downstream target protein transcription levels in the Wnt signaling pathway, both internally and externally, ultimately reduces cell invasion, migration, and overall physiological activity, enhancing the therapeutic effect on tumors.

[0015] Specifically, the nanopeptide material includes hydrophobic fluorescent groups, fibrous deformable peptides, and FZD7 targeting peptides.

[0016] In some implementations, due to the high sequence similarity of the cysteine ​​enrichment domain (CRD) sequences among FZD family members (especially the lipid binding groove region where Wnt binds), traditional small molecules struggle to achieve subtype-selective targeting. Therefore, the FZD7 targeting peptide selected was Fz7-21 (sequence Ac-LPSDDLEFWCHVMY-NH2), obtained through phage display technology. This peptide specifically targets FZD7 and does not bind to other FZD family members, making it the first peptide inhibitor capable of selectively targeting the FZD7 CRD subclass, thus ensuring a foundation for precise targeting.

[0017] In some embodiments, the fibrous deformation peptide can be selected from β-sheet peptides such as QQRFWEFEQQ, VKVKVVKVDPPTKVKVKV, or DFNK, DFNKF.

[0018] Based on the identification of the FZD7 targeting peptide, the peptide backbone was generated using the RFdiffusion diffusion model. Then, proteinmpnn was used to fill in appropriate fibrillation peptide sequences. Finally, the alpha fold model was used to predict the binding affinity between the peptide and protein. After multiple rounds of screening, considering various indicators such as overall hydrophilicity / hydrophobicity, charge distribution, conformational fit, number of binding sites, free binding energy, and binding stability, the KLVFF sequence was selected as the key deformation peptide for the self-assembled peptide system, demonstrating synergistic effects.

[0019] In some embodiments, the hydrophobic fluorescent group is selected from protoporphyrin (PpIX), 7-nitrobenzodiazole (NBD), bispyrene ( Bis-pyrene, BPOne of the molecules. 7-Nitrobenzodiazole is a fluorescent molecule with the same aggregation-caused quenching (ACQ) property as PpIX, exhibiting green fluorescence; bispyrene molecules have aggregation-induced emission (AIE) properties, the opposite of ACQ, with significantly enhanced luminescence intensity under aggregated or solid-state conditions, making them widely applicable in biosensing, cell imaging, OLEDs, and other fields. In specific implementation, considering the compatibility of fluorescent molecule selection with self-assembled peptide systems, protoporphyrin (PpIX) was chosen as an important in vivo fluorescent tracer molecule and hydrophobic core selection, with the following chemical structure: .

[0020] In some embodiments, the hydrophobic fluorescent group is linked to the side chain amino group of the terminal lysine residue of the polypeptide via an amide bond.

[0021] Specifically, receptor-ligand binding is typically achieved through non-covalent interactions such as van der Waals forces, ionic bonds, and hydrogen bonds, with hydrogen bonding being the most common form. Nanopeptide materials used to bind FZD7 protein are based on the binding of FZD7 targeting peptides to FZD7 protein on the surface of breast cancer and liver cancer cell membranes, inducing lateral aggregation of the nanopeptide materials and their transformation into nanofibers. The reason for this fiber deformation is that in aqueous solution, the nanopeptide materials exist as self-assembled spherical nanoparticles dominated by hydrophilic-hydrophobic interactions, essentially belonging to a relatively unstable non-thermodynamic equilibrium state. Under the induction of receptor-ligand interactions on the surface of tumor cell membranes, more hydrogen bond binding sites are exposed, and the self-assembled peptides undergo β-sheeting, ultimately transforming into a more thermodynamically stable fibrous mesh structure.

[0022] The nanopeptide material provided by this invention initially self-assembles into spherical nanoparticles in aqueous solution through hydrophilic-hydrophobic interactions, based on the binding of FZD7 targeting peptides to FZD7 protein on the membrane surface of breast cancer and liver cancer cells. Furthermore, the spherical nanoparticles disaggregate under the induction of "receptor-ligand" interactions, and the fibrous peptides on the tightly packed monomeric peptides undergo "β-sheet" lateral aggregation under intermolecular interactions such as hydrogen bonds and van der Waals forces, transforming into a nanofiber network structure on the membrane surface. This structure is retained for a long time and directly blocks the binding site of Wnt ligands to FZD7 protein, thereby inhibiting signal activation upstream of the pathway.

[0023] In a preferred embodiment, the structural formula of the nanopeptide material that dually inhibits the tumor Wnt signaling pathway is as follows: Figure 1As shown, it consists of a hydrophobic fluorescent group PpIX as the hydrophobic terminator and for fluorescent tracking, a fibrous deformation peptide K(Dde)-KLVFF for linking the hydrophobic fluorescent group to the target peptide, and a target peptide LPSDDLEFWCHVMY as FZD7. The hydrophobic fluorescent group PpIX is linked to the side chain amino group of lysine K(Dde) via an amide bond, while the remaining amino acids are linked to each other on the main chain via amide bonds. The structural formula of the fibrous deformation peptide K(Dde)-KLVFF is as follows: The structural formula of the targeting peptide LPSDDLEFWCHVMY is as follows: .

[0024] In addition, this invention also provides a method for preparing a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, comprising the following steps: Step S10: The amine resin is subjected to swelling treatment, and the Fmoc protecting group of the amino group of the amine resin is removed to obtain the first product; Step S20: Y[Fmoc-Tyr(tBu)-OH], additives, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide are mixed and reacted with the first product to obtain the second product; Step S30: The second product is reacted with a mixed system of acetic anhydride, N,N-diisopropylethylamine, and N,N-dimethylformamide to obtain the third product; Step S40: Couple amino acids to the main chain of the third product in the following order: methionine M, valine V, histidine H, cysteine ​​C, tryptophan W, phenylalanine F, glutamic acid E, leucine L, aspartic acid D, serine S, proline P, leucine L, lysine K (Boc), leucine L, valine V, phenylalanine F, phenylalanine F, lysine K (Dde) to obtain the fourth product; Step S50: Remove the Fmoc protecting group of the main chain amino group from the fourth product, and end the exposed amino group of the main chain with acetic anhydride to obtain the fifth product; Step S60: Remove the protecting group of the side chain amino group Dde of lysine K (Dde) in the fifth product using an N,N-dimethylformamide solution containing hydrazine hydrate to obtain the sixth product; Step S70: The hydrophobic fluorescent group, additive, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide and the sixth product are mixed and subjected to an amidation reaction to obtain nanopeptide material.

[0025] In this embodiment, the nanopeptide material is synthesized using a solid-phase peptide synthesis method. Amine resin is used as the solid-phase support. After the reaction in the reaction tube, only filtration and washing of the reaction solution are required to obtain the peptides cross-linked on the amine resin, making the synthesis operation simple and convenient. Furthermore, all peptide monomers are synthesized using standard solid-phase peptide synthesis technology. Rink Amide-AM amine resin is selected as the solid-phase synthesis support. The first linked amino acid is Y[Fmoc-Tyr(tBu)-OH], and subsequent amino acid dehydration condensations are performed sequentially from right to left according to the amino acid sequence of the peptide monomers. N,N'-diisopropylcarbodiimide (DIC) acts as a condensing agent in peptide synthesis, activating the carboxyl group and promoting peptide bond formation between amino acids. Additives are used to inhibit racemic reactions, improving peptide bond formation efficiency and product purity, thus enhancing the coupling efficiency of Fmoc-protected amino acids. Finally, a special Dde protecting group is removed from the side chain of the terminal amino acid K (Dde), and a hydrophobic fluorescent group is linked. This preparation process is simple, and the product purity is high.

[0026] In some embodiments, step S60, prior to the step of removing the protecting group of the side chain amino group Dde of lysine K (Dde) in the fifth product using an N,N-dimethylformamide solution containing hydrazine hydrate, further includes: Step S61: Remove the Fmoc protecting group on the lysine K (Dde) using N,N-dimethylformamide containing piperidine to obtain lysine K (Dde) containing an exposed amino group; Step S62: Mix carbonic anhydride with the lysine K (Dde) containing exposed amino groups and react to obtain lysine K (Dde) containing inert acyl functional groups.

[0027] Specifically, in terms of precise control of the reaction site, to avoid the simultaneous removal of the side chain protecting group Dde of lysine K (Dde) and the main chain protecting group Fmoc when attaching a hydrophobic fluorescent group, the Fmoc protecting group on lysine K (Dde) is first removed using a mild 20% piperidine DMF solution. Carbonic anhydride is then used to react with the exposed amino group to form an inert acyl functional group. Finally, a more vigorous 2.5% hydrazine hydrate DMF solution is used to remove the Dde protecting group and attach a hydrophobic fluorescent group, ensuring the uniqueness of the reaction site and increasing the yield of the target peptide.

[0028] In some embodiments, step S10 specifically includes: soaking the amine resin in dichloromethane (DCM) for 0.5-1 h, then adding 20% ​​piperidine in N,N-dimethylformamide (DMF) solution and mixing thoroughly, reacting on a rotary mixer for 10-20 min, and repeating once to completely remove the Fmoc protecting groups on the amine resin.

[0029] In some embodiments, after step S10 and before step S20, a step S11 is included: detecting the amino group exposed by the amine resin. Specifically, this includes: vortex washing the first product with DMF solvent, then taking a small amount of the washed first product into a Durham tube, adding 2-4 drops of ninhydrin solution using a dropper, and heating in a 100°C metal bath for 2-5 minutes. If the amine resin turns purple, it indicates that the Fmoc protecting group of the amino group has been removed. If the purple color is very light or there is no color change, the step of removing the Fmoc protecting group of the amino group from the amine resin in step S10 is repeated until the Fmoc group of the amino group is completely removed.

[0030] In some embodiments, in the step of reacting the mixture of Y[Fmoc-Tyr(tBu)-OH], the additive, N,N'-diisopropylcarbodiimide, and N,N-dimethylformamide with the first product, the mass ratio of Y[Fmoc-Tyr(tBu)-OH], the additive, the N,N'-diisopropylcarbodiimide, and the amine resin is (2.5-3.5):(5-7):(5-7):1. Preferably, Oxyma is selected as the additive.

[0031] In a preferred embodiment, in step S20, the mass ratio of Y[Fmoc-Tyr(tBu)-OH], the additive, the N,N'-diisopropylcarbodiimide, and the amine resin is 3:6:6:1; at this mass ratio, the yield of lysine K (Dde) coupled to the first product is the highest.

[0032] Specifically, weigh out 3 equivalents of Y[Fmoc-Tyr(tBu)-OH], 6 equivalents of Oxyma, and 6 equivalents of DIC relative to the amine resin loading, add DMF to completely dissolve them, then pour them into a reaction tube containing the first product, and place it in an incubator at 40°C for 1-2 hours to rotate and mix. This allows the first amino acid Y of the nanopeptide material to be coupled onto the amine resin after the Fmoc protecting group has been removed.

[0033] In some embodiments, after step S20 and before step S30, a step S21 is included: detecting whether the first amino acid Y has been successfully coupled, i.e., detecting whether there are still amino groups exposed due to incomplete reaction in the reaction solution. Specifically, this includes: vortex washing the second product with DMF solvent; then taking a small amount of the washed second product into a Durham tube, adding 2-4 drops of ninhydrin solution using a dropper, and heating it in a 100°C metal bath for 2-5 minutes. If the amine resin does not change color (remains transparent), it indicates that the amino acid coupling is complete. If the color turns purple, step S20 is repeated to ensure that the first amino acid Y is completely coupled.

[0034] In some embodiments, in step S30, in the step of reacting the second product with a mixed system of acetic anhydride, N,N-diisopropylethylamine, and N,N-dimethylformamide, the volume ratio of the acetic anhydride, the N,N-diisopropylethylamine, and the N,N-dimethylformamide is (0.5-1):(0.5-1):(8.5-9).

[0035] In a preferred embodiment, in step S30, the volume ratio of the acetic anhydride, the N,N-diisopropylethylamine, and the N,N-dimethylformamide is 0.5:0.5:9. The mixture is mixed with the second product at this volume ratio and subjected to a rotary mixing reaction at room temperature for 0.5-1 h. This process can end-cap the very small amount of amino groups on the amine resin that have undergone the fish-fishing reaction, thus preventing errors in the starting amino acids that crosslink with the resin.

[0036] In some embodiments, in step S40, amino acids are coupled to the main chain of the third product in the following order: methionine M, valine V, histidine H, cysteine ​​C, tryptophan W, phenylalanine F, glutamic acid E, leucine L, aspartic acid D, serine S, proline P, leucine L, lysine K (Boc), leucine L, valine V, phenylalanine F, phenylalanine F, and lysine K (Dde) to obtain a fourth product that can target and bind to FZD7 protein. For each amino acid coupled, the steps of removing the Fmoc protecting group, step S11, step S20, and step S21 are repeated to link the amino acids through amide bonds.

[0037] In some embodiments, the method for removing the Fmoc protecting group from the lysine K (Dde) backbone amino group in step S50 can be performed with reference to step S10, with the aim of completely removing the Fmoc protecting group. The step of capping the exposed amino group at the terminal lysine K (Dde) backbone with acetic anhydride can be performed with reference to step S30, so that the Fmoc-protected amino group on the lysine K (Dde) backbone becomes an acetyl group.

[0038] In some embodiments, after step S50 and before step S60, the method further includes detecting the exposed amino group; the detection method may refer to step S11.

[0039] In some embodiments, step S60 specifically includes: vortex washing the fifth product with DMF; then adding a 2.5% hydrazine hydrate DMF solution to the vortex-washed fifth product, and reacting to obtain the sixth product.

[0040] Specifically, the fifth product was vortex-washed with DMF solvent. Hydrazine hydrate and DMF were mixed at a volume ratio of 25 μL:1 mL and added to the vortex-washed fifth product. The reaction was carried out at room temperature for 8-10 min, repeated once, until the side-chain amino Dde protecting group of K[Fmoc-lys(Dde)-OH] was completely removed.

[0041] In some embodiments, after step S60 and before step S70, the method further includes the step of detecting the amino group exposed on the K[Fmoc-lys(Dde)-OH] side chain, which can be referred to step S11.

[0042] In some embodiments, in the step of mixing the hydrophobic fluorescent group, the additive, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide with the sixth product, the mass ratio of the hydrophobic fluorescent group, the additive, the N,N'-diisopropylcarbodiimide and the amine resin is (2.5-3.5):(2.5-3.5):(5.5-6.5):1.

[0043] In a preferred embodiment, in step S70, the mass ratio of the hydrophobic fluorescent group, the additive, the N,N'-diisopropylcarbodiimide, and the amine resin is 3:6:6:1. Adding the materials to the sixth product in this mass ratio ensures complete reaction and saves raw materials. Preferably, Oxyma is selected as the additive.

[0044] Specifically, a hydrophobic fluorescent group, an Oxyma equivalent, and a DIC equivalent of 3 times the loading of the amine resin were weighed, and then added to DMF until completely dissolved. The solution was then added to a reaction tube containing the sixth product and placed in a 40°C incubator for 36-48 hours to obtain the seventh product.

[0045] In some embodiments, after obtaining the seventh product, the method further includes the step of detecting whether there are exposed amino groups in the reaction solution, i.e., detecting whether the hydrophobic fluorescent group is successfully connected; the detection method can refer to step S11.

[0046] In some embodiments, after step S70, the hydrophobic fluorescent group, additive, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide, and the sixth product are mixed and subjected to an amidation reaction. The process further includes the following steps: drying the amine resin in the reaction product (i.e., the seventh product), adding a cleavage solution, and then rotary evaporating, precipitating, centrifuging, and drying to obtain the nanopeptide material. The cleavage solution, by volume fraction, comprises: 2.5% triisopropylsilane (TIS), 2.5% ultrapure water, and 95% trifluoroacetic acid (TFA).

[0047] Specifically, the seventh product was vortexed and washed with DMF solvent until the filtrate was colorless, following the order DMF→DCM→DMF→DCM, with each step lasting 1 min and repeated 3 times. The product was then vacuum-dried and transferred to centrifuge tubes. A severing agent was added to the centrifuge tubes, the gas was released, and the reaction was allowed to proceed for 3 hours. The filtrate was collected and transferred to a round-bottom flask. After rotary evaporation, ice-cold ether stored at -20°C was added to precipitate the peptides. The system was then transferred to centrifuge tubes and centrifuged at 3-4°C and 10000-14000 r / min. Ice-cold ether was added, and the vortexing, sonication, and centrifugation were repeated twice. The tubes were sealed with sealing film and vacuum-dried to obtain solid peptide powder, i.e., the nanopeptide material, which was stored under dry conditions at room temperature.

[0048] In this embodiment, after the reaction is completed, the target product is separated from the support by cleaving the amide bond link between the amino acid and the amine resin with a cleaving liquid. Since all soluble impurities can be removed by simple filtration-washing-re-filtration, the product attached to the support can be obtained. Therefore, the intermediate products after solid-phase synthesis are completely exempt from the complicated purification operations such as recrystallization, distillation, column chromatography or plate chromatography in traditional liquid-phase synthesis.

[0049] In addition, the present invention also provides the application of a nanopeptide material that dually inhibits the tumor Wnt signaling pathway in the preparation of drugs for treating breast cancer or liver cancer.

[0050] In this embodiment, after targeting the highly expressed FZD7 protein on the surface of tumor cell membranes, the nanopeptide material transforms in situ into a nanofiber network structure, which retains and directly blocks the binding site of Wnt ligands to FZD7 protein, inhibiting signal activation upstream of the pathway. Simultaneously, the dense fiber network indirectly restricts the autocrine and paracrine processes of mature and modified Wnt protein via vesicles or exosomes, inhibiting signal cascade amplification and transduction between neighboring cells. This dual downregulation of key protein expression and downstream target protein transcription levels in the Wnt signaling pathway, both internally and externally, ultimately reduces cell invasion, migration, and overall physiological activity, enhancing the therapeutic effect on tumors.

[0051] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention are within the scope of protection of the present invention.

[0052] Example 1 This embodiment provides a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, the structure of which is as follows: Figure 1As shown, the amino acid sequence is Ac-K(PpIX)-FFVLK-LPSDDLEFWCHVMY-NH2, including the PpIX hydrophobic fluorescent group, the fiber-deforming peptide, and the FZD7 targeting peptide. This embodiment uses a peptide solid-phase synthesis method to prepare nanopeptide materials, and the specific steps are as follows: 1) Swelling of amine resin: Soak the amine resin in 10 mL of DCM solvent for 0.5 h to allow it to fully swell; 2) Deprotecting the Fmoc groups of the amino groups on the amine resin: Add 8 mL of deprotecting agent (piperidine:DMF=1:4) to step 1), react on a rotary mixing rack for 10 min, and repeat once to completely remove the Fmoc groups of the amino groups on the amine resin to obtain the first product; 3) Detection of exposed amino groups in the amine resin: Vortex wash the resin from step 2) five times with 10 mL of DMF solvent, 1 min each time. Take a small amount of resin into a Durham tube, add 2 drops of ninhydrin solution using a dropper, and heat in a 100°C metal bath for 2 min. If the amine resin turns purple, it indicates that the Fmoc protecting group of the amino group has been removed. If the purple color is very light or there is no color change, repeat step 2) until the Fmoc group of the amino group is completely removed.

[0053] 4) Coupling the first amino acid Y[Fmoc-Tyr(tBu)-OH] in the structure of the nanopeptide material: Weigh 3 equivalents of Y[Fmoc-Tyr(tBu)-OH], 6 equivalents of Oxyma, and 6 equivalents of DIC relative to the amine resin loading, add 10 mL of DMF to completely dissolve, then add it to a reaction tube containing amine resin (after removing the Fmoc group), and place it in an incubator at 40°C for 2 h of rotational reaction to obtain the second product.

[0054] 5) Detect whether the first amino acid Y[Fmoc-Tyr(tBu)-OH] is successfully coupled: This involves checking if an exposed amino group still exists in the reaction solution. The product from step 4) is vortexed five times with 10 mL of DMF solvent, each time for 1 min. A small amount of resin is placed in a Durham tube, and 2 drops of ninhydrin solution are added using a dropper. The tube is then heated in a 100°C metal bath for 2 min. If the amine resin turns its original transparent color, the amino acid coupling is successful. If the color remains purple, step 4 is repeated.

[0055] 6) Acetic anhydride-terminated amine resin: Add 10 mL of a solvent with anhydride:DIEA:DMF = 0.5:0.5:9 to the reaction tube of step 5), and rotate the reaction tube at room temperature for 30 min to obtain the third product. The purpose is to end-cap the very small amount of unreacted amino groups on the amine resin.

[0056] 7) As shown in Table 1, couple amino acids onto the main chain of the third product in the following order: methionine M, valine V, histidine H, cysteine ​​C, tryptophan W, phenylalanine F, glutamic acid E, leucine L, aspartic acid D, serine S, proline P, leucine L, lysine K (Boc), leucine L, valine V, phenylalanine F, phenylalanine F, lysine K (Dde). Repeat steps 2), 3), 4), and 5).

[0057] Table 1

[0058] 8) After coupling the last amino acid K[Fmoc-lys(Dde)-OH], repeat steps 2), 3), 4), and 5). Then, take a small amount of resin into a Durham tube, add 2 drops of ninhydrin solution using a dropper, and heat in a 100°C metal bath for 2 minutes. If the amine resin remains transparent, the amino acid coupling is successful. If the color turns purple, repeat the reaction.

[0059] 9) Remove the protecting group of the amino group Fmoc in the main chain of K[Fmoc-lys(Dde)-OH]: The method is the same as step 2).

[0060] 10) Detection of amino groups exposed in the K[Fmoc-lys(Dde)-OH] backbone: The method is the same as in step 3).

[0061] 11) Exposed amino groups on the K[Fmoc-lys(Dde)-OH] main chain of acetic anhydride-terminated amine resin: The method is the same as in step 6), but the Fmoc-protected amino groups on the K main chain are converted into acetyl groups to avoid the influence of the amino groups removed from the main chain.

[0062] 12) Removal of the K[Fmoc-lys(Dde)-OH] side-chain amino Dde protecting group: The product from step 11) was vortex-washed 5 times with 10 mL of DMF solvent, 1 min each time. 250 μL of hydrazine hydrate was added to 10 mL of DMF and mixed, then added to the vortex-washed product from step 11) and reacted at room temperature for 8 min. This process was repeated once until the K[Fmoc-lys(Dde)-OH] side-chain amino Dde protecting group was completely removed.

[0063] 13) Detection of amino groups exposed on the K[Fmoc-lys(Dde)-OH] side chain: The method is the same as in step 3) until the Dde group is completely removed.

[0064] 14) Coupling PpIX: Weigh PpIX, Oxyma, and DIC with a relative amine resin loading of 3 times, 6 times, and 6 times respectively. Add 10 mL of DMF and dissolve them completely. Then add them to the reaction tube containing the amine resin (step 12) and place it in a 40°C incubator to rotate and react for 48 h.

[0065] 15) Detect the presence of exposed amino groups in the reaction solution: that is, detect whether PpIX is successfully connected, the method is the same as step 3).

[0066] 16) Drying the amine resin: Vortex wash the product from step 14) with DMF solvent until the filtrate is colorless, wash in the order of DMF→DCM→DMF→DCM, 1 min*3 times for each step, vacuum dry, and transfer to a 50 mL centrifuge tube.

[0067] 17) Cutting: Add 10 mL of cutting solution (2.5% triisopropylsilane + 2.5% ultrapure water + 95% trifluoroacetic acid) to a centrifuge tube containing amine resin (the product of step 16). After reacting for 3 h, collect the filtrate, transfer it to a round-bottom flask, and perform rotary evaporation. Then, add ice-cold ether stored at -20℃ to precipitate the peptides. Transfer the system to a centrifuge tube and centrifuge at 3-4℃ and 10000-14000 r / min. Add ice-cold ether and repeat the vortexing, sonication, and centrifugation twice. Seal the tube with sealing film and vacuum dry to obtain solid peptide powder, namely the nanopeptide material TP-Fz, which is stored under room temperature drying conditions.

[0068] The structure of the TP-Fz nanopeptide material prepared in this embodiment was analyzed, and the mass spectrum is shown below. Figure 2 As shown, the size meets the design expectations, confirming the feasibility of the method and the correct synthesis of the materials.

[0069] Example 2 This embodiment verifies the self-assembly tendency of the TP-Fz nanopeptide material prepared in Example 1 under different ratios of water / dimethyl sulfoxide (DMSO) solution systems, as follows: The TP-Fz nanopeptide material prepared in Example 1 was dissolved in DMSO with ultrasonic assistance to make the concentration of the nanopeptide material mother liquor 10 mM. 2 μL of the above solution was added to water / DMSO systems with water contents of 0%, 20%, 40%, 60%, 80%, 90%, 98%, and 99.5%, and the volume was adjusted to 1 mL. The solutions were then shaken, vortexed, and centrifuged to obtain a water content gradient solution group with a peptide concentration of 20 μM.

[0070] Fluorescence intensity was measured in a series of solutions with a water content gradient of 20 μM for the peptides using fluorescence spectroscopy. The PpIX fluorescent molecules in the material, in their monomeric peptide form, are independent and emit high-intensity fluorescence under excitation light. With increasing water content, hydrophilic-hydrophobic interactions drive the self-assembly of peptide monomers into nanoparticles. The aggregation of PpIX leads to the formation of excitokines or excitokines through intermolecular π-π interactions or other non-radiative channels, consuming excited-state energy and thus weakening the fluorescence intensity; this is known as aggregation-caused quenching (ACQ).

[0071] The results are as follows Figure 3 As shown, the fluorescence intensity emitted by PpIX gradually decreases with increasing water content, which indirectly indicates that the degree and intensity of self-assembly of nanopeptide monomers in the system increase, verifying the physicochemical properties of the self-assembly tendency of nanopeptide materials in aqueous solution.

[0072] Example 3 This example studies the initial particle size of the TP-Fz nanopeptide material prepared in Example 1 and the change in particle size with incubation time after induction of fiber deformation by adding FZD7 recombinant protein. The method is as follows: 1. The TP-Fz nanopeptide material prepared in Example 1 was dissolved in DMSO with ultrasonic assistance to make the concentration of the nanopeptide material mother liquor 10mM. 2μL of the above solution and 18μL of dimethyl sulfoxide were added to a 1.5mL centrifuge tube, and then 980μL of ultrapure water was added to the centrifuge tube. The mixture was shaken, vortexed and centrifuged to obtain a nanopeptide material solution with a concentration of 20μM.

[0073] 2. Add FZD7 recombinant protein to the polypeptide nanosolution obtained in step 1 to make the concentration of FZD7 recombinant protein 1.25 μg / mL (protein: polypeptide ratio of 1:1000). 3. The particle size of the nanopeptide material solution and the peptide nanofiber dispersion with added recombinant protein were determined at different time points (0, 0.5, 6, 12, and 24 hours) using a dynamic light scattering instrument. The results are as follows: Figure 4 As shown, the effect of TP-Fz material on fiber aggregation under the induction of FZD7 recombinant protein is that the particle size gradually increases from about 20 nm to about 200 nm over time, indicating that TP-Fz nanopeptide material has good fiber allosteric ability under the induction of target protein FZD7.

[0074] Example 4 This embodiment further builds upon Example 3 by using transmission electron microscopy to observe the specific morphological characteristics of fiber formation over time in the TP-Fz nanopeptide material prepared in Example 1 under the induction of FZD7 recombinant protein. The method is as follows: 1. Based on steps 1 and 2 of Example 3, a polypeptide nanofiber dispersion was added dropwise to a 200-mesh thin carbon support film copper grid at 0, 0.5, 12, 6, and 24 hours, respectively. After drying at room temperature, the material was negatively stained with a 2% (w / v) uranium acetate solution. The morphology of the material was observed using a 120kV transmission electron microscope.

[0075] like Figure 5 As shown, TEM observations revealed that under the induction of recombinant FZD7 protein, the TP-Fz peptide nanoparticles gradually formed a dense fibrous network over time. This more intuitively demonstrates the physicochemical properties of the TP-Fz nanopeptide, which exhibits good fibrous allosteric ability under the induction of the target protein FZD7, providing a stable functional basis for subsequent in vitro cell experiments.

[0076] Example 5 Based on the physicochemical characterization of materials described in the above embodiments, this embodiment screens liver cancer cells and breast cancer cells for subsequent cell experiments. The method is as follows: 1. Using normal liver cells WRL 68 and liver cancer cells HepG2 and Huh7, normal breast cells CCD-1095Sk and breast cancer cells DU4475 and MDA-MB-231, at 5×10⁻⁶... 5 Cells were seeded per well into six-well plates and cultured for 24 hours in a cell culture incubator at 37°C, 5% CO2, and humidified air in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin.

[0077] 2. Discard the culture medium and wash with PBS 2-3 times. Add preheated 1×SDS-PAGE protein loading buffer (5×SDS-PAGE protein loading buffer diluted 1:4 in pure water) to lyse the cells, extract with a homogenizer 30-50 times, and denature in a 100℃ metal bath for 10 min.

[0078] 3. Proteins were separated using 10% polyacrylamide gel electrophoresis (PAGE), and the membrane was transferred on ice at 250 mA for 90 min. The membrane was blocked at room temperature for 1 h in 1×TBST solution containing 5% skim milk (10×TBS / Tween buffer diluted 1:10 in pure water). The PVDF membrane was cut, and primary antibody dilution buffer (diluted appropriately in blocking buffer) was added. The membrane was incubated overnight at 4°C with shake. The membrane was washed three times with 1×TBST for 5 min each time. Secondary antibody dilution buffer (diluted 1:5000 in blocking buffer) was added, and the membrane was incubated at room temperature for 1 h. The membrane was washed three times with 1×TBST for 5 min each time. Enhanced chemiluminescence (ECL) working solution was prepared for exposure imaging.

[0079] The results are as follows Figure 6 As shown in A and B, HepG2 liver cancer cells and MDA-MB-231 breast cancer cells have higher FZD7 expression levels compared with normal cells and other similar cancer cells, and were subsequently selected as model subjects for in vitro cell experiments of TP-Fz nanopeptide materials.

[0080] Example 6 This embodiment examines the co-localization of the TP-Fz nanopeptide material prepared in Example 1 and the anti-FZD7 antibody on the tumor cell membrane, using the following method: 1. Using HepG2 liver cancer cells and MDA-MB-231 breast cancer cells, at a concentration of 2.5 × 10⁻⁶ cells... 4 Cells were seeded per well into confocal dishes and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 37°C, 5% CO2, and humidified air until the cells were fully spread.

[0081] 2. Replace the original culture medium with the culture medium of the TP-Fz nanopeptide material solution (20 μM) prepared in step 1 of Example 3 and culture for 24 h.

[0082] 3. After washing with PBS 2-3 times, fix and block the cells, incubate with anti-FZD7 antibody (antibody: primary antibody dilution = 1:400) at 4°C overnight, and finally incubate with fluorescent secondary antibody at room temperature for 1 hour. Stain the cell nuclei with DAPI dye for 5 minutes, mount with anti-fluorescence quenching mounting medium, and perform CLSM two-photon confocal imaging experiment.

[0083] The results are as follows Figure 7 As shown, where Figure 7 In Figure A, CLSM was used to observe the co-localization of TP-Fz nanopeptide material and FZD7 protein in HepG2 liver cancer cells, and in Figure B, CLSM was used to observe the co-localization of TP-Fz nanopeptide material and FZD7 protein in MDA-MB-231 breast cancer cells. It can be seen that the PpIX fluorescence (red) of TP-Fz peptide nanofibers and the FZD7 antibody (green) both showed good co-localization on the membrane surface of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells. The two overlapped and showed an orange-yellow color, which demonstrated the excellent cell membrane targeting ability of TP-Fz nanopeptide material targeting FZD7 protein at the cellular level.

[0084] Example 7 This embodiment examines the formation of fibers of TP-Fz nanopeptide materials on the cell membrane surface using TEM, and the method is as follows (taking HepG2 liver cancer cells as an example): 1. Using HepG2 liver cancer cells at a concentration of 5 × 10⁻⁶ 5Cells were seeded per well into 6-well plates and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 37°C, 5% CO2 and humidified air until the cells were fully spread.

[0085] 2. Following the method described in step 1 of Example 3, a 20 μM nanopeptide material solution was prepared. The following day, the nanoparticle solution was replaced with the original culture medium and cultured for 24 h. Cells were digested and collected, resuspended in electron microscopy fixation solution (PBS solution containing 2.5% glutaraldehyde), and the cell clumps were dispersed. After fixation at room temperature in the dark for 30 min, the cells were transferred and stored at 4 °C for subsequent resin embedding and ultrathin sectioning.

[0086] The results are as follows Figure 8 As shown in the figure (arrows indicate fibrous structures), TEM observation of cells shows that TP-Fz nanopeptide materials form fibrous structures on the surface of liver cancer cell membranes, proving that TP-Fz nanopeptide materials also have the ability to deform into fibers on the surface of tumor cells, represented by liver cancer cells HepG2.

[0087] Example 8 This embodiment examines the formation of fibers on the cell membrane surface by SEM observation of TP-Fz nanopeptide materials, using the following method: 1. Using HepG2 liver cancer cells and MDA-MB-231 breast cancer cells, at a ratio of 1×10⁻⁶... 4 Cells were seeded per well into 48-well plates with spreaders and cultured in RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in a cell culture incubator at 37°C, 5% CO2 and humidified air until the cells were fully spread.

[0088] 2. Replace the original culture medium with the TP-Fz nanopeptide material solution (20 μM) prepared in step 1 of Example 3 and culture for 24 h. Discard the culture medium, wash 2-3 times with PBS, and then fix overnight with electron microscopy fixation solution (PBS solution containing 2.5% glutaraldehyde).

[0089] 3. Discard the fixative solution, wash 2-3 times with PBS, and then prepare an ethanol gradient solution (30%, 50%, 70%, 80%, 90%, and 99.5%) using purified water for sequential dehydration. Finally, wash twice with hexamethyldisilazane in a fume hood, and after thorough drying in a desiccator, attach the cell slides to the sample stage using conductive adhesive for gold plating, and observe under a SEM scanning electron microscope.

[0090] The results are as follows Figure 9 As shown, where, Figure 9Figure A shows the SEM observation of the fiber formation process of TP-Fz nanopeptide material on the surface of HepG2 cell membrane, and Figure B shows the SEM observation of the fiber formation process of TP-Fz nanopeptide material on the surface of MDA-MB-231 cell membrane. Through SEM observation of cells, TP-Fz nanopeptide material formed fiber structures on the surface of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells that were significantly different from the other two groups (Blank is the blank group without drug, and CTP-Fz is the TP-Fz control drug that does not have fiber deformation ability but retains FZD7 targeting ability, and the following explanation is omitted). This further visually demonstrates the fiber deformation ability of TP-Fz nanopeptide material on the surface of tumor cells (scale bar is 10 μm).

[0091] Example 9 This embodiment investigates the inhibition of β-catenin expression and nuclear translocation, a key protein in the Wnt pathway, by TP-Fz nanopeptide material under Western blot analysis. The method is as follows: 1. Following the method and quantity described in step 1 of Example 5, liver cancer cells HepG2 and breast cancer cells MDA-MB-231 were incubated with TP-Fz nanopeptide material in a six-well plate for 24 hours.

[0092] 2. Discard the culture medium, wash the cells 2-3 times with pre-chilled PBS, and gently collect the cells into a centrifuge tube using a cell scraper. Centrifuge to remove the supernatant. Add pre-chilled cytoplasmic extraction buffer containing PMSF protease inhibitor and incubate on ice for 15 minutes. Add NP-40 lysis buffer, vortex vigorously at maximum speed for 5 seconds, incubate on ice for 1 minute, repeat vortexing for 5 seconds, and then centrifuge to collect the supernatant, which is the cytoplasmic protein extract.

[0093] 3. For precipitation, add pre-cooled nuclear extraction buffer containing PMSF protease inhibitor and vortex vigorously at maximum speed for 30 seconds. Repeat the vortexing step every two minutes for the next 30 minutes. Finally, centrifuge and collect the supernatant, which is the nuclear protein extract.

[0094] 4. The total cell protein extract was prepared as shown in steps 1 and 2 of Example 5. Subsequently, the obtained total cell protein, plasma protein and nuclear protein extracts were subjected to Western blot analysis as shown in step 3 of Example 5.

[0095] Protein immunoblotting assay results of TP-Fz nanopeptide material inhibiting the expression level and nuclear translocation of β-catenin, a key protein in the Wnt pathway. Figure 10 As shown, where, Figure 10In the figures, A and B represent HepG2 liver cancer cells and MDA-MB-231 breast cancer cells, respectively. TP-Fz nanopeptide materials significantly reduced the overall expression level of β-catenin, a key protein in the Wnt signaling pathway, and inhibited its tendency to translocate from the cytoplasm to the nucleus in its active state. This provides strong evidence for further investigation into the effects of TP-Fz nanopeptide materials on the transcription level of downstream target genes of β-catenin.

[0096] Example 10 This embodiment examines the effect of TP-Fz nanopeptide material on the transcriptional level of downstream target genes of β-catenin using qPCR analysis. The method is as follows: 1. Following the method and quantity described in step 1 of Example 5, liver cancer cells HepG2 and breast cancer cells MDA-MB-231 were incubated with TP-Fz nanopeptide material in a six-well plate for 24 hours.

[0097] 2. RNA extraction: Discard the culture medium, wash 2-3 times with PBS, add TRizol for lysis and collect the lysis buffer. Then add chloroform, centrifuge to separate the layers, and collect the upper aqueous phase. Add isopropanol to precipitate the RNA and centrifuge again. Discard the supernatant, wash the precipitate twice with 75% ethanol, discarding the liquid after each centrifugation. Dry the precipitate, dissolve it in DEPC water, check the purity, and prepare for reverse transcription.

[0098] 3. Reverse transcription to cDNA: Using a reverse transcription kit, mix RNA template, Axin2, CyclinD1 and c-Myc primers, dNTP mixture, and RNase-free water in the specified proportions. Incubate at 65°C for 5 minutes, then place on ice. Add reverse transcriptase, buffer, and RNase inhibitor, mix gently, and briefly incubate. Perform reverse transcription according to the following schedule: 25°C for 5 minutes, 42°C for 30 minutes, and 85°C for 5 seconds.

[0099] qPCR: Using a qPCR kit, mix cDNA template, Axin2, CyclinD1 and c-Myc primers, qPCR mixture, and RNase-free water in the specified proportions. A two-step reaction program was used: 95℃ pre-denaturation for 30 seconds, followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 30 seconds. Finally, analyze the melting curve.

[0100] The results of qPCR detection of the inhibition of downstream target gene transcription levels of the Wnt pathway by TP-Fz nanopeptide material are as follows: Figure 11 As shown, where, Figure 11In the examples, A and B represent HepG2 liver cancer cells and MDA-MB-231 breast cancer cells, respectively. TP-Fz nanopeptide materials significantly reduced the transcription levels of downstream target genes Axin2, CyclinD1, and c-Myc in HepG2 liver cancer cells and MDA-MB-231 breast cancer cells. The combined results of Examples 9 and 10 demonstrate the direct inhibitory effect of TP-Fz nanopeptide materials on the Wnt pathway activity of tumor cells.

[0101] Example 11 This embodiment investigates the inhibitory effect of TP-Fz nanopeptide materials on the proliferation of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells. The method is as follows: 1. Following the methods and quantities in steps 1 and 2 of Example 6, liver cancer cells HepG2 and breast cancer cells MDA-MB-231 were incubated with TP-Fz nanopeptide material in a 48-well plate with a climbing plate for 24 hours.

[0102] 2. Add the 2×EdU working solution preheated at 37℃ to a 48-well plate with a spreader, using the same volume as the culture medium, to make the final EdU concentration 1×, and continue incubating the cells for 2 hours.

[0103] 3. After fixing and permeabilizing the cells, prepare the Click reaction solution according to the kit instructions, add 500 μL of the reaction mixture to each well, and incubate at room temperature in the dark for 30 min. Stain the cell nuclei with DAPI dye for 5 min, mount with anti-fluorescence quenching mounting medium, and then perform CLSM two-photon confocal imaging experiments.

[0104] The results of the inhibition of the proliferation of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells by TP-Fz nanopeptide materials are as follows: Figure 12 As shown, where, Figure 12 In the figure, A and B represent HepG2 liver cancer cells and MDA-MB-231 breast cancer cells, respectively. It can be seen that within a time window of only 2 hours, TP-Fz nanopeptide material has a significant inhibitory effect on the proliferation of both HepG2 liver cancer cells and MDA-MB-231 breast cancer cells (scale bar is 5 μm).

[0105] Example 12 This embodiment investigates the inhibitory effect of TP-Fz nanopeptide materials on the migration ability of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells. The method is as follows: 1. Prepared according to conventional methods at a concentration of 5×10⁻⁶. 5Suspensions of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells per mL were added to both sides of the dual-well cell culture insert in a 24-well plate. The cells were incubated at 37°C and 5% CO2 until they were completely adhered. 2 mL of RPMI 1640 complete culture medium was then added to the outside of the insert.

[0106] 2. Replace the cell culture medium in the plug-in with the TP-Fz nanopeptide material solution (20 μM) prepared in step 1 of Example 3 and culture for 24 h.

[0107] 3. Gently remove the insert by holding one corner with sterile forceps. After slowly washing with PBS 2-3 times, add 2 mL of RPMI 1640 complete culture medium to the 24-well plate and continue culturing. During this period, use a CLSM two-photon confocal microscope to photograph the repair of the scratched area at three time points (0, 24, and 48h).

[0108] The results of the inhibition of the migration ability of TP-Fz nanopeptide materials on HepG2 liver cancer cells and MDA-MB-231 breast cancer cells are as follows: Figure 13 As shown, where, Figure 13 In the diagram, A and B represent HepG2 liver cancer cells and MDA-MB-231 breast cancer cells, respectively. During the process of edge cells in the scratched area migrating towards the center for repair within 48 hours, TP-Fz nanopeptide material significantly inhibited the migration ability of both HepG2 liver cancer cells and MDA-MB-231 breast cancer cells.

[0109] Example 13 This embodiment investigates the inhibitory effect of TP-Fz nanopeptide materials on the invasion of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells. The method is as follows: 1. Following the method and quantity described in step 1 of Example 5, liver cancer cells HepG2 and breast cancer cells MDA-MB-231 were incubated with TP-Fz nanopeptide material in a six-well plate for 24 hours.

[0110] 2. Using a refrigerated pipette tip, thoroughly mix serum-free culture medium and matrix gel at a ratio of 8:1, then add the mixture to the appropriate cell culture insert on top of a 24-well plate. Incubate at 37°C for 3 hours.

[0111] 3. Add serum-free RPMI 1640 medium to hydrate the matrix gel for 0.5 hours, and prepare a concentration of 5×10⁻⁶ using standard methods. 5 HepG2 liver cancer cells and MDA-MB-231 breast cancer cells were suspended in a concentration of cells / mL. 200 μL of the suspension was added to the upper layer, and RPMI 1640 medium containing 20% ​​FBS was added to the lower layer. The cells were incubated in an incubator for 24 h.

[0112] 4. After culture, remove the cell culture insert and gently rinse the chamber twice with PBS to remove residual culture medium. Add an appropriate amount of 4% paraformaldehyde to both the upper and lower chambers, fix at room temperature for 20-30 minutes, and then wash 2-3 times with PBS.

[0113] 5. Gently wipe away any unmembrane-bound cells and residual matrix gel from the upper chamber surface with a cotton swab. Add crystal violet staining solution to the lower chamber and stain at room temperature for 15-20 minutes. Finally, rinse the chamber with distilled water until the background is colorless. Invert the chamber, let it air dry, and then observe it under a microscope.

[0114] The results of the inhibition of the invasive ability of TP-Fz nanopeptide materials on HepG2 liver cancer cells and MDA-MB-231 breast cancer cells are as follows: Figure 14 As shown, within 24 hours, TP-Fz nanopeptide materials penetrated the matrix gel from the upper chamber of the cell culture plug to the lower chamber, and the cells showed a significant inhibitory effect on the invasive ability of HepG2 liver cancer cells and MDA-MB-231 breast cancer cells.

[0115] Example 14 This embodiment investigates how TP-Fz nanopeptide materials alter the morphology, number of exosomes, and particle size of HepG2 liver cancer cells (all subsequent embodiments use cells as examples). The method is as follows: 1. In T75 cell culture flasks, HepG2 liver cancer cells are amplified to the required supernatant volume (in this example, 200 mL of cell supernatant is required to amplify to 10 T75 flasks, with 10 mL of culture medium in each flask).

[0116] 2. After washing with PBS 2-3 times, replace the original culture medium with the culture medium of the TP-Fz nanopeptide material solution (20 μM) prepared in step 1 of Example 3 and culture for 24 h (10 mL per bottle).

[0117] 3. After washing 2-3 times with PBS, replace the original culture medium with RPMI 1640 serum-free medium containing 1% penicillin-streptomycin and culture for 48 hours.

[0118] 4. Collect the cell supernatant from each group bottle, and perform pre-centrifugation according to the standard ultracentrifugation method for cell supernatant exosomes at 4℃, centrifuging at 300×g for 10 minutes, 2000×g for 20 minutes, and 10000×g for 30 minutes.

[0119] 5. Pour the pretreated supernatant into an ultracentrifuge tube and centrifuge at 100,000 × g for 70 minutes at 4°C. Discard the supernatant. Gently resuspend the precipitate in 2-3 mL of pre-chilled PBS for purification and washing. Centrifuge again at 100,000 × g for 70 minutes at 4°C. Discard the supernatant; the precipitate is the exosomes after each fraction treatment. Resuspend in a small amount of PBS and aliquot for subsequent experiments.

[0120] 6. Referring to the method in step 1 of Example 4, the morphology of exosomes under each treatment group was photographed using TEM, and the number and particle size distribution of exosomes were analyzed using a nanoparticle tracking analyzer.

[0121] The results of TP-Fz nanopeptide materials altering the morphology, number, and size of exosomes in HepG2 liver cancer cells are as follows: Figure 15 As shown, exosomes were successfully extracted and purified from cell supernatant using the method described in this embodiment. Furthermore, it was observed that the exosomes extracted after treatment with TP-Fz nanopeptide material differed morphologically from the typical "elliptical cup-shaped" structure of the other two groups, losing their regular and uniform morphological characteristics and exhibiting fibrous-like structures surrounding them. Simultaneously, nanoparticle tracking analysis showed that TP-Fz nanopeptide material treatment reduced the number and particle size distribution of HepG2 cell exosomes, preliminarily demonstrating the functional properties of TP-Fz nanopeptide material in inhibiting exosome secretion.

[0122] Example 15 This embodiment further investigates the functional properties of TP-Fz nanopeptide materials in inhibiting exosome secretion by exosome staining and reuptake, as follows: 1. Following the method in step 1 of Example 8, HepG2 liver cancer cells were allowed to fully adhere and spread in a 48-well plate with a climbing slide.

[0123] 2. Take an equal volume of the exosome suspension dispensed in step 5 of Example 14 for each group, stain it with the exosome-specific dye PKH67 according to the method provided in the kit, and then add it to a 48-well plate to induce cell culture for 24 hours until it is fully taken up by the cells.

[0124] 3. Following the method described in step 3 of Example 6, the slide was fixed, sealed, and stained with cell nuclei. After mounting, two-photon confocal imaging was performed using a CLSM.

[0125] The results of exosome staining observation on the effect of TP-Fz nanopeptide material on the overall excretion amount of HepG2 liver cancer cells are as follows: Figure 16As shown, the exosomes extracted after treatment with TP-Fz nanopeptide materials, after PKH67 staining and cellular reuptake, showed a significant reduction compared to the untreated Blank group, further demonstrating the functional properties of TP-Fz nanopeptide materials in inhibiting exosome secretion.

[0126] Example 16 This embodiment investigates whether TP-Fz nanopeptide materials can directly target and induce fibrous deformation in free exosomes of liver cancer cells. The method is as follows: 1. Each group takes an equal amount (1×10). 9 One exosome and 100 μL of the TP-Fz nanopeptide material solution (20 μM) prepared in step 1 of Example 3 were placed in a 300 μL centrifuge tube and allowed to stand for 24 h. The sample was prepared according to the method in step 1 of Example 4 and observed by TEM.

[0127] TEM observation of the direct targeting of TP-Fz nanopeptides to HepG2 exosomes of liver cancer cells is as follows: Figure 17 As shown, TP-Fz nanopeptide materials can directly target free exosomes and produce significant fibrous deformation, while also exhibiting a certain exosome "aggregation" and encapsulation deformation effect. This indicates that since the components of exosomes secreted by cells are largely the same as those on the cell membrane of the cell itself, TP-Fz nanopeptide materials can not only induce fibrous deformation on the cell membrane surface under the induction of FZD7 and play a role in inhibiting exosome secretion, but also have the same effect on exosomes free in blood vessels, achieving a "dual-targeting" inhibitory effect. This provides a certain physicochemical basis for inhibiting the intercellular signaling communication function of exosomes.

[0128] Example 17 This embodiment investigates whether the functional characteristics of exosomes that activate the Wnt signaling pathway are altered after direct targeting with TP-Fz nanopeptide materials. The method is as follows: 1. Following the procedure in step 1 of Example 16, the TP-Fz nanopeptide material was pre-incubated with exosomes secreted by HepG2 liver cancer cells for 24 hours. Simultaneously, HepG2 cells were incubated and cultured in a six-well plate following the procedure in step 1 of Example 5.

[0129] 2. After washing with PBS 2-3 times, replace with new RPMI 1640 medium and add the mixture from step 1 to each well, then induce for 24 hours.

[0130] 3. Perform protein immunoblotting analysis according to the method in step 3 of Example 5.

[0131] Western blot analysis of the effect of exosomes secreted by cells after TP-Fz nanopeptide intervention on the activation capacity of the Wnt pathway is as follows: Figure 18 As shown, compared with other components, the exosomes treated with TP-Fz nanopeptide materials lost the ability to upregulate β-catenin, a key protein in the Wnt signaling pathway, thus inhibiting the overall cascade amplification of signal transduction in the intercellular Wnt pathway.

[0132] It can be seen that the functional characteristics of TP-Fz nanopeptide material in inhibiting the abnormal activation of Wnt signaling pathway in tumor cells by directly targeting the cell membrane and indirectly inhibiting exosome secretion are fully demonstrated.

[0133] In summary, this invention provides a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, its preparation method, and its application. The nanopeptide material comprising a peptide with the amino acid sequence shown in SEQ ID NO:1, and a hydrophobic fluorescent group linked to the terminal lysine residue of the peptide via an amide bond. Upon targeting the highly expressed FZD7 protein on the tumor cell membrane surface, this nanopeptide material transforms in situ into a nanofiber network structure, permanently retaining and directly blocking the binding site of the Wnt ligand to the FZD7 protein, thus inhibiting signal activation upstream of the pathway. Simultaneously, the dense fiber network indirectly restricts the autocrine and paracrine processes of the mature and modified Wnt protein via vesicles or exosomes, inhibiting signal cascade amplification and transduction between neighboring cells. This dual downregulation of key protein expression and downstream target protein transcription levels in the Wnt signaling pathway, both internally and externally, ultimately reduces cell invasion, migration, and overall physiological activity, enhancing the therapeutic effect on tumors.

[0134] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements or modifications should fall within the protection scope of the appended claims.

Claims

1. A nanopeptide material that dually inhibits the tumor Wnt signaling pathway, characterized in that, It includes a polypeptide with an amino acid sequence as shown in SEQ ID NO:1, and a hydrophobic fluorescent group connected to the terminal lysine of the polypeptide via an amide bond.

2. The nanopeptide material with dual inhibition of the tumor Wnt signaling pathway according to claim 1, characterized in that, The hydrophobic fluorescent group is selected from one of protoporphyrin, 7-nitrobenzodiazole, and bispyrene molecules.

3. The nanopeptide material with dual inhibition of the tumor Wnt signaling pathway according to claim 1, characterized in that, The hydrophobic fluorescent group is linked to the amino group of the terminal lysine residue of the polypeptide via an amide bond.

4. A method for preparing a nanopeptide material that dually inhibits the tumor Wnt signaling pathway, characterized in that, Including the following steps: The amine resin was subjected to swelling treatment, and the Fmoc protecting group of the amino group of the amine resin was removed to obtain the first product; Y[Fmoc-Tyr(tBu)-OH], additives, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide were mixed and reacted with the first product to obtain the second product; The second product was reacted with a mixture of acetic anhydride, N,N-diisopropylethylamine, and N,N-dimethylformamide to obtain the third product; The fourth product is obtained by coupling amino acids onto the main chain of the third product in the following order: methionine M, valine V, histidine H, cysteine ​​C, tryptophan W, phenylalanine F, glutamic acid E, leucine L, aspartic acid D, serine S, proline P, leucine L, lysine K (Boc), leucine L, valine V, phenylalanine F, phenylalanine F, and lysine K (Dde). The Fmoc protecting group of the main chain amino group in the fourth product is removed, and the exposed amino group of the main chain is capped with acetic anhydride to obtain the fifth product. The protecting group of the side chain amino group Dde of lysine K (Dde) in the fifth product was removed by using an N,N-dimethylformamide solution containing hydrazine hydrate to obtain the sixth product; The hydrophobic fluorescent group, additives, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide and the sixth product were mixed and subjected to an amidation reaction to obtain nanopeptide materials.

5. The method for preparing the nanopeptide material that dually inhibits the tumor Wnt signaling pathway according to claim 4, characterized in that, Before the step of removing the protecting group of the side chain amino group Dde of lysine K (Dde) in the fifth product using an N,N-dimethylformamide solution containing hydrazine hydrate, the method further includes: The Fmoc protecting group on the lysine K(Dde) was removed using N,N-dimethylformamide containing piperidine to obtain lysine K(Dde) containing an exposed amino group; By mixing carbonic anhydride with the lysine K (Dde) containing exposed amino groups, a reaction is carried out to obtain lysine K (Dde) containing inert acyl functional groups.

6. The method for preparing the nanopeptide material that dually inhibits the tumor Wnt signaling pathway according to claim 4, characterized in that, In the step of reacting the mixture of Y[Fmoc-Tyr(tBu)-OH], additives, N,N'-diisopropylcarbodiimide and N,N-dimethylformamide with the first product, the mass ratio of Y[Fmoc-Tyr(tBu)-OH], the additives, the N,N'-diisopropylcarbodiimide and the amine resin is (2.5-3.5):(5-7):(5-7):

1.

7. The method for preparing the nanopeptide material that dually inhibits the tumor Wnt signaling pathway according to claim 4, characterized in that, In the step of reacting the second product with a mixed system of acetic anhydride, N,N-diisopropylethylamine, and N,N-dimethylformamide, the volume ratio of the acetic anhydride, the N,N-diisopropylethylamine, and the N,N-dimethylformamide is (0.5-1):(0.5-1):(8.5-9).

8. The method for preparing the nanopeptide material that dually inhibits the tumor Wnt signaling pathway according to claim 4, characterized in that, In the step of mixing the hydrophobic fluorescent group, the additive, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide and the sixth product, the mass ratio of the hydrophobic fluorescent group, the additive, the N,N'-diisopropylcarbodiimide and the amine resin is (2.5-3.5):(2.5-3.5):(5.5-6.5):

1.

9. The method for preparing the nanopeptide material that dually inhibits the tumor Wnt signaling pathway according to claim 4, characterized in that, The mixture of hydrophobic fluorescent group, additive, N,N'-diisopropylcarbodiimide, N,N-dimethylformamide and the sixth product is subjected to an amidation reaction. The process further includes the steps of: drying the amine resin in the reaction product, adding the shaving liquid, and obtaining the nanopeptide material by rotary evaporation, precipitation, centrifugation and drying. The cut liquid, by volume fraction, comprises: 2.5% triisopropylsilane, 2.5% ultrapure water, and 95% trifluoroacetic acid.

10. The use of a nanopeptide material that dually inhibits the tumor Wnt signaling pathway as described in claims 1-3 in the preparation of drugs for treating breast cancer or liver cancer.

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