Self-assembled probe and preparation method and application thereof

Abstract

The invention discloses a self-assembled probe as well as a preparation method and application thereof. The self-assembly probe comprises a target recognition unit 1, a self-assembly unit and an imaging group, wherein the self-assembly unit comprises an amino acid sequence as shown in SEQ ID NO: 1; the targeted recognition unit 1 targets carbonic anhydrase IX. The self-assembly probe provided by the invention can recognize and combine target protein overexpressed on the surface of a tumor cell in a targeted manner, and can be triggered to perform in-situ self-assembly on the surface of the tumor to form nanofibers, so that the imaging efficiency and effect of the tumor are enhanced, the residence time is prolonged, and long-acting imaging of the tumor is realized; wide application prospects are realized in preparation of reagents and medicines for diagnosing and / or treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of medical technology, specifically relating to a self-assembled probe, its preparation method, and its application. Background Technology

[0002] Renal cell carcinoma (RCC) is one of the 10 most common malignant tumors and also the leading cause of death among urinary system cancers. It is characterized by rapid progression, a high rate of early metastasis, and insensitivity to treatments such as chemotherapy and radiotherapy, which are considered major reasons for treatment failure. More than 30% of RCC patients have already metastasized at diagnosis, missing the optimal time for surgery. Even patients who initially respond to chemotherapy develop resistance within 10-14 months. These factors contribute to the persistently high mortality rate of RCC, seriously threatening human life and health. Therefore, early diagnosis and accurate imaging of RCC cells would significantly improve the survival rate and quality of life for RCC patients in my country.

[0003] In recent years, small molecule peptides have become the preferred material system due to their bioactivity, high specificity, ease of chemical modification, strong targeting, and biological stability. Among them, targeting peptides are peptides that can specifically bind to tumor cells or tissues, exhibiting advantages such as high affinity, high stability, and low toxicity. Therefore, they can be conjugated with anticancer drugs for the diagnosis and specific drug delivery of early-stage tumor lesions, showing promising prospects in the diagnosis and treatment of cancer. With the deepening of oncology research, significant progress has been made in the screening and synthesis of small molecule tumor-targeting peptide drugs, as well as the discovery, treatment, and imaging of new targets.

[0004] Carbonic anhydrase IX (CAIX) is a transmembrane glycoprotein composed of acidic amino acids, distributed in the cell membrane and nucleus, with relative molecular masses of 58,000 and 54,000, respectively. The CAIX protein consists of 459 amino acids. cDNA and genomic sequence analysis show that the CAIX protein comprises four regions: a 37-amino acid N-terminal signal peptide (SP), a 377-amino acid extracellular region, a 20-amino acid transmembrane region, and a 25-amino acid intracellular C-terminus (IC). The extracellular region is further divided into two parts: the proteoglycan region (PA) and the carbonic anhydrase region (CA). In normal tissues, CAIX expression is extremely low, found only sporadically in the epithelium of the stomach, bile ducts, pancreas, small intestine, and vas deferens. Northern blotting analysis revealed that CAIX is also expressed in the heart, liver, hair follicles, joint cavities, placenta, choroidal vascular plexus, and parotid gland. Fetal lung and muscle tissues show relatively high expression, while adult lungs and muscles do not express CAIX. CAIX is overexpressed in cervical cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, bile duct cancer, colon cancer, bladder cancer, and skin cancer, and is also overexpressed in renal cell carcinoma. Therefore, CAIX has great potential as an imaging target for renal cell carcinoma.

[0005] Furthermore, tumor cells possess strong proliferative and metastatic capabilities, and their proliferation and metastasis are regulated by integrins expressed by the cells. Among these, integrin αvβ3 is one of the most studied and discussed extracellular matrix adhesion receptors. Due to its high expression in tumor angiogenesis, αvβ3 has become an important target for tumor diagnosis and anti-tumor drug research. The natural ligands of αvβ3 all contain an arginine-glycine-aspartic acid (RGD) sequence, which participates in tumor cell proliferation and metastasis. This property of RGD can be used to construct molecular imaging probes for in vitro tumor diagnosis and drugs for targeted therapy. Based on the crucial role of integrin αvβ3 in tumor diagnosis and treatment, using it as another target to construct a bispecific fluorescent molecular probe to further enhance the imaging of renal cell carcinoma shows great promise.

[0006] Currently, supramolecular assembly strategies have been widely used as promising methods for diagnosing renal cell carcinoma. Most of these supramolecular assembly-based nanoparticles or mucosal adhesion biomaterials focus on prolonging the retention time of fluorescent probes and enhancing drug permeability to improve imaging efficacy against renal cell carcinoma. Although improved efficacy has been observed compared to simple drug infusion, its limited targeting ability, potential drug complications, and toxicity restrict its effective application. Therefore, new treatment methods need to be developed to improve imaging time against renal cell carcinoma while reducing complications. The main driving force behind peptide self-assembly is weak forces, such as hydrogen bonds, van der Waals forces, and electrostatic interactions. Furthermore, controllable supramolecular self-assembly with specific morphologies can be achieved through amino acid sequence design. Simultaneously, peptide assembly can be achieved in complex physiological environments. Assembly-induced retention (AIR) can effectively optimize the biodistribution of bioactive molecules in vivo, increase drug tumor permeability, and improve pharmacokinetic and pharmacodynamic behavior, providing new insights for the development of novel, highly efficient, and low-toxicity biomaterials.

[0007] Therefore, how to construct a highly specific probe based on the protein characteristics of tumor cells, and at the same time utilize the advantages of supramolecular assembly strategies to construct a probe that can achieve long-term imaging time and imaging quality for tumors such as renal cell carcinoma, so as to enable early and accurate diagnosis and monitoring of early renal cell carcinoma, and achieve anti-cancer effects through timely surgical treatment, and ultimately prolong the survival of renal cell carcinoma patients, is an urgent problem to be solved. Summary of the Invention

[0008] To address the problems of low targeting efficiency, low imaging quality, and short residence time at the tumor site in existing tumor-targeting probes, this invention provides a self-assembly probe, its preparation method, and its applications. The self-assembly probe is a bispecific polypeptide nanoprobe with dual tumor targeting and self-assembly functions. It targets and binds to carbonic anhydrase IX (CA IX) and integrin receptor αVβ3 overexpressed on the surface of tumor cells. The assembly-capable fragments, due to the binding of the polypeptide molecules to the target protein, reduce the activation entropy required for self-assembly, thereby triggering in-situ self-assembly to form nanofibers. This self-assembly probe, on the one hand, enhances the imaging efficiency and effectiveness of tumors through dual tumor targeting; on the other hand, it prolongs the residence time at the tumor site by forming nanofibers through in-situ self-assembly on the tumor surface, achieving long-term tumor imaging. It has broad application prospects in the preparation of reagents and drugs for the diagnosis and / or treatment of tumors.

[0009] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0010] The first aspect of the present invention provides a self-assembly probe comprising a target recognition unit 1, a self-assembly unit, and an imaging group, wherein the self-assembly unit comprises an amino acid sequence as shown in SEQ ID NO: 1; and the target recognition unit 1 targets carbonic anhydrase IX.

[0011] In this invention, the self-assembly unit is a polypeptide fragment that self-assembles to form a nanofiber-like structure. Under the active targeting of the tumor by the targeting recognition unit, the self-assembly unit's movement is restricted, which reduces the activation entropy required for its self-assembly. This triggers its in-situ self-assembly into nanofibers in tumor cells, prolonging its specific retention time on the tumor surface.

[0012] In this invention, the imaging group emits a signal after the self-assembled probe specifically targets tumor cells, thereby tracing and imaging the tumor cells.

[0013] The bispecific polypeptide nanoprobe provided by this invention contains two targeting recognition units and has a targeting recognition function. It actively targets the tumor site through the specificity of the molecule, and then forms specific nanofibers on the surface of tumor cells to achieve long-term targeting of renal cancer cells, reduce the difficulty and poor quality of renal cancer imaging, and thus achieve accurate diagnosis of renal cancer.

[0014] In some embodiments of the present invention, one of the tumor-targeting recognition units is a molecule that targets and binds to tumor matrix-related proteins, said molecule being selected from any one of a recognition unit targeting carbonic anhydrase IX, AMD3100, BL-8040, LY2510924, POL5551, SP-13786, Talabostat, and Linagliptin.

[0015] In some embodiments of the present invention, another tumor-targeting recognition unit is a polypeptide molecule that targets and binds to proteins on the surface of tumor cells.

[0016] In some preferred embodiments of the present invention, the tumor matrix-associated protein is carbonic anhydrase IX (CAIX) or fibroblast activating protein (FAP); another tumor-targeting recognition unit is chemokine receptor 4 or integrin receptor αvβ3.

[0017] In some preferred embodiments of the present invention, the targeting recognition unit includes a targeting recognition unit that targets carbonic anhydrase IX and / or a targeting recognition unit that targets integrin receptor αVβ3.

[0018] In some embodiments of the present invention, the target recognition unit 1 has a structural formula as shown in formula V or formula X:

[0019] Formula V;

[0020] Formula X; In some embodiments of the present invention, the self-assembled probe further comprises a targeting recognition unit 2, which targets the integrin receptor αVβ3; the targeting recognition unit 2 has a structural formula as shown in Formula VI or Formula IX:

[0021] Formula VI;

[0022] Formula IX.

[0023] In some embodiments of the present invention, the self-assembly probe includes the target recognition unit 1, the self-assembly unit, and the target recognition unit 2.

[0024] In some embodiments of the present invention, the self-assembled probe comprises formula V, an amino acid sequence as shown in SEQ ID NO: 1, and formula VI; or, the self-assembled probe comprises formula V, an amino acid sequence as shown in SEQ ID NO: 1, and formula IX; or, the self-assembled probe comprises formula X, an amino acid sequence as shown in SEQ ID NO: 2, and formula VI; or, an amino acid sequence as shown in SEQ ID NO: 2, and formula IX.

[0025] In some embodiments of the present invention, the imaging group is selected from one or more radionuclides, fluorescent groups, and magnetic groups.

[0026] In some specific embodiments of the present invention, the fluorescent group comprises one or more selected from FIGT, ICG, Cy7, Cy5 and IR-750.

[0027] In some specific embodiments of the present invention, the radionuclide comprises a subset selected from... 68 Ga、 89 Zr、 64 Cu and 82 One or more of Rb.

[0028] In this invention, the connecting unit is a polypeptide fragment or a polyethylene glycol-modified agent that regulates molecular length and hydrophilicity-hydrophobicity balance, which can improve the stability and targeting of the imaging probe and enhance imaging performance. The connecting unit connects the self-assembled sequence to the targeting recognition unit targeting αVβ3; and / or, the connecting unit connects the self-assembled sequence to the targeting recognition unit targeting carbonic anhydrase IX.

[0029] In some preferred embodiments of the present invention, the connecting unit connects the self-assembly unit and the target recognition unit 2.

[0030] In some preferred embodiments of the present invention, the linker unit comprises the amino acid sequence DPGLGYL (SEQ ID NO: 2), (OEG)4, or PEG2.

[0031] In some specific embodiments of the present invention, the connecting unit is PEG2.

[0032] In some embodiments of the present invention, the self-assembly probe further includes a connection unit.

[0033] In some embodiments of the present invention, the radionuclide is connected to the self-assembly unit via a chelating agent.

[0034] In some specific embodiments of the present invention, the chelating agent comprises one or more selected from Nota, DOTA, TETA, PCTA and DTPA.

[0035] In some specific embodiments of the present invention, the self-assembled probe has a structural formula selected from Formula II, Formula IV, and Formula VIII:

[0036] Formula II; Formula IV;

[0037] Formula VII.

[0038] The self-assembly probe provided in this invention binds to proteins and receptors on the surface of tumor cells. The fragments with assembly capabilities are restricted in their movement due to the restriction of polypeptide molecules, which reduces the activation entropy required for self-assembly and triggers their in-situ self-assembly to form nanofibers, thereby achieving long-term imaging of renal cancer cells.

[0039] A second aspect of the present invention provides an intermediate for a self-assembled probe, the intermediate differing from the self-assembled probe of the first aspect in that it does not contain the imaging group.

[0040] In some embodiments of the present invention, the intermediate has a structural formula selected from Formula I, Formula III and Formula XI;

[0041] Formula I;

[0042] Formula III; Formula XI.

[0043] A third aspect of the present invention provides a method for preparing a self-assembled probe or an intermediate of a self-assembled probe, the method comprising the steps of obtaining the self-assembled probe as described in the first aspect or the intermediate as described in the second aspect by polypeptide solid-phase synthesis.

[0044] The preparation method involves synthesizing and connecting the carbonic anhydrase IX targeting recognition unit, integrin receptor αVβ3 targeting unit, self-assembly unit, and linking unit via a polypeptide solid-phase synthesis method to obtain the bispecific polypeptide nanoprobe.

[0045] In this invention, the method for preparing the molecule with the structure shown in Formula I is as follows: (1) Solvent treatment: DMF and methanol should be deionized and dewatered before use.

[0046] (2) Full swelling of resin: Add 15 mL of DMF to every 2.0 g blank Wang resin and activate at 20-37℃ for 20-40 min.

[0047] (3) Inoculation of the first amino acid: At 20-37℃, remove the solvent from step (2), add 1 mmol of 3-5 times molar excess of C-terminal Fmoc-Asp(Oall)-OH, 3-5 times molar excess of DMAP, and 3-5 times molar excess of DIC; react at 20-37℃ for 2-4 h. After the reaction is complete, wash with DMF 4-6 times, 5-6 mL each time. Then add pyridine and acetic anhydride in a volume ratio of 1:(0.5-3), and react for 20-40 min. After the reaction is complete, optionally wash with DMF 4-6 times, 3-10 mL each time.

[0048] (4) Removal of Fmoc protecting group: Remove the solvent from step (3), add 5-20 mL of 15-25% piperidine DMF solution to the resin, stir with N2 for 5-15 min and filter out the solution, add another 5-15 mL of 15-25% piperidine DMF solution, stir with N2 for 2-10 min and filter out the solution again. Repeat this operation one to three times. Optionally, wash with DMF 1-5 times and methanol 1-3 times, 3-10 mL each time.

[0049] Optionally, step (4) may be followed by step (5) to detect the removal effect of ninhydrin.

[0050] (5) Detection of ninhydrin removal effect: Take out a small amount of resin, wash it with methanol two to four times, add ninhydrin, KCN and phenol solution, heat at 105℃-110℃ for 3-5 min, if it turns dark blue, it is a positive reaction, indicating that the removal is complete and the next step of reaction can be carried out; if it is colorless, it means that the protecting group has not been completely removed, and the above deprotection operation needs to be repeated.

[0051] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 2-4 times the molar excess of the next amino acid after the C-terminus, Fmoc-Gly-OH, Fmoc-Arg(pdf)-OH, Fmoc-Lys(DDE)-OH, Fmoc-d-Phe-OH, 2-4 times the molar excess of HBTU and -24 times the molar excess of HOBT into a reaction tube, add an appropriate amount of DMF solution to completely dissolve it, and then add 8-10 times the molar excess of (pure) DIEA. React at room temperature for 30-50 min. Optionally, wash with DMF 4-6 times, 3-10 mL each time.

[0052] Optionally, a small amount of resin is tested with ninhydrin assay reagent; if the result is colorless, then 10 mL of 20% piperidine DMF solution is added to remove Fmoc, repeating twice, for 5-10 min and 3-5 min respectively. Afterward, optionally, the resin is washed 4 times with DMF and 2 times with methanol, 5-6 mL each time. Optionally, a small amount of resin is tested with ninhydrin assay reagent; if the result is blue, the next reaction step can proceed.

[0053] (7) The Oall group was removed by Pd / C (CAS No.: 64741-65-7) catalysis, and DDE was removed by condensation with a condensing agent. The cyclic c(RGDfK) was then synthesized.

[0054] (8) Similarly, the synthesis of other peptides is carried out in the same way. Repeat the steps until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry it.

[0055] Optionally, the preparation method includes steps for resin shedding and pure product separation and detection: (9) Detection of resin shedding and separation of pure product The peptide was cleaved in a trifluoroacetic acid cleavage solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 1-3 h. The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysate was dried as much as possible with nitrogen gas, then precipitated with diethyl ether, centrifuged, and washed with diethyl ether 3-5 times to obtain a white solid. After dissolving in pure water, the solid was desalted and purified by HPLC, lyophilized, and crystals were precipitated. A small amount was used for MS analysis.

[0056] In some specific embodiments of the present invention, the method for preparing the molecule with the structure shown in Formula I is as follows: (1) Solvent treatment: DMF and methanol are soaked overnight in G3 pore molecular sieves to remove impurities and water before use.

[0057] (2) Full swelling of resin: Weigh 2.0 g blank Wang resin into a clean and dry reaction tube, add 15 mL DMF, and activate at room temperature for about 30 min.

[0058] (3) Inoculation of the first amino acid: At room temperature, filter off the solvent from the previous step using a sand filter, add 1 mmol of 5-fold excess C-terminal Fmoc-Asp(Oall)-OH, 5-fold excess DMAP, 5-fold excess DIC, and DMF as solvent, and react at room temperature for 3 h. After the reaction is complete, wash 4-6 times with DMF, 5-6 mL each time. Then add an appropriate amount of pyridine and acetic anhydride in a 1:1 volume ratio, and react for 30 min. After the reaction is complete, wash 4-6 times with DMF, 5-6 mL each time.

[0059] (4) Removal of Fmoc protecting group: Remove the solvent from the previous step by filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir with N2 for 10 min and filter out the solution, add another 10 mL of 20% piperidine DMF solution, stir with N2 for 5 min and filter out the solution again. Repeat this operation twice, wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0060] (5) Detection of ninhydrin removal effect: Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105℃-110℃ for 5 min, and the dark blue color indicates a positive reaction, indicating that the removal is complete and the next step of the reaction can be carried out; if it is colorless, it indicates that the protecting group has not been completely removed, and the above deprotection operation needs to be repeated.

[0061] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the C-terminus amino acid Fmoc-Gly-OH, Fmoc-Arg(pdf)-OH, Fmoc-Lys(DDE)-OH, Fmoc-d-Phe-OH, 3 times the molar excess of HBTU, and 3 times the molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve the resin, then add 10 times the molar excess of (pure) DIEA. React at room temperature for 40 min, and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If the result is colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc. Repeat this process twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and methanol 2 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If the result is blue, the next reaction can proceed.

[0062] (7) The Oall group was removed by Pd / C (CAS No.: 64741-65-7) catalysis, and DDE was removed by condensation with a condensing agent. The cyclic c(RGDfK) was then synthesized.

[0063] (8) Similarly, the synthesis of other peptides is carried out in the same way. Repeat the steps until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry it.

[0064] (9) Detection of resin shedding and separation of pure product

[0065] Finally, the peptide was cleaved for 2 h with trifluoroacetic acid cleavage solution (95% TFA: 2% TIS: 2% EDT: 1% H2O). The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysis buffer was dried as much as possible with nitrogen gas, then precipitated with diethyl ether, centrifuged, and washed with diethyl ether 3-5 times to obtain a white solid. After dissolving in pure water, the solid was desalted and purified by HPLC, lyophilized, and crystals were precipitated. A small amount was taken for MS analysis.

[0066] The synthesis steps of Formulas II-IV and Formula XI in this invention are the same as those of Formula I.

[0067] A fourth aspect of the present invention provides a composition comprising (1) a self-assembled probe as described in the first aspect or an intermediate as described in the second aspect; and (2) a pharmaceutically acceptable carrier.

[0068] The self-assembly probes provided by this invention can be delivered using their own self-assembly properties or targeting capabilities. In some cases, additional vectors may be required for delivery to improve their stability, targeting, and bioavailability in vivo.

[0069] The fifth aspect of the present invention provides the use of one or more of the following in the preparation of medicaments for diagnosing and / or treating tumors: a self-assembled probe as described in the first aspect, an intermediate as described in the second aspect, and a composition as described in the third aspect.

[0070] In some embodiments of the present invention, the tumor is cervical cancer, breast cancer, lung cancer, esophageal cancer, colon cancer, skin cancer, kidney cancer, bladder cancer, pancreatic cancer, or stomach cancer.

[0071] In this invention, the self-assembled probe is administered intravenously.

[0072] In some embodiments of the present invention, the administration concentration is less than 800 μM.

[0073] In some preferred embodiments of the present invention, the administration concentration is 200-500 μM.

[0074] The self-assembled probe provided by this invention has applications in tumor immunotherapy.

[0075] In this invention, the molecule of the structure shown in Formula I is CAIXB-GNNQQNY-K-PEG2-c(RGDfK), the molecule of the structure shown in Formula II is CAIXB-GNNQQNY-K(IR-750)-PEG2-c(RGDfK), the molecule of the structure shown in Formula III is CAIXB-GNNQQNY-K(NOTA)-PEG2-c(RGDfK), and the molecule of the structure shown in Formula IV is CAIXB-GNNQQNY-K(NOTA)-( 68 The molecules with structures shown in Ga)-PEG2-c(RGDfK) and Formula VII are CAIXA-GNNQQNY-K(IR-750)-PEG2-RGD. GNNQQNY (SEQ ID NO: 1) is a self-assembling sequence that enables in-situ assembly on tumor cell membranes, thereby achieving long-term blocking of tumor-related targets. The polypeptide fragment PEG2 is a linker, its purpose being to connect different units of the molecule, regulate molecule length and hydrophilic / hydrophobic balance, thereby improving the molecule's properties.

[0076] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0077] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0078] The reagents and raw materials used in this invention are all commercially available.

[0079] The significant advantages of this invention are as follows: The self-assembled probe provided by this invention targets and binds to CAⅨ and / or αVβ3 proteins overexpressed on the surface of tumor cells, enabling precise localization of tumor cells. After binding to the target protein, the self-assembled probe induces self-assembly on the surface of tumor cells to form nanofibers, thereby achieving long-lasting imaging of cancer cells, such as renal cell carcinoma cells. This provides a new method for cancer diagnostic imaging, and it does not produce significant side effects in vivo, exhibiting good biocompatibility and promising application prospects. Attached Figure Description

[0080] Figure 1 This is a schematic diagram of the structure of a bispecific self-assembled probe.

[0081] Figure 2 These are schematic diagrams illustrating two design principles for bispecific self-assembled probes.

[0082] Figure 3 The figure shows the critical assembly concentration results for the bispecific self-assembled probe CAⅨB-SA-RGD.

[0083] Figure 4 The figure shows the experimental results of the assembly kinetics of the bispecific self-assembly probe CSR-IR-750.

[0084] Figure 5 The image shows the results of the bispecific self-assembly probes CSR-IR-750 / CAⅨ and CSR-IR-750 / Integrin in solution, demonstrating their ability to transform and self-assemble into hydrophobic nanofibers.

[0085] Figure 6 The results are from bio-layer interferometry (BLI) experiments, which show the interaction between the bispecific self-assembled probe CSR-IR-750 and CAⅨ and Integrin protein molecules, respectively.

[0086] Figure 7 This figure shows the retention of the bispecific self-assembled probe CSR-IR-750 on the surface of HELA cell membrane and its co-localization with CAIX and Integrin.

[0087] Figure 8 Morphology of PBS group and bispecific self-assembled probe CSR-IR-750 on the surface of 7860 cells as shown by SEM electron microscopy.

[0088] Figure 9 The image shows the tumor-targeted enrichment fluorescence signals of the bispecific peptide probes CR-IR-750, C1SR1-IR-750, CS1R-IR-750, and CSR-IR-750 in a mouse subcutaneous tumor model.

[0089] Figure 10 CSR-NOTA is a bispecific self-assembled probe. (68Ga) Image of radionuclide scan results in a mouse subcutaneous tumor model.

[0090] Figure 11 The image shows the MS results for CAIXB-GNNQQNY-K-PEG2-c(RGDfK).

[0091] Figure 12 The image shows the MS results for the CSR-IR-750.

[0092] Figure 13 The image shows the MS results for C1SR1-IR-750.

[0093] Figure 14 The image shows the MS results for the CS1R-IR-750.

[0094] Figure 15 The image shows the MS results for CSR-NOTA. Detailed Implementation

[0095] Terminology Definition

[0096] In this invention, the term "self-assembly probe" refers to a polypeptide nanoprobe with tumor-targeting and self-assembly functions and containing groups for detection and imaging. It can target and bind to proteins or receptors overexpressed on the surface of tumor cells. The assembly-capable fragments, due to the binding of the polypeptide molecule to the target protein, reduce the activation entropy required for self-assembly, thereby triggering in-situ self-assembly to form nanofibers.

[0097] In this invention, the term "targeted" refers to specific binding, specifically a non-random binding reaction between two molecules, such as the reaction between a polypeptide sequence and a target protein overexpressed on the surface of tumor cells that it specifically recognizes. In some embodiments, a polypeptide sequence that specifically binds to a target protein means that the polypeptide sequence has a molecular weight of less than about 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or lower affinity (KD) binds to the target protein.

[0098] In this invention, the terms "tumor" or "cancer" refer to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0099] In this invention, the term "targeting recognition unit" refers to a polypeptide fragment that has a specific targeting function for proteins and receptors on the surface of tumors. Its purpose is to specifically and actively deliver self-assembly units with self-assembly capabilities to the tumor site.

[0100] In this invention, the term "self-assembly unit" refers to a polypeptide fragment that has the ability to self-assemble to form a stable nanofiber-like structure.

[0101] In this invention, the term "imaging group" refers to a group used for labeling and detection in imaging technology, enabling qualitative and quantitative studies of biological processes at the tissue, cellular, and molecular levels. Imaging technologies are mainly classified into categories such as optical imaging, radio-nuclear imaging, magnetic resonance imaging (MRI), ultrasound imaging, and computed tomography (CT) imaging.

[0102] In this invention, the term "chelating agent" refers to a class of substances commonly used in the preparation of targeted radiopharmaceuticals that simultaneously chelate radionuclides and connect targeted molecular probes.

[0103] In this invention, the term "radionoid" refers to a class of atoms with excess nuclear energy, making them unstable; they are also known as unstable nuclides. The excess nuclear energy contained in a radionuclide can generate or emit new radiation (gamma radiation) or new ions (alpha or beta particles) from the atomic nucleus, or it can be transferred to electrons within the atom and emitted (internal electron conversion), a process known as radioactive decay. Radionuclides with suitable half-lives are commonly used in nuclear medicine for diagnosis and treatment.

[0104] In this invention, the term "fluorescent group" refers to a group structure capable of absorbing photons of a certain energy (wavelength) and emitting fluorescence. Fluorescent groups can be prepared from independent small organic molecules or are fluorescent components of a functional system. Based on their chemical and physical properties, the sources of fluorescent groups can generally be divided into four categories: proteins and polymorphic substances, such as fluorescent proteins, which are commonly used for labeling and imaging in biological research; small organic molecule compounds, such as certain dyes, which exhibit fluorescence properties due to their small molecular size and specific chemical structures; synthetic oligomers and polymers, such as quantum dots, which are used in multiple fields due to their unique photoelectric properties; and multi-component systems, composed of multiple components that generate fluorescence through interactions.

[0105] In this invention, the term "magnetic group" refers to groups containing magnetic materials (such as iron oxide nanoparticles) that can be used in magnetic resonance imaging (MRI) and other magnetic detection technologies. These groups, through specific chemical treatments, can be used for the extraction, separation, analysis, and identification of biological materials. Magnetic groups have a wide range of applications, including but not limited to targeted drug delivery, separation and detection of biomolecules, and as contrast enhancers in magnetic resonance imaging.

[0106] In this invention, the term "connecting unit" refers to a polypeptide fragment or polyethylene glycol modification agent that regulates molecular length and hydrophilicity / hydrophobicity balance, thereby enhancing the stability and biocompatibility of self-assembled probes while reducing immunogenicity and nonspecific binding.

[0107] In this invention, the terms "intermediate for self-assembling probes" and "intermediate" have the same meaning, referring to polypeptide molecules that possess specific targeting capabilities for tumor cells and the ability to self-assemble in situ on the tumor surface, and can be further linked with imaging groups to form self-assembling probes. These intermediates have the potential to be used to connect drugs, thereby achieving specific killing of tumor cells.

[0108] In this invention, the term "FITC" stands for Fluorescein Isothiocyanate, a fluorescein derivative characterized by high absorbance, excellent fluorescence quantum yield, and good water solubility. It is one of the most widely used green fluorescein derivatives in biology. The isothiocyanate group of FITC can react with the amino terminus or primary amine of proteins to achieve protein labeling, including antibodies and lectins. In addition to its use as a protein marker, FITC can also be used as a protein fluorescent tracer, to label antibodies for rapid identification of pathogens, and for microsequencing of proteins and peptides (HPLC).

[0109] In this invention, the term "ICG" refers to a near-infrared I-region fluorescent dye approved by the U.S. Food and Drug Administration (FDA) for in vivo application. ICG possesses specific pharmacological properties; after injection, it binds to serum proteins and is primarily excreted via the liver, with a half-life of approximately 3 to 4 minutes. Clinically, ICG is widely used in assessing liver reserve function, tumor localization, and sentinel lymph node tracing and navigation. It can also be used in intracranial angiography (ICGA) and gastrointestinal surgery to evaluate lymph node and vascular perfusion.

[0110] In this invention, the term "Cy7" stands for Cyanine 7, a near-infrared anthocyanin fluorescent dye. Cy7 typically has an excitation wavelength around 750 nm and an emission wavelength around 770 nm. This characteristic allows Cy7 to penetrate deep biological tissues and avoid interference from background fluorescence, thus exhibiting unique advantages in in vivo imaging and experiments. Cy7 is commonly used in small animal in vivo imaging and experiments, such as cell imaging and vascular imaging, to help researchers observe and analyze physiological and pathological processes within organisms. Furthermore, Cy7 can also be used as a marker in drug delivery systems to track drug distribution and metabolism in vivo, as well as for the labeling and detection of biomolecules such as proteins and nucleic acids.

[0111] In this invention, the term "Cy5" stands for Cyanine 5, a far-red fluorescent dye belonging to the anthocyanin fluorescent dye series. Its excitation wavelength is approximately 640-650 nm, and its emission wavelength is approximately 660-670 nm. This makes Cy5 usable in most fluorescence instruments, especially in the long-wavelength spectral range where sample background fluorescence is low, and CCD cameras / detectors have the highest detection sensitivity in this region, thus resulting in high sensitivity. Cy5 is widely used in fields such as biolabeling, biosensors, and imaging research. It can be used to label proteins, antibodies, peptides, nanoparticles, etc., and is particularly commonly used to label nucleic acid molecules (DNA and RNA). In immunohistochemistry, Cy5 is often bound to antibodies to detect the location and expression level of target proteins or cells. In gene expression studies, Cy5 can be used to label DNA or RNA probes to determine the location and expression level of specific sequences.

[0112] In this invention, the term "IR-750" refers to a symmetrical dye with a peak excitation of 756 nm and a maximum emission of 776 nm in 1×PBS. It is spectrally similar to other 750 dyes used in flow cytometry, microscopy, and other applications.

[0113] In this invention, the term "(OEG)4" refers to oligo(ethylene glycol) containing four ethylene glycol units. Oligo(ethylene glycol) is a polymer composed of multiple ethylene glycol units linked by ether bonds. When used as a side chain, (OEG)4 can be used to modify various polymers to improve their physicochemical properties and biocompatibility.

[0114] In this invention, the term "PEG2" refers to a polyethylene glycol (PEG) derivative with good water solubility and low immunogenicity, thus having significant application value in drug formulation modification, drug delivery, and biochemical experiments. For example, it can help antibody-drug conjugates (ADCs) remain stable in the bloodstream and effectively release the drug after reaching tumor cells.

[0115] In this invention, the term "NOTA" refers to a bifunctional chelating agent with the chemical formula C. 12 H 21 N3O6 can be used as a framework for constructing PET imaging tools and can also be used for probe design and signal amplification through multivalent effects. NOTA has a strong affinity for metal ions, forms stable complexes, and is non-toxic and harmless to biomolecules.

[0116] In this invention, the term "Integrin" or "integrin" refers to a class of substances that are ubiquitous on the surface of vertebrate cells and are dependent on Ca2+.2+ or Mg 2+ These heterophilic cell adhesion molecules mediate mutual recognition and adhesion between cells and between cells and the extracellular matrix, thus connecting external cell processes with internal cell structures.

[0117] In this invention, the term "peptide" refers to a compound composed of amino acid residues covalently linked by peptide bonds. Peptides include, for example, bioactive fragments, substantially homologous peptides, homodimers, heterodimers, variants of peptides, modified peptides, derivatives, analogs, fusion proteins, etc. Peptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0118] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0119] Example 1

[0120] This embodiment provides a bispecific self-assembled nanoprobe CAIXB-GNNQQNY-K-PEG2-c(RGDfK), hereinafter abbreviated as CAIXB-SA-RGD or CSR, whose structural formula can be represented by Formula I, and the probe is prepared by peptide solid-phase synthesis.

[0121]

[0122] Formula I

[0123] The steps for synthesizing the probe using a polypeptide solid-phase synthesis method are as follows: (1) Synthesis of the polypeptide sequence CAIXB-GNNQQNY-K-PEG2-c(RGDfK) (hereinafter referred to as "polypeptide 1") Experimental instruments and materials: Dimethylformamide (DMF), piperidine, Wang resin, dichloromethane (DCM), ninhydrin reagent (ninhydrin, vitamin C, and phenol), benzotriazole -N,N,N',N' -Tetramethylurea hexafluorophosphate (HBTU), hexahydropyridine, triisopropylsilane (TIS), ethylene dithiol (EDT), anhydrous diethyl ether, trifluoroacetic acid (TFA). N 1-Methylmorpholine (NMM) N-Fluorenylmethoxycarbonyl-6-aminohexanoic acid (Fmoc-e-Acp-OH), methanol, Fmoc-alanine (Fmoc-Ala-OH), Fmoc-cysteine ​​(Fmoc-Cys(Trt)-OH), Fmoc-aspartic acid (Fmoc-Asp(OtBu)-OH), Fmoc-phenylalanine (Fmoc-Phe-OH), Fmoc-glycine (Fmoc-Gly-OH), Fmoc-lysine (Fmoc-Lys(Boc)-OH), Fmoc -Leucine (Fmoc-Leu-OH), Fmoc-Aspartic acid (Fmoc-Asn(Trt)-OH), Fmoc-Proline (Fmoc-Pro-OH), Fmoc-Arginine (Fmoc-Arg(Pbf)-OH), Fmoc-Threonine (Fmoc-Thr(tBu)-OH), Fmoc-Valine (Fmoc-Val-OH), Fmoc-Tyrosine (Fmoc-Tyr(Trt)-OH), CAIXB, and polypeptide solid-phase synthesis tubes, etc.

[0124] Preparation of experimental solutions: Deprotection solution – a mixture of hexahydropyridine and DMF at a volume ratio of 1:4; The reaction solution is a mixture of NMM and DMF at a volume ratio of 1:24. The lysis buffer is a mixture of TFA, TIS, and EDT, with each solution having a volume fraction of 92.5% TFA, 2.5% TIS, and 2.5% EDT. Ninhydrin test solution – one drop each of ninhydrin, vitamin C, and phenol; Specific operation methods: (1) Solvent treatment: DMF and methanol are soaked overnight in G3 pore molecular sieves to remove impurities and water before use.

[0125] (2) Full swelling of resin: Weigh 2.0 g blank Wang resin into a clean and dry reaction tube, add 15 mL DMF, and activate at room temperature for about 30 min.

[0126] (3) Inoculation of the first amino acid: At room temperature, filter off the solvent from the previous step using a sand filter, add 1 mmol of 5-fold excess C-terminal Fmoc-Asp(Oall)-OH, 5-fold excess DMAP, 5-fold excess DIC, and DMF as solvent, and react at room temperature for 3 h. After the reaction is complete, wash 4-6 times with DMF, 5-6 mL each time. Then add an appropriate amount of pyridine and acetic anhydride in a 1:1 volume ratio, and react for 30 min. After the reaction is complete, wash 4-6 times with DMF, 5-6 mL each time.

[0127] (4) Removal of Fmoc protecting group: Remove the solvent from the previous step by filtration, add 10 mL of 20% piperidine DMF solution to the resin, stir with N2 for 10 min and filter out the solution, add another 10 mL of 20% piperidine DMF solution, stir with N2 for 5 min and filter out the solution again. Repeat this operation twice, wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0128] (5) Detection of ninhydrin removal effect: Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN and phenol solution, heat at 105℃-110℃ for 5 min, and the dark blue color indicates a positive reaction, indicating that the removal is complete and the next step of the reaction can be carried out; if it is colorless, it indicates that the protecting group has not been completely removed, and the above deprotection operation needs to be repeated.

[0129] (6) Removal of the second amino acid and Fmoc protecting group: Weigh 3 times the molar excess of the C-terminus amino acid Fmoc-Gly-OH, Fmoc-Arg(pdf)-OH, Fmoc-Lys(DDE)-OH, Fmoc-d-Phe-OH, 3 times the molar excess of HBTU, and 3 times the molar excess of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve the resin, then add 10 times the molar excess of (pure) DIEA. React at room temperature for 40 min, and wash with DMF 4-6 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If the result is colorless, add 10 mL of 20% piperidine DMF solution to remove Fmoc. Repeat this process twice, for 10 min and 5 min respectively. Then wash with DMF 4 times and methanol 2 times, 5-6 mL each time. Take a small amount of resin and test it with ninhydrin reagent. If the result is blue, the next reaction can proceed.

[0130] (7) The Oall group was removed by Pd / C (CAS No.: 64741-65-7) catalysis, and DDE was removed by condensation with a condensing agent. The cyclic c(RGDfK) was then synthesized.

[0131] (8) Similarly, the synthesis of other peptides is carried out in the same way. Repeat the steps until the last amino acid at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry it.

[0132] (9) Resin shedding and pure product separation detection: The peptide was cleaved for 2 h with trifluoroacetic acid cleavage solution (95% TFA: 2% TIS: 2% EDT: 1% H2O). The reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The lysate was dried as much as possible with nitrogen gas, then precipitated with ether, centrifuged, and washed 3-5 times with ether to obtain a white solid. After dissolving in pure water, the solid was desalted and purified by HPLC, lyophilized, and crystals were precipitated. A small amount was analyzed by MS. The MS analysis results are as follows: Figure 11As shown, the measured molecular weight result is the same as the target molecular weight.

[0133] Finally, polypeptide 1 was obtained, with the structure as follows: Figure 1 As shown, CAⅨB is the CAⅨ targeting unit, the polypeptide sequence RGDfK is the integrin receptor αvβ3 targeting unit, PEG2 is the linking unit, GNNQQNY (SEQ ID NO: 1) is the self-assembly unit, and the R group is the part for further linking, such as the imaging group and / or chelating agent. The resulting polypeptide 1 is lyophilized and stored at -20℃ for later use.

[0134] The design principle of bispecific self-assembled peptide nanoprobes is as follows: Figure 2 As shown, the bispecific self-assembling peptide nanoprobe targets and binds to the CAIX and αvβ3 proteins of tumor cells via the CAIX targeting unit CAIXB and the integrin receptor αvβ3 targeting unit RGDfK, respectively. The assembly-capable fragments, due to the binding of the peptide molecules to the target proteins, reduce the activation entropy required for self-assembly, thereby triggering in-situ self-assembly to form nanofibers. This drug's self-assembling probe, on the one hand, enhances the imaging efficiency and effect of small molecule peptides on renal cell carcinoma tumors through dual tumor-targeting effects; on the other hand, it prolongs the residence time at the tumor site by forming nanofibers in situ on the tumor surface, achieving long-term tumor imaging. The bispecific self-assembling probe provided by this invention can be used in different imaging systems when connected to different imaging groups. For example, when connected to IR-750, it can be used for fluorescence imaging technology; when connected to NOA... (68Ga) It can be used in PET / CT imaging technology.

[0135] Example 2

[0136] This embodiment provides a bispecific self-assembled nanoprobe: CAⅨB-GNNQQNY-K(IR-750)-PEG2-c(RGDfK), hereinafter abbreviated as CSR-IR-750. This bispecific glycopeptide nanomedicine can be represented by Formula II. The probe preparation method is the same as in Example 1, wherein IR-750 is IRDye®750 NHS Ester (Q-0127571r), purchased from Xi'an Qiyue Biotechnology, China.

[0137] The polypeptide sequence represents the tumor targeting unit, CAIXB represents the CAIX targeting unit, RGDfK represents the integrin receptor αvβ3 targeting unit, PEG2 represents the linker unit, GNNQQNY represents the self-assembly unit, and IR-750 represents the imaging unit. MS results from CSR-IR-750 are shown below. Figure 12 As shown.

[0138]

[0139] Formula II

[0140] Example 3

[0141] This embodiment provides a bispecific self-assembled nanoprobe: CAIXB-GNNQQNY-K(NOTA)-PEG2-c(RGDfK), hereinafter referred to as CSR-NOTA, which can be represented by Formula III, and its preparation method is the same as that in Example 1.

[0142] The polypeptide sequence represents the tumor targeting unit, CAIXB represents the CAIX targeting unit, RGDfK represents the integrin receptor αvβ3 targeting unit, PEG2 represents the linker unit, GNNQQNY represents the self-assembly unit, and NOA represents the chelating agent. MS results for CSR-NOTA are as follows: Figure 15 As shown. CSR-NOTA can further chelate radionuclides for tumor imaging.

[0143]

[0144] Formula III

[0145] Example 4

[0146] This embodiment provides a bispecific polypeptide nanoprobe: CAⅨB-GNNQQNY-K(NOTA)-( 68 Ga)-PEG2-c(RGDfK), hereinafter referred to as CSR-NOTA ( 68 Ga), can be represented by formula IV, and its preparation method is the same as in Example 1.

[0147] The polypeptide sequence represents the tumor-targeting unit; CAIXB represents the CAIX-targeting unit; the polypeptide sequence RGDfK represents the integrin receptor αvβ3-targeting unit; PEG2 represents the linker unit; GNNQQNY represents the self-assembly unit; and NOA represents the chelating agent. 68 Ga is a radionuclide imaging group.

[0148]

[0149] Formula IV

[0150] Example 5

[0151] Critical assembly concentration experiments were conducted using the probe CAⅨB-SA-RGD prepared in Example 1, either alone or in combination with CAⅨ (Carbonic AnhydraselX / CA9, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., China, catalog number: 10107-H08H) or with Integrin (Integrin alpha V beta 3, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., China, catalog number: CTO98-H08H), with 1×PBS as the buffer.

[0152] The experimental group includes: CAIXB-SA-RGD group: 20 μM CAIXB-SA-RGD; CAIXB-SA-RGD / CAIX group: 20 μM CAIXB-SA-RGD and 100 nM CAIX; CAIXB-SA-RGD / Integrin group: 20 μM CAIXB-SA-RGD and 100 nM Integrin.

[0153] 200 μL of pyrene dye was added to each experimental group to make the concentration of pyrene dye in the solution 20 μM. Then, the dye was added to cuvettes and the fluorescence intensity was scanned and measured using a fluorescence spectrometer F-7000. The emission start wavelength was 360 nm and the stop wavelength was 450 nm.

[0154] Plotting the I3 / I1 ratios obtained from different peptide solutions on the ordinate and the concentrations on the abscissa, the critical assembly concentrations for CAⅨB-SA-RGD, CAⅨB-SA-RGD / CAIX, and CAⅨB-SA-RGD / Integrin were determined to be 29.7 μM, 6.3 μM, and 7.2 μM, respectively (e.g., ...). Figure 3 (As shown).

[0155] Example 6

[0156] Assembly kinetics experiments were conducted using the probe CSR-IR-750 prepared in Example 2, either alone or with the addition of CAⅨ (Carbonic AnhydraseⅨ / CA9, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., China, catalog number: 10107-H08H) or with the addition of Integrin (Integrin alpha V beta 3, purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd., China, catalog number: CTO98-H08H), with 1×PBS as the buffer.

[0157] The experimental group includes: CSR-IR-750 group: 20 μM CSR-IR-750; CSR-IR-750 / CAⅨ group: 20 μM CSR-IR-750 and 100 nM CAⅨ; CSR-IR-750 / Integrin group: 20 μM CSR-IR-750 and 100 nM Integrin.

[0158] In each experimental group, ANS (8-anilino-1-naphthalenesulfonic acid) solution was added to a concentration of 20 µM. The microplate reader was set to kinetic testing mode, with an emission wavelength of 390 nm and a stop wavelength of 470 nm. The duration was set to 8 hours with 5-minute intervals. Fluorescence intensity was measured using a fluorescence microplate reader and plotted. The results are as follows: Figure 4 As shown, CSR-IR-750 with added CAⅨ and Integrin proteins underwent self-assembly over time, resulting in changes in fluorescence intensity. In contrast, CSR-IR-750 without added CAⅨ and Integrin proteins did not undergo assembly, and its fluorescence intensity did not change significantly.

[0159] Example 7

[0160] The solutions containing CAⅨ (Carbonic Anhydrase Ⅸ / CA9, purchased from Beijing E.T. Inc., China, catalog number: 10107-H08H) or Integrin (Integrin alpha Vbeta 3, purchased from Beijing E.T. Inc., China, catalog number: CTO98-H08H) proteins were observed after CSR-IR-750 was exposed to the solution. The solutions underwent conformational changes and self-assembled into water-insoluble nanofibers.

[0161] CSR-IR-750 solutions were prepared using 1×PBS solution, and CAⅨ or Integrin protein was added to them to a final concentration of 100 µM. Samples were prepared 2 hours later and observed using transmission electron microscopy.

[0162] The results are as follows Figure 5 As shown in the results, CSR-IR-750 / CAⅨ and CSR-IR-750 / Integrin can undergo metamorphism in solution and self-assemble to form hydrophobic nanofibers.

[0163] Example 8: Bio-layer Interferometry (BLI) Experiment

[0164] The interaction between CSR-IR-750 protein molecules and CAⅨ and Integrin protein molecules was carried out to quantitatively detect the interaction forces between CSR-IR-750 protein molecules and CAⅨ and Integrin protein molecules, respectively.

[0165] First, initialize the Fortebio Octet K2 instrument and its operating software, Data Acquisition. Then, pre-wet the sensor by adding 200 μL of PBST to each well of the Greiner 96-well black plate (catalog number 655209) for 15 min. Next, program the Data Acquisition software to immobilize CAIX and Integrin proteins and bind to the CSR-IR-750 protein molecule. Then, prepare samples at different concentrations (12.5 nM, 25 nM, 50 nM, 100 nM, and 200 nM) according to the programmed settings and add them to the detection wells. Finally, place the sensor and sample plate into the instrument and perform detection sequentially. Apparent Kd represents the affinity of the combination. Results are as follows: Figure 6 As shown, the surface affinity K of CSR-IR-750 / CAⅨ d The surface affinity K of CSR-IR-750 / Integrin is 36 nM. d The value is 75 nM.

[0166] Example 9

[0167] The probe CSR-IR-750, prepared in Example 2, was used to perform experiments on cell membrane surface-level retention and co-localization with CAIX and Integrin proteins. The cells selected for the experiment were CAIX and integrin receptor α. V HELA cells with high expression of β3.

[0168] The pLenti-Puro+CA9 / CAⅨ(h) plasmid (catalog number: CTCC-DZ-0202, sourced from Zhejiang Meisen Cell Technology Co., Ltd.) was transfected into HELA cells using lentiviral packaging to achieve stable expression of the CAⅨ gene (NM_001216.3) in the genome, thereby constructing CAIX and integrin receptor α. V HELA cells with high β3 expression were then incubated with the CSR-IR-750 probe (20 μM) for 30 minutes. After washing three times with 1×PBS, the cell nuclei were stained with Hoechst 33342 (purchased from LABLEAD, Beijing, China, catalog number B2662), followed by washing three times with 1×PBS. Finally, the cells were observed using a multibeam laser confocal imaging system (U-Vox), and fluorescence images of HELA cells were observed under a 40× objective lens. Figure 7 As shown, the fluorescence of probe CSR-IR-750 was observed to colocalize with the fluorescence of CAIX and Integrin on the surface of HELA cells, indicating that probe CSR-IR-750 can target and bind to the cell membrane of HELA cells. Therefore, this study preliminarily verifies that probe CSR-IR-750 has the ability to colocalize with CAIX and Integrin and remain on the cell membrane.

[0169] Example 10

[0170] Cell surface morphology was observed using PBS and probe CSR-IR-750. The cells used in the experiment were 786o cells.

[0171] Cells were seeded into 24-well plates containing silica wafers and incubated for 24 h. Subsequently, the PBS group and experimental group were co-incubated with 1×PBS solution or probe CSR-IR-750 for 6 h, respectively. After treatment, both groups were fixed with 4% paraformaldehyde for 30 min, washed three times with 1×PBS solution for 5 min each time, and then dehydrated using a gradient of 10%, 30%, 50%, 70%, 90%, and 100% (v / v) ethanol solutions, with each concentration for 10 min. Finally, after drying, the cells were sputter-coated with gold and observed using a traceable metrological scanning electron microscope (JC-Zeiss). The results are as follows: Figure 8 As shown, it was found that there were no other structures on the cell membrane surface in the PBS group, while the probe CSR-IR-750 formed short rod-shaped fibrous structures on the cell membrane surface.

[0172] Example 11

[0173] The CR-IR-750 probe is a probe without a self-assembly sequence. The only difference between it and CSR-IR-750 is the absence of a self-assembly sequence. The C1SR1-IR-750 probe differs from CSR-IR-750 in that it targets carbonic anhydrase IX and integrin receptor αVβ3 with different peptide sequences. The structural formula of C1SR1-IR-750 is CAIXA-GNNQQNY-K(IR-750)-PEG2-RGD (as shown in Formula VII). MS results are shown in the figure below. Figure 13 As shown.

[0174]

[0175] Formula VII

[0176] The difference between CS1R-IR-750 and CSR-IR-750 lies in their self-assembly sequence; the structural formula is CAⅨB-FF-K(IR-750)-PEG2-c(RGDfK) (as shown in Equation VIII). The MS results for CS1R-IR-750 are shown below. Figure 14 As shown.

[0177]

[0178] Formula VIII

[0179] The prepared CR-IR-750 probe, CSR-IR-750 probe, C1SR1-IR-750 probe and CS1R-IR-750 probe were used to conduct specific recognition and long-term retention experiments at the animal level. The animals selected for the experiment were BALB / c nude mice.

[0180] Construction of a mouse subcutaneous tumor model: Subcutaneous xenografts were established in mice using renal cell carcinoma cells (786o cells). 5 × 10⁶ cells were collected. 6 7860 cells were injected subcutaneously into the right leg of a mouse, and a tumor formed after 2 weeks, thus obtaining a mouse subcutaneous tumor model.

[0181] Probes CR-IR-750, CSR-IR-750, CS1R-IR-750, and C1SR1-IR-750 were injected intravenously into the tail vein of mice. Three mice were used. Imaging was performed using an IVIS Spectrum imaging system at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, and 96h post-injection. The imaging results are shown below. Figure 9 As shown, the probe CSR-IR-750 provided by this invention exhibits more significant signal aggregation at tumor tissue compared to other control groups, and CSR-IR-750 has a long-lasting retention of up to 96 hours. This long-lasting retention effect is significantly superior to probes without self-assembly sequences, probes with different targeting recognition polypeptide sequences for carbonic anhydrase IX or targeting integrin receptor αVβ3, and probes with assembly sequences different from those designed in this invention.

[0182] Example 12

[0183] The probe CSR-NOTA (prepared using the experiment) 68 Ga) conducted an animal-level radionuclide PET-CT tumor imaging experiment, using BALB / c nude mice as the selected animals.

[0184] Construction of a mouse subcutaneous tumor model: Subcutaneous xenografts of renal cell carcinoma were established in mice, using 5 × 10⁶ cells. 6 786o cells were injected into the left axilla of mice, and tumors formed after 2 weeks, thus obtaining a mouse subcutaneous tumor model.

[0185] Using probe CSR-NOTA ( 68Three mice were injected via the tail vein into a PET / CT scanner (Super Nova® PET / CT) for microvessels. Imaging was performed at 20 min, 40 min, 60 min, 90 min, 120 min, and 240 min post-injection. The imaging results are shown below. Figure 10 As shown (images were formed in all three mice with similar results; one mouse is used as an example), the probe CSR-NOTA ( 68 Ga) exhibits significant signal accumulation in tumor tissue. Similar to the probe CSR-IR-750, it can be inferred that CSR-NOTA ( 68 Ga) also has a long-lasting retention effect.

[0186] Based on the above embodiments and comparative analyses, it can be seen that the bispecific polypeptide nanoprobe provided by the present invention can recognize and bind to CAⅨ and integrin receptor α overexpressed on tumor cells. V The β3 protein, a fragment with assembly capabilities, reduces the activation entropy required for self-assembly due to the binding of polypeptide molecules to the target protein, thereby triggering its in-situ self-assembly to form nanofibers. This enables long-term imaging of renal cell carcinoma cells and significantly improves the quality of the imaging, ultimately achieving highly accurate imaging and monitoring of renal cell carcinoma cells.

Claims

1. A self-assembled probe, characterized in that, The self-assembled probe comprises a target recognition unit 1, a self-assembly unit, and an imaging group. The self-assembly unit comprises an amino acid sequence as shown in SEQ ID NO:

1. The target recognition unit 1 targets carbonic anhydrase IX.

2. The self-assembled probe as described in claim 1, characterized in that, The target recognition unit 1 has a structural formula as shown in formula V or formula X: Formula V; Formula X; And / or, the self-assembled probe further comprises a targeting recognition unit 2, which targets the integrin receptor αVβ3; the targeting recognition unit 2 has a structure as shown in Formula VI or Formula IX: Formula VI; Formula IX.

3. The self-assembled probe as described in claim 1, characterized in that, The imaging group is selected from one or more of radionuclides, fluorescent groups, and magnetic groups; and / or, The self-assembly probe further includes a connection unit; and / or, The radionuclide is connected to the self-assembly unit via a chelating agent.

4. The self-assembled probe as described in claim 3, characterized in that, The fluorescent group comprises one or more selected from FIGT, ICG, Cy7, Cy5, and IR-750; and / or, The radionuclides include those selected from... 68 Ga、 89 Zr、 64 Cu and 82 One or more of Rb; and / or, The connection unit connects the self-assembly unit and the target recognition unit 2; and / or, The linker unit is one or more selected from the amino acid sequence shown in SEQ ID NO: 2, (OEG)4, and PEG2; and / or, The chelating agent comprises one or more selected from Nota, DOTA, TETA, PCTA, and DTPA.

5. The self-assembled probe according to any one of claims 1-4, characterized in that, The self-assembly probe has a structure selected from Formula II, Formula IV and Formula VII: Formula II; Formula IV; Formula VII.

6. An intermediate for a self-assembled probe, characterized in that, The intermediate differs from the self-assembled probe as described in any one of claims 1-5 in that it does not contain the imaging group.

7. The intermediate as described in claim 6, characterized in that, The intermediate has a structural formula selected from Formula I, Formula III and Formula XI; Formula I; Formula III; Formula XI.

8. A method for preparing a self-assembled probe or an intermediate of a self-assembled probe, characterized in that, The preparation method includes the step of obtaining the self-assembled probe as described in any one of claims 1-5, or the intermediate as described in claim 6 or 7, by polypeptide solid-phase synthesis.

9. A composition, characterized in that, The composition comprises (1) a self-assembled probe as described in any one of claims 1-5, or an intermediate as described in claim 6 or 7; and (2) a pharmaceutically acceptable carrier.

10. The use of one or more of the self-assembled probe as described in any one of claims 1-5, the intermediate as described in claim 6 or 7, and the composition as described in claim 9 in the preparation of medicaments for diagnosing and / or treating tumors.

11. The application as described in claim 10, characterized in that, The tumors mentioned are cervical cancer, breast cancer, lung cancer, esophageal cancer, colon cancer, skin cancer, kidney cancer, bladder cancer, pancreatic cancer, or stomach cancer.

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