A fluorescent / magnetic resonance enhanced bimodal breast cancer imaging polypeptide probe, a preparation method thereof and application thereof

Abstract

The present application relates to breast cancer diagnostic probes, in particular to a fluorescence / magnetic resonance enhanced dual-mode breast cancer imaging polypeptide probe, a preparation method thereof and application thereof. The polypeptide probe of the present application is made of a fluorescence imaging unit, a magnetic resonance imaging unit, a self-assembly imaging unit and a TROP-2 targeting recognition unit. The present application constructs a polypeptide probe which can enhance fluorescence / magnetic resonance signals in situ self-assembly. Through modular optimization of the synthesized polypeptide probe, in situ self-assembly mediated by high expression of TROP-2 protein in triple-negative breast cancer cells can be realized. The self-assembly is accompanied by aggregation of fluorescence and magnetic resonance units, which can significantly enhance the fluorescence and magnetic resonance dual-mode imaging signals of triple-negative breast cancer tissue, improve the imaging sensitivity and enhance the imaging effect of triple-negative breast cancer.

Description

Technical Field

[0001] This invention relates to breast cancer diagnostic probes, and more particularly to a fluorescence / magnetic resonance enhanced dual-modality breast cancer imaging polypeptide probe, its preparation method, and its applications. Background Technology

[0002] Breast cancer is the most common malignant tumor among women worldwide. Triple-negative breast cancer (TNC) is the most aggressive subtype of breast cancer, characterized by negativity of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for 15-20% of all breast cancers. Compared to other types of breast cancer, TNC is more prone to recurrence and metastasis, has a low late-stage survival rate, and a poor prognosis, earning it the nickname "the king of breast cancers." Early-stage TNC has a cure rate of over 60%, while the 5-year survival rate for late-stage TNC is only 11%. Therefore, developing effective early imaging strategies for TNC is crucial.

[0003] Early imaging of triple-negative breast cancer (TNC) remains a challenging clinical problem due to the lack of characteristic molecular subtyping biomarkers. The main issues are: 1) Current research on potential biomarkers for TNC through molecular subtyping is insufficient, and there is a lack of effective targeted diagnostic probes; 2) Diagnostic probes are subject to strong biological background interference in vivo, and if the probe signal is weak, false negative results are likely. Therefore, developing targeted imaging probes for TNC and enhancing the imaging signal to minimize the impact of background interference are urgent issues that need to be addressed in the early imaging of TNC.

[0004] Fluorescence imaging detects the emission signal of a fluorescent imaging probe at a specific excitation wavelength, offering advantages such as high sensitivity, high resolution, non-invasiveness, and real-time imaging. Fluorescence imaging probes are typically organic compound molecules that can be modified with various potential triple-negative breast cancer targeting ligands, enabling specific imaging of triple-negative breast cancer tissue in vivo. Therefore, multifunctional fluorescent imaging probes that combine triple-negative breast cancer targeting with enhanced fluorescence signal aggregation can be designed to address the challenges of targeted imaging and background interference in triple-negative breast cancer. Magnetic resonance imaging (MRI) offers advantages such as no ionizing radiation, no bone artifacts, good imaging depth, and avoidance of interference from gaseous organs. MRI contrast agents can shorten the longitudinal relaxation time (T1) or transverse relaxation time (T2) of water protons, thereby improving the imaging contrast between the lesion site and the background, providing important evidence for the early diagnosis of soft tissue lesions such as tumors.

[0005] However, while fluorescence imaging offers high sensitivity and resolution, its imaging signal is easily affected by tissue scattering, resulting in weak imaging capabilities for deep tissues. It is suitable for real-time imaging and behavioral studies of superficial triple-negative breast cancer tissue or individual triple-negative breast cancer cells. Magnetic resonance imaging (MRI) has excellent tissue penetration and imaging depth, enabling imaging of deep (centimeter-level) tissues, but it cannot image structures within individual living cells. Integrating complementary magnetic resonance and fluorescence signals into a single probe molecule broadens its application scenarios. This integrated probe molecule can utilize fluorescence signals for real-time behavioral studies and intraoperative navigation of triple-negative breast cancer cells, and also utilize MRI to detect triple-negative breast cancer in deep living tissues. Therefore, constructing a fluorescence / magnetic resonance-enhanced dual-modal imaging probe targeting triple-negative breast cancer is of great significance for early imaging of this disease. Summary of the Invention

[0006] To address the problem that biological background signals can severely interfere with the early diagnosis of triple-negative breast cancer, leading to false negative signals and affecting the diagnosis and treatment of triple-negative breast cancer, this invention aims to develop a probe to enhance magnetic resonance / fluorescence signals using peptide in situ self-assembly technology, thereby reducing interference from biological backgrounds in imaging and enhancing the imaging effect of triple-negative breast cancer.

[0007] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows:

[0008] In a first aspect of the invention, a fluorescence / magnetic resonance enhanced dual-modality breast cancer imaging polypeptide probe is provided, the polypeptide probe being made of a fluorescence imaging unit, a magnetic resonance imaging unit, a self-assembled imaging unit, and a TROP-2 target recognition unit;

[0009] The fluorescence imaging unit is a bispyrene;

[0010] The magnetic resonance imaging unit is Gd-DOTA, which is 1,4,7,10-tetraacetyl-1,4,7,10-tetraazacyclododecanoic acid (DOTA) coordinated with gadolinium (Gd);

[0011] The self-assembled imaging unit is a self-assembled polypeptide with an amino acid sequence as shown in SEQ ID NO: 1; specifically, FFVLK (F: phenylalanine; V: valine; L: leucine; K: lysine).

[0012] The TROP-2 targeting recognition unit is a TROP-2 recognition peptide with an amino acid sequence as shown in SEQ ID NO: 2; specifically, GGLPYEAYC (G: glycine; L: leucine; P: proline; Y: tyrosine; E: glutamic acid; A: alanine; C: cysteine).

[0013] The structural formula of the bispyrene BP is shown in Formula I;

[0014] Formula I;

[0015] In Formula I, R is selected from alkyl groups having 4-10 carbon atoms;

[0016] The structural formula of Gd-DOTA is shown in Formula II:

[0017] Formula II.

[0018] Preferably,

[0019] The structural formula of the polypeptide probe is shown in Formula III:

[0020] Formula III.

[0021] In a second aspect of the invention, the use of the fluorescence / magnetic resonance enhanced dual-modality breast cancer imaging peptide probe as described in the first aspect is provided in the preparation of breast cancer diagnostic reagents.

[0022] Preferably, the breast cancer is triple-negative breast cancer.

[0023] In a third aspect of the invention, a method for preparing a fluorescence / magnetic resonance enhanced dual-modality breast cancer imaging polypeptide probe as described in the first aspect, Formula III, is provided.

[0024] The BP-FFVLKK(DOTA)GGLPYEAYC polypeptide sequence designed in this invention was synthesized by Shanghai ChuTai Biotechnology Co., Ltd., and an equal molar mass fraction of gadolinium ion solution was added to form a macrocyclic coordination compound with DOTA to prepare BP-FFVLKK(DOTA-Gd)GGLPYEAYC, abbreviated as BP-FDG.

[0025] The specific preparation method and steps are as follows:

[0026] 1) Synthesis of peptide chain: The peptide chain FFVLKKGGLPYEAYC was synthesized using a solid-phase method.

[0027] The lysine used is protected with a double protecting group for Fmoc-Lys(DDE)-OH;

[0028] 2) Condensation of BP: Take BP with a molar amount of 1-1.5 times relative to the peptide chain, add HBTU with a molar amount of 2-4 times the molar amount of HBTU and HOBT with an excess of 2-4 times the molar amount of HOBT to the reaction tube, add DMF solution to completely dissolve BP, then add 8-12 times the molar amount of DIEA, react with the peptide chain on the resin at room temperature for 30-60 min, and then wash with DMF.

[0029] 3) Removal of protecting groups and condensation of DOTA: The lysine protecting group DDE was removed using 1.5-2.5% hydrazine hydrate to expose the amino group. A 1-1.5 molar excess of DOTA-tbu3-COOH relative to the peptide chain was added, along with 2-4 molar excesses of HBTU and HOBT. A suitable amount of DMF solution was added to completely dissolve the DOTA-tbu3-COOH, followed by 8-12 molar excesses of DIEA. The reaction was carried out at room temperature for 30-50 minutes. Finally, the peptide was cleaved with trifluoroacetic acid cleavage buffer, and the reaction solution was filtered to obtain a trifluoroacetic acid solution of the peptide. The trifluoroacetic acid solution was dried under nitrogen, precipitated with diethyl ether, centrifuged, and washed 3-5 times with diethyl ether to obtain a white solid. After dissolving in pure water, the solid was desalted by HPLC, lyophilized, and crystals were obtained to yield the peptide. The synthesized peptide was then dissolved in a small amount of DMSO to aid dissolution, followed by water. Subsequently, the same molar amount of gadolinium nitrate aqueous solution was added to coordinate with DOTA to obtain the BP-GDG polypeptide solution.

[0030] Preferably, in step 1), the specific method for peptide chain synthesis is as follows:

[0031] 1) After activating the resin with a 1 molar amount of the first amino acid Fmoc-Cys(Trt)-OH to remove the solvent, add 1-2 molar amounts of the first C-terminal amino acid cysteine, 3-8 molar amounts of DMAP, 3-8 molar amounts of DIC, and DMF as the solvent. React at room temperature 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 1:1 volume ratio and react for 20-50 min. After the reaction is complete, wash with DMF 4-6 times. The reaction is colorless when tested with ninhydrin test reagent. After removing the solvent, remove the Fmoc protecting group.

[0032] 2) Removal of the second amino acid and Fmoc protecting group: Weigh 2-4 molar amounts of the second C-terminal amino acid, 2-4 molar amounts of HBTU, and 2-4 molar amounts of HOBT into a reaction tube. Add DMF solution to dissolve them completely, then add 8-12 molar amounts of DIEA. React at room temperature for 30-60 minutes, and wash with DMF 4-6 times. Detect with ninhydrin test reagent; if the result is colorless, add piperidine DMF solution to remove Fmoc. After washing, take a small amount of resin and detect with ninhydrin test reagent; if the result is blue, proceed to the next step of the reaction.

[0033] 3) Peptide chain elongation: Repeat the above steps in this manner until the last amino acid Fmoc-Phe-OH at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry; lysine is used for special protection in the method.

[0034] The concept of this invention is as follows: This invention provides a fluorescence / magnetic resonance enhanced dual-modality breast cancer imaging peptide probe, which mainly consists of four parts: a fluorescence imaging unit, a magnetic resonance imaging unit, a self-assembly imaging unit, and a TROP-2 targeting recognition unit. The fluorescence imaging unit is selected from bispyrene with aggregation-induced excitatory emission function; the magnetic resonance imaging unit is selected from Gd-DOTA, which is 1,4,7,10-tetraacetyl-1,4,7,10-tetraazacyclododecanoic acid (DOTA) coordinated with gadolinium (Gd); the self-assembly imaging unit is selected from the core peptide sequence derived from β-amyloid protein assembly: FFVLK (F: phenylalanine; V: valine; L: leucine; K: lysine); and the targeting unit for triple-negative breast cancer is selected from the TROP-2 recognition unit: GGLPYEAYC (G: glycine; L: leucine; P: proline; Y: tyrosine; E: glutamate; A: alanine; C: cysteine). The aggregation-induced fluorescence enhancement of this invention is due to the restriction of molecular rotation or vibration after aggregation, resulting in reduced radiative transitions and increased non-radiative transitions between molecules, thus greatly enhancing the fluorescence signal. The key indicator for evaluating contrast agent performance is the relaxation rate, which can be adjusted by modifying the structural characteristics of the contrast agent, such as size, morphology, crystal structure, and assembly structure, thereby enhancing the magnetic resonance signal. This invention designs a magnetic resonance contrast agent that specifically aggregates in triple-negative breast cancer tissue, increasing the relaxation rate, improving magnetic resonance imaging contrast, and solving the problem of background interference in triple-negative breast cancer imaging.

[0035] This invention also provides a method for synthesizing and modifying a polypeptide probe. The polypeptide probe's peptide backbone (self-assembled polypeptide + TROP-2 recognition peptide) is synthesized by solid-phase synthesis. The fluorescent bispyrene molecule is coupled to the amino group of the phenylalanine at the starting end of the self-assembled polypeptide via an amide bond formed by its carboxyl group. Due to the large molecular weight of the polypeptide chain and the bispyrene (BP) molecule, the steric hindrance effect is strong. In order to couple bispyrene to the polypeptide, this invention utilizes an alkane chain (R) to extend the side chain of bispyrene to reduce the steric hindrance effect. The structural formula of the BP used in this invention is shown in Formula I.

[0036] In addition, the present invention couples magnetic resonance Gd-DOTA by forming an amide bond between its carboxyl group and the amino group of lysine at the end of the self-assembled peptide, that is, by modifying the magnetic resonance unit at the middle position of the assembled peptide, thus avoiding signal interference between the fluorescence imaging unit and the magnetic resonance imaging unit.

[0037] Compared with existing technologies, this invention has the following advantages: This invention constructs a peptide probe BP-FDG that enhances fluorescence / magnetic resonance signals through in-situ self-assembly. The modularly optimized synthesized peptide probe enables in-situ self-assembly mediated by the high expression of TROP-2 protein in triple-negative breast cancer cells. The self-assembly, accompanied by the aggregation of fluorescence and magnetic resonance units, significantly enhances the fluorescence and magnetic resonance signals of triple-negative breast cancer tissue, improving imaging sensitivity and enhancing the imaging effect of triple-negative breast cancer. The BP-FDG peptide probe constructed in this invention has advantages such as good biocompatibility and signal stability, enabling in-situ self-assembly of peptides mediated by the high expression of Trop2 protein in triple-negative breast cancer, as well as assembly-enhanced fluorescence and magnetic resonance dual-modal imaging of triple-negative breast cancer. Attached Figure Description

[0038] Figure 1 This diagram illustrates the chemical structures of the BP-FDG, BP-GDG, and AC-FDG peptide probes and their assembly enhancing fluorescence / magnetic resonance signals after interaction with triple-negative breast cancer cells. Specifically, a represents the peptide probe BP-FDG (full name: BP-FFVLKK(Gd-DOTA)GGLPYEAYC); b represents the control sequence BP-GDG (full name: BP-GGGGGK(Gd-DOTA)GGLPYEAYC); c represents the control sequence AC-FDG (full name: AC-FFVLKK(Gd-DOTA)GGLPYEAYC); and d represents the assembly enhancing fluorescence / magnetic resonance signals after interaction of BP-FDG with triple-negative breast cancer cells.

[0039] Figure 2 This is the mass spectrum of the BP-FDG peptide probe.

[0040] Figure 3 This is a high-performance liquid chromatogram of the BP-FDG peptide probe.

[0041] Figure 4 This is the mass spectrum of the BP-GDG peptide probe.

[0042] Figure 5 This is a high-performance liquid chromatogram of the BP-GDG peptide probe.

[0043] Figure 6 This is the mass spectrum of the AC-FDG peptide probe.

[0044] Figure 7 This is a high-performance liquid chromatogram of the AC-FDG peptide probe.

[0045] Figure 8 Electron micrographs of in-situ self-assembly of BP-FDG, BP-GDG, and AC-FDG peptide probes mediated by Trop2 protein.

[0046] Figure 9The images show electron micrographs of the in situ self-assembly of the BP-FDG peptide probe in triple-negative breast cancer cell lines. a is a scanning electron micrograph of cells in the control group, b is a scanning electron micrograph of cells in the BP-FDG group, and c is an electron micrograph of ultrathin sections of cells in the control group and the BP-FDG group.

[0047] Figure 10 The fluorescence spectrum of the BP-FDG peptide probe in the presence of Trop2 protein is shown.

[0048] Figure 11 The fluorescence spectrum of the BP-GDG peptide probe in the presence of Trop2 protein is shown.

[0049] Figure 12 Magnetic resonance imaging of the BP-FDG peptide probe in the presence of Trop2 protein.

[0050] Figure 13 Magnetically resonant imaging of BP-FDG, BP-GDG, and AC-FDG peptide probes mediated by Trop2 protein. (a) Magnetically resonant mapping images of different concentrations of BP-FDG, BP-GDG, and AC-FDG peptide probes mediated by Trop2 protein; (b) Longitudinal relaxation rate (r1) of different concentrations of BP-FDG, BP-GDG, and AC-FDG peptide probes mediated by Trop2 protein.

[0051] Figure 14 The images show in vivo fluorescence imaging of the BP-FDG peptide probe. a) shows the in vivo fluorescence imaging results of mice at each time point, b) shows the fluorescence imaging results after mouse dissection, and c) shows the fluorescence section results of mouse tumor tissue.

[0052] Figure 15 This is an in vivo magnetic resonance imaging image of the BP-FDG peptide probe.

[0053] Figure 16 Hemolysis test for BP-FDG peptide probe.

[0054] Figure 17 This study aimed to perform routine blood tests on mice after intravenous injection of the BP-FDG peptide probe, with PBS injection serving as a control group.

[0055] Figure 18 Images of visceral sections of mice after intravenous injection of the BP-FDG peptide probe, with PBS injection serving as the control group. Detailed Implementation

[0056] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solutions.

[0057] The English abbreviations or words involved in this invention are shown in Table 1.

[0058] .

[0059] The sources of the experimental materials, reagents, and instruments involved in this invention are explained below:

[0060] 1) Experimental materials and reagents

[0061] ;

[0062] .

[0063] 2) Experimental instruments

[0064] .

[0065] Example 1: Construction of a fluorescence / magnetic resonance enhanced triple-negative breast cancer imaging peptide probe

[0066] The structure of the fluorescence / magnetic resonance enhanced dual-modality breast cancer imaging peptide probe BP-FDG of the present invention is as follows: Figure 1 As shown, it consists of a fluorescence imaging unit, a magnetic resonance imaging unit, a self-assembly imaging unit, and a TROP-2 target recognition unit.

[0067] The BP-FFVLKK(DOTA)GGLPYEAYC polypeptide sequence designed in this invention was synthesized by Shanghai ChuTai Biotechnology Co., Ltd., and an equal molar mass fraction of gadolinium ion solution was added to form a macrocyclic coordination compound with DOTA to prepare BP-FFVLKK(DOTA-Gd)GGLPYEAYC, abbreviated as BP-FDG.

[0068] The specific steps for synthesizing the peptide probe BP-FDG are as follows:

[0069] 1.1 Solvent Treatment

[0070] Before use, DMF and methanol should be soaked overnight in a G3-pore molecular sieve to remove impurities and water.

[0071] 1.2 Sufficient swelling of the resin

[0072] Weigh 2.0 g of blank Wang resin into a clean, dry reaction tube, add 15 mL of DMF, and activate at room temperature for about 30 min.

[0073] 1.3 Connect the first amino acid Fmoc-Cys(Trt)-OH

[0074] At room temperature, the solvent from the previous step was removed by filtration through a sand filter. Based on a resin nitrogen loading of approximately 1 mmol (2 g resin), 1.5 molar amounts of the first C-terminal amino acid, 3 molar amounts of DMAP, and 3 molar amounts of DIC were added. DMF was used as the solvent, and the reaction was carried out at room temperature for 3 h. After the reaction was complete, the resin was washed 4–6 times with 5–6 mL of DMF each time. Then, an appropriate amount of pyridine and acetic anhydride (volume ratio 1:1) was added, and the reaction was carried out for 30 min. After the reaction was complete, the resin was washed 4–6 times with 5–6 mL of DMF each time. (Purpose: To block the active sites on unreacted empty resin.)

[0075] 1.4 Departure of Fmoc Protecting Group

[0076] Remove the solvent from the previous step by filtration. Add 10 mL of 20% piperidine DMF solution to the resin. Stir under N2 protection for 10 min and then filter out the solution. Add another 10 mL of 20% piperidine DMF solution and stir under N2 for 5 min. Filter out the solution again. Repeat this operation twice. Wash with DMF 4 times and methanol 2 times, 5-6 mL each time.

[0077] 1.5 Ninhydrin Removal Effect

[0078] Take out a small amount of resin, wash it three times with methanol, add one drop each of ninhydrin, KCN, and phenol solution, and heat at 105℃–110℃ for 5 minutes. A deep blue color indicates a positive reaction, meaning that the protection group has been completely removed and the next step can be carried out. If the color is colorless, it means that the protecting group has not been completely removed and the above deprotection operation needs to be repeated.

[0079] 1.6 Removal of the second amino acid and Fmoc protecting group

[0080] Weigh out 3 molar excess of the second C-terminal amino acid, 3 molar amounts of HBTU, and 3 molar amounts of HOBT into a reaction tube. Add an appropriate amount of DMF solution to completely dissolve them, then add 10 molar amounts of (pure) DIEA. React at room temperature for 40 min, washing 4-6 times with DMF, 5-6 mL each time. Test a small amount of resin with ninhydrin; a colorless result is observed. Then add 10 mL of 20% piperidine DMF solution to remove Fmoc, repeating twice, for 10 min and 5 min respectively. Afterward, wash 4 times with DMF and 2 times with methanol, 5-6 mL each time. Test a small amount of resin with ninhydrin; a blue result indicates the reaction is ready for the next step.

[0081] 1.7 Repeat steps 1.6 (in which the special amino acid Fmoc-Lys(DDE)-OH is synthesized) until the last amino acid Fmoc-Phe-OH at the N-terminus is synthesized, remove the Fmoc protecting group, and then dry the mixture.

[0082] 1.8 Condensation of BP small molecules: Take BP with a molar amount of 1.5 times that of the peptide chain, add HBTU with a molar amount of 3 times that of the peptide chain and HOBT with a molar excess of 3 times to the reaction tube, add DMF solution to completely dissolve BP, then add DIEA with a molar amount of 10 times, react with the peptide chain on the resin at room temperature for 30-60 min, and then wash with DMF 4-6 times, 5-6 mL each time.

[0083] 1.9 Remove the specific amino acid Lys(DDE) with 2% hydrazine hydrate to expose the amino group. Add 1.5 molar amounts of DOTA-tbu3-COOH (DOTA is protected with tert-butyl ester groups before condensation with the peptide to obtain DOTA-tbu3-COOH) relative to the peptide chain. Add 3 molar amounts of HBTU and 3 molar amounts of HOBT to the reaction tube. Add an appropriate amount of DMF solution to completely dissolve them, and then add 10 molar amounts of (pure) DIEA. React at room temperature for 40 min. Wash with DMF 4-6 times, 5-6 mL each time.

[0084] 1.10 Finally, the peptide was cleaved with trifluoroacetic acid cleavage solution (95% TFA: 2% TIS: 2% EDT: 1% H2O) for 2 h. 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.

[0085] 1.11 Take the synthesized peptide, add a small amount of DMSO to aid dissolution, and then add water to dissolve it. Subsequently, add the same molar amount of gadolinium nitrate aqueous solution to coordinate with DOTA to obtain the BP-GDG peptide solution.

[0086] Chemical structure characterization of the prepared BP-FDG peptide probe: (1) Mass spectrometry test: nebulizer gas flow rate 1.5 L / min, flow rate 0.2 ml / min, CDL Temp. 250℃, Block Temp 200℃, B. Conc. 50%H2O / 50%ACN. (2) High performance liquid chromatography test, chromatographic column: 4.6×250 mm, Sinochrom ODS-BP 5μm, solvent A: acetonitrile solution containing 0.1% trifluoroacetic acid, solvent B: aqueous solution containing 0.1% trifluoroacetic acid, gradient elution mode: 0.0 min 60% solvent A + 40% solvent B, 25.0 min 85% solvent A + 15% solvent B, 26 min 100% solvent A, stop at 30 min, sample volume: 5 μL, detection wavelength: 220 nm, flow rate: 1.0 mL / min.

[0087] The results are as follows Figure 2-3As shown. Result analysis: Figure 2 This is the mass spectrum of the peptide probe BP-FDG, [M+3H]. 3+ =923.2, [M+2H] 2+ =1384.2, indicating that the molecular weight of the peptide is 2766.4, which is consistent with the designed molecular structure, proving the successful construction of the polypeptide; Figure 3 The high-performance liquid chromatogram of the peptide probe shows that the purity of the peptide is 94.48%.

[0088] Example 2 Probes constructed from different components

[0089] Previously, this application also synthesized BP-GDG and AC-FDG peptide probes. The structures of the BP-GDG and AC-FDG peptide probes are as follows: Figure 1 As shown, the synthesis methods of BP-GDG and AC-FDG peptide probes are the same as those in Example 1, and the effects are verified by fluorescence spectroscopy and magnetic resonance imaging experiments, respectively.

[0090] The only difference between the BP-GDG peptide probe synthesis method and the BP-FDG peptide probe synthesis method in Example 1 is that the peptide sequence GGGGG (amino acid as shown in SEQ ID NO: 3) is used instead of the peptide sequence FFVLK in Example 1.

[0091] The only difference between the AC-FDG peptide probe synthesis method and the BP-FDG peptide probe synthesis method in Example 1 is that the BP molecule is replaced by an acetyl group (AC).

[0092] Mass spectrometry and high-performance liquid chromatography results of BP-GDG and AC-FDG peptide probes are as follows: Figures 4-7 As shown, Figure 4 This is the mass spectrum of the BP-GDG peptide probe; Figure 5 The high-performance liquid chromatogram of the BP-GDG peptide probe is shown. Figure 6 This is the mass spectrum of the AC-FDG peptide probe; Figure 7 The images show high-performance liquid chromatograms of the AC-FDG peptide probe. Both chromatograms confirm the successful synthesis of the two peptides mentioned above.

[0093] Example 3: Self-assembly test, enhanced fluorescence performance test, and enhanced magnetic resonance performance test of the peptide probes from Example 1 and Example 2.

[0094] (a) Self-assembly test in solution: First, prepare 100 μg / mL BP-FDG, BP-GDG and AC-FDG peptide solutions respectively, then add 10 μg Trop2 protein respectively, and finally incubate for 0.5 h, 2 h and 6 h respectively. Take about 5 μL of the above peptide reaction solution (the peptide solution without Trop2 protein is used as the control group) and drop it on copper grid to stop the reaction. After drying, observe it under transmission electron microscope.

[0095] The results are as follows Figure 8 As shown in the results, before the addition of Trop2 protein, the above-mentioned peptide probes all appeared as amorphous nanoparticles in aqueous solution. With the addition of Trop2 protein, the BP-FDG peptide probe gradually transformed from a particulate assembly into nanofibers, demonstrating that Trop2 protein can effectively mediate the fibrillation and self-assembly of the BP-FDG peptide probe. In contrast, with the addition of Trop2 protein, the BP-GDG peptide probe gradually aggregated into amorphous macroaggregates. Compared with the BP-FDG peptide probe as a control, the AC-FDG peptide probe also gradually deformed and assembled into shorter peptide fibers under the action of Trop2 protein, demonstrating that the degree of fibrillation assembly was lower than that of BP-FDG.

[0096] (II) In-situ self-assembly test of BP-FDG peptide probe on cell surface: (1) Scanning electron microscopy was used to observe the assembly performance of the peptide probe on the surface of triple-negative breast cancer cells. 1×10 3 100 4T1 cells (4T1-TACSTD2 stable transgenic line) were incubated with 10 μg of peptide for 4 h after 12 h. The cells were then fixed with 4% paraformaldehyde and anhydrous ethanol, dehydrated and dried, and then observed under a scanning electron microscope. (2) The assembly performance of the peptide probe on the cell surface was observed by transmission electron microscopy. 1×100 cells were seeded in a 6 cm cell culture dish. 6 Four T1 cells were incubated with 500 μg of peptide for 4 h. Cells were then collected using a cell scraper and cross-linked in 2.5% glutaraldehyde for 24 h. Cell structure was stained with 0.5% osmium tetroxide using electron microscopy. Cells were then dehydrated using an ethanol gradient and embedded in epoxy resin. The epoxy resin containing the cells was cut into approximately 70 nm thin slices using an ultramicrotome. Transmission electron microscopy was used to observe the morphology of cell membrane self-assemblies and changes in cell membrane structure. Control groups were treated with PBS.

[0097] The results are as follows Figure 9 As shown, the results analysis is as follows: The scanning electron microscope results show ( Figure 9(ab) In the control group, cells exhibited normal morphology, intact structure, and smooth surface. However, after incubation with the aforementioned polypeptide probe, breast cancer cells showed in situ growth of numerous nanofiber peptides on their cell surface, resulting in disrupted cell morphology and a roughened cell membrane surface. Simultaneously, electron microscopy results of ultrathin cell sections indicated ( Figure 9 c) The cell membrane structure of the control group was relatively intact, while a large number of polypeptide fibers grew on the surface of the cell membrane incubated with the polypeptide probe, and the cell membrane was significantly damaged. All of the above results confirm that the BP-FDG polypeptide probe can effectively assemble nanofiber polypeptides in situ on the breast cancer cell membrane.

[0098] (III) Test of enhanced fluorescence performance of peptide probe assembly: Prepare 10 μg / mL BP-FDG and BP-GDG peptide solutions respectively, add 1 μg, 5 μg and 10 μg Trop2 protein respectively, and then perform fluorescence spectral scanning on the above samples.

[0099] The results are as follows Figure 10-11 As shown, the results indicate that with increasing Trop2 protein concentration, the fluorescence intensity of BP-FDG at 508 nm gradually increases, and the fluorescence intensity (at 508 nm) increases fourfold compared to the concentration without Trop2 protein. Figure 10 In contrast, the BP-GDG group showed almost no enhancement in fluorescence at 508 nm after the addition of different concentrations of Trop2 protein. Figure 11 The above results demonstrate that the ordered assembly of the BP-FDG peptide probe is the main reason for the enhanced exciton emission induced by bispyrene aggregation, and that peptide self-assembly can significantly enhance the fluorescence intensity of the bispyrene fluorescent unit.

[0100] (iv) Testing of Peptide Probe Assembly Enhanced Magnetic Resonance Performance: A 10 μg / mL BP-FDG peptide solution was prepared, and 1 μg, 5 μg, 10 μg, and 20 μg of Trop2 protein were added, respectively. Magnetic resonance imaging in T1 mode was then performed on the above samples. BP-FDG, BP-GDG, and AC-FDG peptide probe solutions were prepared, and 10 μg of Trop2 protein was added. The peptide probes were diluted to 40 μM, 20 μM, 10 μM, 5 μM, and 2.5 μM (based on gadolinium concentration), respectively. Magnetic resonance imaging in T1 mode was then performed on the above samples.

[0101] The results are as follows Figure 12-13 As shown, the results of BP-FDG in vitro magnetic resonance imaging (MRI) indicate that ( Figure 12As the TROP-2 concentration increased, the T1-weighted signal of the BP-FDG solution was significantly enhanced, confirming that TROP-2 mediates peptide self-assembly to enhance the magnetic resonance signal. Furthermore, this invention also compared the magnetic resonance imaging effects of two control peptides, BP-GDG and AC-FDG. The relationship between the relaxation rates was found to be... Figure 13 ): BP-FDG (32.32×10 -3 mM -1 s -1 AC-FDG(10.36×10) -3 mM -1 s -1 )>BP-GDG(6.16×10 -3 mM -1 s -1 This confirms that the self-assembled polypeptide proposed in this invention can enhance magnetic resonance signals.

[0102] Example 4: In vivo fluorescence imaging, in vivo magnetic resonance imaging, and biocompatibility testing of the peptide probe from Example 1.

[0103] (I) In vivo fluorescence imaging test of BP-FDG peptide probe: 18 healthy mice were prepared and randomly divided into 6 groups. Each mouse was subcutaneously injected with 1×10 6 Four T1 cells were used to establish a mouse subcutaneous breast cancer model. One week later, mice were injected with the BP-FDG peptide probe (10 mg / Kg) via the tail vein. In vivo fluorescence imaging was performed on mice at 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h. Mice were dissected at each time point, and tumors, hearts, livers, spleens, lungs, and kidneys were collected for fluorescence imaging. Simultaneously, tumor tissue was collected for fluorescent sectioning and scanning (blue fluorescence indicates cell nuclear staining, and green fluorescence indicates the peptide probe signal).

[0104] The results are as follows Figure 14 As shown, the results analysis: the in vivo fluorescence imaging results of mice at each time point showed ( Figure 14 (a) After being injected via the tail vein, the polypeptide probe gradually accumulates in the tumor area through blood circulation, reaching its maximum accumulation at 48 hours. Over time, the BP-FDG polypeptide probe is gradually metabolized and excreted from the body. Post-dissection fluorescence imaging also showed ( Figure 14 (b) With prolonged administration, the BP-FDG peptide probe gradually accumulates within tumor tissue and is primarily metabolized and excreted via the liver and kidneys. Tissue fluorescence section results show ( Figure 14 c) The BP-FDG peptide probe is mainly enriched in the tumor region, further demonstrating the tumor targeting ability of the BP-FDG peptide probe.

[0105] (II) In vivo magnetic resonance imaging test of BP-FDG peptide probe: 18 healthy mice were prepared, and each mouse was subcutaneously injected with 1×10 6 4T1 cells were used to establish a mouse subcutaneous breast cancer model. One week later, the mice were injected with BP-FDG peptide probe (10 mg / Kg) via the tail vein, and whole-body in vivo magnetic resonance imaging was performed at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h.

[0106] The results are as follows Figure 15 As shown, the results analysis: in vivo magnetic resonance imaging (MRI) scans at different times show ( Figure 15 This BP-FDG peptide probe can effectively target and accumulate in tumor tissue, resulting in strong imaging contrast. These results demonstrate that this peptide probe possesses excellent magnetic resonance imaging performance and has significant potential for targeted imaging of triple-negative breast cancer.

[0107] (III) Biocompatibility test of BP-FDG peptide probe:

[0108] (1) Test of hemolytic effect induced by peptide probe. Red blood cells were extracted from mouse blood, and a 4% (w / w) red blood cell suspension was prepared. 10 μM, 50 μM, 100 μM, 200 μM, and 400 μM peptide probes were added, respectively. Deionized water and physiological saline were added as positive and negative controls. After incubation at 37°C for 6 hours, the above samples were centrifuged at high speed, and the supernatant was used to measure the ultraviolet absorbance at a wavelength of 450 nm to calculate the hemolysis rate.

[0109] (2) Blood routine test: Ten healthy mice were randomly divided into two groups of five each. On days 0, 2, 4 and 6, the mice were intravenously injected with a polypeptide probe (10 mg / Kg) and physiological saline, respectively. On day 8, blood was collected from the orbital sinus of the mice to measure the hematocrit, hemoglobin, lymphocytes and mean corpuscular hematocrit in the blood.

[0110] (3) Ten healthy mice were prepared and randomly divided into two groups of five mice each. On days 0, 2, 4 and 6, the mice were intravenously injected with a polypeptide probe (10 mg / Kg) and physiological saline, respectively. On day 8, the mice were sacrificed and the heart, liver, spleen, lung and kidney were collected and treated with tissue fixative. Then, sections were prepared and H&E staining was performed to analyze the pathological tissues of the five organs.

[0111] The results are as follows Figure 16-18 As shown, the results analysis: the red blood cell hemolysis test results showed ( Figure 16 Compared with the PBS control group, the addition of 10 μM, 50 μM, 100 μM, 200 μM, and 400 μM peptides to the red blood cell suspension did not cause hemolysis. Meanwhile, treatment with deionized water, used as the positive control group, resulted in significant hemolysis. Mouse blood routine analysis showed... Figure 17 After treatment with BP-FDG peptides, several indicators in the blood of mice, including hematocrit, hemoglobin, lymphocytes, and mean erythrocyte hematocrit, were not significantly different from those in the PBS-treated group, and all were within the normal range. Results of visceral tissue sections showed... Figure 18 The pathological sections of the five internal organs in the BP-FDG peptide-treated group showed no significant abnormalities. These results demonstrate that the BP-FDG peptide probe has good biocompatibility and great potential in in vivo imaging of triple-negative breast cancer.

[0112] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries.

Claims

1. A fluorescence / magnetic resonance-enhanced dual-modality breast cancer imaging peptide probe, characterized in that, The polypeptide probe is made of a fluorescence imaging unit, a magnetic resonance imaging unit, a self-assembled imaging unit, and a TROP-2 target recognition unit; The structural formula of the polypeptide probe is shown in Formula III: Formula III.

2. The application of the fluorescence / magnetic resonance enhanced dual-modal breast cancer imaging peptide probe as described in claim 1 in the preparation of breast cancer diagnostic reagents, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

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