Metal-free polypeptide probe and its use in magnetic resonance imaging

Abstract

The present application relates to the biomedical technology field, especially to a kind of metal-free polypeptide probe and its application in magnetic resonance imaging.The present application provides a kind of metal-free polypeptide probe capable of in situ self-assembly forming nanofiber network under tumor microenvironment, the metal-free polypeptide probe can regulate water molecule state and then respond MRI signal, in vivo and in vitro experiment verification, the metal-free polypeptide probe of the present application has good blood long-acting circulation stability, imaging window is long and can stably respond MRI signal, and biological safety is high, can be used for high expression integrin receptor tumor imaging and monitoring, in the field of tumor magnetic resonance imaging has wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a metal-free polypeptide probe and its application in magnetic resonance imaging. Background Technology

[0002] Enhanced magnetic resonance imaging (MRI) is a crucial imaging assessment tool for postoperative and post-treatment follow-up in pancreatic cancer. Gadolinium-based contrast agents (GBCAs) play a vital role in tumor detection by altering relaxation time to improve signal contrast between lesions and normal tissues. However, as extracellular liquid-type small molecule contrast agents, GBCAs have limitations such as short circulating half-life, lack of active targeting ability, and limited selective enrichment and retention efficiency within tumors. Furthermore, the use of certain classes of GBCAs in patients with impaired renal function carries the risk of renal systemic fibrosis, and repeated use can lead to gadolinium deposition in vivo (e.g., in brain tissue). Although their clinical significance is still under investigation, it has prompted more cautious contrast agent selection and usage strategies. To reduce potential metal-related risks, metal-free MRI contrast enhancement strategies (such as organic free radical contrast agents and CEST probes) have gained attention, but currently face challenges such as poor in vivo stability, short imaging window, strong dependence on imaging sequences and hardware, and poor cross-platform reproducibility, limiting their clinical application.

[0003] Peptides have become important materials for constructing novel molecular probes due to their excellent biocompatibility, modular design, and ease of chemical modification. Peptides can self-assemble into nanostructures driven by hydrogen bonding and hydrophobic interactions, improving in vivo stability and prolonging cycle time. At the same time, peptides can be specifically cleaved or have conformational changes triggered by tumor-associated enzymes, inducing in-situ self-assembly and enabling responsive imaging and precise delivery to the tumor microenvironment.

[0004] Based on the above advantages, how to combine the self-assembly ability of peptides with enzyme response mechanisms to construct a safe, long imaging window, and stable response to MRI signals has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical challenges, this invention provides a metal-free polypeptide probe that can be cleaved by fibroblast activation protein-α, yielding a chemical structure as shown in formula ii: Formula ii (R-BSPP).

[0006] This invention designs a metal-free polypeptide probe that targets integrin receptors on tumor cell membranes. This metal-free polypeptide probe can be cleaved by fibroblast activation protein-α (FAP-α). After cleavage, nanoparticles with the above structure can self-assemble to form a nanofiber network, thereby regulating the state of water molecules to achieve MRI signal response.

[0007] In some embodiments, the metal-free polypeptide probe is cleaved and then self-assembles in situ to form a nanofiber network.

[0008] In some embodiments, the nanofiber network is characterized by β-folds.

[0009] In some embodiments, the metal-free polypeptide probe is cleaved and then self-assembles in situ into a nanofiber network in the presence of calcium ions.

[0010] In some embodiments, after the metal-free polypeptide probe is cleaved, it utilizes the calcium ion-dependent carboxyl binding behavior of integrin to simulate the interaction between integrin and ligand, and forms a nanofiber network through in situ self-assembly.

[0011] In some implementations, after the aforementioned metal-free peptide probe specifically recognizes and binds to integrin receptors highly expressed on tumor cell membranes, fibroblast activation protein-α, highly expressed in the tumor microenvironment, can cleave the metal-free peptide probe, generating nanoparticles as shown in Formula ii. This increases the local peptide concentration at the tumor site and, by utilizing the calcium-dependent carboxyl-binding behavior of integrins, mimics the interaction between integrins and ligands, further triggers a self-assembly effect, causing the nanoparticles to rapidly transform into a nanofiber network with β-sheet characteristics. This, in turn, regulates the local water molecule state to achieve an MRI signal response. A schematic diagram of the working mechanism is shown below. Figure 1 As shown.

[0012] In some implementations, the nanofiber network is capable of modulating the state of water molecules to respond to magnetic resonance imaging signals.

[0013] In some embodiments, the chemical formula of the metal-free polypeptide probe is shown in Formula i: Formula i (BSPP).

[0014] In Formula i, 1000 is the number-average molecular weight of PEG. Other structural formulas with similar molecular weights are also within the scope of protection of this invention (e.g., number-average molecular weight ranges of 500~1500, 800~1200, 900~1100, etc.).

[0015] In practice, the aforementioned metal-free peptide probes can be synthesized using the standard peptide solid-phase synthesis method.

[0016] Formulas ii and i of the present invention can self-assemble into nanoparticles in aqueous solution (e.g., at a molar concentration of 100 μM or higher), and the addition of calcium ions (which can be derived from calcium salts, such as calcium chloride) can induce the nanoparticles to self-assemble into nanofibers.

[0017] Furthermore, the present invention provides a formulation containing the aforementioned metal-free polypeptide probe.

[0018] In practice, the aforementioned metal-free polypeptide probes can be prepared into an aqueous solution for use in magnetic resonance imaging.

[0019] Furthermore, the present invention provides a contrast agent containing the aforementioned metal-free polypeptide probe.

[0020] Furthermore, the present invention provides the use of the metal-free polypeptide probe, the formulation, and the contrast agent in at least one of the following aspects: (1) Magnetic resonance imaging; (2) Prepare reagents or kits for magnetic resonance imaging; Preferably, the purpose of the magnetic resonance imaging is to monitor or diagnose tumors; Preferably, the tumor is a tumor expressing an integrin receptor; Preferably, the tumors expressing integrin receptors include pancreatic cancer, glioma, melanoma, breast cancer, or prostate cancer.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a metal-free polypeptide probe capable of in-situ self-assembling to form a nanofiber network. This metal-free polypeptide probe can regulate the state of water molecules and thus respond to MRI signals. Through in vitro and in vivo experiments, the metal-free polypeptide probe of this invention has good long-term blood circulation stability, a long imaging window, and can stably respond to MRI signals. Moreover, it has high biosafety and can be used for tumor imaging and monitoring with high expression of integrin receptors. It has broad application prospects in the field of tumor magnetic resonance imaging. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the working mechanism of the metal-free polypeptide probe of the present invention.

[0023] Figure 2 Is it R-BSPP and BSPP in the presence / absence of Ca 2+ Transmission electron microscopy images of morphological transformation at 0h and 4h under induced conditions, scale bar 200nm.

[0024] Figure 3 These are the CD spectra of R-BSPP and BSPP; where a represents the presence / absence of Ca in BSPP at 0h and 4h.2+ Induced CD spectrum; b represents R-BSPP with / without Ca at 0h and 4h. 2+ Induced CD spectrum; c represents BSPP and R-BSPP in the presence / absence of Ca 2+ The percentage change of secondary structure composition under induced conditions.

[0025] Figure 4 R-BSPP and BSPP in Ca 2+ Particle size distribution at 0 h, 4 h, 8 h, and 12 h after induction.

[0026] Figure 5 These are fluorescence confocal microscopy images of R-BSPP and BSPP after interaction with KPC cells. Red represents Cy5-labeled self-assembled peptides, blue represents cell nuclei, and the scale bar is 10 μm.

[0027] Figure 6 These are scanning electron microscope images of KPC cells after 4 h of R-BSPP and BSPP binding, with scale bars at 10 μM and 2 μM.

[0028] Figure 7 These are in vitro MRI images and pseudocolor images of R-BSPP and BSPP.

[0029] Figure 8 These are in vivo MRI images and pseudocolor images of metal-free polypeptide probes.

[0030] Figure 9 This is an HE staining image of the major organs of a mouse treated with a metal-free polypeptide probe.

[0031] Figure 10 This is the result of the hemolytic activity evaluation of the metal-free polypeptide probe.

[0032] Figure 11 The results are the detection results of three renal function indicators: creatinine, blood urea nitrogen, and uric acid in UUO mice after treatment with metal-free polypeptide probes and Manevist.

[0033] Figure 12 These are photographs of the kidneys of UUO mice treated with metal-free peptide probes and Manevist.

[0034] Figure 13 This is a high-magnification HE staining image of UUO mouse kidney tissue after treatment with metal-free polypeptide probes and Manevist. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] In the embodiments provided in this specification, unless specific techniques or conditions are specified, the techniques or conditions described in the literature in this field, or the product instructions, shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0037] Example 1 This embodiment provides a metal-free polypeptide probe (BSPP), as shown in Formula i. This metal-free polypeptide probe can target integrin receptors on tumor cell membranes and can be cleaved by fibroblast activation protein-α, which is highly expressed in the tumor microenvironment, resulting in the chemical structure shown in Formula ii. The metal-free polypeptide probe of Formula i is synthesized using a standard solid-phase polypeptide synthesis method (i.e., synthesis from the C-terminus to the N-terminus of the polypeptide chain, a process of repeatedly adding amino acids), as follows: 1. Weigh 1g of Fmoc-Wang Resin into a 50ml peptide synthesis tube, add 25ml of N,N-dimethylformamide (DMF) and allow to swell overnight. The resin has been pre-linked with the first amino acid (D, Asp) as the C-terminal starting point.

[0038] 2. After rinsing the resin three times alternately with dichloromethane (DCM) and DMF, add 25 ml of deprotecting agent (prepared as a mixed solution containing 2% DBU and 5% anhydrous piperazine using DMF as solvent), shake for 15 minutes to remove the protecting Fmoc group and expose the N-terminal primary amino group of the amino acid. The deprotection result is detected by the Kaiser test (add a small amount of resin to the ninhydrin test reagent, boil for 1 minute; if the resin particles turn purple, deprotection is successful).

[0039] 3. Weigh 10 equivalents of the next Fmoc-amino acid and the catalyst O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) into a coupling agent (a mixed solution containing 5% N-methylmorpholine in DMF as solvent) and pre-react for 10 minutes. Then add the mixture to a synthesis tube and react for 1 hour. After the reaction, use the Kaiser test (add a small amount of resin to the ninhydrin test reagent, boil for 1 minute; if the resin particles do not turn purple, the coupling is successful).

[0040] 4. Repeat steps 2 and 3 in a cycle to protect and couple, sequentially coupling all amino acids in the designed sequence from the C-terminus to the N-terminus.

[0041] 5. After deprotecting the last amino acid, couple with mPEG. 1000 Add 5 times the equivalent of mPEG 1000 10 times the equivalent of HBTU was dissolved in the coupling agent and reacted overnight. Finally, the Kaiser test was used to confirm the reaction (this step is not required if the cleaved sequence is synthesized).

[0042] 6. Remove the reaction solution, rinse the resin three times alternately with DCM and DMF, then add methanol to shrink the resin for 15 minutes to facilitate subsequent pyrolysis and product collection.

[0043] 7. Add 10 ml of lysis buffer (a mixed solution containing 2.5% triisopropylsilane (TIS) and 2.5% ultrapure water, with trifluoroacetic acid (TFA) as the main solvent), stir in an ice bath for 2.5 hours, filter, collect the solution, dry the TFA with nitrogen, add ice-cold diethyl ether to precipitate, and collect the peptide product in an EP tube. Centrifuge at 8000 rpm for 3 minutes, repeat the washing with ice-cold diethyl ether three times, and evaporate the diethyl ether to obtain the solid crude peptide. Finally, purify using preparative liquid chromatography. The molecular weight and purity of the peptide were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC), finally obtaining the metal-free peptide probe BSPP shown in formula i.

[0044] This embodiment further prepares R-BSPP. R-BSPP is the corresponding structure of the product obtained by cleavage of BSPP by fibroblast activation protein-α. The sample was not obtained by in vitro enzymatic digestion of BSPP, but was directly synthesized according to the chemical structure shown in Formula ii using the standard Fmoc solid-phase polypeptide synthesis method, as follows: 1. Weigh an appropriate amount of Fmoc-Wang Resin and place it in a polypeptide synthesis tube. Add N,N-dimethylformamide (DMF) and allow it to swell completely. Pre-link the resin to the terminal amino acid D (Asp) as the C-terminal starting point.

[0045] 2. After washing the resin alternately with dichloromethane (DCM) and DMF, a deprotecting agent is added to remove the Fmoc protecting group, exposing the N-terminal amino group of the amino acid, and the Kaiser test is used to detect whether the deprotection is complete.

[0046] 3. Following the design sequence of R-BSPP, the corresponding Fmoc-amino acids were sequentially coupled from the C-terminus to the N-terminus. For each coupling, the corresponding Fmoc-amino acid and HBTU were dissolved in a DMF coupling system containing N-methylmorpholine for activation before being added to the resin for reaction. After the reaction, the resin was washed, and the Kaiser test was used to check whether the coupling was complete.

[0047] 4. Repeat the deprotection and coupling steps until the solid-phase synthesis of the full R-BSPP sequence is complete. Since R-BSPP corresponds to the structure generated after BSPP digestion, it will not be modified with mPEG1000 again.

[0048] 5. After synthesis, the resin was washed alternately with DCM and DMF, and the resin was shrunken with methanol. Then, a pyrolysis buffer containing trifluoroacetic acid (TFA), triisopropylsilane (TIS) and ultrapure water was added for pyrolysis. After collecting the pyrolysis buffer, TFA was removed, and crude R-BSPP was obtained by precipitation with ice-cold diethyl ether.

[0049] 6. The crude R-BSPP was purified by preparative reversed-phase high-performance liquid chromatography and then lyophilized to obtain pure R-BSPP. The molecular weight and purity of R-BSPP were identified using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography (HPLC). The R-BSPP used in subsequent in vitro assembly, cell binding, magnetic resonance imaging, and safety evaluation experiments were all samples synthesized in the above solid-phase phase and purified as described above.

[0050] Example 2: Characterization of the assembly morphology of metal-free peptide probes The self-assembly morphology of the R-BSPP and BSPP peptide samples from Example 1 was observed using transmission electron microscopy (TEM). First, the peptide sample was prepared to a specified concentration (100 μM, with or without equimolar concentration of CaCl2). 10 μL of the sample was added to a copper grid and incubated at room temperature for 10 minutes. Excess solution was blotted away with filter paper, and 10 μL of 2% phosphotungstic acid staining solution was added for staining for 2 minutes. The staining solution was then removed. Finally, the copper grid was quickly rinsed with deionized water, blotted dry with filter paper, and thoroughly dried. The grid was then observed under a TEM, and representative images were acquired for morphology analysis.

[0051] Considering that the recognition process of receptors such as integrins on the surface of tumor cells usually relies on the participation of divalent cations, this embodiment adds CaCl2 to the peptide solution to simulate the changes in the local ionic microenvironment that may be formed during the receptor-ligand interaction process and the resulting conformational constraints, and further evaluates the effect of this condition on peptide assembly behavior.

[0052] Depend on Figure 2 As shown, in Ca 2+ After co-incubation for 4 hours under the presence of Ca, BSPP further transformed from initial nanoparticles into a nanofiber network structure; R-BSPP also assembled from nanoparticles to form a nanofiber network under the same conditions. In contrast, without the addition of Ca... 2+ In the control group, only a small amount of nanofiber formation was observed in both groups of samples.

[0053] Further comparison revealed that, within the same incubation period, the fiber network formed by R-BSPP was richer and more compact than that of BSPP, indicating that the assembly units exposed after BSPP enzymatic digestion can significantly promote its self-assembly process.

[0054] Example 3: Characterization of the secondary structure of assembled metal-free peptide probes In this embodiment, a circular dichroism (CD) spectrometer was used to analyze the secondary structures of the R-BSPP and BSPP peptides from Example 1. A 100 μM aqueous solution of the peptides (with or without equimolar concentration of CaCl2) was prepared beforehand. Measurements were performed using a quartz cuvette with a 1 mm path length, within the range of 185 to 240 nm, with a resolution of 2.0 nm and a scan rate of 500 nm / min. The spectral data were processed and analyzed using Spectra Manager software.

[0055] Depend on Figure 3 As shown, in relation to Ca 2+ After co-incubation for 4 hours, the CD spectrum of BSPP showed a positive absorption peak at approximately 205 nm and a significant negative absorption peak at approximately 215 nm, exhibiting typical β-sheet secondary structure characteristics, indicating that BSPP underwent ordered assembly under these conditions. Similarly, R-BSPP also exhibited similar characteristic peaks under the same conditions, indicating that it also formed a secondary conformation dominated by β-sheets.

[0056] To further quantitatively analyze the changes in secondary structure composition, a statistical analysis was conducted on the proportions of secondary structures in BSPP and R-BSPP, in Ca... 2+ Under induction conditions, the β-sheet content of BSPP was significantly increased compared to its initial state, indicating that this condition can promote its conformational change and ordered assembly. In contrast, R-BSPP, due to the exposure of the assembly core unit after enzyme cleavage, is more likely to form a β-sheet structure within the same timescale; under Ca... 2+ When present, this change is more pronounced, with both the increase in the magnitude and proportion of β-sheet components being higher than that of BSPP.

[0057] Example 4: Determination of particle size of metal-free peptide probes In this embodiment, dynamic light scattering (DLS) was used to measure the changes in particle size and zeta potential of BSPP and R-BSPP peptides before and after calcium ion induction. A 100 μM aqueous solution of the peptides (with or without equimolar concentrations of CaCl2) was prepared beforehand. DLS measurements were performed at 25°C for 10 seconds, repeated five times. The experiments were performed in triplicate, and the data were analyzed using Zetasizer software.

[0058] like Figure 4As shown, BSPP mainly exists as small and concentrated nanoparticles at 0 h. After 4 h of incubation, the hydrated particle size increases significantly, indicating that BSPP begins to transform from initial particles into higher-order assemblies. With the extension of incubation time to 8 h and 12 h, the particle size further increases, and the distribution peak gradually shifts to the larger size region, indicating that its assemblies have time-dependent growth characteristics and gradually form larger-scale aggregate structures.

[0059] R-BSPP also exhibited well-dispersed nanoparticles at 0 h, with particle size gradually increasing at 4 h, 8 h, and 12 h, indicating that the enzymatic digestion products could continue to self-assemble and form higher-order structures. Overall, both R-BSPP and BSPP showed a trend of increasing particle size with prolonged incubation time, but R-BSPP differed from BSPP in terms of the growth rate and final particle size distribution, suggesting that the enzymatically digested peptides may have a stronger tendency for self-assembly.

[0060] Example 5: Fluorescent colocalization of metal-free peptide probes bound to cells This embodiment uses KPC (pancreatic ductal adenocarcinoma cells from a mouse model, with a density of approximately 5 × 10⁻⁶) cells. 3 Cells were seeded in confocal culture dishes 24 hours in advance, and the culture medium containing Cy5-labeled peptides was replaced the next day for co-incubation. Excess material was then washed away with PBS, and cells were fixed with paraformaldehyde and counterstained with DAPI. Images were acquired using a laser scanning confocal microscope (CLSM) to observe the distribution and localization of the peptides in the cells.

[0061] like Figure 5 As shown, the Cy5 fluorescence signals (red) of both peptides are mainly enriched in the pericellular region of KPC cells, exhibiting a continuous and clear ring or arc-shaped distribution, while almost no obvious diffuse fluorescence signal is observed in the cytoplasm or nucleus. This distribution pattern clearly indicates that both BSPP and R-BSPP can specifically recognize and bind to receptors such as integrins on the cell membrane surface, thereby achieving efficient enrichment at the membrane interface.

[0062] Example 6: Binding of metal-free peptide probes to cell surfaces and in-situ assembly morphology characterization This embodiment uses scanning electron microscopy (SEM) to analyze the binding of peptides to the cell surface and their in-situ assembly morphology. Mouse pancreatic cancer cells (KPC) were pre-seeded on sterile silicon wafers and cultured for 24 hours. The cells were then co-incubated with peptide-containing culture medium, followed by washing with PBS to remove unbound peptides. Cells were fixed with paraformaldehyde and then dehydrated stepwise with gradient concentrations of ethanol solutions, followed by further drying with tert-butanol. After complete drying, the samples were sputter-coated with gold to enhance conductivity and finally observed under a scanning electron microscope.

[0063] like Figure 6As shown, the first row is an image observed at low magnification, and the second row is an image observed at high magnification. The images reveal that the cells in the blank control group exhibit a relatively porous and smooth microstructure, with no obvious fibrous attachments. In contrast, after 4 hours of BSPP treatment, a network structure composed of interwoven nanofibers is visible on the cell surface. This network is relatively loose but uniformly distributed, preliminarily confirming the ability of BSPP to assemble in situ at the membrane interface. In the R-BSPP treatment group, a denser, continuous, and more interwoven fiber layer forms on the cell surface, with significantly higher density and thickness than the BSPP treatment group.

[0064] Example 7 In vitro magnetic resonance imaging with metal-free peptide probes In this embodiment, a 1.5T magnetic resonance imaging (MRI) head coil was used to evaluate the in vitro MRI performance of metal-free peptide probes. The scanning parameters were: T2-weighted sequence, TR / TE = 4400 ms / 90.4 ms, FOV = 160 mm × 160 mm, slice thickness = 1.5 mm, and number of slices = 12. The final concentration of BSPP or R-BSPP was 20 mM. The final volume of all samples was uniformly 2 mL. BSPP or R-BSPP (with or without equimolar concentration of CaCl2) was incubated at room temperature for 4 h before scanning.

[0065] like Figure 7 As shown, compared with the H2O group and the CaCl2 group, regardless of whether Ca was added... 2+ Reagents containing BSPP or R-BSPP showed a significant decrease in T2 signal, appearing as darker signals on T2WI images. This result indicates that BSPP and its enzyme digestion product R-BSPP can induce significant changes in the magnetic resonance signal of the system at reagent levels. Further comparison revealed that the T2 signal attenuation was more pronounced after the addition of CaCl2 to the BSPP or R-BSPP groups.

[0066] Example 8: In vivo magnetic resonance imaging using metal-free peptide probes KPC cells in good growth condition were cultured to the logarithmic growth phase, and digested when the cell confluence reached approximately 80%. After digestion, the cells were resuspended in PBS and mixed with matrix gel at a 1:1 volume ratio to adjust the cell density to 1×10⁶ cells / day. 7 Cells / mL. Balb / c nude mice were then anesthetized in an induction box containing 3% isoflurane. After the mice were fully anesthetized, the inoculation area on their backs was disinfected with 75% alcohol. 100 μL of cell suspension was drawn up using an insulin syringe and slowly injected subcutaneously into the back of the mice at approximately a 45° angle, successfully constructing Balb / c tumor-bearing mice.

[0067] In vivo magnetic resonance imaging (MRI) was performed using a 1.5T MRI scanner. Balb / c nude tumor-bearing mice were anesthetized by intraperitoneal injection of tribromoethanol at a concentration of 1.25% at a dose of 50 μL / 10g body weight. After anesthesia, the tumor-bearing mice were randomly divided into three groups: PBS, BSPP, and R-BSPP, with six mice in each group (n=6). The BSPP and R-BSPP groups were administered 100 μL and 20 mM of BSPP or R-BSPP solution, respectively; the PBS group received an equal volume of PBS as a control. Continuous MRI scans were performed at preset time points after drug administration. Head coils and small animal coils were used for scanning depending on the imaging agent and imaging requirements. The BSPP group used a head coil to acquire T2-weighted imaging (T2WI) images with the following scanning parameters: TR=4400ms, TE=90.4ms, FOV=160mm×160mm, slice thickness of 1mm, and number of slices of 12.

[0068] In vivo magnetic resonance imaging results as follows Figure 8 As shown, the T2-weighted signal in the tumor region significantly decreased immediately after BSPP injection. Two hours after injection, the signal intensity began to decline from baseline; over time, the signal intensity gradually decreased, reaching its lowest level at 6 hours post-injection, and then maintained a stable low-signal plateau for the next 4 hours (6-10 hours). This sustained low-signal state indicates that BSPP can achieve efficient enrichment and long-term retention in the tumor site. The signal in the tumor region only began to slowly recover 12 hours after injection, indicating that the probe was gradually metabolized or cleared from the tumor site. This signal change pattern may be attributed to the unique enzyme-responsive self-assembly characteristics of BSPP. After injection, BSPP undergoes specific enzymatic cleavage under the action of highly expressed proteases in the tumor microenvironment, exposing assembly units and self-assembling in situ to form a nanofiber network structure. This fibrous network structure restricts water molecule movement, resulting in a decrease in the proportion of free water and an increase in the proportion of bound water, thereby altering the relaxation time and leading to a sustained decrease in the T2-weighted signal. The low signal plateau period of 6-10 hours reflects the stability of the nanofiber network structure, while the signal rebound after 12 hours may be related to the gradual degradation of the assembly and the metabolic clearance of the probe.

[0069] Example 9: In vivo safety of metal-free peptide probes 1. Histopathological evaluation of major organs Based on Example 8, mice in the PBS and BSPP groups were euthanized, and major organs such as the heart, liver, spleen, lungs, and kidneys were rapidly separated. After removing excess surrounding tissue, residual blood was gently rinsed off with physiological saline, and the gross morphology of each organ was observed. Subsequently, the tissues were fixed in 4% paraformaldehyde solution for subsequent HE staining.

[0070] like Figure 9 As shown, compared with the PBS control group, no significant abnormal pathological changes were observed in the major organs of mice treated with BSPP. Specifically, the myocardial fibers were neatly arranged and clearly structured; the hepatocyte cords in the liver tissue were relatively well-arranged, with no obvious hepatocyte degeneration or necrosis; the white and red pulp structures of the spleen were clear, and the splenic nodules were normal in morphology; the alveolar structure of the lung tissue was intact, with no obvious thickening of the alveolar walls or significant inflammatory cell infiltration; the glomeruli and renal tubules in the kidney tissue were intact, with no obvious atrophy, necrosis, or inflammatory response. These results indicate that BSPP injection did not cause significant tissue damage or pathological abnormalities in major organs such as the heart, liver, spleen, lungs, and kidneys, suggesting that the self-assembled polypeptide probe BSPP has good overall in vivo biocompatibility.

[0071] 2. Evaluation of hemolytic activity To evaluate the blood compatibility of BSPP, an in vitro hemolysis assay was used to detect its membrane-damaging effect on erythrocytes. Blood from the orbital venous plexus of Balb / c mice was collected in EDTA anticoagulant tubes. Whole blood was diluted 6-fold with PBS, centrifuged at 1500 rpm for 15 min, the supernatant was discarded, and the erythrocyte pellet was collected. The erythrocytes were then washed three times with PBS and resuspended in PBS. 500 μL of the diluted erythrocyte suspension was mixed with different concentrations of BSPP solution (1, 10, 100, 1000 μg / mL), gently vortexed, and incubated at 37°C for 4 h. After incubation, the supernatant was collected by centrifugation at 1500 rpm for 15 min and transferred to a 96-well plate. The absorbance was measured at 540 nm. Erythrocytes incubated with Triton-100 (2%) and PBS served as positive and negative controls, respectively. The hemolysis rate was calculated using the following formula.

[0072] like Figure 10 As shown, no significant hemolysis was observed after BSPP treatment within the concentration range of 1-15 mM; when the concentration was increased to 20 mM, the hemolysis rate remained below the safety threshold of 5%. The results indicate that BSPP did not exhibit significant hemolytic activity over a wide concentration range, suggesting that BSPP did not cause significant hemolysis under these experimental conditions and demonstrated good blood compatibility.

[0073] 3. Kidney injury assessment Six-week-old female Balb / c mice were selected. Mice were randomly divided into four groups (n=3 per group): healthy control group (Cont.), UUO model group, UUO+Magnevist group, and UUO+BSPP group. BSPP was prepared at a concentration of 20 mM and administered at 100 μL per mouse via tail vein. Magnevist, as a clinically approved commercial contrast agent, was administered at a standard dose of 0.1 mmol Gd / kg. Before administration, Magnevist was diluted with sterile saline to 12.5 mmol / L Gd and administered at 8 mL / kg via tail vein. Mice were fixed supine on a temperature-controlled operating table. Abdominal hair was removed with a depilatory agent, and the skin was disinfected three times with povidone-iodine and twice with 75% alcohol. Sterile surgical drapes were applied. A 0.8–1.0 cm longitudinal incision was made in the left abdomen of the mouse, sequentially incising the skin, subcutaneous tissue, and abdominal muscles. The incision was gently opened to expose the abdominal cavity. Using sterile microforceps, the intestines were gently pushed aside to expose the kidneys and ureters. The ureter on one side was dissected approximately 0.5 cm, taking care not to damage the surrounding blood vessels and kidney tissue. Double ligation was performed on the proximal (near the kidney) and distal ends of the ureter using 4-0 silk sutures. The ureter was then severed between the two ligatures to prevent recanalization. Sterile gauze was applied to the incision to stop bleeding for 3 minutes. After confirming no active bleeding, the abdominal muscles, subcutaneous tissue, and skin were sutured layer by layer. The incision was then disinfected again with povidone-iodine. In the control group, mice underwent only anesthesia, abdominal incision, abdominal cavity exposure, and ureteral dissection; ureteral ligation and transection were not performed. All other procedures were identical to those in the model group.

[0074] After administration of BSPP or Magnevist, blood samples were collected from mice in each group for renal function testing. Blood was collected by enucleation, allowed to coagulate at room temperature, and then centrifuged at 3000 rpm for 10 min to separate serum. The serum was used to detect renal function indicators such as uric acid, creatinine, and blood urea nitrogen.

[0075] The levels of renal function-related indicators, including blood urea nitrogen (BUN), serum creatinine (Scr), and uric acid (UA), in the serum of mice in each group were detected using a fully automated biochemical analyzer. All tests were performed according to the instrument's operating instructions. The effects of BSPP and Magnevis on renal function were evaluated by comparing changes in BUN, Scr, and UA levels among the groups. All experiments were repeated three times, and results are expressed as mean ± standard deviation (mean ± SD).

[0076] Kidney function test results as follows Figure 11As shown in the figure, compared with the healthy control group, the serum Cre, BUN, and UA levels in the UUO group mice were all elevated, indicating that unilateral ureteral obstruction successfully induced kidney injury, demonstrating the successful establishment of the model. Based on this, after Magnevist treatment, the Cre, BUN, and UA levels in the UUO+Mag. group were further elevated, with Cre and UA showing particularly significant increases, indicating that Magnevist further aggravated the renal burden in the context of obstructive kidney injury. In contrast, the Cre, BUN, and UA levels in the UUO+BSPP group did not show a significant further increase, remaining similar to the UUO group and significantly lower than the UUO+Mag. group, indicating that BSPP did not exhibit a significant additional kidney injury effect under this model condition.

[0077] Further, mice were euthanized by cervical dislocation, and both kidneys were harvested, with the capsule and connective tissue removed. Kidney tissue was fixed in 4% paraformaldehyde for pathological section preparation. The gross morphology of the kidneys in each group was then observed and photographed, comparing size, color, surface morphology, and the presence of significant atrophy or swelling. Subsequently, the kidney tissue was fixed in 4% paraformaldehyde, dehydrated routinely, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). The effects of BSPP and Magnevis on kidney tissue under conditions of kidney injury were observed using an optical microscope.

[0078] Results of gross morphological observation of the kidneys as follows Figure 12 As shown. Compared with the healthy control group (Cont.), the obstructed kidneys in all UUO-related groups showed varying degrees of abnormal appearance. The kidneys in the control group were intact, with a relatively smooth surface and uniform color distribution. The kidneys in the UUO group were enlarged, with increased surface irregularity, and showed signs of congestion and swelling. The kidneys in the UUO+Mag. group were further darker in color than those in the UUO group, generally dark red, with more obvious local congestion, and further increased surface irregularity. In contrast, the kidneys in the UUO+BSPP group had a more uniform color distribution than those in the UUO+Mag. group, and the overall appearance was similar to that of the UUO group, with no further worsening trend observed.

[0079] The results of renal tissue pathological analysis further support the above conclusions. For example... Figure 13 As shown, under low-power HE vision, the renal parenchyma structure in the control group was intact, and the renal pelvis / collecting system cavities were relatively small. In contrast, the UUO group, UUO+Mag. group, and UUO+BSPP group all showed significant dilation of the renal pelvis / collecting system, forming large, obvious cavities, accompanied by ring-like thinning of the renal parenchyma, consistent with the morphological characteristics of obstructive hydronephrosis, further demonstrating the successful establishment of the UUO model. Comparison among the three groups revealed that the UUO+Mag. group showed more pronounced heterogeneity in the morphology of the renal parenchyma tissue bands under low-power vision, with focal areas showing deeper staining; while the UUO+BSPP group exhibited more uniform morphology of the renal parenchyma tissue bands.

[0080] The above results demonstrate that the metal-free polypeptide probe of the present invention has good in vivo safety.

[0081] In summary, the metal-free peptide probe of this invention can specifically target tumor cells and achieve rapid local accumulation of tumor cells through in-situ transformation from nanoparticles to nanofibers. This, in turn, modulates the local water molecule state, generating a magnetic resonance imaging (MRI) signal response. In a mouse subcutaneous pancreatic cancer xenograft model, based on a clinically relevant 1.5T MRI platform and T2-weighted imaging (T2WI) sequences, the probe significantly altered the tumor region signal, exhibited a long imaging window, and demonstrated good biocompatibility. Furthermore, in a mouse model of unilateral ureteral obstruction (UUO), the probe also showed low nephrotoxicity. Therefore, the metal-free peptide probe of this invention provides a feasible strategy for the development of metal-free MRI contrast agents and has good application potential in tumor imaging and monitoring.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A metal-free polypeptide probe, characterized in that, The metal-free polypeptide probe can be cleaved by fibroblast activation protein-α, resulting in a chemical structure as shown in formula ii: Formula ii.

2. The metal-free polypeptide probe according to claim 1, characterized in that, The metal-free polypeptide probe will self-assemble in situ to form a nanofiber network after being cleaved.

3. The metal-free polypeptide probe according to claim 2, characterized in that, The nanofiber network is characterized by β-folds.

4. The metal-free polypeptide probe according to any one of claims 1 to 3, characterized in that, After being cleaved, the metal-free polypeptide probe self-assembles in situ into a nanofiber network in the presence of calcium ions.

5. The metal-free polypeptide probe according to claim 4, characterized in that, After the metal-free polypeptide probe is cleaved, it utilizes the calcium ion-dependent carboxyl group binding behavior of integrin to simulate the interaction between integrin and ligand, and forms a nanofiber network through in situ self-assembly.

6. The metal-free polypeptide probe according to any one of claims 2 to 5, characterized in that, The nanofiber network can regulate the state of water molecules and respond to magnetic resonance imaging signals.

7. The metal-free polypeptide probe according to any one of claims 1 to 6, characterized in that, The chemical formula of the metal-free polypeptide probe is shown in formula i: Formula i.

8. A formulation, characterized in that, It contains any one of the metal-free polypeptide probes according to claims 1 to 7.

9. A contrast agent, characterized in that, It contains any one of the metal-free polypeptide probes according to claims 1 to 7.

10. The use of the metal-free polypeptide probe according to any one of claims 1 to 7, the formulation according to claim 8, and the contrast agent according to claim 9 in at least one of the following aspects: (1) Magnetic resonance imaging; (2) Prepare reagents or kits for magnetic resonance imaging; Preferably, the purpose of the magnetic resonance imaging is to monitor or diagnose tumors; Preferably, the tumor is a tumor expressing an integrin receptor; Preferably, the tumor expressing the integrin receptor includes: Pancreatic cancer, glioma, melanoma, breast cancer, or prostate cancer.

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