A magnetic resonance imaging agent for targeted detection of collagen molecular structure degeneration, its preparation method and application

Abstract

This invention discloses a magnetic resonance imaging (MRI) contrast agent for targeted detection of collagen molecular structural denaturation, its preparation method, and its application. The general chemical formula of this MRI contrast agent is: [X] m -L-(GfO) n Among them, (GfO) n [n] is a collagen hybrid peptide, where n is a positive integer between 6 and 12; L is a linker, comprising amino acids and / or amino acid derivatives; [X] is a paramagnetic metal complex, composed of a chelating agent and paramagnetic metal ions; m represents the number of paramagnetic metal complexes, where m is a positive integer between 1 and 12. The magnetic resonance contrast agent of this invention can target and detect degenerated collagen, exhibiting higher lesion specificity and detection sensitivity compared to conventional targeted contrast agents that rely on differences in collagen content for imaging. Furthermore, this magnetic resonance contrast agent can significantly enhance the magnetic resonance signal of damaged and degenerated cartilage, Achilles tendon, and abdominal aortic aneurysm, facilitating the early diagnosis of collagen degeneration-related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a magnetic resonance imaging agent for targeted detection of collagen molecular structural degeneration, its preparation method, and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) is a diagnostic technique that utilizes the nuclear magnetic resonance phenomenon of certain atomic nuclei in human tissues. The resulting radio frequency signals are processed by a computer to reconstruct an image of a specific layer of the human body. MRI is a non-invasive imaging technique that provides high spatial resolution and three-dimensional imaging of anatomical structures, and can also provide functional and physiological information about tissues within the body.

[0003] Magnetic resonance imaging (MRI) contrast agents are the primary reagents used in MRI. They alter the MR signal of local tissues, shorten relaxation time, and improve the contrast between lesions and normal tissues, thereby enhancing image clarity and lesion detection rates. Currently, Magendieconazole (Gd-DTPA) is the most commonly used MRI contrast agent in clinical practice. Its main component, gadolinium ions, are strongly paramagnetic metal ions that can significantly shorten the relaxation time of surrounding tissues, aiding in the detection of small lesions and weakly enhancing lesions. However, currently used MRI contrast agents lack targeting affinity for disease biomarkers in lesions, failing to specifically target and enrich them at the lesion site. Furthermore, the differentiation between lesions and normal tissues relies on morphological changes, making precise and early lesion identification difficult.

[0004] In related technologies, imaging for the clinical diagnosis of musculoskeletal system injuries and diseases mainly relies on magnetic resonance imaging (MRI), such as for osteoarthritis (OA) and Achilles tendinopathy (AT). Osteoarthritis is the most common degenerative joint disease, presenting a "window of opportunity" for early diagnosis and treatment. However, while conventional MRI contrast agents can provide imaging, they can only detect morphological changes in cartilage tissue and cannot detect early OA. Furthermore, there are currently no drugs to reverse or halt the progression of OA. Once OA reaches an advanced stage, joint replacement surgery is required, significantly increasing the risks. Achilles tendinopathy is a common disease, with collagen fiber destruction being a key pathological manifestation closely related to disease progression. However, due to the lack of blood supply to the Achilles tendon and low MRI signal, early Achilles tendinopathy is often undetectable, greatly hindering early intervention and treatment.

[0005] Therefore, there is an urgent need for new biomarkers and corresponding magnetic resonance imaging targeted contrast agents to enable accurate characterization and non-invasive examination of early musculoskeletal system-related diseases, thereby achieving early intervention and treatment. Summary of the Invention

[0006] This invention aims to address at least one of the technical problems existing in the prior art. To this end, this invention proposes a magnetic resonance imaging (MRI) contrast agent that targets and detects denatured collagen. Compared to conventional targeted MRI-near-infrared fluorescence dual-modal probes that rely on differences in type II collagen content for imaging, this agent exhibits higher lesion specificity and detection sensitivity. Furthermore, the MRI contrast agent of this invention has a superior relaxation rate compared to conventional Magendie contrast agents, significantly enhancing the MRI signals of damaged articular cartilage and ligaments, as well as the MRI signals of damaged Achilles tendons, thus facilitating the early diagnosis of joint and Achilles tendon diseases.

[0007] The present invention also proposes a method for preparing a magnetic resonance imaging contrast agent.

[0008] The present invention also proposes a reagent kit for detection.

[0009] This invention also proposes the application of magnetic resonance contrast agents in the preparation of products for imaging detection of cartilage damage, degenerative diseases of bone and muscle soft tissues, cardiovascular diseases, tumors, or organ fibrosis.

[0010] In a first aspect, the present invention provides a magnetic resonance imaging (MRI) contrast agent, the general chemical formula of which is as follows:

[0011] [X] m -L-(GfO) n ;

[0012] Wherein, the (GfO) n It is a collagen hybrid peptide, where n is taken from a positive integer between 6 and 12;

[0013] L is a connector, and the connector includes amino acids and / or amino acid derivatives;

[0014] [X] is a paramagnetic metal complex, which is composed of a chelating agent and paramagnetic metal ions. m represents the number of paramagnetic metal complexes, and m is a positive integer between 1 and 12.

[0015] The magnetic resonance imaging agent according to embodiments of the present invention has at least the following beneficial effects:

[0016] (1) The magnetic resonance contrast agent of the present invention can target and detect denatured collagen. Collagen molecules are composed of a Gly-Xaa-Yaa triplet repeating amino acid sequence, where Xaa and Yaa are usually fluoroproline (flp, f) and hydroxyproline (Hyp, O), respectively. The triple helix is ​​a unique secondary structure of collagen, which intertwines the three chains together through intramolecular hydrogen bonds. Under pathological conditions, the collagen triple helix molecule is broken by matrix metalloproteinases or mechanical force, and the broken collagen molecule is unstable at body temperature and spontaneously unwinds. The collagen hybridizing peptide (CHP) in the magnetic resonance contrast agent of the present invention acts as a molecular probe. It can form a heterotrihelical structure with the unwinding collagen chain through interchain hydrogen bonds. Since normal collagen triple helix molecules lack binding sites, CHP will not bind to normal collagen molecules. Therefore, the magnetic resonance contrast agent of the present invention has excellent targeting of denatured collagen.

[0017] (2) The magnetic resonance contrast agent of the present invention can target and detect cartilage damage. Compared with conventional targeted magnetic resonance-near-infrared fluorescence dual-modal probes that rely on differences in type II collagen content for imaging, the magnetic resonance contrast agent of the present invention can target and bind to denatured collagen, thus exhibiting higher lesion specificity and detection sensitivity. Furthermore, experiments have shown that the magnetic resonance contrast agent of the present invention can significantly enhance the magnetic resonance signal of damaged cartilage, ligaments, and tendons, which is helpful for the early diagnosis of cartilage diseases.

[0018] (3) The magnetic resonance contrast agent of the present invention can realize non-invasive early detection of osteoarthritis and Achilles tendon disease. The present invention has experimentally confirmed that the magnetic resonance contrast agent designed by the present invention has a better relaxation rate (8.45 times that of the Magneto contrast agent) compared with the Magneto contrast agent. It can significantly increase the magnetic resonance T1 signal of thermally degenerated cartilage and tendon, the magnetic resonance T1 signal of knee cartilage and ligament in osteoarthritis model (DMM rat), and the T1 signal of Achilles tendon disease model, which helps to improve the early diagnosis effect of collagen damage-related diseases.

[0019] (4) The magnetic resonance contrast agent of the present invention is used for non-invasive clinical detection. The magnetic resonance contrast agent of the present invention has a better penetration depth than near-infrared fluorescent dye-labeled CHP probes and can be used for non-invasive clinical detection.

[0020] In some embodiments of the present invention, the magnetic resonance contrast agent further comprises a fluorescent dye group, which is connected to the collagen hybrid peptide via the linker.

[0021] In some embodiments of the present invention, the magnetic resonance contrast agent has the structural formula shown in formula (I) or formula (II), wherein:

[0022]

[0023]

[0024] In the formula, [X] is a paramagnetic metal complex, which is composed of a chelating agent and paramagnetic metal ions; and [Y] is a fluorescent dye.

[0025] In some embodiments of the present invention, the fluorescent dye group is selected from at least one of anthocyanin, rhodamine, BODIPY, FITC, erythrosine, phthalocyanine, phycocyanin, phycoerythrin, and Alexa Fluor.

[0026] In some embodiments of the present invention, the anthocyanin is selected from any one of sulfo-Cy3, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7, and sulfo-Cy7.5.

[0027] In some embodiments of the present invention, the chelating agent is selected from any one of DOTA chelating agent, DTPA chelating agent, DO3A chelating agent, NOA chelating agent, DOTMA chelating agent, M4DOTA chelating agent, M4DO3A chelating agent, BOPTA chelating agent, NODAGA chelating agent, HYNIC chelating agent, HPDO3A chelating agent, and EDTA chelating agent.

[0028] Preferably, the DOTA chelating agent is p-SCN-Bn-DOTA.

[0029] Preferably, the DTPA chelating agent is p-SCN-Bn-DTPA.

[0030] Preferably, the DO3A chelating agent is p-SCN-Bn-oxo-DO3A.

[0031] Preferably, the NOA chelating agent is p-SCN-Bn-NOTA.

[0032] In some embodiments of the present invention, the paramagnetic metal ion is selected from at least one of gadolinium ions, dysprosium ions, neodymium ions, and manganese ions.

[0033] Preferably, the paramagnetic metal ion is a gadolinium ion.

[0034] In some embodiments of the present invention, the connector comprises lysine and / or 6-aminocaproic acid.

[0035] A second aspect of the present invention provides a method for preparing a magnetic resonance imaging contrast agent as described in any one of the first aspects, comprising the following steps:

[0036] S1. A precursor probe containing the collagen hybrid peptide and the linker is prepared by solid-phase synthesis.

[0037] S2. The product of step S1 is reacted sequentially with the chelating agent and the paramagnetic metal ions, and after purification, the product is obtained.

[0038] The preparation method according to the embodiments of the present invention has at least the following beneficial effects: the preparation method of the magnetic resonance contrast agent of the present invention is simple, has high yield and low cost, and is suitable for industrial production.

[0039] In some embodiments of the present invention, step S1, after obtaining the precursor probe, further includes: performing a deprotection treatment on the precursor probe, and then labeling it with a fluorescent dye to obtain a precursor probe containing the fluorescent dye group.

[0040] In some embodiments of the present invention, in step S1, the solid-phase synthesis method includes Fmoc chemical solid-phase synthesis or BOC chemical solid-phase synthesis.

[0041] Preferably, the solid-phase synthesis method is the Fmoc chemical solid-phase synthesis method.

[0042] A third aspect of the invention provides a kit for detection, comprising the magnetic resonance contrast agent as described in any of the first aspects.

[0043] A fourth aspect of the invention provides the use of the magnetic resonance imaging agent as described in any one of the first aspects in any one of (A) to (D).

[0044] (A) To develop products for imaging detection of cartilage damage-related diseases;

[0045] (B) To develop products for imaging detection of degenerative diseases of musculoskeletal and soft tissues;

[0046] (C) Prepare products for imaging detection of cardiovascular lesions;

[0047] (D) Prepare products for imaging detection of tumors or organ fibrosis.

[0048] In some embodiments of the present invention, the cartilage injury-related diseases include, but are not limited to, arthritis, tendinopathy, Achilles tendinopathy, rotator cuff injury, and ligament injury.

[0049] In some embodiments of the present invention, the degenerative diseases of the musculoskeletal system include, but are not limited to, intervertebral disc injury, intervertebral disc degeneration, paraspinal muscle degeneration, sacroiliitis, ankylosing spondylitis, and spinal degenerative diseases.

[0050] Collagen is a primary component of articular cartilage, tendons, and the annulus fibrosus of intervertebral discs. Dense collagen networks maintain the structural integrity of these tissues and bear mechanical loads. With the progression of diseases such as arthritis, Achilles tendinopathy, and intervertebral disc degeneration, the accumulation of mechanical damage, and the increased degradation of collagen by matrix metalloproteinases induced by inflammatory cytokines and abnormal cell metabolism, gradually destroy the collagen matrix, leading to the failure of the tissue's mechanical structure. Therefore, targeted recognition of degenerated collagen molecules can enable accurate imaging and detection of these degenerative diseases of musculoskeletal soft tissues.

[0051] In some embodiments of the present invention, the cardiovascular lesions include, but are not limited to, abdominal aortic aneurysm, intracranial aneurysm, aortic dissection, atherosclerosis, valvular lesions, etc.

[0052] The structure and mechanical stability of blood vessels are also primarily maintained by collagen fiber networks. In common vascular diseases such as aneurysms and atherosclerosis, the degradation and destruction of collagen in the vessel wall is not only a prominent feature of the lesions but also a direct cause of highly critical cardiovascular events such as aneurysm and atherosclerotic plaque rupture. Therefore, targeted recognition of degenerated collagen molecules can enable accurate imaging and detection of these cardiovascular lesions.

[0053] In some embodiments of the present invention, the tumor includes, but is not limited to, pancreatic tumors, breast tumors, prostate tumors, digestive tract tumors, lung tumors, liver tumors, etc.

[0054] A prominent feature of various tumors (such as pancreatic cancer and breast cancer) and organ fibrosis (such as pulmonary fibrosis) is the abundant proliferation of dense collagen-rich extracellular matrix. Matrix metalloproteinases, expressed in large quantities during tumor and fibrosis progression, cause collagen structures to denature and unwind. Researchers have found a large number of structurally denatured and unwound collagen molecules in the dense matrix of pancreatic cancer and pulmonary fibrosis lesions. Therefore, by targeting and recognizing denatured collagen molecules, accurate imaging and detection of tumors and organ fibrosis lesions with collagen matrix abnormalities can be achieved.

[0055] Specifically, the pancreatic tumors include, but are not limited to, pancreatic ductal adenomas, pancreatic endocrine tumors, mucinous cystadenomas, serous cystadenomas, pancreatic angiomyomas, and pancreatic lymphoepithelial cysts.

[0056] In some embodiments of the present invention, the organ fibrosis includes, but is not limited to, fibrosis of the liver, kidneys, skin, epidermis, endothelium, muscles, tendons, cartilage, heart, pancreas, lungs, uterus, nervous system, testes, penis, ovaries, adrenal glands, arteries, veins, colon, intestines, bile ducts, soft tissues, bone marrow, joints, and stomach, particularly fibrosis of the liver, digestive tract, lungs, heart, kidneys, muscles, skin, soft tissues, bone marrow, intestines, eyes, and joints.

[0057] Other features and advantages of the present invention will be set forth in the following description. Attached Figure Description

[0058] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0059] Figure 1 This is a schematic diagram illustrating the binding of collagen hybrid peptides to denatured collagen molecular chains.

[0060] Figure 2 The chemical structure and synthetic route of the magnetic resonance contrast agent Gd-CHP of the present invention are shown, where A: chemical structure diagram and B: synthetic route diagram.

[0061] Figure 3 This is the mass spectrum of the magnetic resonance contrast agent Gd-CHP of this invention.

[0062] Figure 4 The magnetic resonance contrast agent Gd of this invention n The chemical structure of -Cy5-CHP and Gd n Synthetic route diagram of -Cy5-CHP, where A: chemical structure diagram, B: synthetic route diagram.

[0063] Figure 5 For the magnetic resonance imaging (MRI) model of this invention, Gd-DTPA and Gd n -Cy5-CHP、Gd n -Cy5- S A standard T1-weighted image of relaxation rate measured by CHP.

[0064] Figure 6 Gd is used in this invention n Magnetic resonance and fluorescence imaging results of thermally denatured patellofemoral cartilage plugs in the knee joint of normal pigs treated with the Cy5-CHP probe.

[0065] Figure 7 Gd is used in this invention n In vitro magnetic resonance and fluorescence imaging results of the Achilles tendon of rats with thermal denaturation treated with the Cy5-CHP probe.

[0066] Figure 8The results of in vivo detection of damaged tibial cartilage and ligaments in the knee joint of rats with osteoarthritis using the magnetic resonance contrast agent Gd-CHP of this invention.

[0067] Figure 9 The magnetic resonance contrast agent Gd of this invention n -Cy5-CHP in vivo detection results of damaged cartilage and ligaments in the knee joint of rats with osteoarthritis, including: A: Representative in vivo T1WI images of the knee joint on the sham-operated side (Sham) or DMM-operated side of the same rat 7 weeks after surgery; B: Contrast-to-noise ratio of tibial plateau cartilage in the knee joint of the rat with DMM model before and after contrast enhancement (n=4, statistical analysis using paired t-test); C: Representative in vivo T1WI images of the knee joint of the rat with DMM model before and after contrast enhancement 7 weeks after modeling, and T2WI image before enhancement; D: Staining and imaging results of cartilage sections in the box-shaped area in Figure C; E: In vivo T1WI and T2WI scan results of the same DMM-injured rat knee joint repeated at 3 and 7 weeks after DMM surgery.

[0068] Figure 10 The magnetic resonance contrast agent Gd of this invention n -Cy5-CHP assay results of isolated osteoarthritis patients' knee cartilage damage, including A: gross appearance of cartilage tissue and characterization of cartilage degeneration; B: Gd... n - Safranin-Fix-Green staining of cartilage plug specimens treated with Cy5-CHP; C: T1WI magnetic resonance imaging results; D: Fluorescence imaging results; E: Fluorescence intensity in cartilage samples of groups i-iii; F: In situ fluorescence map of adjacent frozen sections in B.

[0069] Figure 11 The results of routine sagittal scans of normal and diseased Achilles tendons in mice using the T2W sequence are presented in this invention.

[0070] Figure 12 The magnetic resonance contrast agent Gd of this invention n -Cy5-CHP in vivo detection results of Achilles tendon tendinopathy, including: A: Comparison of T1 signal in normal Achilles tendons before and after 6 hours of subcutaneous injection of contrast agent; B: Comparison of T1 signal in diseased (collagenase model) Achilles tendons before and after 6 hours of subcutaneous injection of contrast agent; C: Statistical analysis of MRI signal values ​​in the normal group before and after injection; D: Gd injection results in the model group. n -Statistical analysis of signal values ​​in MRI images before and after Cy5-CHP; E: Gd injection into the modeling group n -Cy5- S Statistical analysis of signal values ​​in MRI images before and after CHP.

[0071] Figure 13 The magnetic resonance contrast agent Gd of this invention n -Cy5-CHP (positive sequence) and control contrast agent Gdn -Cy5- S MRI imaging results of the abdominal aorta in mice with abdominal aortic aneurysms and sham-operated control mice before and after CHP (randomized) tail vein injection.

[0072] Figure 14 The magnetic resonance contrast agent Gd of this invention n -Cy5-CHP (positive sequence) and control contrast agent Gd n -Cy5- S Fluorescence imaging results of isolated abdominal aortic tissue 1 h after CHP (randomized) tail vein injection in mice with abdominal aortic aneurysms and sham-operated control mice. Detailed Implementation

[0073] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0074] The terms "preferred," "more preferably," etc., used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0075] When a numerical range is disclosed herein, the range is considered continuous and includes the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0076] In the description of this invention, the reference term "and / or" includes all and any combination of one or more of the associated listed items.

[0077] In the description of this invention, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0078] In the embodiments of the present invention, all experimental protocols involving knee cartilage specimens from OA patients were approved by the Ethics Committee of Nanjing Drum Tower Hospital.

[0079] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0080] Example 1: Gd-CHP contrast agent

[0081] This embodiment provides a magnetic resonance imaging contrast agent, Gd-CHP, designed based on the collagen hybrid peptide CHP. A schematic diagram illustrating the binding of the collagen hybrid peptide CHP to denatured collagen is shown below. Figure 1 As shown, the sequence of the collagen hybrid peptide CHP is GfOGfOGfOGfOGfOGfOGfOGfOGfO (hereinafter referred to as (GfO)9). In this embodiment, the magnetic resonance imaging contrast agent Gd-CHP can specifically target unwound collagen undergoing structural denaturation. Its probe sequence structure is simplified as follows:

[0082] DOTA(Gd)-Ahx-(GfO)9;

[0083] Wherein, G represents a glycine residue; f represents a fluoroproline residue; O represents a hydroxyproline residue; Ahx is 6-aminohexanoic acid; and DOTA is p-SCN-Bn-DOTA. This targeting probe chelates only one gadolinium ion.

[0084] The above magnetic resonance imaging contrast agent Gd-CHP and its preparation flowchart are as follows: Figure 2 As shown, A represents the chemical structure of the magnetic resonance imaging contrast agent Gd-CHP, and B represents the synthetic route, which includes the following:

[0085] (1) Ahx-(GfO)9-Rink Amide AM resin was obtained by standard Fmoc chemical solid-phase synthesis on Rink Amide AM resin using a PurePep Chorus peptide synthesizer. The Fmoc amino acids used included Fmoc-Gly-OH, Fmoc-flp-OH, Fmoc-Hyp-(tBu)-OH, and Fmoc-Ahx-OH. The specific reaction conditions are as follows:

[0086] Rink Amide AM resin was shaken in 20% piperidine DMF solution for 90 s at 50 °C to deprotect Fmoc; then Fmoc-amino acids, condensing agents HATU, HOAT and DIEA were dissolved in DMF and coupled, wherein resin:amino acid:HATU:HOAT:DIEA = 1:5:5:5:10 (equivalent ratio), and each amino acid was reacted once every 5 minutes at 50 °C.

[0087] (2) At room temperature, a mixture of trifluoroacetic acid / triisopropylsilane / water (volume ratio of 95:2.5:2.5) was added to the resin and stirred for 3 hours. The peptide was then sheared off the resin and purified by HPLC and lyophilized to obtain the peptide Ahx-(GfO)9-NH2. The product was confirmed by mass spectrometry.

[0088] (3) The lyophilized peptide (10 μmol), p-SCN-Bn-DOTA (20 μmol), and DIEA (100 μmol) were dissolved in anhydrous DMSO (2 mL) and stirred in the dark at room temperature for 24 h. The peptide was then purified by HPLC and lyophilized to obtain the DOTA-labeled peptide.

[0089] (4) Dissolve the above-mentioned DOTA-labeled peptide (10 μmol) and GdCl3 (22 μmol) in 2.5 M sodium acetate buffer (pH = 6.4) and stir overnight at 60 °C. After the reaction is complete, remove excess GdCl3 by repeated centrifugation using an ultrafiltration tube with a molecular weight cutoff of 3000 Da. 3+ until Gd could no longer be detected in the filtrate using xylenol orange. 3+ Finally, the sample was freeze-dried to obtain the target product.

[0090] The target product was identified by mass spectrometry, and its mass spectrum is shown below. Figure 3 As shown, MALDI-MS: m / z calcd for Gd-CHP[M+K] + 3440.2, found 3443.5.

[0091] Example 2: Gd n -Dye-CHP contrast agent

[0092] To further improve the relaxation rate of CHP contrast agents, this embodiment provides a magnetic resonance contrast agent Gd based on collagen hybrid peptide CHP that chelates multiple gadolinium ions. n -Cy5-CHP, the magnetic resonance imaging agent Gd in this embodiment n -Cy5-CHP chelates eight gadolinium ions and can specifically target unwound collagen undergoing structural denaturation. The simplified structural formula of its probe sequence is as follows:

[0093]

[0094] Wherein, G represents glycine residue; f represents fluoroproline residue; O represents hydroxyproline residue; Ahx is 6-aminohexanoic acid; DOTA is p-SCN-Bn-DOTA chelating agent, and the chelating agent in this embodiment can also be replaced with other metal chelating agents, such as DOTAGA chelating agent; K is lysine residue; Cy5 is a fluorescent dye group, which can be replaced with other fluorescent dyes, such as other anthocyanin dyes Cy3, Cy7, etc., or common fluorescent dyes such as rhodamine, BODIPY, FITC, erythrosine, phthalocyanine, phycocyanin, phycoerythrin, Alexa Fluor, etc., depending on the actual situation.

[0095] The aforementioned magnetic resonance imaging contrast agent Gd n The chemical structure and preparation flowchart of -Cy5-CHP are shown below. Figure 4 As shown, the specific preparation process includes the following steps:

[0096] (1) Using a PurePep Chorus peptide synthesizer, [(BocAhx-K(Boc))2K]2-KK(Dde)-Ahx-(GfO)9-Rink Amide AM resin was synthesized on Rink Amide AM resin using standard Fmoc chemical solid-phase synthesis. The Fmoc amino acids included Fmoc-Gly-OH, Fmoc-flp-OH, Fmoc-Hyp-(tBu)-OH, Fmoc-Ahx-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Fmoc)-OH, Fmoc-Lys(Boc)-OH, and Boc-Ahx-OH. Specific reaction conditions are as follows:

[0097] At 50°C, the resin was shaken in a 20% piperidine DMF solution for 1.5 minutes to deprotect Fmoc. Fmoc-amino acids, condensing agents HATU, HOAT, and DIEA were dissolved in DMF and coupled, with a resin:amino acid:HATU:HOAT:DIEA ratio of 1:5:5:5:10 (equivalent). Each amino acid was reacted twice at 50°C for 5 minutes. The deprotection of the last two amino acids, Boc-Ahx-OH and Fmoc-Lys(Boc)-OH, was repeated twice, and the coupling reaction was repeated three times.

[0098] (2) The Dde protecting group was removed by 3% hydrazine hydrate to expose the amino group. Then, DIEA (5.0 equivalents) and sulfonated Cy5-NHS cyanine dye (0.5 equivalents) were dissolved in DMSO and reacted with the resin for more than 24 hours to label the peptide with fluorescent dye. After the reaction was complete, a mixture of trifluoroacetic acid / triisopropylsilane / water (volume ratio of 95:2.5:2.5) was added to the resin at room temperature and stirred for 3 hours. The peptide was then cleaved off the resin, purified by HPLC, and lyophilized to obtain [(Ahx-K)2K]2-KK(Cy5)-Ahx-(GfO)9-NH2, i.e., the Cy5-labeled peptide.

[0099] The product was confirmed by mass spectrometry, with the following parameters: MALDI-MS: m / z calcd [M+H]. + 4798.2, found4798.3.

[0100] (3) The above-mentioned lyophilized Cy5-labeled peptide (94.6 mg, 19.7 μmol), p-SCN-Bn-DOTA (542.4 mg, 788.3 μmol) and DIEA (651 μL, 3942 μmol) were dissolved in anhydrous DMSO (2 mL), stirred at room temperature in the dark for 24 h, and then the obtained peptide was purified by HPLC and lyophilized to obtain DOTA-labeled peptide.

[0101] (4) The DOTA-labeled peptide (184.5 mg, 21.8 μmol) and GdCl3 (68.8 mg, 261.0 μmol) were dissolved in 2.5 M sodium acetate buffer (25 mL, pH = 6.4) and stirred overnight at 60 °C. After the reaction was complete, the excess GdCl3 was removed by repeated centrifugation using an ultrafiltration tube with a molecular weight cutoff of 3000 through a high-speed centrifuge. 3+ until Gd could no longer be detected in the filtrate using xylenol orange. 3+ Finally, the sample was freeze-dried to obtain the target product.

[0102] Comparative Example 1: Gd- S CHP contrast agent

[0103] This comparative example provides a collagen hybrid peptide-based... S CHP-designed magnetic resonance imaging contrast agent Gd- S CHP differs from the magnetic resonance contrast agent Gd-CHP in Example 1 in that it contains different collagen hybrid peptides. The collagen hybrid peptides in this comparative example... S The sequence of CHP is OfGGOfGfGfOfOGOfGOOfGGOOffG (hereinafter referred to as...) S (GfO)9), which is obtained by randomly shuffling the target sequence (GfO)9, does not have the ability to target denatured collagen.

[0104] Comparative Example 2: Gd n -Cy5- S CHP

[0105] This comparative example provides a magnetic resonance imaging contrast agent, Gd, designed based on collagen hybrid peptide CHP. n -Cy5- S CHP, which is similar to the magnetic resonance contrast agent Gd from Example 2 n The difference between -Cy5-CHP and others lies in the collagen hybrid peptide; everything else is the same. This comparative example shows the collagen hybrid peptide... S The sequence of CHP is OfGGOfGfGfOfOGOfGOOfGGOOffG (hereinafter referred to as...) S (GfO)9), which is obtained by randomly shuffling the target sequence (GfO)9, does not have the ability to target denatured collagen.

[0106] Example of testing: Relaxation rate test

[0107] This test example examines the longitudinal relaxation rate of the magnetic resonance contrast agents prepared in Examples 1-2 and Comparative Examples 1-2. The results were obtained using a 9.4T small animal MRI scanner at room temperature with a 20mm surface coil. The specific methods are as follows:

[0108] (1) Gd-CHP and Gd- S CHP relaxation rate detection:

[0109] probes Gd-CHP and Gd- S CHP is formulated into Gd 3+200 μL of aqueous solutions with concentrations of 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM were prepared, with magnusin (gadopentetate diglucamine) as a control. To obtain T1-weighted images of these sample solutions, the following parameters were used: echo time (TE) = 7.0 ms; repetition time (TR) = 5500, 3000, 1500, 800, 400, and 200 ms; slice thickness: 0.5 mm; field of view (FOV) = 30 × 25 mm; matrix dimension = 128 × 128; bandwidth = ±610.4 kHz; echo train length (ETL) = 2; scan time: 12 minutes and 9 seconds. The reciprocal of the relaxation time (1 / T1, s) was used to construct the image. -1 ) and Gd 3+ The linear curve of concentration has a slope equal to the longitudinal relaxation rate (r1).

[0110] (2)Gd n -Cy5-CHP and Gd n -Cy5- S CHP relaxation rate detection:

[0111] probe Gd n -Cy5-CHP and Gd n -Cy5- S CHP is formulated into Gd 3+ 200 μL of aqueous solutions were prepared at concentrations of 14, 28, 56, and 112 μM, with magnusin (gadopentetate dimethylamine) as a control. T1-weighted images of these sample solutions were obtained (e.g., Figure 5 As shown), its parameters are set as follows: echo time (TE) = 7.0 ms; repetition time (TR) = 5500, 3000, 1500, 800, 400 and 200 ms; slice thickness: 0.5 mm; field of view (FOV) = 30 × 25 mm; matrix dimension = 128 × 128; bandwidth = ±610.4 kHz; echo train length (ETL) = 2; scan time is 12 minutes and 9 seconds. The reciprocal of the relaxation time (1 / T1, s) is used to create the... -1 ) and Gd 3+ A linear graph of peptide concentration, with the slope being the longitudinal relaxation rate (r1) value.

[0112] The test results are shown in Table 1.

[0113] Table 1: Longitudinal relaxation rate of magnetic resonance contrast agents

[0114]

[0115] As can be seen from the table above, the magnetic resonance imaging contrast agents Gd-CHP and Gd- S The longitudinal relaxation rate of CHP was slightly higher than that of Magendie, while that of the magnetic resonance contrast agent Gdn -Cy5-CHP has a stronger relaxation rate than the clinical MR contrast agent Magenvis and the magnetic resonance contrast agent Gd-CHP, being 8.45 times that of the clinical MR contrast agent Magenvis and 7.74 times that of the magnetic resonance contrast agent Gd-CHP prepared in Example 1.

[0116] Application Example 1: Application in isolated magnetic resonance imaging of thermally degenerated cartilage

[0117] This application example demonstrates the use of the magnetic resonance contrast agents prepared in Examples 1 and 2 and Comparative Example 2 in ex vivo magnetic resonance imaging of thermally degenerated cartilage. The specific method is as follows:

[0118] First, a pig tibial plateau was taken and placed in 10 times its volume of 4% PFA solution. After fixing at room temperature for 48 hours, it was washed with PBS for 30 minutes, repeated 3 times. Then, a bone-harvesting electric drill was used to prepare a cylindrical cartilage plug with a diameter of 0.6 cm and a height of 0.5 cm. The plug was then placed in PBS solution and heated in a 100°C water bath for 2 hours to denature the cartilage collagen.

[0119] Then, the heat-denatured porcine cartilage plugs were taken and divided into three groups: PBS group, Gd group, and Gd group. n -Cy5-CHP and Gd n -Cy5- S The CHP group was prepared in PBS solution and Gd... n -Cy5-CHP solution or Gd n -Cy5- S The samples were incubated in CHP solution (probe concentration: 5 μM, three replicates per group) at 4 °C for 96 hours. Then, the samples were washed with PBS for 3 hours, repeated three times, to remove unbound contrast agent. The cartilage samples were then imaged using an IVIS small animal in vivo imaging system with the following IVIS imaging parameters: excitation / emission wavelength: 620 / 670 nm, pixels: 4, exposure time: 1 s, field of view: A.

[0120] In addition, cartilage samples were scanned on a 9.4T small animal MRI scanner using a 20mm inner diameter surface coil and a T1-mapped rapid acquisition (T1-RARE) sequence with relaxation enhancement. Heat-denatured cartilage plugs incubated in PBS were used as blank controls. Major imaging parameters: T1-RARE: echo time (TE) = 6.0 ms, repetition time (TR) = 200 ms, slice thickness: 0.7 mm, field of view (FOV) = 20 × 10 mm, matrix size = 192 × 96, bandwidth = 1400 Hz, scan time 48 s. Each group was repeated three times.

[0121] Use Gd nMagnetic resonance and fluorescence imaging results of a thermodegenerated cartilage plug (diameter: 6 mm) in the patellofemoral joint of a normal porcine knee joint treated with a Cy5-CHP probe are shown below. Figure 6 As shown, under bright field conditions, due to Cy5 shading, Gd n The heat-denatured cartilage plug incubated with Cy5-CHP was stained blue, and Gd can be seen in the image. n Cy5-CHP stained porcine cartilage exhibits extremely high fluorescence intensity. Similarly, in the sagittal plane of samples scanned with T1-RARE sequences, only Gd... n Strong MRT1 enhancement can be observed in the -Cy5-CHP treated group (see arrow markings in the figure). Meanwhile, the group treated with Gd... n -Cy5- S The lack of magnetic resonance T1 signal and fluorescence signal in CHP-stained samples indicates that the binding of the contrast agent to denatured collagen requires triple helix hybridization. Each image represents similar results from three samples within each group. These results suggest that Gd n -Cy5-CHP can significantly enhance the magnetic resonance signal of isolated thermally denatured cartilage.

[0122] Application Example 2: Application in ex vivo magnetic resonance imaging of thermally degenerated Achilles tendons

[0123] This application example demonstrates the use of the magnetic resonance contrast agents prepared in Examples 1 and 2 and Comparative Example 2 in ex vivo magnetic resonance imaging of the Achilles tendon of a thermally degenerated rat. The specific method is as follows:

[0124] Rat Achilles tendon samples were heated in boiling water for 1 minute to denature collagen (as a positive control), and divided into 3 groups, each in 5 mL PBS, Gd... n -Cy5-CHP and Gd n -Cy5- S Incubate overnight at 4°C in CHP (probe concentration: 5 μM, 3 replicates per group). After probe incubation, wash with PBS to remove unbound contrast agent. Then, perform fluorescence imaging of the Achilles tendon samples using an IVIS Spectrum imager, with the following IVIS imaging parameters: excitation / emission wavelength: 620 / 670 nm, pixels: 4, exposure time: 1 s, field of view: A.

[0125] In addition, heat-denatured rat Achilles tendon samples were scanned on a 9.4T small animal MRI scanner using a 20mm inner diameter surface coil and a T1-mapped rapid acquisition (T1-RARE) sequence with relaxation enhancement. Heat-denatured Achilles tendons incubated in PBS were used as a blank control. Key imaging parameters: T1-RARE: echo time (TE) = 6.0 ms, repetition time (TR) = 200 ms, slice thickness: 0.5 mm, field of view (FOV) = 20 × 20 mm, matrix size = 136 × 136, scan time: 2 min 16 s.

[0126] Test results as follows Figure 7 As shown, under bright field conditions, using Gd n Achilles tendons incubated with Cy5-CHP were dyed blue, and Gd n Cy5-CHP staining of the Achilles tendon showed extremely high fluorescence intensity. In cross-sections of samples scanned using T1-RARE sequences on MR, only Gd... n Strong MR signals are visible in the Cy5-CHP processed groups. Each image represents the similarity results of three samples within each group.

[0127] Application Example 3: Application in Magnetic Resonance Imaging of the Knee Joint in Osteoarthritis

[0128] This application example provides the application of the magnetic resonance contrast agent described in the above embodiments and comparative examples in the magnetic resonance imaging of the knee joint in osteoarthritis, specifically including the following:

[0129] 1. Constructing an animal model

[0130] Medial meniscus transection (DMM) was performed on 8-week-old male SD rats to induce osteoarthritis (OA). Under general anesthesia with isoflurane, both knee joints of the rats were shaved, disinfected, and draped. A longitudinal incision of approximately 1.5 cm was made on the medial side of the right knee joint. The skin was dissected, and sterile surgical scissors were used to longitudinally dissect along the medial side of the patellar ligament to open the joint capsule and expose the joint cavity. The anteromedial meniscus ligament was transected, freeing the anterior portion of the medial meniscus. Simultaneously, the freed portion of the meniscus was removed using microsurgical scissors. The wound was repeatedly rinsed with sterile saline. The joint capsule, muscle, and skin tissue were sutured in layers with 5-0 absorbable sutures, and the joint cavity and skin were closed. The knee joint on the sham-operated side was treated in the same way as the surgically created joint in mice, but the anteromedial meniscus ligament was not transected. Postoperatively, the wound was disinfected again, and the rats were kept in standard feeding conditions and closely observed to prevent infection.

[0131] 2. Gd-CHP magnetic resonance imaging

[0132] Osteoarthritis (OA) rats were imaged on a 9.4T small animal MRI scanner using a 20mm inner diameter surface receiving coil. Rats were anesthetized with 2% isoflurane oxygen to maintain a constant respiratory rate, and temperature was maintained using a heating pad and monitored via a small animal physiological monitoring system. Images were acquired before and after contrast agent administration for comparison during imaging. Contrast agents (Gd-CHP or Gd-) were used. S CHP) with 1 nmol Gd 3+ The contrast agent was injected intra-articularly into the knee of rats at a dose of / g. Images were acquired 3 hours post-injection using a Turbo Rapid Acquisition Sequence (TurboRARE, TSE) with relaxation enhancement.

[0133] Key imaging parameters: T1WI (sequence: T1WI-3D-flash): Echo time (TE) = 3.5ms, repetition time (TR) = 16ms, slice thickness: 0.2mm, field of view (FOV) = 22×22×8mm, matrix size = 192×192×40, scan time: 18 minutes 50 seconds. T2WI (sequence: T2WI-2D-RARE): Echo time (TE) = 27ms, repetition time (TR) = 4000ms, slice thickness: 0.2mm, field of view (FOV) = 25×25mm, matrix size = 228×228, scan time: 7 minutes 36 seconds. MR images were processed and analyzed using RadiAnt DICOM Viewer software.

[0134] The results are as follows Figure 8 As shown, compared with the pre-contrast scan, intra-articular injection of Gd-CHP three hours later enhanced the T1 signal in the tibial cartilage and ligaments of the knee joint of rats on the DMM model side (see details). Figure 8 (The orange arrow in the image shows an increase in T1 signal in the knee cartilage and ligaments of the sham-operated rat), while the same dose did not enhance the T1 signal. These results indicate that Gd-CHP significantly enhances the T1 signal of the knee cartilage and ligaments on the DMM side compared to the sham-operated knee.

[0135] 3. Gd n -Cy5-CHP magnetic resonance imaging

[0136] Since Gd-CHP with a single gadolinium ion enhances the T1 signal in the knee cartilage of rats with DMM modeling, this application example further investigated the effect of Gd-CHP. n The enhancement effect of Cy5-CHP on cartilage in OA knee joint injuries is shown in the following methods:

[0137] (1) Magnetic Resonance Imaging: OA rats were imaged on a 9.4T small animal MRI scanner using a 20mm inner diameter surface receiving coil. Rats were anesthetized with 2% isoflurane oxygen to maintain a constant respiratory rate, and temperature was maintained using a heating pad and monitored via a small animal physiological monitoring system. Images were acquired before and after contrast agent administration for comparison during imaging. Contrast agent (Gd...) n -Cy5-CHP or Gd n -Cy5- S CHP) with 1 nmol Gd 3+ The contrast agent was injected intra-articularly into the rat knee at a dose of / g. Images were acquired 6 hours post-injection using Turbo Rapid Acquisition Sequences with relaxation enhancement (TurboRARE, TSE). The main imaging parameters were set as follows: T1WI (sequence: T1WI-3D-flash): Echo time (TE) = 3.5 ms, repetition time (TR) = 16 ms, slice thickness: 0.2 mm, field of view (FOV) = 22 × 22 × 8 mm, matrix size = 192 × 192 × 40, scan time: 18 min 50 s. T2WI (sequence: T2WI-2D-RARE): Echo time (TE) = 27 ms, repetition time (TR) = 4000 ms, slice thickness: 0.2 mm, field of view (FOV) = 25 × 25 mm, matrix size = 228 × 228, scan time: 7 min 36 s. MR images were processed and analyzed using RadiAnt DICOM Viewer software.

[0138] (2) Histopathological staining and imaging: SD rats that underwent the above magnetic resonance imaging were sacrificed. Both knee joints were removed, the joints were opened, the tibial plateau was separated, and the joints were fixed for 48 hours. After fixation, the samples were washed with PBS, decalcified with 20% EDTA for 14 days until complete decalcification, dehydrated with 30% sucrose solution for 12 hours, treated with 30% sucrose + 30% OCT solution for 12 hours, and then embedded in OCT. Frozen sections were prepared perpendicular to the tibial plateau cartilage plane, with a section thickness of 10 μm. The sections were stained with safranin and fast green and imaged under a microscope. Adjacent sections were used for fluorescence imaging.

[0139] The results are as follows Figure 9As shown, A represents representative in vivo T1WI images of the knee joint on the sham-operated or DMM-operated side of the same rat 7 weeks post-surgery; B represents the contrast-to-noise ratio of the tibial plateau cartilage in the DMM-inducing knee joint before and after contrast enhancement (n=4, statistical analysis was performed using paired t-tests); C represents representative in vivo T1WI images of the DMM-inducing knee joint in rats before and after contrast enhancement 7 weeks post-modeling, as well as a T2WI image before enhancement; D represents the staining and imaging results of cartilage sections within the box-shaped area in image C; E represents the in vivo T1WI and T2WI scans of the same DMM-injured rat knee joint repeated at 3 and 7 weeks post-DMM surgery. The results show that compared with the pre-contrast scan, intra-articular injection of Gd... n Six hours after Cy5-CHP injection, T1 signal in the tibial cartilage and ligaments of the knee joint of rats on the DMM model side was significantly enhanced, while the T1 signal in the sham-operated cartilage showed almost no enhancement after the same dose was injected. T1 signal measurements also showed the same results (e.g., Figure 9 (As shown in A and B in the figure). The location of the high signal in the cartilage shown in the T1WI imaging is in excellent agreement with the fluorescence results of frozen sections of the knee joint after rat sacrifice, both indicating Gd n -Cy5-CHP binds to cartilage sites in vivo where proteoglycans (GAGs) are lost (e.g., ...). Figure 9 (As shown in C and D).

[0140] Furthermore, continuous observation of the same DMM-modeled rat revealed Gd n -Cy5-CHP can detect changes in the level and location of collagen loss in cartilage and ligaments over several weeks during early OA progression (e.g., Figure 9 As shown in E), these subtle lesions are not easily detected in T2WI scans, which are routinely used in clinical practice to examine soft tissue injuries (e.g., Figure 9 (As shown in C and E).

[0141] Application Example 4: Application in isolated magnetic resonance imaging of femoral cartilage in knee joints of OA patients

[0142] This application example demonstrates the use of the magnetic resonance contrast agent prepared in Example 2 above in ex vivo magnetic resonance imaging of femoral cartilage in patients with osteoarthritis (OA), as detailed below:

[0143] Femoral cartilage samples removed during joint replacement surgery in OA patients were fixed in 4% PFA solution at room temperature for 48 hours and washed three times with PBS for 30 minutes each. Using a bone-harvesting drill, cartilage plugs with a diameter of 1 cm and a height of 5 mm were obtained from samples with mild to severe cartilage damage. These cartilage plugs were then treated with Gd... n Cy5-CHP (10 μM, 5 mL) was incubated at 4 °C for 2 days. After washing with PBS for 3 hours (repeated 3 times), fluorescence and MR imaging were performed according to the above parameters. Following imaging, Gd...n Cy5-CHP-labeled samples were decalcified in 20% EDTA for 7 days, dehydrated with 30% sucrose solution, and frozen sections with a thickness of 10 μm were prepared. Sections were stained with safranin and fast green and imaged under a microscope. MR images were processed and analyzed using RadiAnt DICOM Viewer software.

[0144] The results are as follows Figure 10 As shown, A represents cartilage tissue removed from an OA patient undergoing knee replacement surgery, and i-iii represent areas with mild to severe cartilage degeneration; B represents areas treated with Gd... n -Cy5-CHP stained cartilage plug specimens with Safranin-Fix-Green staining; C is T1WI magnetic resonance imaging result, D is fluorescence imaging result, E is fluorescence intensity in cartilage samples of groups i-iii; F is in situ fluorescence map of adjacent frozen sections in B. Detection results show that using Gd n Cy5-CHP incubation of ex vivo cartilage samples, T1WI MRI and fluorescence imaging showed that group II columnar cartilage samples exhibited the most pronounced CHP signal near the less smooth cartilage surface. In situ fluorescence and histology of frozen cartilage sections after MR imaging showed Gd n Cy5-CHP was mainly enriched in group ii in areas near the cartilage surface where GAG ​​was significantly lost, while in group i (where collagen was not yet damaged) and group iii (where collagen was severely eroded), Gd... n -Cy5-CHP showed poor efficacy in targeting cartilage. These results are highly consistent with those in animal models, indicating that CHP can effectively detect collagen molecule damage in moderately damaged cartilage in OA patients.

[0145] Application Example 5: Application in Magnetic Resonance Imaging of Achilles Tendon Disease

[0146] This application example provides the application of the magnetic resonance contrast agent prepared in Example 2 and Comparative Example 2 in magnetic resonance imaging of Achilles tendinopathy, specifically including the following:

[0147] 1. Constructing an animal model

[0148] Adult SD rats were used. A type I collagenase solution was subcutaneously injected into the middle of the left Achilles tendon using a disposable sterile insulin syringe, with an injection volume of 60 μL (approximately 31.8 U) per tendon. The same method was used to subcutaneously inject 60 μL of physiological saline solution into the right Achilles tendon of the SD rats as a sham-operated group. Injections were given every other day for a total of 7 times. After 14 days, the SD Achilles tendinopathy model (AT) was established. Two weeks later, after the swelling around the Achilles tendon subsided, in vivo imaging and tissue sampling were performed.

[0149] Male C57BL / 6 mice aged 8-10 weeks were selected for Achilles tendinopathy modeling. Type I collagenase (100 mg, 256 U / mg, Solarbio) was dissolved in 50 mL of TESCA buffer solution (pH 7.4, Solarbio). 10 μL of collagenase (approximately 5.3 U) was subcutaneously injected into the middle region of the left Achilles tendon using a microsyringe, and 10 μL of physiological saline was injected into the right Achilles tendon. Injections were repeated daily for 3 days, and the Achilles tendinopathy model was obtained 30 days after the last injection.

[0150] 2. Gd n -Cy5-CHP magnetic resonance imaging

[0151] MR imaging of a mouse model of Achilles tendinopathy was performed using a 9.4T small animal MRI scanner with a 20mm inner diameter surface coil. Signals were acquired via T1-weighted rapid acquisition and relaxation enhancement (T1-RARE) sequences. Bilateral Achilles tendons of each mouse were pre-scanned before contrast agent injection. Then, Gd... n -Cy5- S CHP (50 nmol, 10 μL) PBS solution was subcutaneously injected into the affected Achilles tendon area, and a T1-weighted scan was performed 6 hours after injection. One week later, both the affected and healthy legs of the same mouse received Gd injections into the Achilles tendon area. n Subcutaneous injection of Cy5-CHP (50 nmol, 10 μL) was performed, and scanning was conducted 6 hours post-injection. The main imaging parameters were set as follows: T1-FS: Echo Time (TE) = 6.1 ms; Repetition Time (TR) = 400 ms; Slice Thickness: 0.3 mm; Field of View (FOV) = 20 × 20 mm; Matrix Dimension = 256 × 256; Bandwidth = 434.0 kHz; Echo Train Length (ETL) = 2; Scan Time: 10 minutes 14 seconds. MR images were processed and analyzed using RadiAnt DICOM Viewer software.

[0152] The results of routine sagittal scans of normal and diseased Achilles tendons (in a collagenase model) in mice using T2W sequences are as follows: Figure 11 As shown, the T2-FSE sequence settings were as follows: TE = 30.0 ms; TR = 1500 ms; slice thickness: 0.3 mm; field of view = 20 × 20 mm; matrix dimension = 228 × 228; bandwidth = 219.3 kHz; ETL = 8. The results showed that conventional sagittal scans with T2WI sequences did not show either enhanced Achilles tendon signal or significant thickening of the Achilles tendon, indicating that T2 signal did not suggest a lesion.

[0153] Furthermore, T1WI pre-scans were performed on normal and Achilles tendinopathy ankle joints, and the results showed no difference in signal intensity (e.g., Figure 12As shown in A), compared to before the scan, Gd n Six hours after Cy5-CHP probe injection, T1 signal enhancement was observed in the diseased Achilles tendon, but no signal enhancement was observed in the normal Achilles tendon, and Gd without targeting ability was also observed. n -Cy5- S CHP showed virtually no enhancement of the T1 signal in the Achilles tendon of the model group. Figure 12 (As shown in A and B in the diagram). Meanwhile, with Gd n -Cy5- S After CHP administration, the T1WI signal of the affected Achilles tendon showed almost no enhancement. Figure 12 As shown in Figure B (pseudocolor), the enhanced signal of Gdn-Cy5-CHP mainly originates from the triple helix hybridization of CHP with degenerated collagen in the diseased Achilles tendon. Quantitative data indicate that Gdn-Cy5-CHP... n Following Cy5-CHP injection, the average T1WI signal of each affected Achilles tendon almost doubled, while Gd n Normal Achilles tendon and Gd after Cy5-CHP injection n -Cy5- S The T1WI signal of the diseased tendon remained unchanged after CHP injection (as shown in C-E in the figure). This demonstrates that by using the CHP-based MR probe of this invention to target denatured collagen, MRI can be used to visualize Achilles tendinopathy in vivo.

[0154] Application Example 6: Application in Abdominal Aortic Aneurysm Imaging

[0155] This application example provides the use of the magnetic resonance contrast agents prepared in Example 2 and Comparative Example 2 in the imaging of abdominal aortic aneurysms (AAA), specifically including the following:

[0156] First, a mouse model of AAA disease, induced by porcine pancreatic elastase (PPE), was established. C57BL / 6 mice were anesthetized with 3% isoflurane and then injected intraperitoneally with 10 μL / g of 4% sodium pentobarbital. The hair on the mouse abdomen was shaved, and the skin was incised approximately 1.0 cm along the midline. The subrenal abdominal aorta was exposed under a stereomicroscope, and a gauze soaked in 10 μL of porcine pancreatic elastase (E1250, Sigma-Aldrich) was wrapped around it for 10 minutes. The gauze was then removed, and the mice were rinsed three times with saline. In the control group (the sham-operated Sham group), after exposing the abdominal aorta, a gauze soaked in saline was wrapped around it for 10 minutes, then the gauze was removed, the mice were rinsed with saline, and the skin was intermittently sutured.

[0157] Magnetic resonance imaging (MRI) was performed on the abdominal aorta of mice (1 week after PPE modeling) using a 9.4T MRI scanner equipped with a 30mm transmit / receive cage coil. The mice were then anesthetized with isoflurane (0.5-2%), and the MRI contrast agent was injected at a concentration of 60 μmol Gd / kg. n -Cy5-CHP and Gd n -Cy5- S CHP was injected into the animals via live injection through the tail vein. Scanning images were performed before injection and 1 hour after injection.

[0158] The scanning and imaging methods specifically included: identifying the abdominal aorta in coronal sections using a localization sequence, and performing T1-weighted (T1W) magnetic resonance imaging using a black blood turbine spin echo sequence with an arterial saturation band. Sixteen consecutive 500 μm thick slices were obtained using a spin echo sequence with a matrix size of 256 × 256, achieving an in-plane microscale resolution of 101 μm. The repetition time (TR) and echo time (TE) of the T1W images were 800 ms and 8.6 ms, respectively. A 3 mm inflow saturation band and a 3 mm slice gap were used to obtain additional arterial flow suppression. The total imaging time per scan was 28 minutes, using the average of 16 signals. Saturation pulses were used to eliminate signals from adipose tissue, better delineate the boundaries of the aortic wall, and minimize chemical shift artifacts.

[0159] Gd n MRI imaging results of mice with abdominal aortic aneurysms before and after Cy5-CHP injection are as follows: Figure 13 As shown, compared with the pre-contrast scan, mice injected with Gd via the tail vein... n Cy5-CHP (orthogonal sequence group) significantly enhanced the T1 signal of the abdominal aortic aneurysm model segment 1 hour later (yellow arrow indicates the abdominal aorta), while the T1 signal of the sham surgery group and the disordered sequence group (magnetic resonance contrast agent corresponding to Comparative Example 2) showed almost no enhancement, and T1 signal measurements also showed the same results.

[0160] Furthermore, NIRF imaging of isolated tissue was performed 1 hour after tail vein injection in mice with abdominal aortic aneurysms. The results are as follows: Figure 14 As shown, the location of the high signal intensity of the aneurysm after tail vein injection of the magnetic resonance contrast agent of this invention is co-localized with the IVIS fluorescence results of the abdominal aorta after mouse sacrifice. Both indicate that Gd n -Cy5-CHP binds to sites in vivo where the extracellular matrix remodels the vessel wall, and these subtle damages are not easily detected in T2WI scans, which are routinely used in clinical practice to examine tissue damage. Therefore, the magnetic resonance contrast agent of this invention has higher sensitivity for imaging abdominal aortic aneurysms.

[0161] In summary, this invention provides a magnetic resonance imaging (MRI) contrast agent for targeted detection of collagen molecular structure denaturation, its preparation method, and its application. The MRI contrast agent of this invention is based on denatured collagen. This invention creatively proposes further chelating gadolinium ions and modifying fluorescent groups on the basis of collagen hybrid peptide CHP. Compared to near-infrared fluorescently labeled CHP, the CHP-based MRI contrast agent designed in this invention does not have the problem of penetration depth and can provide high spatial resolution and three-dimensional imaging, which is beneficial for clinical translation.

[0162] Furthermore, the magnetic resonance contrast agent based on this invention images cartilage by detecting denatured collagen. This method offers higher sensitivity and accuracy compared to conventional imaging methods based on changes in type II collagen content (such as a targeted magnetic resonance-near-infrared fluorescence dual-modal probe based on 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetamide (DOTAM)). For example, in the detection of osteoarthritis, since the dual-modal probe TCA images cartilage based on changes in type II collagen content, the content of type II collagen in the cartilage of osteoarthritis-affected rats is reduced, resulting in a lower magnetic resonance signal compared to normal rat cartilage, which is unfavorable for clinical examination. In contrast, imaging based on differences in type II collagen content (present in both normal and osteoarthritis-affected cartilage) targets only structurally degenerated collagen present in disease sites, providing higher lesion specificity and detection sensitivity.

[0163] Finally, it is worth noting that the magnetic resonance contrast agent of this invention can also achieve non-invasive early detection of osteoarthritis and Achilles tendinopathy. Experiments have demonstrated that, compared to Magendie contrast agents, the magnetic resonance contrast agent designed in this invention has a superior relaxation rate and can significantly enhance the T1 signal of thermally degenerated cartilage and tendons, the T1 signal of knee cartilage and ligaments in osteoarthritis models, and the T1 signal of damaged Achilles tendons in Achilles tendinopathy models, thus improving the early diagnostic efficacy of cartilage diseases. Furthermore, the magnetic resonance contrast agent of this invention has been applied to abdominal aortic aneurysm imaging to help examine subtle damage that is difficult to detect clinically, thereby improving diagnostic efficiency.

[0164] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A magnetic resonance contrast agent, characterized in that, The structural formula of the magnetic resonance contrast agent is shown in formula (II): ； In the formula, [X] is a paramagnetic metal complex, which is composed of a chelating agent and a paramagnetic metal ion; [Y] is a fluorescent dye group; The chelating agent is selected from any one of DOTA, DTPA, DO3A, NOTA, DOTMA, M4DOTA, M4DO3A, BOPTA, NODAGA, HYNIC, HPDO3A, and EDTA chelating agents; the paramagnetic metal ion is selected from at least one of gadolinium ions, dysprosium ions, neodymium ions, and manganese ions; The fluorescent dye group is selected from one of sulfo-Cy3, sulfo-Cy5, sulfo-Cy5.5, sulfo-Cy7, and sulfo-Cy7.

5.

2. A process for the preparation of a magnetic resonance contrast agent as claimed in claim 1, characterized in that, Includes the following steps: S1. A precursor probe containing a collagen hybrid peptide and a linker is prepared by solid-phase synthesis. The precursor probe is deprotected and then labeled with a fluorescent dye to obtain a precursor probe containing the fluorescent dye group. The collagen hybrid peptide is (GfO)9, and the linker includes lysine and 6-aminohexanoic acid. S2. The product of step S1 is reacted sequentially with the chelating agent and the paramagnetic metal ions, and after purification, the product is obtained.

3. The preparation method according to claim 2, characterized in that, In step S1, the solid-phase synthesis method includes Fmoc chemical solid-phase synthesis or BOC chemical solid-phase synthesis.

4. A reagent kit for detection, characterized in that, Includes the magnetic resonance imaging agent as described in claim 1.

5. The use of the magnetic resonance contrast agent as described in claim 1 in any one of (A) to (D), (A) To prepare products for the detection of imaging-related diseases of cartilage damage; (B) To prepare products for the detection and imaging of degenerative diseases of musculoskeletal soft tissues; (C) Preparation of products for detecting and imaging cardiovascular lesions; (D) Prepare products for detecting and imaging tumors or organ fibrosis.

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