FAPα-responsive magnetic resonance molecular imaging probe and preparation method and application thereof

Abstract

The present invention provides a FAPα-responsive magnetic resonance molecular imaging probe and a preparation method and application thereof. The magnetic resonance molecular imaging probe of the present invention comprises a quencher, an enhancer and a connecting polypeptide, wherein the quencher and the enhancer are connected by a connecting polypeptide, and the connecting polypeptide is a polypeptide having an amino acid sequence of a FAPα-specific cleavage site. The FAPα-responsive magnetic resonance molecular imaging probe of the present invention can be used for non-invasive and accurate grading detection of patients with liver fibrosis.

Description

Technical Field

[0001] The invention belongs to the field of nano-biomedicine, and specifically relates to a FAPα-responsive magnetic resonance molecular imaging probe and a preparation method and application thereof. Background Art

[0002] China is a country with a high incidence of liver disease, with a higher incidence and mortality rate than the world average. Most patients with liver disease suffer from chronic liver disease, which is characterized by inflammation and progressive fibrosis.

[0003] It is worth noting that liver fibrosis is a key intermediate link in the progression of various chronic liver diseases to cirrhosis. Usually, low-grade liver fibrosis has mild pathological changes, and individualized treatment targeting the cause is expected to reverse the course of the disease; intermediate and advanced liver fibrosis causes severe tissue damage and patients have a poor prognosis. Therefore, as a common clinical chronic disease, it is very important to achieve accurate graded diagnosis of liver fibrosis.

[0004] At present, the gold standard for the clinical diagnosis of liver fibrosis grading is tissue puncture biopsy, but there are defects such as sampling that is difficult to reflect the overall level of fibrosis activity and large tissue trauma. Patients have certain concerns when accepting it, and the target accuracy of puncture sampling has a certain impact on the results of pathological diagnosis. Hematological examinations reflect lesions through the expression of extracellular matrix or fibrosis metabolites. Their specificity is affected by hepatocyte necrosis, inflammation, and other organ diseases, and their diagnostic value is low. Traditional imaging examinations rely on changes in liver morphology and structure to identify lesions. The diagnostic time window is late, the sensitivity is low, and there is a lack of targeted contrast agents for the core pathological links of liver fibrosis. In summary, this field still lacks non-invasive grading methods that can accurately evaluate the degree of liver fibrosis.

[0005] Therefore, there is an urgent need in this field to develop non-invasive detection methods that can be used for accurate grading and diagnosis of liver fibrosis with high sensitivity. Summary of the invention

[0006] The purpose of the present invention is to provide a highly sensitive non-invasive detection method for accurate graded diagnosis of liver fibrosis and a diagnostic reagent - a magnetic resonance molecular imaging probe.

[0007] In the first aspect of the present invention, a magnetic resonance molecular imaging probe for the graded detection of liver fibrosis is provided, which comprises a quencher, an enhancer and a connecting polypeptide, wherein the quencher and the enhancer are connected via a connecting polypeptide, and the connecting polypeptide is a polypeptide having an amino acid sequence with a FAPα-specific cleavage site.

[0008] In another preferred example, when the quencher and enhancer are connected via a connecting polypeptide, the detectable signal of the enhancer is reduced or quenched by the quencher; and when the connecting polypeptide is specifically cleaved so that the quencher and enhancer are no longer connected via the connecting polypeptide, the detectable signal of the enhancer is no longer reduced or quenched by the quencher.

[0009] In another preferred embodiment, the detectable signal emitted by the enhancer includes a relaxation enhancement signal.

[0010] In another preferred embodiment, when the quencher and enhancer are connected via a connecting polypeptide, the relaxation enhancement signal is quenched, and the T1 magnetic resonance signal of the enhancer is in an OFF state; and when the connecting polypeptide is specifically cleaved so that the quencher and enhancer are no longer connected via the connecting polypeptide, the T1 relaxation enhancement signal of the enhancer is no longer quenched, thereby restoring the magnetic resonance signal and being in an ON state.

[0011] In another preferred embodiment, the magnetic resonance molecular imaging probe has a structure of Formula I:

[0012] Q-(LE)n(I)

[0013] In the formula,

[0014] Q is a quencher;

[0015] L is a FAPα-responsive connecting polypeptide;

[0016] E is an enhancer;

[0017] n is a positive integer ≥1.

[0018] In another preferred embodiment, n is 10-10000, preferably 1000-10000.

[0019] In another preferred embodiment, the quencher is superparamagnetic nanoparticles.

[0020] In another preferred embodiment, the superparamagnetic nanoparticles are magnetic transition metals or their oxides.

[0021] In another preferred embodiment, the quencher is amorphous iron nanoparticles, preferably superparamagnetic amorphous iron nanoparticles.

[0022] In another preferred embodiment, the enhancing agent is a paramagnetic T1 contrast agent.

[0023] In another preferred embodiment, the paramagnetic T1 contrast agent is selected from the following group: (i) magnetic transition metal complexes or oxides thereof, (ii) magnetic lanthanide rare earth metal complexes or oxides thereof; or (iii) any combination of the above (i) and (ii).

[0024] In another preferred embodiment, the connecting polypeptide comprises one or more amino acid sequences having FAPα specific cleavage sites, that is, it has one or more FAPα specific cleavage sites or one or more FAPα responsive specific cleavage units.

[0025] In another preferred embodiment, in the connecting polypeptide, a plurality of FAPα-responsive specific cleavage units are connected in series.

[0026] In another preferred embodiment, the length of the connecting polypeptide is 6-30 amino acids, preferably 7-20 amino acids, and more preferably 7-15 amino acids.

[0027] In another preferred embodiment, the polypeptide having an amino acid sequence of a FAPα-specific cleavage site is a peptide containing proline.

[0028] In another preferred example, the amino acid sequence having a FAPα specific cleavage site is selected from the following group: ASGPAGPA, VGPAGK, PPGPPGPA, DKGESGPA, FQGLPGPA, AGPVGPPA, and PPGAVGPA.

[0029] In another preferred embodiment, the magnetic resonance molecular imaging probe is a nanoparticle.

[0030] In another preferred embodiment, the particle size of the magnetic resonance molecular imaging probe is 2-900 nm, preferably 5-500 nm, and more preferably 10-100 nm.

[0031] In another preferred embodiment, the linker polypeptide is connected to the quencher and / or enhancer via a surface ligand molecule.

[0032] In another preferred embodiment, the superparamagnetic nanoparticles are superparamagnetic nanoparticles modified with surface ligand molecules.

[0033] In a second aspect of the present invention, there is provided a method for preparing the magnetic resonance molecular imaging probe for liver fibrosis grading detection according to the first aspect of the present invention, comprising the steps of:

[0034] (1) providing a quencher, an enhancer and a connecting polypeptide;

[0035] (2) The quencher and enhancer are connected via a connecting polypeptide to form the magnetic resonance molecular imaging probe according to the first aspect of the present invention.

[0036] In another preferred embodiment, the connection includes connection via chemical bonds (such as covalent bonds), hydrogen bonds, ligand molecules, and / or affinity.

[0037] In another preferred embodiment, the quencher is superparamagnetic nanoparticles.

[0038] In another preferred embodiment, the enhancing agent is a paramagnetic T1 contrast agent.

[0039] In another preferred embodiment, in step (2), the connection is carried out by chemical reaction, physical adsorption or physical coating.

[0040] In the third aspect of the present invention, there is provided a use of the magnetic resonance molecular imaging probe as described in the first aspect of the present invention for preparing a diagnostic reagent for the graded detection of liver fibrosis.

[0041] In a fourth aspect of the present invention, a device for grading the degree of liver fibrosis is provided, the device comprising:

[0042] (a) a data input module, wherein the data input module is configured to input data of magnetic resonance molecular imaging of the liver, wherein the magnetic resonance molecular imaging is a magnetic resonance molecular imaging of the liver obtained by performing magnetic resonance molecular imaging on a subject to which the magnetic resonance molecular imaging probe according to the first aspect of the present invention is applied;

[0043] (b) a data processing module, the data processing module being configured to compare the data of the magnetic resonance molecular imaging with a predetermined judgment standard, thereby obtaining a graded detection result of the degree of liver fibrosis of the subject;

[0044] (c) A data output module, wherein the data output module is configured to output the graded detection result of the degree of liver fibrosis.

[0045] In another preferred embodiment, the device also includes: (d) a magnetic resonance molecular imaging module, which is configured to perform magnetic resonance molecular imaging on the liver of a subject, thereby obtaining a magnetic resonance molecular image of the liver of the subject, wherein the patient is administered the magnetic resonance molecular imaging probe described in the first aspect of the present invention.

[0046] In a fifth aspect of the present invention, a method for grading the degree of liver fibrosis is provided, the method comprising:

[0047] (a) administering the magnetic resonance molecular imaging probe for liver fibrosis grading detection described in the first aspect of the present invention to a subject;

[0048] (b) performing magnetic resonance molecular imaging on the subject, thereby obtaining a magnetic resonance molecular image of the subject's liver;

[0049] (c) obtaining a graded detection result of the degree of liver fibrosis of the subject based on magnetic resonance molecular imaging.

[0050] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The figure shows a transmission electron microscope (TEM) image of a magnetic resonance molecular imaging probe (AFe-ASGPAGPA-Gd nanoparticles) in one embodiment of the present invention.

[0052] Figure 2 The figure shows an X-ray diffraction (XRD) pattern of a magnetic resonance molecular imaging probe (AFe-ASGPAGPA-Gd nanoparticles) in one embodiment of the present invention.

[0053] Figure 3 The hydrated particle size (DLS) of the magnetic resonance molecular imaging probe (AFe-ASGPAGPA-Gd nanoparticles) in one embodiment of the present invention is shown.

[0054] Figure 4 The magnetization curves of AFe nanoparticles and AFe-ASGPAGPA-Gd nanoparticles are shown.

[0055] Figure 5 An image showing the imaging effect of a magnetic resonance imaging nanoprobe in aqueous solution in one embodiment of the present invention is shown.

[0056] Figure 6 The graph shows the image of the magnetic resonance imaging nanoprobe AFe-ASGPAGPA-Gd for graded detection of different grades of liver fibrosis and the quantitative evaluation of the diagnostic effect (AUC curve) in one embodiment of the present invention. DETAILED DESCRIPTION

[0057] After extensive and in-depth research and a large number of screenings, the inventors have developed for the first time a method and detection reagent (i.e., a FAPα-responsive magnetic resonance molecular imaging probe) that can accurately grade and diagnose liver fibrosis non-invasively. The magnetic resonance molecular imaging probe of the present invention has a unique structure and can respond to FAPα protein with high sensitivity and specificity. Experiments have shown that the FAPα-responsive magnetic resonance molecular imaging probe of the present invention can accurately grade and detect the degree of liver fibrosis in patients. In addition, the graded detection of the FAPα-responsive magnetic resonance molecular imaging probe provided by the present invention is a non-invasive detection, which can eliminate the trauma caused to patients by traditional detection methods such as tissue puncture biopsy. The present invention was completed on this basis.

[0058] FAPα-responsive magnetic resonance molecular imaging probe

[0059] As used in the present invention, the terms "the magnetic resonance molecular imaging probe of the present invention", "the magnetic resonance imaging nanoprobe of the present invention", "the FAPα-responsive magnetic resonance molecular imaging probe", "the probe of the present invention", "the nanoprobe of the present invention", and "the molecular imaging probe of the present invention" are used interchangeably to refer to the FAPα-responsive magnetic resonance molecular imaging probe in the first aspect of the present invention. The magnetic resonance molecular imaging probe of the present invention can achieve graded detection of liver fibrosis by responding to FAPα linearly expressed in livers with different grades of fibrosis.

[0060] FAPα is a fibroblast activation protein. There is a significant linear correlation between the expression of FAPα protein and the fibrosis grade, and it can be used as a biomarker for the graded detection of liver fibrosis. The inventors' research shows that there is a significant linear correlation between the protein expression and mRNA expression level of FAPα in clinical liver fibrosis samples and the fibrosis grade. 2 The values ​​reached 0.89 and 0.91 respectively, indicating that FAPα has the best correlation with the degree of liver fibrosis and can be used as an indicator of liver fibrosis.

[0061] The magnetic resonance molecular imaging probe of the present invention comprises (i) superparamagnetic nanoparticles (as a quencher), (ii) a polypeptide having an amino acid sequence with a cleavage site specific to FAPα (as a connecting polypeptide), and (iii) a paramagnetic T1 contrast agent (as an enhancer).

[0062] In a preferred example of the present invention, the FAPα-responsive magnetic resonance molecular imaging probe uses superparamagnetic amorphous iron (AFe) nanoparticles as a quencher and a paramagnetic gadolinium agent (such as Gd-DOTA) as an enhancer. The two are connected by a FAPα-responsive broken polypeptide chain (such as ASGPAGPA, SEQ ID No: 1), thereby constructing a magnetic resonance imaging nanoprobe based on the distance-dependent magnetic resonance tuning principle.

[0063] In the present invention, the representative quencher is superparamagnetic nanoparticles, including (but not limited to): magnetic transition metals and their oxides, preferably iron and its oxides.

[0064] Preferably, in the present invention, the particle size of the superparamagnetic nanoparticles is 1-200 nanometers, preferably 2-30 nanometers.

[0065] In the present invention, the connecting polypeptide is a polypeptide having an amino acid sequence of a FAPα-specific cleavage site (ie, a FAPα-responsive specific cleavage sequence).

[0066] The FAPα-responsive specific cleavage unit sequence (or cleavage sequence) is an amino acid sequence having a cleavage site specific to FAPα. Representative amino acid sequences having a cleavage site specific to FAPα include (but are not limited to) the following cleavage units:

[0067] ASGPAGPA SEQ ID No:1 VGPAGK SEQ ID No:2 PPGPPGPA SEQ ID No:3 DKGESGPA SEQ ID No:4 FQGLPGPA SEQ ID No:5 AGPVGPPA SEQ ID No:6 PPGAVGPA SEQ ID No:7

[0068] Since the cleavage site of FAPα is after proline (Pro), the preferred FAPα cleavage unit in the present invention is a peptide segment having proline (Pro).

[0069] In the present invention, representative enhancement agents are paramagnetic T1 contrast agents, including (but not limited to): magnetic transition metal complexes or oxides thereof, magnetic lanthanide rare earth metal complexes or oxides thereof, preferably complexes and oxides of manganese and gadolinium.

[0070] In the present invention, the enhancer emits a detectable signal, such as a relaxation enhancement signal.

[0071] In the present invention, when the quencher and enhancer are connected via a connecting polypeptide, the relaxation enhancement signal emitted by the enhancer is quenched, and the T1 magnetic resonance signal of the enhancer is in an OFF state; and when the connecting polypeptide is specifically cleaved so that the quencher and enhancer are no longer connected together via the connecting polypeptide (such as in the presence of fibrotic liver tissue), the relaxation enhancement signal is no longer quenched, thereby restoring the T1 magnetic resonance signal of the enhancer to an ON state.

[0072] Preparation method

[0073] The present invention also provides a method for preparing a FAPα-responsive magnetic resonance molecular imaging probe that can be used for liver fibrosis grading detection.

[0074] In the present invention, the superparamagnetic nanoparticles and the paramagnetic T1 contrast agent can be connected / compounded by chemical reaction or physical action to form the FAPα responsive magnetic resonance molecular imaging probe of the present invention.

[0075] A preferred method is to directly or indirectly connect, couple and / or complex the superparamagnetic nanoparticles with the paramagnetic T1 contrast agent via a linker polypeptide via a surface ligand molecule.

[0076] Representative surface ligand molecules include (but are not limited to) small molecules (such as protocatechuic acid, dithiothioate, etc.), linear polymers (such as polyethylene glycol carboxyl, polyacrylic acid, etc.), block copolymers and their derivatives. In the present invention, the surface ligand is water-soluble or amphiphilic. The surface ligand contains one or more coordination groups and is connected to the surface of superparamagnetic nanoparticles through coordination.

[0077] In the present invention, a representative preparation method comprises the following steps:

[0078] Scheme 1: Superparamagnetic nanoparticles-FAPα responsive peptide chain-paramagnetic T1 contrast agent, including the following steps:

[0079] (a1) dissolving the superparamagnetic nanoparticles modified with surface ligand molecules in MES buffer and connecting them with FAPα responsive peptide chains through chemical reaction;

[0080] (a2) reacting the product of step (a1) with a paramagnetic T1 contrast agent through chemical reaction or physical adsorption / coating.

[0081] Scheme 2: Paramagnetic T1 contrast agent-FAPα responsive peptide chain-superparamagnetic nanoparticles, including the following steps:

[0082] (b1) connecting the paramagnetic T1 contrast agent to the FAPα responsive peptide chain through chemical reaction;

[0083] (b2) connecting the product obtained in (b1) with superparamagnetic nanoparticles modified with surface ligand molecules through chemical reaction.

[0084] Scheme 3: Surface ligand molecule / FAPα responsive peptide chain-paramagnetic T1 contrast agent / superparamagnetic nanoparticles, including the following steps:

[0085] (c1) The FAPα responsive peptide chain reacts with the surface ligand molecule (polymer) through chemical reaction to form a long-chain macromolecule of the surface ligand molecule-FAPα responsive peptide chain-surface ligand molecule structure;

[0086] (c2) Physically mixing the product obtained in (c1) with paramagnetic T1 contrast agent / superparamagnetic nanoparticles, and stirring to obtain a magnetic resonance molecular imaging probe containing paramagnetic T1 contrast agent / superparamagnetic nanoparticles coated with surface ligand molecules containing FAPα responsive peptide chains.

[0087] In a preferred embodiment of the present invention, a method for preparing an imaging probe (AFe-ASGPAGPA-Gd) is provided, which uses superparamagnetic amorphous iron nanoparticles (AFe) as a quencher, alanine-serine-glycine-proline-alanine-glycine-proline-alanine (ASGPAGPA) polypeptide chains as connecting polypeptides, and a magnetic resonance imaging contrast agent gadolinium (Gd) as an enhancer. The method comprises the steps of:

[0088] (1) In a solvent, iron carbonyl compounds are pyrolyzed at high temperature to obtain AFe nanoparticles.

[0089] (2) Dispersing the AFe nanoparticles obtained in step (1) in a solvent, then adding a carboxyl ligand, and stirring to obtain AFe-PA nanoparticles.

[0090] (3) Dispersing the AFe-PA nanoparticles obtained in step (2) in a solvent, then adding the polypeptide chain ASGPAGPA, and stirring to obtain AFe-ASGPAGPA nanoparticles.

[0091] (4) Dispersing the AFe-ASGPAGPA nanoparticles obtained in step (3) in a solvent, then adding a Gd agent, and stirring to obtain a magnetic resonance molecular imaging nanoprobe AFe-ASGPAGPA-Gd.

[0092] In step (1), the solvent is selected from one of 1-hexadecene, octyl ether, 1-octadecene, 1-eicosene, trioctylamine, water, etc.; preferably, it is 1-octadecene.

[0093] In step (1), the iron carbonyl ligand is selected from one or more of tetracarbonyl iron diiodide, pentacarbonyl iron, nonacarbonyl iron, dodecacarbonyl iron, cyclooctatetraene tricarbonyl iron, etc.; preferably, pentacarbonyl iron.

[0094] In step (1), the reaction temperature is 160-190°C; preferably, 160°C, or 170°C, or 190°C; more preferably, 180°C.

[0095] In step (1), the reaction time is 30-60 min; preferably, 30 min, 40 min, or 60 min; more preferably, 40 min.

[0096] Step (1) also includes a subsequent treatment step: after the reaction is completed, the AFe nanoparticles are washed 1-2 times with a large amount of anhydrous ethanol and dispersed in n-hexane.

[0097] Step (2) also includes a pretreatment step: adding a large amount of anhydrous ethanol to the n-hexane solution of AFe nanoparticles, removing the supernatant after centrifugation, and obtaining AFe nanoparticles.

[0098] In step (2), the solvent is selected from one of methanol, chloroform, toluene, dimethyl sulfoxide, N,N-dimethylformamide, etc., preferably toluene.

[0099] In step (2), the carboxyl compound is selected from one of protocatechuic acid, disuccinic acid, polyethylene glycol carboxyl, etc., preferably, protocatechuic acid.

[0100] In step (2), the reaction temperature is 25-40°C; preferably, 25°C.

[0101] In step (2), the reaction time is 5-30 min; preferably, 10 min.

[0102] Step (2) also includes a post-treatment step: after the reaction is completed, the supernatant is discarded, deionized water and ethanol are added, and centrifuged 2-3 times to obtain AFe-PA nanoparticles.

[0103] In step (3), the solvent is selected from one or more of deionized water, PBS buffer, MES buffer, methanol, etc., preferably, MES buffer.

[0104] In step (3), the reaction temperature is 25-40°C; preferably, 25°C.

[0105] In step (3), the reaction time is 12-24 hours; preferably, 24 hours.

[0106] Step (3) also includes a post-treatment step: after the reaction is completed, centrifuge with deionized water for 2-3 times, and disperse the obtained AFe-ASGPAGPA nanoparticles in deionized water and store them for later use.

[0107] In step (4), the solvent is selected from one or more of deionized water, PBS buffer, MES buffer, methanol, etc., preferably, MES buffer.

[0108] In step (4), the gadolinium agent is selected from one or more of Gd-DOTA, Gd-DTPA, Gd-DTPA-BMA, Gd-BOPTA, etc., preferably, it is Gd-DOTA.

[0109] In step (4), the reaction temperature is 25-40°C; preferably, 25°C.

[0110] In step (4), the reaction time is 12-24 hours; preferably, 24 hours.

[0111] Step (4) also includes a post-treatment step: after the reaction is completed, centrifuge and wash with deionized water for 2-3 times, and disperse the obtained magnetic resonance molecular imaging nanoprobe AFe-ASGPAGPA-Gd in deionized water for storage.

[0112] application

[0113] The magnetic resonance molecular imaging nanoprobe (such as AFe-ASGPAGPA-Gd) of the present invention can be used for non-invasive detection and classification of patients with liver fibrosis.

[0114] Taking AFe-ASGPAGPA-Gd as an example, when the nanoprobe of the present invention is in normal liver tissue, the strong magnetic moment of the AFe nanoparticles causes the relaxation enhancement signal of the adjacent Gd-DOTA to be quenched, and the magnetic resonance signal is turned off (OFF state); while in the fibrotic liver, the ASGPAGPA in the nanoprobe can be specifically cut off by FAPα, resulting in an increase in the distance between the AFe nanoparticles and Gd-DOTA, thereby restoring the magnetic resonance signal of Gd-DOTA (ON state).

[0115] In the present invention, since the content of FAPα is positively correlated with the degree of liver fibrosis, the responsiveness of the nano-imaging probe of the present invention to FAPα can be utilized to achieve accurate and non-invasive graded detection of liver fibrosis by recording changes in magnetic resonance imaging signals.

[0116] The main advantages of the present invention include:

[0117] (1) The magnetic resonance molecular imaging nanoprobe of the present invention has high imaging sensitivity and good magnetic resonance signal stability. Compared with existing contrast agents, it has significantly improved MR signal enhancement and diagnostic sensitivity, and can be used for accurate quantitative detection and evaluation of liver fibrosis at different levels.

[0118] (2) The method of evaluating liver fibrosis grading using magnetic resonance molecular imaging nanoprobes described in the present invention is a non-invasive detection method that can achieve non-invasive detection and eliminate the trauma caused to patients during traditional detection methods.

[0119] (3) The synthesis steps of the magnetic resonance molecular imaging nanoprobe of the present invention are simple, the reaction conditions are mild, and the yield is high, and it can be used for large-scale industrial production.

[0120] (4) The raw materials and products of the magnetic resonance molecular imaging nanoprobes of the present invention have high biosafety and biocompatibility, thus ensuring safety and effectiveness during clinical application.

[0121] (5) The AUCs of the magnetic resonance molecular imaging nanoprobe AFe-ASGPAGPA-Gd for grades 1-4 liver fibrosis were 85.2%, 74.1%, 59.3% and 99.8%, respectively. Compared with magnetic resonance scanning without the use of nanoprobes, the diagnostic accuracy was improved by 25.9%, 14.8%, 7.4% and 23.6%, respectively.

[0122] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0123] Example 1: Preparation of amorphous iron (AFe) nanoparticles

[0124] 0.7 mL of oleylamine was added to the reaction solution of 20 mL of 1-octadecene. Under gentle magnetic stirring, the solution was heated to 80 ° C. The reaction bottle was evacuated by a vacuum pump for half an hour, and then filled with argon. After the reaction solution was heated to 180 ° C at a heating rate of 0.5 ° C / min, 0.5 mL of pentacarbonyl iron was quickly injected into the reaction solution and the reaction was continued for 40 min. After the reaction was completed, the temperature was cooled to room temperature, 20 mL of n-hexane solution and 100 mL of anhydrous ethanol were added, and the mixture was centrifuged at 13000 rpm for 10 minutes, the supernatant was discarded, and n-hexane and anhydrous ethanol were centrifuged 1-2 times. The obtained AFe nanoparticles were dispersed in 20 mL of n-hexane solution and stored for later use.

[0125] Example 2: Preparation of AFe-PA nanoparticles

[0126] (1) Preparation of protocatechuic acid (PA) solution: Weigh 0.4 g of protocatechuic acid (as a surface ligand molecule) and dissolve it in a mixed solution of 3 mL of toluene and 1 mL of pyridine to obtain a PA solution.

[0127] (2) Preparation of Na2CO3 solution: Weigh 1.0 g of Na2CO3 solid and dissolve it in 100 mL of deionized water to obtain 1% w / v Na2CO3.

[0128] (3) Take 10 mL of the AFe nanoparticles in Example 1, add 20 mL of anhydrous ethanol, centrifuge and discard the supernatant, and disperse the precipitated AFe nanoparticles in 40 mL of toluene solution. Slowly drop the prepared PA solution into the toluene solution containing the AFe nanoparticles, stir at room temperature for 10 min and then let stand for 30 min, discard the supernatant, add 10 mL of 1% w / v Na2CO3 solution and 30 mL of anhydrous ethanol, 13000 rpm / min, quickly centrifuge to collect the product, wash it again with deionized water 2-3 times, and disperse it in 50 mL of deionized water to obtain AFe-PA nanoparticles.

[0129] Example 3: Preparation of AFe-ASGPAGPA Nanoparticles

[0130] (1) Preparation of 2-morpholineethanesulfonic acid (MES) buffer: Weigh 19.5 g of 2-morpholineethanesulfonic acid, dissolve in 500 mL of deionized water, adjust the pH to 6.0 with NaOH, and dilute to 1 L to obtain 0.1 M MES buffer, which is then placed in a 4°C refrigerator for later use.

[0131] (2) Preparation of PBS buffer.

[0132] (3) Take 30 mL of the AFe-PA nanoparticles in Example 2, collect the product (about 50 mg) by rapid centrifugation, disperse it in 30 mL of MES buffer at pH 6.0, add 400.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir at room temperature for 10 min; adjust the pH of the system to 7.2-7.5 with PBS buffer, add 1.1 g of N-hydroxysuccinimide sulfonic acid sodium salt, continue stirring for 30 min, and then add 500 mg of the polypeptide chain ASGPAGPA (SEQ ID No: 1, artificially synthesized). After reacting for 24 h, collect the product by centrifugation, wash it with deionized water 2-3 times, and then disperse the product in 30 mL of MES buffer to obtain AFe-ASGPAGPA nanoparticles (about 65 mg).

[0133] Example 4: Preparation of AFe-ASGPAGPA-Gd

[0134] Take 30 mL of the AFe-ASGPAGPA nanoparticles in Example 3 (about 65 mg of AFe-ASGPAGPA nanoparticles), add 300.0 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir at room temperature for 10 min; adjust the pH of the system to 7.2-7.5 with PBS buffer, add 825.0 mg of N-hydroxysuccinimide sulfonic acid sodium salt, continue stirring for 30 min, then add 300 mg of Gd-DOTA, react for 24 h, collect the product by centrifugation, wash with deionized water 2-3 times, and then disperse the obtained magnetic resonance molecular imaging probe AFe-ASGPAGPA-Gd (about 40 mg) in 10 mL of deionized water.

[0135] Example 5: Magnetic resonance imaging performance test of aqueous solution

[0136] In this example, the performance of the magnetic resonance molecular imaging probe of the present invention in response to FAPα cleavage was tested by in vitro experiments. The method is as follows:

[0137] (1) Preparation of samples: AFe, Gd-DOTA, and AFe-ASGPAGPA-Gd were prepared with PBS buffer solution to concentrations of 0.5000, 0.2500, 0.1250, 0.0625, 0.0312, and 0.0015 mM (corresponding to the concentration of Fe in AFe, the concentration of Gd in Gd-DOTA, and the concentration of Gd in AFe-ASGPAGPA-Gd, respectively). 1.5 mL of each solution was placed in a 2 mL centrifuge tube for later use.

[0138] (2) Detection of T1 relaxation performance of magnetic resonance molecular imaging probe AFe-ASGPAGPA-Gd: The above test solutions were divided into 4 groups, namely AFe, Gd-DOTA, AFe-ASGPAGPA-Gd, and AFe-ASGPAGPA-Gd+FAPα, where the concentration of FAPα was 1000 ng / mL. Next, the 4 groups of samples were cultured at 37°C for 24 h, and then the supernatant of each group was scanned by 3.0T MRI (Discovery MR750, General Motors, USA) to obtain T1 plain scan images.

[0139] (3) Experimental results are as follows Figure 5 shown.

[0140] The results show that Gd-DOTA exhibits effective T1-MRI contrast performance (r1 = 6.51 mM -1 s -1 ), while AFe nanoparticles showed limited T1 relaxation characteristics (r1 = 0.49 mM -1 s -1Similarly, due to the strong magnetic field confinement of AFe on Gd-DOTA, the T1 relaxation characteristics of AFeAGd are still limited (r1 = 0.53 mM -1 s -1 However, in the presence of FAPα (1000 ng / mL), the T1-MRI contrast performance of AFeAGd became 16.01 mM -1 s -1 After responding to FAPα, the T1 relaxation time of AFeAGd (0.05 mM) changed from 1814.70 ms to 752.80 ms. The results showed that AFeAGd can be cleaved in response to FAPα.

[0141] Example 6: Magnetic resonance imaging nanoprobe AFe-ASGPAGPA-Gd for graded detection of liver fibrosis

[0142] In this embodiment, animal experiments were conducted to test whether the magnetic resonance molecular imaging probe of the present invention can detect the grade of liver fibrosis non-invasively and accurately through FAPα responsive cleavage. The method is as follows:

[0143] (1) Establishment of animal model of liver fibrosis: 30 C57BL6 female mice were randomly divided into a control group and a model group, with 6 mice in the control group and 24 mice in the model group. The mice in the control group were fed with standard rodent chow and water, and the mice in the model group were fed with a NASH diet for 2 months. The feeding conditions of the two groups were completely consistent. After the animal model was established, the 30 mice were further divided into a control group, grade I-II liver fibrosis, and grade III-IV liver fibrosis.

[0144] (2) In vitro magnetic resonance scanning: 150 μL of AFe-ASGPAGPA-Gd (0.05 mM Gd / Kg) prepared in Example 4 of the present invention was injected into the three groups of mice through the tail vein. One hour later, the mice were subjected to magnetic resonance scanning. The images obtained by processing the T1W-FSE sequence were used to obtain T1 plain scan and T1 mapping images, respectively, to evaluate the nanoprobe's ability to detect liver fibrosis graded by the test.

[0145] (3) Experimental results are as follows Figure 6 shown.

[0146] As the degree of fibrosis deepens, the brightness of T1 plain scan and T1 mapping images gradually increases. For livers with grade 1-4 fibrosis, the Area Under Curve (AUC) values ​​are 85.2%, 74.1%, 63.0% and 96.3%, respectively. The results show that the nanoprobe has achieved good diagnostic effects on different grades of liver fibrosis and can achieve graded detection of different grades of liver fibrosis.

[0147] discuss

[0148] Magnetic resonance molecular imaging has the characteristics of being not limited by tissue depth, having high spatial resolution, being sensitive and non-invasive, and has been widely used in the diagnosis of central nervous system, soft tissue lesions, tumors and inflammatory diseases. Targeting specific liver fibrosis markers with magnetic resonance molecular imaging probes can achieve quantitative molecular imaging and graded detection of liver fibrosis.

[0149] However, existing clinical magnetic resonance molecular imaging probes are extracellular contrast agents, and there is currently a lack of reliably expressed biomarkers that are closely related to the pathological degree of liver fibrosis in clinical practice. Therefore, it is impossible to assess the degree of pathological changes in liver cells, and there is a lack of guiding value for the detection and grading of the degree of liver fibrosis.

[0150] The inventors have developed for the first time a magnetic resonance molecular imaging probe with a unique structure that can respond to FAPα protein with high sensitivity and specificity. Experiments have shown that the magnetic resonance molecular imaging detection technology developed based on the FAPα-responsive magnetic resonance molecular imaging probe of the present invention can accurately perform non-invasive grading detection of the degree of liver fibrosis in patients.

[0151] Compared with existing tissue puncture biopsy, the method of the present invention can detect and evaluate the overall fibrosis level of the liver.

[0152] Compared with existing hematological examinations, the method of the present invention directly detects liver tissue and is therefore substantially unaffected by confounding effects of other organs or diseases, and has a high diagnostic value.

[0153] Compared with traditional imaging examinations, the method of the present invention does not rely on changes in liver morphology and structure, but identifies lesions through the level of the highly sensitive biomarker FAPα. Therefore, on the one hand, it has high sensitivity, and on the other hand, it is conducive to early diagnosis or earlier detection of liver fibrosis, so as to take corresponding treatment measures as early as possible.

[0154] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A magnetic resonance molecular imaging probe for liver fibrosis grading detection, characterized in that: It comprises a quencher, an enhancer and a connecting polypeptide, wherein the quencher and the enhancer are connected by a connecting polypeptide, and the connecting polypeptide is a polypeptide having an amino acid sequence of a FAPα-specific cleavage site; The magnetic resonance molecular imaging probe has a structure of Formula I: Q-(LE)n(I) In the formula, Q is a quencher; the quencher is amorphous iron nanoparticles; L is a FAPα responsive connecting polypeptide; the sequence of the polypeptide is ASGPAGPA; E is an enhancer; the enhancer is Gd-DOTA; n is a positive integer ≥1.

2. The magnetic resonance molecular imaging probe according to claim 1, characterized in that: When the quencher and enhancer are linked via a connecting polypeptide, the detectable signal of the enhancer is reduced or quenched by the quencher; and when the connecting polypeptide is specifically cleaved so that the quencher and enhancer are no longer linked together via the connecting polypeptide, the detectable signal of the enhancer is no longer reduced or quenched by the quencher.

3. The magnetic resonance molecular imaging probe according to claim 2, characterized in that: The detectable signal of the enhancer includes a relaxation enhancement signal.

4. The magnetic resonance molecular imaging probe according to claim 3, characterized in that: When the quencher and enhancer are connected via a connecting polypeptide, the relaxation enhancement signal is quenched, and the T1 magnetic resonance signal of the enhancer is in an OFF state; and when the connecting polypeptide is specifically cleaved so that the quencher and enhancer are no longer connected via the connecting polypeptide, the T1 relaxation enhancement signal of the enhancer is no longer quenched, thereby restoring the magnetic resonance signal and being in an ON state.

5. The magnetic resonance molecular imaging probe according to claim 1, wherein: n is 10-10000.

6. The magnetic resonance molecular imaging probe according to claim 1, characterized in that: The linker polypeptide is linked to the quencher and / or enhancer via a surface ligand molecule.

7. The magnetic resonance molecular imaging probe according to claim 1, wherein: The particle size of the magnetic resonance molecular imaging probe is 2-900nm.

8. The magnetic resonance molecular imaging probe according to claim 1, wherein: The particle size of the magnetic resonance molecular imaging probe is 5-500nm.

9. The magnetic resonance molecular imaging probe according to claim 1, wherein: The particle size of the magnetic resonance molecular imaging probe is 10-100 nm.

10. A method for preparing the magnetic resonance molecular imaging probe for liver fibrosis grading detection according to claim 1, characterized in that: Includes steps: (1) providing a quencher, an enhancer and a connecting polypeptide; (2) The quencher and enhancer are connected via a connecting polypeptide to form the magnetic resonance molecular imaging probe of claim 1.

11. A use of the magnetic resonance molecular imaging probe as claimed in claim 1, characterized in that: Used to prepare diagnostic reagents for grading detection of liver fibrosis.

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