Enzyme response magnetic resonance imaging nano probe and preparation method and application thereof

Abstract

The application discloses an enzyme-responsive magnetic resonance imaging nano probe and a preparation method and application thereof. The enzyme-responsive magnetic resonance imaging nano probe comprises a surface functional group A metal nanoparticle as an imaging basic unit, and a functional group B polypeptide sequence as a crosslinking agent and an enzyme-responsive unit; and the enzyme-responsive magnetic resonance imaging nano probe is a metal nanoparticle crosslinking body formed by crosslinking the metal nanoparticle through the polypeptide sequence. The enzyme-responsive magnetic resonance imaging nano probe can be specifically cut by an enzyme highly expressed in a cell differentiation process, and forms a monodispersed metal nanoparticle, so that a signal change is presented on magnetic resonance imaging. Meanwhile, the enzyme-responsive magnetic resonance imaging nano probe can establish an effective correlation among a probe existing form, a cell state and an imaging signal, and is used for real-time tracking of a directional differentiation process of mesenchymal stem cells, neural stem cells and promyelocytic leukemia cells.

Description

Technical Field

[0001] This invention belongs to the field of medical imaging, specifically relating to an enzyme-responsive magnetic resonance imaging nanoprobe, its preparation method, and its application. Background Technology

[0002] Cell differentiation has significant clinical value in disease treatment. For example, mesenchymal stem cells need to differentiate into osteoblasts to achieve repair in treating bone defects; neural stem cells need to differentiate into neurons or glial cells to replace non-regenerating neurons and treat nerve damage in treating neurodegenerative diseases. Furthermore, HL60 differentiation-inducing therapy, as a novel strategy, has been applied in the clinical treatment of acute promyelocytic leukemia (APL) (e.g., retinoic acid, low-dose arsenic trioxide), shifting the goal from the traditional elimination of cancer cells to inducing their differentiation into functionally normal cells. This approach holds great potential.

[0003] Current research on in vivo visualization of transplanted stem cells focuses on tracking physiological processes such as migration, homing, and apoptosis, lacking imaging techniques to visualize their differentiation in vivo. Furthermore, the evaluation of the therapeutic effect of APL differentiation relies on in vitro and endpoint detection methods (such as CCK-8, flow cytometry, and Western blotting). These methods cannot achieve real-time, non-invasive, and intuitive monitoring of the dynamic and continuous process of cell differentiation in vivo, thus having certain limitations. Therefore, developing imaging probes combined with in vivo imaging technology to achieve in vivo visualization of cell differentiation has significant research value.

[0004] Magnetic resonance imaging (MRI) is a non-ionizing imaging technique that is not limited by the depth of tissue penetration. It can non-invasively present the anatomical details and pathological information of organs and soft tissues. In particular, it can utilize responsive magnetic resonance probes to change MRI signals through changes in probe structure, thereby dynamically visualizing the occurrence, development, and treatment process of diseases. Yang et al. developed an MRI nanoprobe based on Fe3O4, FFT NPs, which can display tumor location through structural MRI and respond to high concentrations of ATP to activate T1 MRI signals in malignant tumors, enabling differential diagnosis of benign and malignant tumors. Song et al. constructed a NO-responsive magnetic probe composed of superparamagnetic iron oxide nanoparticles with breakable connecting arms sensitive to NO, which can be used for in vivo high-sensitivity and high-selectivity NO magnetic resonance imaging.

[0005] A large amount of research focuses on using MRI technology in conjunction with responsive probes for the diagnosis and treatment of tumors or the detection of signaling molecules in vivo, but there is currently a lack of in vivo imaging technology that visualizes cell differentiation in vivo. Summary of the Invention

[0006] The main objective of this invention is to provide an enzyme-responsive magnetic resonance imaging nanoprobe, its preparation method, and its application, in order to overcome the shortcomings of the prior art.

[0007] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0008] This invention provides an enzyme-responsive magnetic resonance imaging nanoprobe, comprising: surface functionalized metal nanoparticles with A-type functional groups as basic imaging units, and peptide sequences with B-type functional groups at both ends as crosslinking agents and enzyme-responsive units; the enzyme-responsive magnetic resonance imaging nanoprobe is a metal nanoparticle crosslink formed by crosslinking the metal nanoparticles through the peptide sequences.

[0009] Wherein, in the surface-functionalized metal nanoparticles, functional group A is selected from any one of double bond, azide group, alkynyl group, and 6-amino-2-cyanobenzothiazole residue, wherein the double bond includes maleimide residue containing a double bond; the metal nanoparticles include any one or more combinations of iron-based, gadolinium-based, and manganese-based metal nanoparticles;

[0010] The functional group B in the polypeptide sequence with B-functionalized ends is selected from any one of thiol, alkynol, and azide, wherein the thiol includes cysteine ​​residues; the polypeptide sequence includes any one or more combinations of cathepsin digestion sequences, trypsin digestion sequences, and matrix metalloproteinase digestion sequences.

[0011] This invention also provides a method for preparing the aforementioned enzyme-responsive magnetic resonance imaging nanoprobe, comprising:

[0012] Enzyme-responsive magnetic resonance imaging nanoprobes were prepared by clicking reactions between surface-functionalized A-type metal nanoparticles and end-functionalized B-type polypeptide sequences under activating conditions.

[0013] This invention also provides the application of the aforementioned enzyme-responsive magnetic resonance imaging nanoprobe in the visualization of cell differentiation; wherein the cells include any one of human promyelocytic leukemia cells, mesenchymal stem cells, and neural stem cells.

[0014] This invention also provides a method for visualizing cell differentiation for non-diagnostic purposes, comprising: inducing cell differentiation in a system containing cells and the aforementioned enzyme-responsive magnetic resonance imaging nanoprobe, and simultaneously performing detection using MRI imaging, thereby achieving visualization of cell differentiation; wherein the cells include any one of human promyelocytic leukemia cells, mesenchymal stem cells, and neural stem cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] (1) The enzyme-responsive magnetic resonance imaging nanoprobe in this invention has good stability, can be stored for a long time after freeze-drying, and its particle size and other properties remain stable after solvent reconstitution.

[0017] (2) The enzyme-responsive magnetic resonance imaging nanoprobe in this invention has good biosafety;

[0018] (3) The enzyme-responsive magnetic resonance imaging nanoprobe in this invention has good enzyme responsiveness and can achieve decrosslinking of nanoaggregates through specific enzyme cleavage.

[0019] (4) The enzyme-responsive magnetic resonance imaging nanoprobe in this invention has a large change in magnetic resonance signal before and after enzyme digestion, and can achieve a strong signal contrast transition before and after enzyme digestion in aqueous solution and cells;

[0020] (5) The enzyme-responsive magnetic resonance imaging nanoprobe in this invention establishes an effective correlation between the form of the imaging probe, the cell state, and the imaging signal, and is used to track the directed differentiation process of mesenchymal stem cells, neural stem cells and HL60 in real time. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figures 1a-1b Morphology and particle size distribution of the SPIO-mal product obtained in Example 1 of this invention;

[0023] Figure 2 This is an MRI result image of the SPIO-mal product obtained in Example 1 of the present invention;

[0024] Figure 3 The mass spectrum of the polypeptide sequence CFRPKC, which has thiol functional groups at both ends, synthesized by solid-phase synthesis in Example 2 of the present invention.

[0025] Figures 4a-4b The morphology and particle size diagrams of the M-SPIO prepared in Example 3 of this invention are shown.

[0026] Figures 5a-5b The images show the morphology and particle size of M-SPIO after digestion in Example 4 of this invention.

[0027] Figure 6 These are the T2-weighted magnetic resonance images of the three groups SPIO-mal, M-SPIO, and M-SPIO+Cat B in Embodiment 4 of the present invention;

[0028] Figure 7 The images shown are T2-weighted magnetic resonance images of M-SPIO-labeled HL60 cells before and after differentiation in Example 5 of this invention.

[0029] Figure 8 This is a diagram showing the cell safety results of M-SPIO in Example 6 of the present invention;

[0030] Figure 9 This is a T2-weighted magnetic resonance image of M-SPIO-labeled HL60 cells in the left tibial muscle group of mice before and after differentiation in Example 7 of the present invention;

[0031] Figure 10 The mass spectrum of the polypeptide sequence CGKTGC, which has thiol functional groups at both ends, synthesized by solid-phase synthesis in Example 8;

[0032] Figure 11 This is a schematic diagram of the process for preparing enzyme-responsive magnetic resonance imaging nanoprobes and performing MR imaging in a typical embodiment of the present invention. Detailed Implementation

[0033] In view of the deficiencies of the prior art, the inventors of this case, through long-term research and extensive practice, have proposed the technical solution of this invention. The technical solution of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0034] Specifically, as one aspect of the technical solution of the present invention, the enzyme-responsive magnetic resonance imaging nanoprobe includes: metal nanoparticles with surface functional groups A-modified as the basic imaging unit, and a polypeptide sequence with end functional groups B-modified as a crosslinking agent and enzyme-responsive unit (B-cleaved peptide-B); the enzyme-responsive magnetic resonance imaging nanoprobe is a metal nanoparticle crosslink formed by crosslinking the metal nanoparticles through the polypeptide sequence.

[0035] Wherein, in the surface-functionalized metal nanoparticles, functional group A is selected from any one of double bond, azide group, alkynyl group, and 6-amino-2-cyanobenzothiazole residue, wherein the double bond includes maleimide residue containing a double bond; the metal nanoparticles include any one or more combinations of iron-based, gadolinium-based, and manganese-based metal nanoparticles;

[0036] The functional group B in the polypeptide sequence with B-terminal functional groups is selected from any one of thiol, alkynol, and azide, wherein the thiol includes cysteine ​​residues; the polypeptide sequence includes any one or more combinations of cathepsin restriction enzyme sequences, mesotrypsin restriction enzyme sequences, and matrix metalloproteinase (MMP) restriction enzyme sequences.

[0037] In some preferred embodiments, when functional group A is a double bond, functional group B is a thiol group.

[0038] In some preferred embodiments, when functional group A is an azide group, functional group B is an alkynyl group.

[0039] In some preferred embodiments, when functional group A is an alkynyl group, functional group B is an azide group.

[0040] In some preferred embodiments, when functional group A is a 6-amino-2-cyanobenzothiazole residue, functional group B is a cysteine ​​residue.

[0041] In some preferred embodiments, the metal nanoparticles are selected from MRI iron-based iron oxide nanoparticles (SPIO), wherein the particle size of the MRI iron-based iron oxide nanoparticles is less than 5 nm.

[0042] In some preferred embodiments, functional group A is a maleimide residue containing a double bond, and functional group B is a cysteine ​​residue.

[0043] In some preferred embodiments, the cathepsin cleavage sequence includes a cathepsin B cleavage polypeptide sequence and / or a cathepsin L cleavage polypeptide sequence. The cathepsin B cleavage polypeptide sequence includes, but is not limited to, any one or more combinations of B-Gly-Phe-Lys-Phe-Trp-B, B-Gly-Phe-Arg-Ser-Trp-B, B-Phe-Arg-Lys-Trp-B, B-Phe-Arg-Phe-Lys-B, B-Gly-Ile-Val-Arg-Ala-Lys-B, and B-Val-Cit-B. The cathepsin L cleavage polypeptide sequence includes, but is not limited to, any one or more combinations of B-Arg-Arg-B, B-Phe-Arg-B, and B-Lys-Lys-B.

[0044] The trypsin digestion sequence mentioned includes, but is not limited to, any one or more combinations of B-Lys-Ser-B, B-Lys-Ser-B, B-Gly-Pro-Arg-B, and B-Gly-Lys-Thr-Gly-B.

[0045] The metalloproteinase cleavage sequences include, but are not limited to, the cleavage substrate of matrix metalloproteinase 2 (MMP2) B-Gly-Pro-Leu-Gly—Leu-Lys-Ala-Arg-B and / or the cleavage site of matrix metalloproteinase 13 (MMP13) B-Gly-Pro-Leu-Gly—Val-Arg-Gly-Lys-B;

[0046] In this context, "—" represents the enzyme cleavage site, and B represents the functional group B.

[0047] Furthermore, the cathepsin digestion sequence includes B-Phe-Arg—Phe-Lys-B, wherein B is a cysteine ​​residue containing a thiol group, namely Cys-Phe-Arg—Phe-Lys-Cys.

[0048] Another aspect of the present invention provides a method for preparing the aforementioned enzyme-responsive magnetic resonance imaging nanoprobe, comprising:

[0049] Enzyme-responsive magnetic resonance imaging nanoprobes were prepared by clicking reactions between surface-functionalized A-type metal nanoparticles and end-functionalized B-type polypeptide sequences under activating conditions.

[0050] In some preferred embodiments, the preparation method includes: preparing a 1-100 mM solution of surface functionalized metal nanoparticles, adding 1-10 equivalents of a B-cleaved peptide sequence with end functionalized groups B and the corresponding activating reagent to obtain a metal nanoparticle cross-link, i.e., an enzyme-responsive magnetic resonance imaging nanoprobe.

[0051] In some preferred embodiments, the molar ratio of the surface-functionalized A-type metal nanoparticles to the end-functionalized B-type polypeptide sequences is 1:1 to 10.

[0052] In some preferred embodiments, the activator includes, but is not limited to, any one or more combinations of tris(2-carboxyethyl) phosphate hydrochloride for activating thiol groups, glutathione for activating thiol groups, and CuBr for activating alkynyl groups.

[0053] In some preferred embodiments, the particle size of the enzyme-responsive magnetic resonance imaging nanoprobe is 10 nm to 1000 nm.

[0054] In some preferred embodiments, the preparation method further includes: dialysis of the obtained product after the click reaction is completed.

[0055] In some preferred embodiments, the preparation method specifically includes: modifying functional group A on the surface of metal nanoparticles by means of ligand exchange or covalent bonding with a bifunctional crosslinking agent.

[0056] Furthermore, dicarboxylic acids such as citric acid and tartaric acid are used to react with metal ions M on the surface of metal nanoparticles. n+ Coordination bonds are formed to modify carboxyl groups on the surface of metal nanoparticles.

[0057] Furthermore, polyethyleneimine, polylysine, polydopamine, etc., are used to react with metal ions M on the surface of metal nanoparticles. n+ Coordination bonds are formed to modify amino groups on the surface of metal nanoparticles.

[0058] Furthermore, a bifunctional crosslinking agent, maleimide-polyethylene glycol-amino (NH2-PEG-MAL), is used to form amide bonds with surface-active carboxylated metal nanoparticles, thereby modifying the surface of the metal nanoparticles with maleimide containing double bonds.

[0059] Furthermore, bifunctional crosslinking agents such as α-succinimide carboxylate-ω-maleimide (amide bond) polyethylene glycol (NHS-PEG-MAL) or maleimide-polyethylene glycol-carboxylic acid (MAL-PEG-COOH) are used to form amide bonds covalently linked with surface-aminated metal nanoparticles, thereby modifying the surface of metal nanoparticles with maleimide containing double bonds.

[0060] Furthermore, the surface of the metal nanoparticles was modified with azide by forming amide bonds covalently with the bifunctional crosslinking agent succinimide carboxylate-polyethylene glycol-azide (NHS-PEG-N3) to form a amide bond.

[0061] Furthermore, the surface of the metal nanoparticles is modified with alkynyl groups by forming amide bonds covalently with the bifunctional crosslinking agent succinimide carboxylate-polyethylene glycol-alkynyl (NHS-PEG-CCH) to form amide bonds.

[0062] Furthermore, 6-amino-2-cyanobenzothiazole (CBT) and its derivatives are covalently linked to surface-carboxylated metal nanoparticles by forming amide bonds, thereby modifying the surface of the metal nanoparticles with CBT.

[0063] In some preferred embodiments, the preparation method specifically includes: synthesizing a polypeptide sequence with B-terminal functional groups using a solid-phase synthesis method.

[0064] Furthermore, the amino acid containing the active functional group B was first attached to 2-chlorotriphenylmethyl chloride resin. Then, the resin was blocked with a blocking solution of N,N-dimethylformamide (DMF):methanol (CH3OH) = 19:1. The protection was then removed with 20% piperidine. The amino acids in the enzyme-digested peptide sequence were coupled in order from the C-terminus to the N-terminus using benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT) / N-ethyldiisopropylamine (DIPEA) as coupling reagents. The last amino acid was also coupled with an amino acid containing the active functional group B. The protection was removed using a mixture of trifluoroacetic acid (TFA): benzyl sulfide: phenol: triisopropylsilane (TIPS): ultrapure water = 82.5:7.5:5:3:2 (v / v). After purification, a peptide sequence forming B-cleaved peptide-B was obtained, i.e., a peptide sequence with B-functionalized peptide groups at both ends.

[0065] In this invention, superparamagnetic iron oxide nanoparticles (SPIO) are preferentially used, and citric acid is used to react with Fe on the surface of the iron oxide nanoparticles. 3+ Coordination bonds are formed to modify carboxyl groups on the surface of iron oxide nanoparticles. Then, the bifunctional crosslinking agent NH2-PEG-MAL is used to form amide bonds with the carboxylated iron oxide nanoparticles on the surface, and maleimide containing double bonds is modified on the surface.

[0066] Another aspect of the present invention provides the application of the aforementioned enzyme-responsive magnetic resonance imaging nanoprobe in the visualization of cell differentiation; wherein the cells include any one of human promyelocytic leukemia cells (HL60), mesenchymal stem cells (MSCs), and neural stem cells (NSCs).

[0067] Furthermore, the imaging technology, combined with the enzyme-responsive magnetic resonance imaging nanoprobe, is used to visualize the differentiation of human promyelocytic leukemia cells (HL60), mesenchymal stem cells (MSCs), and neural stem cells (NSCs).

[0068] Furthermore, the enzyme-responsive magnetic resonance imaging nanoprobe is used to track the directed differentiation process of mesenchymal stem cells, neural stem cells, and HL60 in real time, especially the directed differentiation process of HL60.

[0069] Another aspect of the present invention provides a method for decrosslinking enzyme-responsive magnetic resonance imaging nanoprobes, comprising: adding a protease to a solution containing the aforementioned enzyme-responsive magnetic resonance imaging nanoprobes to cleave the probes and react, thereby achieving decrosslinking of the enzyme-responsive magnetic resonance imaging nanoprobes.

[0070] Another aspect of the present invention provides a method for visualizing cell differentiation for non-diagnostic purposes, comprising: inducing cell differentiation in a system containing cells and the aforementioned enzyme-responsive magnetic resonance imaging nanoprobe, and simultaneously performing detection using MRI imaging, thereby achieving visualization of cell differentiation; wherein the cells include any one of human promyelocytic leukemia cells, mesenchymal stem cells, and neural stem cells.

[0071] In some more specific embodiments, the specific solutions of the present invention are as follows:

[0072] a. Superparamagnetic iron oxide nanoparticles (SPIO) were dissolved in tetrahydrofuran, and citric acid was added to react with Fe on the surface of the iron oxide nanoparticles. 3+ Coordination bonds were formed to modify carboxyl groups on the surface of iron oxide nanoparticles, yielding the intermediate product SPIO-COOH. The intermediate product was dissolved in DMSO, and a bifunctional crosslinking agent NH2-PEG-MAL was added to form amide bonds with the carboxylated iron oxide nanoparticles on the surface, which were then modified with maleimide containing double bonds. After precipitation with anhydrous diethyl ether, the precipitate was dissolved in ultrapure water and dialyzed to finally obtain SPIO-mal.

[0073] b. A polypeptide sequence with thiol groups at both ends was synthesized by solid-phase synthesis, and its amino acid sequence is Cys-Phe-Arg—Phe-Lys-Cys:

[0074] First, cysteine ​​containing the active functional group thiol is used as the first amino acid and attached to 2-chlorotriphenylmethyl chloride resin. Then, the resin is blocked with a blocking solution DMF:CH3OH=19:1. Deprotection is performed with 20% piperidine. The amino acids in the enzyme-digested peptide sequence are coupled in order from the C-terminus to the N-terminus using HBTU / HOBT / DIPEA as coupling reagents. The last amino acid is also coupled with cysteine ​​containing the active functional group thiol. A mixture of TFA: benzyl sulfide: phenol: TIPS: ultrapure water = 82.5: 7.5: 5: 3: 2 (v / v) is used as the deprotection reagent. After purification, a peptide sequence forming SH-cleaved peptide-SH is obtained, abbreviated as CFRPKC.

[0075] c. Prepare a 1 mM solution of SPIO-mal using PBS buffer. Add the solid CFRPKC obtained from solid-phase synthesis to the solution, and simultaneously add tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) as an activator to activate the thiol groups at both ends of the peptide. React at room temperature for 4–8 h. After the reaction, transfer the resulting solution to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyze for 36–48 h to obtain a solution with uniform particle size. The final product is the enzyme-responsive imaging nanoprobe M-SPIO. The particle size of the enzyme-responsive magnetic resonance imaging nanoprobe is related to the degree of cross-linking and can be controlled by adjusting the ratio of surface functionalized A-type metal nanoparticles to B-cleaved peptide-B sequences with B-type functionalized ends. The particle size of the enzyme-responsive magnetic resonance imaging nanoprobe constructed according to this method can be controlled between 10 nm and 1000 nm.

[0076] d. Add cathepsin B enzyme to the enzyme-responsive magnetic resonance imaging nanoprobe solution M-SPIO. Cathepsin B can specifically recognize and cleave the R-F site of the short peptide Cys-Phe-Arg-Phe-Lys-Cys, thereby decrosslinking the crosslinked enzyme-responsive magnetic resonance imaging nanoprobe and reducing the particle size after enzyme digestion.

[0077] e. The application scenarios of the enzyme-responsive magnetic resonance imaging nanoprobe include, but are not limited to, the visualization of the differentiation of human promyelocytic leukemia cells (HL60), the differentiation of mesenchymal stem cells (MSCs), and the differentiation of neural stem cells (NSCs) by combining imaging technology with nanoprobes.

[0078] This invention uses enzyme-responsive polypeptide sequences as cross-linking agents to cross-link monodisperse SPIO into nanoaggregates M-SPIO. After labeling cells, differentiation is induced. The difference in protease expressed by cells before and after differentiation can be used to decrosslink the nanoaggregates M-SPIO, thereby realizing the signal change from dark to bright on magnetic resonance T1-weighted and T2-weighted images. Thus, the visualization of cell differentiation can be achieved by combining magnetic nanoparticles with magnetic resonance imaging technology.

[0079] In this invention, MR imaging was used to determine the relaxation rate of SPIO-mal in aqueous solution and M-SPIO after cross-linking with CFRPKC peptide. The feasibility of M-SPIO cross-linking with aqueous solution was verified by measuring MRI changes before and after enzyme digestion using cathepsin B enzyme. Simultaneously, DLS was used to characterize the particle size before and after enzyme digestion, validating the MRI results. After 24 h of 1 mM M-SPIO labeling, cells were divided into two groups: one group was used to induce HL60 cell differentiation with TPA, and the other group was cultured normally as a control group. MRI was used to test and record signal changes.

[0080] A schematic diagram of the process for preparing enzyme-responsive magnetic resonance imaging nanoprobes and performing MR imaging in this invention is shown below. Figure 11 As shown.

[0081] The technical solution of the present invention will be further described in detail below with reference to several preferred embodiments and accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides detailed implementation methods and specific operation processes. However, the protection scope of the present invention is not limited to the following embodiments.

[0082] The following specific implementation plan takes the construction and application of enzyme-responsive MR nanoprobe M-SPIO as an example. Taking advantage of the high resolution and non-invasiveness of MRI, the probe is disassembled by cathepsin B enzyme specifically cleaving the peptide FRFK, realizing the "dark-to-bright" signal transition of MRI T2-weighted image, and visualizing the in vivo differentiation of human promyelocytic leukemia cells in real time.

[0083] Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0084] Example 1: Construction of SPIO-mal metal nanoparticles with surface functional group A

[0085] (1) Superparamagnetic iron oxide nanoparticles (SPIO) (30 mg) were dissolved in 5 mL THF. Citric acid (230 mg, 1.2 mmol) was added to the solution. The mixture was refluxed at 50 °C overnight under nitrogen protection. After the reaction was completed, excess THF was removed by rotary evaporation. 30 mL DMSO was added to remove excess citric acid from the product. The product was centrifuged at 13000 rpm for 30 minutes to obtain a black solid. The product was washed again with DMSO. This process was repeated three times to obtain the intermediate product SPIO-COOH, which was dissolved in DMSO for later use.

[0086] (2) SPIO-COOH (10 mg) was dissolved in a mixture of 5 mL DMSO and 5 mL water, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS) were added as reaction solution A; NH2-PEG-MAL (80 mg) was dissolved in 10 mL DMSO solution as reaction solution B; after complete dissolution, solution B was added to solution A, and the mixture was stirred at room temperature for 24 h. After the reaction was completed, the reaction mixture was slowly added dropwise to anhydrous ice-cold ether for precipitation treatment to obtain a precipitate. The precipitate was collected, dissolved in a small amount of ultrapure water, and the resulting solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da. The solution was dialyzed in ultrapure water for 3 days to obtain SPIO-PEG-MAL (abbreviated as SPIO-mal) for later use. The obtained products were characterized in morphology and particle size by electron microscopy (TEM) and dynamic light scattering (DLS). The T1 and T2 weighted signals at concentrations of 0.5, 1, 2, 4, and 8 mM were measured using an MRI system to analyze the quantitative relationship between concentration and T1 weighted signal. The results showed that the average particle size of SPIO-mal was approximately 5 nm (e.g., ...). Figure 1a Its hydrated particle size is 14 nm (e.g. Figure 1b The product exhibits good dispersibility. The T1-weighted signal intensity of SPIO-mal increases with increasing concentration (e.g., Figure 2 This indicates that SPIO-mal has a clear T1-dominant relaxation characteristic and can be used as a "positive contrast agent".

[0087] Example 2: The peptide sequence Cys-Phe-Arg—Phe-Lys-Cys, with functional groups B-terminated at both ends, was obtained by solid-phase synthesis.

[0088] Cysteine ​​Fmoc-Cys(trt)-OH, containing the active functional group thiol, was used as the first amino acid and attached to 2-chlorotriphenylmethyl chloride resin. The resin was then blocked with a blocking solution DMF:CH3OH = 19:1, and deprotected with 20% piperidine. Using HBTU / HOBT / DIPEA as coupling reagents, the amino acids in the enzyme-digested polypeptide sequence were sequentially coupled from the C-terminus to the N-terminus: Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH, Fmoc-Arg(pbf)-OH, and Fmoc-Phe-OH. The reaction was carried out under nitrogen atmosphere for 2 h. Subsequently, the resin lysis buffer TFA: benzyl sulfide: phenol: TIPS: ultrapure water = 82.5:7.5:5:3:2 was slowly stirred and reacted at low temperature for 0.5 h, followed by 2 h at room temperature. h. The FRFK fragment was cleaved from the resin, and the lysate was obtained by filtration. The lysate was slowly added to anhydrous ice-cold ether and stirred. The crude peptide was separated by filtration and washed three times with ice-cold ether to obtain the crude peptide. The molecular weight of the crude peptide was confirmed by mass spectrometry. It was then purified by high-performance liquid chromatography (HPLC), lyophilized, and the pure peptide CFRPKC was obtained. HPLC purification yielded a product purity >95%. The molecular weight of CFRPKC was 802.3, and 803.2 in the mass spectrometry results indicates [M+H]. + The molecular ion peak at 825.2 is [M+Na]. + The molecular ion peak at 402.2 is [M+2H]. 2+ peaks (such as) Figure 3 This indicates that the prepared enzyme digestion substrate CFRPKC, which serves as a cross-linking agent, has the correct structure.

[0089] Example 3: Construction of enzyme-responsive MRI nanoprobe M-SPIO

[0090] The SPIO-mal obtained in Example 1 was prepared into a 1 mM 3300 μL solution using PBS buffer, which was used as reaction solution A. 0.033 mmol (26.478 mg) of CFRPKC solid obtained in Example 2 was added to solution A, along with 0.099 mmol (28.378 mg) of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP). The reaction was carried out at room temperature for 4–8 h. After the reaction, the resulting solution was transferred to a dialysis bag with a molecular weight cutoff of 3000 Da and dialyzed in 25 mM MES hydrate solution (pH=5.0) for 36–48 h to obtain the enzyme-responsive MRI nanoprobe (M-SPIO). The morphology and particle size of M-SPIO were measured. The size of M-SPIO was 550 nm ± 50 nm (e.g., ...). Figure 4a , Figure 4b The results showed that the crosslinking agent CFRPKC successfully crosslinked SPIO into nano-aggregates.

[0091] Example 4: Solution digestion of enzyme-responsive MRI nanoprobe M-SPIO

[0092] After the dialysis in Example 3 was completed, the reaction liquid was collected and divided into two groups, one as the control group and the other as the experimental group. Cathepsin B enzyme (0.2 μg) was added to a small amount of 25 mM MES and 5 mM DTT solution (pH=5.0) and activated at room temperature for 30 min for later use.

[0093] The enzyme-containing activation solution was added to the experimental group, while the control group received an equal volume of MES buffer solution. The experimental group was then transferred to a 37°C water bath for enzyme digestion for 48 h. After the experiment, both the control and experimental groups underwent DLS characterization and MRI imaging. The aqueous solution enzyme digestion experiment, as shown by DLS, revealed that the M-SPIO particle size decreased from 550 nm ± 50 nm before digestion to 100 nm ± 20 nm after digestion (e.g., ...). Figure 5a , Figure 5b This confirms that cathepsin B enzyme effectively cleaves CFRPKC, achieving the enzymatic cleavage and cross-linking process.

[0094] MRI relaxation time and weighted images were measured in three groups (4 mM SPIO-mal, M-SPIO, and M-SPIO+Cat B). The results showed that the T2-weighted images exhibited a significant "bright-dark-bright" transition (e.g., Figure 6 This further demonstrates that crosslinking and decrosslinking of SPIO were achieved, and the crosslinking and decrosslinking processes were monitored using MRI.

[0095] Example 5: Enzyme-responsive MRI nanoprobe M-SPIO visualizes HL60 cell differentiation

[0096] After 2 days of culture, HL60 cells seeded in T25 cell culture flasks were cultured at a density of approximately 1 × 10⁻⁶ cells per dish. 6Cells were collected by centrifugation according to the above method. 2 mL of complete culture medium was added to the cell pellet, and the pellet was thoroughly dispersed. The pellet was then transferred to two wells of a 12-well plate, with 1 mL of complete culture medium added to each well. The synthesized enzyme-responsive magnetic resonance imaging nanoprobe M-SPIO was added to the cell culture medium to a final concentration of 50 μg / mL, and the cells were incubated for 4–8 h. After cell labeling, the cells were collected by centrifugation and washed three times with 2–3 mL of PBS to remove residual material. The cells were then divided into two groups: one group was incubated in IMDM complete culture medium with 48 nM TPA to induce differentiation of HL60 cells labeled with the enzyme-responsive MRI nanoprobe M-SPIO into macrophages; the other group served as a control, cultured normally in IMDM complete culture medium only. After co-incubation for 24 h, the cells were collected and washed. The cell pellet was immediately measured on a 7T small animal MRI scanner. On T2-weighted images, M-SPIO-labeled HL60 cells showed a significant dark signal, while the induced differentiated cells showed a significant bright signal, with a 175% increase in signal intensity on T2-weighted images (e.g., 175%). Figure 7 Experimental results showed that the enzyme-responsive MRI nanoprobe M-SPIO achieved enzymatic cleavage and cross-linking during the differentiation of HL60 cells into macrophages. MRI results provided clear imaging information on the cell differentiation process and outcome.

[0097] Example 6: Cell safety evaluation of enzyme-responsive MRI nanoprobe M-SPIO

[0098] M-SPIO was dissolved in IMDM medium to prepare solutions with concentrations of 0, 1, 2, 4, and 8 mM. HL60 cells were collected, and the cells were counted at a ratio of 10 cells per well. 5 Cells were added to 12-well cell culture plates and incubated at 37°C with 5% CO2 for 4–6 h. After incubation, the liquid in the 12-well plates was aspirated, and the plates were centrifuged twice at 800 rpm for 5 min to remove the material. The cells were then transferred to 96-well cell culture plates, with 200 μL of complete culture medium containing cells in each well, and 5 replicates for each concentration. Subsequently, 20 μL of a cell counting kit (CCK-8) reagent was added to each well, and the plates were incubated at 37°C with 5% CO2 for 2 h. A cytotoxicity test was then performed at a wavelength of 450 nm. Even at a concentration of 8 mM, the cell viability remained above 95% (e.g., ...). Figure 8 This indicates that the nanoprobe has low cytotoxicity and high safety.

[0099] Example 7: Enzyme-responsive MRI nanoprobe M-SPIO visualizes in vivo differentiation of HL60 cells

[0100] Six 4-6 week old Balb / c nude mice were randomly divided into a control group and a differentiation group, with three mice in each group. Their weight was recorded, and they were then anesthetized with isoflurane at a concentration of 5 mL / kg body weight. Subsequently, 10 mL / kg of isoflurane was injected into the left tibialis muscle group of each mouse. 6 HL60 cells were labeled and immediately injected with 100 μL of 5 mM TPA to induce in vivo differentiation. The control group received an equal volume of saline as a control. Immediately after transplantation, T2-weighted images of the labeled cells were measured on a 7T small animal MRI scanner, as 0D. Subsequently, 1D and 2D T2-weighted images were acquired, and signal changes in the T2-weighted images were observed.

[0101] At day 0, there was no significant difference in the transplantation site between the two groups on T2-weighted imaging. At days 1 and 2, high-signal areas appeared at the transplantation site in the TPA-induced differentiation group, while no significant signal changes were observed in the control group (e.g., Figure 9 MRI results showed that HL60 cells successfully differentiated into macrophages in vivo, and the highly expressed Cat B cleaved M-SPIO, resulting in signal enhancement in the local area and a "dark-to-bright" MRI signal transition.

[0102] Example 8: The peptide sequence Cys-Gly-Lys–Thr-Gly-Cys, with functional groups B-terminated at both ends, was obtained by solid-phase synthesis.

[0103] Cysteine ​​Fmoc-Cys(trt)-OH, containing the active functional group thiol, was used as the first amino acid and attached to 2-chlorotriphenylmethyl chloride resin. The resin was then blocked with a blocking solution DMF:CH3OH = 19:1, and deprotected with 20% piperidine. Using HBTU / HOBT / DIPEA as coupling reagents, the amino acids in the enzyme-digested polypeptide sequence were sequentially coupled from the C-terminus to the N-terminus: Fmoc-Gly-OH, Fmoc-Thr(OtBu)-OH, Fmoc-Lys(boc)-OH, Fmoc-Gly-OH, and Fmoc-Cys(trt)-OH. The reaction was carried out under nitrogen atmosphere for 2 h. Subsequently, the resin lysis buffer TFA: benzyl sulfide: phenol: TIPS: ultrapure water = 82.5:7.5:5:3:2 was slowly stirred and reacted at low temperature for 0.5 h, followed by 2 h at room temperature. h. The FRFK fragment was cleaved from the resin, and the lysate was obtained by filtration. The lysate was slowly added to anhydrous ice-cold ether and stirred. The crude peptide was separated by filtration and washed three times with ice-cold ether to obtain the crude peptide. The molecular weight of the crude peptide was confirmed by mass spectrometry. It was then purified by high-performance liquid chromatography (HPLC), lyophilized, and the pure peptide was obtained. HPLC purification yielded a product purity >95%, and the molecular weight of CGKTGC was 567.2. Mass spectrometry results showed that 568.2 is [M+H]. +The molecular ion peak at 284.7 is [M+2H]. 2+ peaks (such as) Figure 10 This indicates that the polypeptide sequence structure of the mesotrypsin enzyme response is correct.

[0104] Example 9

[0105] (1) Preparation of surface functionalized metal nanoparticles Gd2O3-N3

[0106] Gadolinium-based metal nanoparticles were prepared by ultrasound-assisted coprecipitation. First, gadolinium chloride hexahydrate (GdCl3·6H2O) was dissolved in tetraethylene glycol (TeEG) to prepare a 5 mM solution. The solution was sonicated for 5 minutes to form a clear solution. NaOH was added dropwise to this solution to adjust the pH to 11, resulting in a white precipitate of gadolinium hydroxide. Then, polylysine was dissolved in TeEG and added to the suspension. The final solution was sonicated at 350 W for 2 h at 60°C to obtain a surface-aminated Gd2O3 solution, which was dialyzed against 0.1 M PBS buffer for later use.

[0107] The ligand NHS-PEG-N3 was dissolved in 0.1M PBS buffer to prepare a 10 mM ligand solution. The Gd2O3 nanoparticle solution was then mixed with the ligand solution. The mixture was stirred at room temperature for 12 h, and then purified by dialysis in ultrapure water using a 10-20 kDa dialysis bag to obtain surface-azidized gadolinium trioxide nanoparticles Gd2O3-N3.

[0108] (2) Preparation of the peptide sequence Gly(CCH)-Pro-Leu-Gly—Leu-Lys-Ala-Arg-Gly(CCH) with β-functionalized ends

[0109] The polypeptide sequence Gd2O3-CCH-Pro-Leu-Gly—Leu-Lys-Ala-Arg-Gly(CCH) with B-terminal functional groups (B being alkyne) was obtained by solid-phase synthesis. Glycine (Fmoc-propargyl-Gly-OH), containing the active functional group alkynyl, was used as the first amino acid and attached to 2-chlorotriphenylmethyl chloride resin. The resin was then blocked with a blocking solution DMF:CH3OH = 19:1, and deprotected with 20% piperidine. Using HBTU / HOBT / DIPEA as coupling reagents, the amino acids in the enzyme-digested polypeptide sequence were sequentially coupled from the C-terminus to the N-terminus with Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Lys(boc)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, and Fmoc-propargyl-Gly-OH. The reaction was carried out under nitrogen atmosphere for 2 h. Subsequently, the reaction was carried out with resin lysis buffer TFA: benzyl sulfide: phenol: TIPS: ultrapure water = 82.5:7.5:5:3:2, with slow stirring, at low temperature for 0.5 h, and then at room temperature for 2 h. h, the polypeptide fragments were cut off from the resin, filtered to obtain the lysis buffer, the lysis buffer was slowly added to anhydrous ice-cold ether and stirred, the crude polypeptide was separated by filtration, washed 3 times with ice-cold ether to obtain the crude peptide, the molecular weight of the crude peptide was confirmed by mass spectrometry, purified and separated by high performance liquid chromatography, lyophilized, and then the pure polypeptide was obtained.

[0110] (3) Preparation of enzyme-responsive magnetic resonance imaging nanoprobes

[0111] The surface-azidized Gd-based nanoparticles obtained in the previous step were prepared into a 1 mM solution using ultrapure water, which was designated as reaction solution A. Gly(CCH)-Pro-Leu-Gly—Leu-Lys-Ala-Arg-Gly(CCH) solid was added to solution A to bring the final concentration to 5 mM, along with a catalytic amount of cuprous bromide (CuBr). The mixture was immediately evacuated and purged with nitrogen, and reacted at room temperature for 24 h. After the reaction, the resulting solution was transferred to a dialysis bag with a molecular weight cutoff of 10-20 kDa and dialyzed in 50 mM hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES) (pH=7.5) for 36-48 h to obtain the nanoprobe M-Gd.

[0112] Example 10

[0113] (1) Preparation of surface functionalized metal nanoparticles Mn3O4-CBT

[0114] Gadolinium-based metal nanoparticles were prepared by an oxidation precipitation method. First, manganese sulfate (MnSO4) and citric acid were dissolved in ultrapure water at a molar ratio of 1:2 to form a clear solution. NaOH was then added dropwise to the solution to adjust the pH to 11, resulting in a white precipitate of manganese hydroxide (Mn(OH)2). The solution was then heated to reflux at 90 °C, and hydrogen peroxide was added to the suspension until it turned dark brown. Reflux was continued for another 4 h to obtain a surface-carboxylated Mn3O4 solution, which was dialyzed with ultrapure water and then freeze-dried for later use.

[0115] Mn3O4 nanoparticles were dissolved in tetrahydrofuran to form a 5 mM solution. Equimolar amounts of isobutyl chloroformate and 4-methylmorpholine were added, and the mixture was stirred in an ice bath for 0.5 h. An equimolar amount of the ligand 6-amino-2-cyanobenzothiazole (CBT) was then added to the solution, and the reaction was stirred in an ice bath for 1 h. The mixture was then purified by dialysis in 0.1 M PBS buffer using a 10-20 kDa dialysis bag to obtain manganese oxide nanoparticles Mn3O4-CBT with 6-amino-2-cyanobenzothiazole residues on their surface.

[0116] (2) Preparation of Cys-Arg-Arg—Cys polypeptide sequence with β-functionalized ends

[0117] The Cys-Arg-Arg—Cys polypeptide sequence with β-functionalized ends was obtained by solid-phase synthesis. The amino acid containing the active functional group Fmoc-Cys(trt)-OH was attached to 2-chlorotriphenylmethyl chloride resin as the first amino acid. The resin was then blocked with a blocking solution DMF:CH3OH = 19:1, and deprotected with 20% piperidine. Using HBTU / HOBT / DIPEA as coupling reagents, the amino acids in the enzymatically digested polypeptide sequence were sequentially coupled from the C-terminus to the N-terminus with Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, and Fmoc-Cys(trt)-OH. The reaction was carried out under nitrogen atmosphere for 2 h. Subsequently, the reaction was carried out at low temperature for 0.5 h with slow stirring using a resin lysis buffer TFA: benzyl sulfide: phenol: TIPS: ultrapure water = 82.5:7.5:5:3:2. The reaction was carried out at room temperature for 2 hours. The peptide fragments were cut off from the resin and filtered to obtain the lysis buffer. The lysis buffer was slowly added to anhydrous ice-cold ether and stirred. The crude peptide was separated by filtration and washed three times with ice-cold ether to obtain the crude peptide. The molecular weight of the crude peptide was confirmed to be correct by mass spectrometry. It was purified and separated by high performance liquid chromatography, lyophilized, and then the pure peptide was obtained.

[0118] (3) Preparation of enzyme-responsive magnetic resonance imaging nanoprobes: Mn3O4-CBT nanoparticles with surface modification of 6-amino-2-cyanobenzothiazole residues obtained in the previous step were prepared into a 1 mM solution using PBS buffer solution, which is reaction solution A. An equimolar amount of Cys-Arg-Arg-Cys solid and an equimolar amount of tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) were added to solution A, and the pH was adjusted to 7.4. The reaction was carried out at room temperature for 4-8 h. After the reaction was completed, the resulting solution was transferred to a dialysis bag with a molecular weight cutoff of 10-20 kDa and dialyzed in 25 mM MES hydrate solution (pH=5.0) for 36-48 h to obtain the nanoprobe M-Mn.

[0119] In addition, the inventors of this case also conducted experiments with other raw materials, process operations, and process conditions described in this specification, referring to the aforementioned embodiments, and obtained relatively ideal results in all cases.

[0120] It should be understood that the technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made to the technical solutions of the present invention without departing from the spirit and scope of the claims are within the scope of protection of the present invention.

Claims

1. An enzyme-responsive magnetic resonance imaging nanoprobe, characterized in that, include: The metal nanoparticles with surface functional groups A serve as the basic imaging unit, and the polypeptide sequences with end functional groups B serve as the crosslinking agent and enzyme response unit; the enzyme-responsive magnetic resonance imaging nanoprobe is a metal nanoparticle crosslink formed by crosslinking the metal nanoparticles through the polypeptide sequences. Wherein, in the surface-functionalized metal nanoparticles, functional group A is selected from maleimide residues containing double bonds; the metal nanoparticles are selected from MRI iron-based iron oxide nanoparticles; the particle size of the MRI iron-based iron oxide nanoparticles is less than 5 nm. The functional group B in the polypeptide sequence with B-functionalized ends is selected from cysteine ​​residues; the polypeptide sequence includes a cathepsin digestion sequence, which includes B-Phe-Arg—Phe-Lys-B, wherein B is a sulfhydryl-containing cysteine ​​residue.

2. The method for preparing enzyme-responsive magnetic resonance imaging nanoprobes as described in claim 1, characterized in that, include: Enzyme-responsive magnetic resonance imaging nanoprobes were prepared by clicking reactions between surface-functionalized A-type metal nanoparticles and end-functionalized B-type polypeptide sequences under activating conditions.

3. The preparation method according to claim 2, characterized in that: The molar ratio of the surface-functionalized metal nanoparticles (A-type) to the peptide sequences with end-functionalized peptides (B-type) is 1:1~10.

4. The preparation method according to claim 2, characterized in that: The activator includes any one or more combinations of tris(2-carboxyethyl) phosphate hydrochloride for activating thiol groups, glutathione for activating thiol groups, and CuBr for activating alkynyl groups.

5. The preparation method according to claim 2, characterized in that: The particle size of the enzyme-responsive magnetic resonance imaging nanoprobe is 10 nm to 1000 nm.

6. The preparation method according to claim 2, characterized in that... Also includes: After the click reaction is completed, the obtained product is subjected to dialysis.

7. The preparation method according to claim 2, characterized in that, Specifically, it includes: Functional group A is modified on the surface of metal nanoparticles by ligand exchange or covalent bonding with bifunctional crosslinking agents.

8. The preparation method according to claim 2, characterized in that, Specifically, it includes: A polypeptide sequence with β-functionalized functional groups at both ends was synthesized using a solid-phase synthesis method.

9. A method for visualizing cell differentiation for non-diagnostic purposes, characterized in that, include: The system comprising cells and the enzyme-responsive magnetic resonance imaging nanoprobe of claim 1 is used to differentiate cells, and MRI imaging is used for detection to visualize the cell differentiation; wherein the cells are human promyelocytic leukemia cells.

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