Copper ion response type magnetic resonance imaging probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological imaging probes, and discloses a copper ion response type magnetic resonance imaging probe, a preparation method of the copper ion response type magnetic resonance imaging probe and application of the copper ion response type magnetic resonance imaging probe in preparation of a tumor magnetic resonance imaging contrast agent. The preparation method of the probe comprises the following steps: firstly, preparing oil-phase superparamagnetic iron oxide nanoparticles (SPIONs), and then carrying out water-soluble ligand modification on the oil-phase superparamagnetic iron oxide nanoparticles to obtain water-phase dispersed superparamagnetic iron oxide nanoparticles; then covalently linking the micromolecule ligand with the SPIONs through amidation coupling reaction, and complexing with gadolinium ions to obtain the probe. The probe has high sensitivity and dynamic response capability, is suitable for accurate diagnosis of copper ion steady state imbalance related diseases (such as tumors, cardiovascular diseases and neurodegenerative diseases), and especially can be used for malignant tumor identification and treatment effect monitoring.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological imaging probes, and relates to a copper ion responsive magnetic resonance imaging probe and a preparation method thereof, and application of the probe in preparation of a tumor magnetic resonance imaging contrast agent. BACKGROUND

[0002] Copper ions (Cu 2+ ) play a key role in various cell physiological processes and are significantly involved in the progression of malignant tumors. Imbalance of copper homeostasis accelerates tumorigenesis by promoting key processes such as angiogenesis, metastasis and cell proliferation. In many high-incidence malignant tumors such as breast cancer, lung cancer, colorectal cancer and hepatocellular carcinoma, the phenomenon of abnormal increase in copper ion concentration in tumor tissues is observed. Therefore, accurate imaging of endogenous Cu 2+ has great significance for in-depth understanding of tumor biological behavior, establishment of an effective diagnosis and prognosis biomarker system, and guidance for the development and evaluation of copper-targeted treatment strategies. However, existing technologies are difficult to achieve non-invasive in vivo detection of Cu 2+ in tumors at a physiologically relevant concentration.

[0003] Although fluorescence imaging technology exhibits high sensitivity at the cellular level, its tissue penetration depth is limited and it is difficult to be applied to in vivo research of large animal models or human bodies. In contrast, magnetic resonance imaging (MRI) has become an ideal tool for monitoring the spatiotemporal dynamic changes of Cu 2+ in tumors due to its excellent tissue contrast and deep tissue penetration capability. However, the application of MRI imaging probes faces many challenges, including inefficient delivery of the probes in vivo, complex interference of the biological molecular environment, etc. This requires Cu 2+ MRI probes for tumor diagnosis and monitoring to have extremely high sensitivity to detect small concentration changes of key biomarkers in the physiological range. The current mainstream Cu 2+ MRI probes are mainly designed based on coordination binding mechanism, and their performance (especially sensitivity) is highly dependent on the binding constant of the probe and Cu 2+ . This mechanism has inherent defects: the binding behavior of the probe to Cu 2+ itself consumes the local ion concentration, causing fluctuations in the actual Cu 2+ concentration at the detection site, and thus affecting the accuracy of detection. Therefore, existing probes based on binding often exhibit insufficient sensitivity in in vivo applications, especially in the detection of extremely low concentration differences required for tumor diagnosis.

[0004] Recent advances in the field of reaction-active chemical probes provide a new approach to the design of Cu 2+The realization of high sensitivity and high selectivity imaging of metal ions in vivo brings great hope. The core principle of such probes is to use the inherent catalytic activity of the target metal ion (such as Cu 2+ ) to drive specific chemical reactions, thereby triggering significant structural or conformational changes in the probe molecules (for example, cleavage of chemical bonds, molecular assembly or release / activation of signal reporting groups). Compared with traditional binding-type probes that rely on a single binding event to generate a static signal, this reaction activity-based strategy has a significant advantage: a catalytically active metal ion can trigger the conversion reaction of multiple probe molecules, achieving dynamic amplification of the signal, thereby theoretically greatly improving the detection sensitivity. Therefore, this method is considered an effective way to achieve high-sensitivity detection of analytes with reaction activity (such as Cu 2+ ). However, although reaction activity-based chemical probes conceptually exhibit great potential, the concept has not yet been fully realized as an imaging system that can effectively and practically visualize endogenous Cu 2+ in vivo. There is still a significant gap in this technical field: the lack of practical probe design that can detect endogenous Cu 2+ ions (especially for extremely low concentration differences present in malignant tumors) with high accuracy and high sensitivity in a complex in vivo environment. SUMMARY

[0005] To solve the above problems of the prior art, the present application provides a copper ion responsive magnetic resonance imaging probe and a preparation method thereof, which combines active chemical probe design with distance-dependent magnetic resonance energy transfer, thereby improving the response sensitivity of the nanoprobe to Cu 2+ and achieving high-sensitivity MR imaging of the malignancy degree of Cu 2+ related tumors in vivo.

[0006] The present application also provides the use of the probe in the preparation of a tumor magnetic resonance imaging contrast agent. The probe is composed of superparamagnetic iron oxide nanoparticles (SPIONs) and paramagnetic gadolinium-based small molecule complexes connected by covalent bonds, and its signal response characteristics are dynamically related to the concentration of copper ions. Under normal physiological copper ion concentration, the magnetic interaction with SPIONs inhibits the T1 signal of the gadolinium-based complex; when the copper ion concentration pathologically increases, the covalent bond breaks, causing the gadolinium-based complex to be released, the distance between SPIONs and the gadolinium-based molecule increases, and the T1 signal enhancement ability is restored.

[0007] The preparation method of the copper ion responsive magnetic resonance imaging probe of the present application comprises the following steps:

[0008] S1: under inert atmosphere, a solution of iron oleate and 1-octadecene is placed at 200-320℃ for 10-60 minutes, and after treatment, oil phase superparamagnetic iron oxide nanoparticles are obtained;

[0009] S2: under inert atmosphere, the oil phase superparamagnetic iron oxide nanoparticles are added to a polar-nonpolar mixed solvent, and a hydrophilic molecule is added, and the mixture is reacted at 90-200℃ for 8-24 hours, and after treatment, water phase superparamagnetic iron oxide nanoparticles are obtained, wherein the hydrophilic molecule is one or more of polyethylene glycol, polyvinylpyrrolidone, polydopamine, citric acid or mercaptopropionic acid;

[0010] S3: the water phase superparamagnetic iron oxide nanoparticles and a catalyst are added to a good solvent, and a small molecule ligand is added, and the mixture is stirred at room temperature to obtain functionalized iron oxide nanoparticles; wherein the catalyst is one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexyl carbodiimide or N-hydroxysuccinimide, and the small molecule ligand is one or more of maleimide, adipic acid dihydrazide, diethylenetriamine pentaacetic acid or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid;

[0011] S4: the functionalized iron oxide nanoparticles and a gadolinium ion compound are added to a good solvent, and the mixture is stirred at room temperature to obtain a copper ion responsive magnetic resonance imaging probe.

[0012] In an embodiment of the present application, preferably, in step S1, the feeding ratio of iron oleate, oleic acid and 1-octadecene is 1-10 mmol: 0.025-10 mL: 10-200 mL.

[0013] In an embodiment of the present application, preferably, the after-treatment of step S1 includes first precipitation with a polar solvent, and then centrifugation and washing to obtain the oil phase superparamagnetic iron oxide nanoparticles, wherein the polar solvent is one or more of anhydrous ethanol, acetone, methanol, isopropanol or acetic acid.

[0014] In an embodiment of the present application, preferably, in step S2, the feeding ratio of the oil phase superparamagnetic iron oxide nanoparticles and the hydrophilic molecule is 10-50 mg: 20-300 mg.

[0015] In an embodiment of the present application, preferably, in the polar-nonpolar mixed solvent of step S2, the volume ratio of the polar solvent to the nonpolar solvent is 1:1-1:3; wherein the polar solvent is one or more of acetone, ethanol, N,N dimethylformamide, dimethyl sulfoxide or tetrahydrofuran, and the nonpolar solvent is one or more of o-dichlorobenzene, chloroform, n-hexane, cyclohexane or toluene.

[0016] In an embodiment of the present application, preferably, the post-processing of step S2 comprises precipitation with ether, followed by centrifugation and washing with acetone to obtain the aqueous superparamagnetic iron oxide nanoparticles.

[0017] In an embodiment of the present application, preferably, the gadolinium ion compound of step S4 is one or more of gadolinium acetylacetonate, gadolinium acetate, gadolinium nitrate or gadolinium chloride hexahydrate.

[0018] In an embodiment of the present application, preferably, the good solvent of steps S3 and S4 is one or more of 2-(N-morpholino)ethanesulfonic acid buffer, phosphate buffer solution, deionized water, dimethyl sulfoxide, N,N dimethylformamide, ethanol or acetonitrile.

[0019] The present application also provides a copper ion responsive magnetic resonance imaging probe prepared by the above preparation method.

[0020] The present application also provides the use of the above copper ion responsive magnetic resonance imaging probe in the preparation of a tumor magnetic resonance imaging contrast agent, such as tumor diagnosis, malignancy differentiation or monitoring of copper chelation therapy for tumors.

[0021] Compared with the prior art, the present application has the following advantages:

[0022] The copper ion responsive magnetic resonance imaging probe of the present application, by combining active chemical probe design with distance-dependent magnetic resonance energy transfer, improves the response sensitivity of the nanoprobe to Cu 2+ , and realizes high-sensitivity MR imaging of Cu 2+ related tumor malignancy. First, after the probe binds to Cu 2+ , the hydrazine structure is destroyed by Cu 2+ catalyzed chemical reaction, and the paramagnetic gadolinium-based small molecules released gradually move away from the superparamagnetic iron oxide nanoparticles due to random thermal motion, resulting in the failure of the magnetic resonance energy transfer mechanism and the restoration of the T1 signal enhancement ability of the paramagnetic center. Second, as a catalytic component, the released Cu 2+ can participate in catalytic reactions multiple times, driving signal amplification based on reaction activity. Therefore, the probe has extremely high copper ion detection sensitivity and dynamic response capability, can respond to abnormal pathological copper ion levels, accelerate the T1 relaxation of water protons, and has significant magnetic resonance T1 imaging effects in copper ion imbalance related disease diagnosis, and is suitable for precise diagnosis of copper ion homeostasis imbalance related diseases (such as tumors, cardiovascular diseases and neurodegenerative diseases), and can be used for tumor malignancy differentiation and monitoring of the efficacy of copper ion chelation therapy for tumors.

[0023] The preparation method of the present application has controllable reaction conditions, and the prepared product has good biocompatibility and extremely high stability. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Cu for the embodiment 1 of the present application 2+ Schematic diagram of the mechanism of the ion-responsive magnetic resonance imaging probe;

[0025] Figure 2 Transmission electron micrograph of the oil phase superparamagnetic iron oxide nanoparticles for the embodiment 1 of the present application;

[0026] Figure 3 Transmission electron micrograph of the water phase superparamagnetic iron oxide nanoparticles for the embodiment 1 of the present application;

[0027] Figure 4 Transmission electron micrograph of the functionalized iron oxide nanoparticles for the embodiment 1 of the present application;

[0028] Figure 5 Cu for the embodiment 1 of the present application 2+ Transmission electron micrograph of the ion-responsive magnetic resonance imaging probe;

[0029] Figure 6 Cu for the embodiment 1 of the present application 2+ Hydrodynamic particle size diagram of the ion-responsive magnetic resonance imaging probe;

[0030] Figure 7 Cu for the test example 1 2+ Magnetic resonance imaging diagram of the ion-responsive magnetic resonance imaging probe in different metal ion solutions;

[0031] Figure 8 Cu for the test example 2 2+ Magnetic resonance imaging diagram of the ion-responsive magnetic resonance imaging probe in different concentrations of copper ion solutions;

[0032] Figure 9 Magnetic resonance imaging diagram of the ion-responsive magnetic resonance imaging probe and physiological saline for the tumor-bearing mouse in the application example 1 2+ Magnetic resonance imaging diagram of the ion-responsive magnetic resonance imaging probe and physiological saline for the tumor-bearing mouse in the application example 1

[0033] Figure 10 Magnetic resonance imaging diagram of the ion-responsive magnetic resonance imaging probe and physiological saline for the tumor-bearing mouse in the application example 2 2+ Magnetic resonance imaging diagram of the ion-responsive magnetic resonance imaging probe and physiological saline for the tumor-bearing mouse in the application example 2

[0034] Figure 11 Transmission electron micrograph of the oil phase superparamagnetic iron oxide nanoparticles for the embodiment 2 of the present application;

[0035] Figure 12 Transmission electron micrograph of the oil phase superparamagnetic iron oxide nanoparticles for the embodiment 3 of the present application. DETAILED DESCRIPTION

[0036] In order to make the above objectives, features and advantages of the present application more obvious and comprehensible, the specific embodiments of the present application are described in detail below with reference to the drawings.

[0037] In the following description, a lot of specific details are set forth in order to provide a thorough understanding of the present application. However, the present application can be practiced in a variety of ways beyond those described herein, and it is understood that similar modifications of the present application will occur to the skilled in the art. Therefore, the present application is not intended to be limited to the particular embodiments disclosed in the following description.

[0038] Embodiment 1

[0039] The preparation method of the copper ion responsive magnetic resonance imaging probe according to the embodiment 1 of the present application comprises the following steps:

[0040] S1: 1.0 mmol of iron oleate and 0.032 mL of oleic acid are dissolved in 10 mL of 1-octadecene, and reacted under the condition of inert atmosphere at 300℃ for 20 minutes. After the reaction, the product is precipitated with acetone, and then centrifuged and washed to obtain oil-phase superparamagnetic iron oxide nanoparticles;

[0041] The oil-phase superparamagnetic iron oxide nanoparticles prepared are subjected to transmission electron microscopy for morphology characterization, as shown in FIG. 1, which shows that the oil-phase superparamagnetic iron oxide nanoparticles are spherical nanostructures with a size of about 8.5 nm; Figure 2

[0042] S2: 50 mg of the oil-phase superparamagnetic iron oxide nanoparticles are added to a mixed solution of o-dichlorobenzene and N,N-dimethylformamide (volume ratio of 1:1), followed by the addition of 100 mg of citric acid. The mixture is ultrasonically dissolved until the solution is clear and transparent, and then reacted under anaerobic condition at 100℃ for 24 hours. After the reaction, the product is first precipitated with ether and centrifuged, and then washed with acetone for multiple times and dissolved in water to obtain water-phase superparamagnetic iron oxide nanoparticles;

[0043] The water-phase superparamagnetic iron oxide nanoparticles prepared are subjected to transmission electron microscopy for morphology characterization, as shown in FIG. 2, which shows that the water-phase superparamagnetic iron oxide nanoparticles maintain the basic structure of the above oil-phase iron oxide nanoparticles, and have good dispersibility in water medium; Figure 3

[0044] ​​S3: 10 mg of aqueous superparamagnetic iron oxide nanoparticles, 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 10 mg of N-hydroxysuccinimide were added to 10 mL of 2-(N-morpholino)ethanesulfonic acid buffer and stirred at room temperature for 1 hour. Subsequently, 10 mg of adipic acid dihydrazide and 20 mg of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid were added to the above mixture, and the reaction was stirred at room temperature for 24 hours. Functionalized iron oxide nanoparticles were obtained by dialysis and centrifugation.

[0045] The morphology of the prepared functionalized iron oxide nanoparticles was characterized by transmission electron microscopy, such as... Figure 4 As shown, the functionalized iron oxide nanoparticles maintain the basic structure of the above-mentioned aqueous iron oxide nanoparticles, while exhibiting good dispersibility in aqueous media.

[0046] S4: 10 mg of functionalized iron oxide nanoparticles and 2 mg of gadolinium chloride hexahydrate were dispersed in 10 mL of deionized water and reacted at room temperature for 12 hours. Cu was obtained by dialysis and centrifugation. 2+ Ion-responsive magnetic resonance imaging probe.

[0047] For the obtained Cu 2+ Ion-responsive magnetic resonance imaging probes were used for morphological characterization using transmission electron microscopy, such as... Figure 5 As shown, this Cu 2+ The ion-responsive magnetic resonance imaging probe maintained the basic structure of the aforementioned aqueous iron oxide nanoparticles while exhibiting good dispersibility in aqueous media. Its hydrodynamic particle size was measured using dynamic light scattering, and the results are as follows: Figure 6 As shown, the imaging probe's size increases slightly (26.3 nm) after stepwise modification with ligands, and it exhibits good dispersibility in aqueous media.

[0048] The Cu 2+ The structure and mechanism of action of ion-responsive magnetic resonance imaging probes are as follows: Figure 1 As shown, the probe consists of superparamagnetic iron oxide nanoparticles linked by an acylhydrazine structure and a paramagnetic small molecule Gd complex. Due to the magnetic interaction between the superparamagnetic iron oxide nanoparticles and the neighboring paramagnetic small molecule Gd complex, magnetic resonance energy transfer and T1 magnetic resonance signal quenching occur. When this probe interacts with Cu... 2+ After ion contact, Cu 2+ Ions specifically catalyze the hydrolytic breakage of the hydrazide structure, thereby releasing a small Gd-based complex, which gradually moves away from the superparamagnetic iron oxide nanoparticles due to thermal motion. When the distance between the superparamagnetic iron oxide nanoparticles and the paramagnetic small Gd-based complex increases and exceeds the critical distance for magnetic interaction, the T1 signal of the small Gd-based complex can be recovered.

[0049] Test Example 1: Cu of the magnetic resonance imaging probe 2+ Specificity verification

[0050] To verify the selectivity of the probe of the present invention for different metal ions, Cu from Example 1 was used. 2+ Ion-responsive magnetic resonance imaging probes are dispersed in 20 μM of common metal ions (K+). + Na + Cu + Ca 2+ Mg 2+ ,Zn 2+ Mg 2+ Cu 2+ ,Fe 3+ In solution, the effects of different types of metal ion treatments on Cu were tested. 2+ T1 magnetic resonance signal of ion-responsive magnetic resonance imaging probe solution.

[0051] Cu 2+ The magnetic resonance imaging performance of ion-responsive magnetic resonance imaging probes in different metal ion solutions is as follows: Figure 7 As shown, this indicates that the probe can specifically respond to Cu. 2+ Ions, and produce changes in magnetic resonance signals.

[0052] Test Example 2: Cu of the magnetic resonance imaging probe 2+ Concentration sensitivity test

[0053] To evaluate the probe of the present invention in different Cu 2+ Relaxation properties under concentration conditions, comparing Cu from Example 1 2+ Ion-responsive magnetic resonance imaging probes were dispersed in CuCl2 aqueous solutions of different concentrations (0, 0.1, 1, 5, 10, 20, 40, and 80 μM). A 3T magnetic resonance scanner was used to test the CuCl2 concentrations. 2+ Under ion treatment conditions, Cu 2+ T1 magnetic resonance signal of ion-responsive magnetic resonance imaging probe solution.

[0054] Cu 2+ Ion-responsive magnetic resonance imaging probes at different concentrations of Cu 2+ Magnetic resonance imaging performance in ionic solutions, such as Figure 8 As shown, this indicates that the probe is capable of detecting Cu. 2+ Ions produce concentration-dependent magnetic resonance signal changes, and this is possible even for Cu atoms as low as 10 μM. 2+ Ionic solutions produce highly sensitive response signal changes.

[0055] Application Example 1

[0056] Cu of the embodiment 1 of the application 2+ Application of the copper ion responsive magnetic resonance imaging probe in magnetic resonance imaging of triple negative breast cancer: first establish a triple negative breast cancer model in female BALB / c mice, and the tumor size is less than 500mm 3 . Inject Cu 2+ ion responsive magnetic resonance imaging probe through the tail vein, and observe the change of the magnetic resonance imaging signal in the breast cancer lesion area after injection by using a 9.0T magnetic resonance imaging device, as shown in Figure 9 . After 8 hours of injection of the probe, the magnetic resonance signal in the tumor area is obviously different from that in the surrounding normal muscle tissue, which proves that the Cu 2+ ion responsive magnetic resonance imaging probe can realize accurate detection of malignant tumors.

[0057] Application example 2

[0058] Cu of the embodiment 1 of the application 2+ Application of the copper ion responsive magnetic resonance imaging probe in magnetic resonance imaging of benign and malignant tumors: first establish a benign and malignant tumor model in female BALB / c nude mice, and the tumor size is less than 500mm 3 . Inject Cu 2 + ion responsive magnetic resonance imaging probe through the tail vein, and observe the change of the magnetic resonance imaging signal in the benign and malignant tumor lesion area after injection by using a 9.4T magnetic resonance imaging device, as shown in Figure 10 . After 8 hours of injection of the probe, the magnetic resonance signal in the malignant tumor area is obviously different from that in the benign tumor area, which proves that the Cu 2+ ion responsive magnetic resonance imaging probe can realize accurate identification of malignant tumors.

[0059] Embodiment 2

[0060] The preparation method of the copper ion responsive magnetic resonance imaging probe in the embodiment 2 of the application comprises the following steps:

[0061] S1: dissolve 2.0mmol of iron oleate and 0.032mL of oleic acid in 20mL of 1-octadecene, and react under the condition of inert atmosphere at 300℃ for 20 minutes; after the reaction is completed, precipitate with acetone, and then centrifuge and wash to obtain oil-phase superparamagnetic iron oxide nanoparticles;

[0062] Perform morphology characterization on the prepared oil-phase superparamagnetic iron oxide nanoparticles by transmission electron microscopy, as shown in Figure 11 , which shows that the nanoparticles are uniform spherical nanostructures with a size of about 15nm, meeting the particle size requirement of the nano-probe;

[0063] S2: Take 50 mg of oil phase superparamagnetic iron oxide nanoparticles and add to a mixed solution of o-dichlorobenzene and N,N dimethylformamide (volume ratio of both is 1:1), then add 300 mg of citric acid, and fully dissolve by ultrasonic to make the solution clear and transparent, and then react under anaerobic conditions at 110 DEG C for 24 hours. After the reaction is completed, first precipitate with ether and centrifuge, then wash with acetone several times, and then dissolve in water to obtain water phase superparamagnetic iron oxide nanoparticles;

[0064] S3: Take 10 mg of water phase superparamagnetic iron oxide nanoparticles, 10 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 10 mg of N-hydroxysuccinimide and add to 10 mL of 2-(N-morpholine)ethanesulfonic acid buffer solution, and stir at room temperature for 1 hour. Then add 10 mg of adipic acid dihydrazide and 20 mg of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the above mixture, and stir at room temperature for 24 hours. Centrifuge and dialyze to obtain functionalized iron oxide nanoparticles;

[0065] S4: Take 10 mg of functionalized iron oxide nanoparticles and 2 mg of gadolinium chloride hexahydrate and disperse in 10 mL of deionized water, and react at room temperature for 12 hours. Centrifuge and dialyze to obtain Cu 2+ ion responsive magnetic resonance imaging probes.

[0066] According to the test method of the above application example 1 of embodiment 1, the copper ion responsive magnetic resonance imaging probe of embodiment 2 is respectively applied to the magnetic resonance imaging of triple negative breast cancer and benign and malignant tumors, and the experimental method is the same as that of application examples 1-2. The test results show that the Cu 2+ ion responsive magnetic resonance imaging probe of embodiment 2 realizes precise detection and identification of malignant tumors.

[0067] Embodiment 3

[0068] The preparation method of the copper ion responsive magnetic resonance imaging probe of embodiment 3 of the application comprises the following steps:

[0069] S1: Dissolve 1.0 mmol of iron oleate and 0.032 mL of oleic acid in 10 mL of 1-octadecene, and react under inert gas conditions at 300 DEG C for 20 minutes. After the reaction is completed, precipitate with acetone, and then centrifuge and wash to obtain oil phase superparamagnetic iron oxide nanoparticles;

[0070] The oil phase superparamagnetic iron oxide nanoparticles prepared are subjected to transmission electron microscopy for morphology characterization, as shown in FIG. 1, which shows that the oil phase iron oxide nanoparticles are cubic nanostructures with uniform morphology and size, and the size is about 13 nm, which meets the requirements of nano probe morphology and particle size; Figure 12

[0071] ​S2: Take 50 mg of oil phase superparamagnetic iron oxide nanoparticles into a mixed solution of o-dichlorobenzene and N, N dimethylformamide (volume ratio of 1:1), then add 100 mg of mercaptopropionic acid, and fully dissolve by ultrasonic until the solution is clear and transparent. React at 100℃ under anaerobic conditions for 24 hours. After the reaction is completed, first precipitate with ether and centrifuge, then wash with acetone several times, and then dissolve in water to obtain water phase superparamagnetic iron oxide nanoparticles;

[0072] S3: Take 10 mg of water phase superparamagnetic iron oxide nanoparticles and 10 mg of dicyclohexyl carbodiimide into 10 mL of 2-(N-morpholine) ethanesulfonic acid buffer, and stir at room temperature for 1 hour. Then add 10 mg of adipic acid dihydrazide and 20 mg of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the above mixture, and stir at room temperature for 24 hours. Centrifuge and dialyze to obtain functionalized iron oxide nanoparticles;

[0073] S4: Take 10 mg of functionalized iron oxide nanoparticles and 4 mg of gadolinium nitrate and disperse in 10 mL of deionized water, and react at room temperature for 12 hours. Centrifuge and dialyze to obtain Cu 2+ ion responsive magnetic resonance imaging probe.

[0074] According to the test method of the above-mentioned test example 1, the Cu 2+ specificity and sensitivity of the copper ion responsive magnetic resonance imaging probe of example 3 were tested respectively. The test results show that the Cu 2+ ion responsive magnetic resonance imaging probe of the present example 3 can specifically respond to Cu 2+ ions and produce changes in magnetic resonance signals, and can produce concentration-dependent changes in magnetic resonance signals for Cu 2+ ions, and can produce high sensitivity response signal changes for a minimum of 10 μM Cu 2+ ion solution.

[0075] The preferred embodiments of the present application disclosed above are only used to help explain the present application. The preferred embodiments do not describe all the details, nor limit the present application to the specific embodiments described. Obviously, many modifications and changes can be made according to the content of the present specification. The present specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present application, so that those skilled in the art can well understand and utilize the present application. The present application is limited only by the claims and their full scope and equivalents.

Claims

1. A method for preparing a copper ion-responsive magnetic resonance imaging probe, characterized by, The method comprises the following steps: S1: under inert atmosphere, a solution of iron oleate and oleic acid in 1-octadecene is placed at 200-320℃ for 10-60 minutes, and after treatment, oil-phase superparamagnetic iron oxide nanoparticles are obtained; S2: under inert atmosphere, the oil-phase superparamagnetic iron oxide nanoparticles are added to a polar-nonpolar mixed solvent, and a hydrophilic molecule is added, and the mixture is reacted at 90-200℃ for 8-24 hours, and after treatment, water-phase superparamagnetic iron oxide nanoparticles are obtained, wherein the hydrophilic molecule is one or more of polyethylene glycol, polyvinylpyrrolidone, polydopamine, citric acid or mercaptopropionic acid; S3: the water-phase superparamagnetic iron oxide nanoparticles and a catalyst are added to a good solvent, and a small-molecule ligand is added, and the mixture is stirred at room temperature to obtain functionalized iron oxide nanoparticles; wherein the catalyst is one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexyl carbodiimide or N-hydroxysuccinimide, and the small-molecule ligand is one or more of maleimide, adipic acid dihydrazide, diethylenetriamine pentaacetic acid or 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid; S4: the functionalized iron oxide nanoparticles and a gadolinium ion compound are added to a good solvent, and the mixture is stirred at room temperature to obtain the copper ion responsive magnetic resonance imaging probe.

2. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. In step S1, the feeding ratio of iron oleate, oleic acid and 1-octadecene is 1-10 mmol: 0.025-10 mL: 10-200 mL.

3. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. The after-treatment of step S1 comprises first precipitation with a polar solvent, and then centrifugation and washing to obtain the oil-phase superparamagnetic iron oxide nanoparticles, wherein the polar solvent is one or more of anhydrous ethanol, acetone, methanol, isopropanol or acetic acid.

4. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. In step S2, the feeding ratio of the oil-phase superparamagnetic iron oxide nanoparticles and the hydrophilic molecule is 10-50 mg: 20 mg-300 mg.

5. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. In the polar-nonpolar mixed solvent of step S2, the volume ratio of the polar solvent to the nonpolar solvent is 1:1-1:3; wherein the polar solvent is one or more of acetone, ethanol, N,N dimethylformamide, dimethyl sulfoxide or tetrahydrofuran, and the nonpolar solvent is one or more of o-dichlorobenzene, chloroform, n-hexane, cyclohexane or toluene.

6. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. The after-treatment of step S2 comprises first precipitation with diethyl ether, and then centrifugation and acetone washing to obtain the water-phase superparamagnetic iron oxide nanoparticles.

7. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. The gadolinium ion compound of step S4 is one or more of gadolinium acetylacetonate, gadolinium acetate, gadolinium nitrate or gadolinium chloride hexahydrate.

8. The method of claim 1, wherein the copper ion responsive MRI probe is prepared by the following steps: (a) synthesizing a copper ion responsive MRI probe precursor; (b) mixing the copper ion responsive MRI probe precursor with a copper ion source; and (c) purifying the copper ion responsive MRI probe. The good solvent of steps S3 and S4 is one or more of 2-(N-morpholine)ethanesulfonic acid buffer, phosphate buffer solution, deionized water, dimethyl sulfoxide, N,N dimethylformamide, ethanol or acetonitrile.

9. A copper ion responsive magnetic resonance imaging probe prepared by the method of any one of claims 1-8.

10. Use of the copper ion responsive magnetic resonance imaging probe of claim 9 in the preparation of a tumor magnetic resonance imaging contrast agent.

Images