A single-atom metal magnetic resonance imaging contrast agent, its preparation method and application

Abstract

The present application discloses a single-atom metal magnetic resonance contrast agent, a preparation method thereof, and an application thereof, relating to the field of medical technology. The contrast agent has a hollow bowl-shaped structure with a nitrogen-doped carbon loaded with single-atom metal as the inner layer and SiO2 as the outer layer; the contrast agent is prepared by first coating the surface of a templating agent nanosphere with metal ions and a nitrogen-containing polymer, then coating with SiO2, and then successively undergoing high-temperature pyrolysis and chemical etching; the metal is selected from rare earth metals or / and transition metals. The present application solves the problems of poor stability, low relaxation rate, and non-specificity of existing gadolinium-based contrast agents. The single-atom metal magnetic resonance contrast agent provided by the present application has excellent T1 / T2 dual-modal MRI capabilities, and at the same time has excellent stability and tumor targeting capabilities.

Description

Technical Field

[0001] The present application relates to the field of magnetic resonance technology, and particularly relates to a single-atom metal magnetic resonance contrast agent, a preparation method thereof, and an application thereof. Background Art

[0002] Currently, magnetic resonance contrast agents (CAs) widely used in clinical practice are mainly Gd-based T1 contrast agents in the form of molecular complexes, such as Gd-DTPA and Gd-DOTA. However, these commercial Gd-based complexes have problems such as short blood circulation time, low relaxation rate, and non-specificity in the body. Especially in terms of toxicity, it may cause nephrogenic systemic fibrosis and brain deposition in patients. In addition, free Gd 3+ has a similar radius to Ca 2+ and will interfere with the calcium-mediated signaling pathway. Subsequently, Gd-based inorganic nanoparticles such as oxides (Gd2O3), fluorides (NaGdF4), and carbonates (Gd2(CO3)3) have been developed, which can capture Gd ions into the host crystal, thereby effectively overcoming the leaching of Gd ions and improving the stability of the contrast agent. However, the metal ions inside the nanoparticles cannot directly undergo chemical exchange with hydrogen protons, resulting in a decrease in the signal of magnetic resonance imaging (MRI). Summary of the Invention

[0003] In view of the deficiencies of the above technologies, the present application provides a single-atom metal magnetic resonance contrast agent, a preparation method thereof, and an application thereof, which solve the problems of poor stability, low relaxation rate, and non-specificity of existing Gd-based contrast agents. The single-atom metal magnetic resonance contrast agent provided by the present application has excellent T1 / T2 dual-modal MRI ability, and at the same time has excellent stability and tumor targeting ability.

[0004] To achieve the above object, the present application mainly provides the following technical solutions:

[0005] The present application provides a single-atom metal magnetic resonance contrast agent, which has a hollow bowl-shaped structure with a nitrogen-doped carbon loaded with single-atom metal as the inner layer and SiO2 as the outer layer;

[0006] The contrast agent is prepared by first coating the surface of the template agent nanospheres with metal ions and a nitrogen-containing polymer, then coating with SiO2, and then successively undergoing high-temperature pyrolysis and chemical etching;

[0007] The metal is selected from rare earth metals or / and transition metals.

[0008] Preferably, the metal is selected from one or more of Gd, Dy, Ho, Mn, Fe, and Co.

[0009] Preferably, the weight content of the metal in the contrast agent is 0.1% to 5.4%, more preferably 0.27% to 5.4%.

[0010] Preferably, the templating agent nanospheres are polymer nanospheres. Specifically, the polymer nanospheres are selected from polystyrene nanospheres and styrene-acrylic acid copolymer nanospheres.

[0011] Preferably, the nitrogen-containing polymer is polydopamine.

[0012] The single-atom metal magnetic resonance contrast agent provided in this application forms an ultrathin polydopamine layer by the oxidative polymerization of dopamine in a buffer solution and coats it on the surface of the templating agent nanospheres. At the same time, metal ions form coordination bonds with the nitrogen on the polydopamine, enabling the metal ions to be anchored on the polydopamine. When pyrolyzed at high temperature, nitrogen-doped carbon loaded with single-atom metal, that is, the structure of M-N-C (M is metal), can be formed.

[0013] After coating metal ions and a nitrogen-containing polymer on the surface of the templating agent nanospheres, a silica precursor can be hydrolyzed to generate silica gel, which is then coated on the outermost layer of the templating agent nanospheres to form a SiO2 layer. When pyrolyzed at high temperature, the templating agent nanospheres can be pyrolyzed and volatilized, and the polydopamine layer loaded with metal ions on the surface of the templating agent nanospheres forms a nitrogen-doped carbon sphere with a hollow interior loaded with single-atom metal. When chemically etched, the outer SiO2 layer and the inner hollow nitrogen-doped carbon sphere form a hollow bowl-like structure.

[0014] In the single-atom metal magnetic resonance contrast agent provided in this application, the structure of the nitrogen-doped carbon loaded with single-atom metal can utilize the strong interaction between metal atoms and nitrogen atoms to anchor the metal ions on the nitrogen-doped carbon sphere and prevent them from easily falling off, thereby improving the biosafety of the single-atom metal contrast agent; at the same time, the metal atoms are evenly and isolatedly dispersed on the nitrogen-doped carbon sphere, forming single-atom metal sites, which can ensure sufficient contact between the single-atom metal and water and effectively utilize the single-atom metal to enhance the relaxation performance of water protons. The hollow bowl-like structure can maximize the exposure of the metal sites loaded on the nitrogen-doped carbon on the inner side, enabling sufficient contact with water molecules, thereby improving its relaxation performance; at the same time, the SiO2 outside the coated nitrogen-doped carbon layer serves as a spatial confinement layer and a hydrophilic layer, which can not only improve the dispersibility between the contrast agent particles, prevent aggregation from affecting the relaxation performance, but also improve the water solubility and biocompatibility of the contrast agent particles, thereby reducing chemical toxicity.

[0015] Preferably, the particle size of the single-atom metal magnetic resonance contrast agent is 65-200 nm. The above single-atom metal magnetic resonance contrast agent provided by the present application can control the size of the contrast agent by controlling the size of the template agent nanospheres. In the particle size range of 65-200 nm, the contrast agent has strong permeability and retention effect on tumor tissues, improving its aggregation at the tumor site. At the same time, there is a good dispersant between the contrast agent particles to prevent the influence on the relaxation performance due to aggregation.

[0016] Due to the special hollow bowl-like structure and the double-layer structure with nitrogen-doped carbon loaded with single-atom metal in the inner layer and SiO2 in the outer layer, the above single-atom metal magnetic resonance contrast agent provided by the present application has more excellent kinetic inertness and thermal stability than the commercial Gd-DTPA contrast agent, and can achieve passive targeting of tumor sites; at the same time, it has excellent T1 / T2 dual-mode MRI ability, which can avoid false positive signals and provide accurate and complementary diagnostic information.

[0017] The present application also provides a preparation method of the above single-atom metal magnetic resonance contrast agent, including the following steps:

[0018] (1) In a buffer solution, a template agent nanosphere, a metal salt, and a nitrogen-containing organic compound are added, and after reaction, a template agent nanosphere with a metal ion and a nitrogen-containing polymer coated on the outer layer is obtained, denoted as the first nanosphere; the nitrogen-containing polymer is polymerized from the nitrogen-containing organic compound;

[0019] (2) The first nanosphere obtained in step (1) is dispersed in a solvent, and a silica precursor and ammonia water are added, and after reaction, a first nanosphere with SiO2 coated on the outer layer is obtained, denoted as the second nanosphere;

[0020] (3) Under a protective atmosphere, the second nanosphere obtained in step (2) is pyrolyzed at high temperature, then dispersed in an alkaline solution for chemical etching to form a hollow bowl-like structure, and finally centrifuged, washed, and dried to obtain a single-atom metal magnetic resonance contrast agent.

[0021] The preparation method of the above contrast agent provided by the present application is suitable for preparing transition metal or / and rare earth metal-based MRI contrast agents. Among them, the transition metal can be selected from Mn, Fe, Co, and the rare earth metal can be selected from Gd, Dy, Ho.

[0022] In step (1), the metal salt is selected from water-soluble salts of at least one of the following metals: Gd, Dy, Ho, Mn, Fe, C o , that is, the water-soluble salts of these metals are used to form single-atom metals in the contrast agent. Specifically, the metal salt can be chlorides, nitrates, etc. of the above metals.

[0023] In step (1), preferably, the templating agent nanospheres are polymer nanospheres. By using this templating agent, it can volatilize during subsequent high-temperature pyrolysis to form a hollow structure. Specifically, the polymer nanospheres can be selected from polystyrene nanospheres and styrene-acrylic copolymer nanospheres.

[0024] In step (1), preferably, the nitrogen-containing organic compound is dopamine. Dopamine can undergo oxidative polymerization in a buffer solution to form an ultrathin polydopamine layer, which coats the surface of the polymer nanospheres. That is, the first nanospheres are polymer nanospheres coated with metal ions and polydopamine. At the same time, the metal ions form coordination bonds with the nitrogen on the polydopamine, enabling the metal ions to be anchored on the polydopamine. When pyrolyzed, nitrogen-doped carbon loaded with single-atom metal, that is, the structure of M-N-C (M is metal), can be formed.

[0025] In step (2), the silica precursor can hydrolyze to form silica gel under alkaline conditions and coat the surface of the first nanospheres to form the second nanospheres. The second nanospheres have a structure with an outer silica layer, an intermediate layer of polydopamine anchored with metal ions, and an inner polymer nanosphere layer.

[0026] Specifically, the silica precursor can be selected from tetraethoxysilane, tetramethoxysilane, and 3-aminopropyltriethoxysilane. These silica precursors can all hydrolyze to form silica gel under alkaline conditions.

[0027] In step (3), when the second nanospheres undergo high-temperature pyrolysis, the innermost polymer nanospheres volatilize through pyrolysis to form a hollow, and the polydopamine anchored with metal ions in the intermediate layer forms nitrogen-doped carbon loaded with single-atom metal through pyrolysis. Therefore, after high-temperature pyrolysis, the second nanospheres form nitrogen-doped carbon spheres loaded with single-atom metal with a hollow interior and SiO2 coating. Then, through chemical etching with an alkali solution, a hollow bowl-shaped structure can be formed.

[0028] Specifically, during the chemical etching process, as Figure 1 shown, the outer SiO2 coating layer first becomes thinner from thick, and then the inner nitrogen-doped carbon spheres are etched to form an opening at the thinnest part of the SiO2 layer. Since the SiO2 and nitrogen-doped carbon at the opening are more easily etched away, the opening gradually becomes larger, and finally a hollow bowl-shaped structure is formed.

[0029] In this application, the degree of chemical etching of the SiO2-coated nitrogen-doped carbon spheres loaded with single-atom metals determines the morphology and relaxation properties of the obtained single-atom metal magnetic resonance contrast agents. When the etching is less, only the SiO2 layer on the outer layer of the nitrogen-doped carbon spheres can be thinned from thick to thin or the opening on the nitrogen-doped carbon spheres is small, so that the single-atom metals loaded on the inner layer of the nitrogen-doped carbon spheres cannot be fully exposed, and thus cannot come into full contact with water molecules. Therefore, the single-atom metals cannot be effectively used to enhance the relaxation efficiency of water protons. When the etching is more, less SiO2 layer remains on the outer layer of the nitrogen-doped carbon spheres, making the contrast agent particles irregular and prone to aggregation, thereby affecting their relaxation properties. When the single-atom metal magnetic resonance contrast agent with a hollow bowl-shaped structure is formed by etching, it has excellent relaxation properties. The hollow bowl-shaped structure can maximize the exposure of the metal sites loaded on the inner nitrogen-doped carbon, enabling full contact with water molecules, thereby improving its relaxation properties. At the same time, on the one hand, the SiO2 layer outside the coated nitrogen-doped carbon layer serves as a spatial confinement layer, which can not only prevent the collapse of the carbon sphere structure during high-temperature pyrolysis, resulting in the aggregation of contrast agent particles, but also prevent the formation of nanoparticles by metal ions during high-temperature pyrolysis, leading to the aggregation of metal ions. Therefore, the SiO2 layer improves the dispersibility between contrast agent particles and prevents their aggregation from affecting the relaxation properties. On the other hand, the SiO2 layer outside the coated nitrogen-doped carbon layer serves as a hydrophilic layer, which can improve the water solubility and biocompatibility of the contrast agent particles, thereby reducing chemical toxicity.

[0030] In this application, by controlling the reaction conditions of the chemical etching process, the degree of chemical etching of the SiO2-coated nitrogen-doped carbon spheres loaded with single-atom metals can be controlled. Specifically, the degree of chemical etching is controlled by controlling the pH of the alkali solution used, the etching temperature, i.e., the temperature of the alkali solution, and the etching time.

[0031] Preferably, the pH used for chemical etching is >11; when the temperature of the alkali solution is 40 - 60 °C, the etching time is 50 - 200 min; when the temperature of the alkali solution is 60 - 100 °C, the etching time is 20 - 100 min. Under these etching conditions, the SiO2-coated nitrogen-doped carbon spheres loaded with single-atom metals can be etched into a bowl-shaped structure with the optimal performance. Specifically, the alkali solution can be an inorganic strong alkali solution such as sodium hydroxide or potassium hydroxide.

[0032] Preferably, the size of the template agent nanospheres is 50 - 180 nm. In this application, the size of the single-atom metal magnetic resonance contrast agent particles can be controlled by controlling the size of the template agent nanospheres. When template agent nanospheres with a size of 50 - 180 nm are used, single-atom metal magnetic resonance contrast agents with a size of 65 - 200 nm can be prepared.

[0033] In step (1), preferably, the mass ratio of the templating agent nanospheres to the nitrogen-containing organic compound is 100∶(30 - 100); the molar ratio of the metal salt to the nitrogen-containing organic compound is (0.5 - 10)∶100.

[0034] In the above preparation method of the present application, the mass ratio of the templating agent nanospheres to the nitrogen-containing organic compound determines the thickness of the polydopamine layer coated on the templating agent nanospheres and the thickness of the nitrogen-doped carbon layer in the resulting contrast agent particles. When the mass ratio of the templating agent nanospheres to the nitrogen-containing organic compound is 100∶(30 - 100), the thickness of the nitrogen-doped carbon layer in the resulting contrast agent particles is 5 - 10 nm.

[0035] In step (2), preferably, the mass ratio of the silica precursor to the templating agent nanospheres is (15 - 75)∶10.

[0036] In step (2), preferably, the solvent is water, methanol, ethanol or a mixture thereof. More preferably, the solvent is a mixture of methanol and water or a mixture of ethanol and water. More preferably, the volume ratio of methanol or ethanol to water in the solvent is 25∶(3 - 6). Using a mixture of methanol or ethanol and water as the solvent can slow down the hydrolysis of the silica precursor, enabling the formed silica gel to slowly coat the surface of the first nanospheres, improving the uniformity and stability of the silica coating layer. After subsequent pyrolysis and etching, the formed hollow bowl-shaped structure is more stable.

[0037] In step (2), preferably, the temperature of the high-temperature pyrolysis is 500 - 900 °C, the pyrolysis time is 2 - 4 h, and the heating rate during pyrolysis is 2 - 10 °C / min. The protective atmosphere used includes but is not limited to N2, Ar, etc.

[0038] The present application also provides the use of the above single-atom metal magnetic resonance contrast agent in disease diagnosis.

[0039] Specifically, the above single-atom metal magnetic resonance contrast agent provided by the present application can be used as a T1 / T2 dual-modal nuclear magnetic resonance imaging contrast agent.

[0040] In the present application, a range can be expressed as a range from “about” one specific value and / or to “about” another specific value. When expressing such a range, examples include starting from a certain specific value and / or ending at another specific value. Similarly, when using the antecedent “about” to indicate that a numerical value is an approximation, it should be understood that the specific numerical value constitutes another aspect. It should also be understood that each endpoint value of a range is meaningful both in relation to and independent of another endpoint value.

[0041] Unless otherwise stated, none of the methods described herein are intended to be understood as requiring their steps to be performed in a specific order. Accordingly, when method claims do not actually recite their steps as following a certain order or when they are not otherwise specifically indicated in the claims or the specification to be limited to a specific order, no particular order is intended to be implied.

[0042] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages:

[0043] (1) The single-atom metal magnetic resonance contrast agent provided in the present application uses a template agent nanosphere coated with metal ions and nitrogen-containing organic matter on its surface. During the high-temperature pyrolysis process, the metal has a strong local electron interaction with N in the nitrogen-containing organic matter, and a nitrogen-doped carbon loaded with single-atom metal, that is, an M-N-C structure (M is a metal), can be formed. Thus, the strong interaction between metal atoms and nitrogen atoms can be used to anchor metal ions on the nitrogen-doped carbon sphere and prevent them from easily falling off, thereby improving the biological safety of the single-atom metal contrast agent. At the same time, metal atoms are uniformly and isolatedly dispersed on the nitrogen-doped carbon sphere, forming single-atom metal sites, which can ensure sufficient contact between single-atom metal and water and effectively utilize single-atom metal to enhance the relaxation performance of water protons.

[0044] (2) The single-atom metal magnetic resonance contrast agent provided in the present application has a hollow bowl-like structure, which can enable the metal sites loaded on the inner nitrogen-doped carbon to fully contact with water molecules, thereby improving its relaxation performance. At the same time, on the one hand, the SiO2 layer outside the coated nitrogen-doped carbon layer serves as a spatial confinement layer, which can not only prevent the collapse of the carbon sphere structure during high-temperature pyrolysis, resulting in aggregation between contrast agent particles, but also prevent the formation of nanoparticles by metal ions during high-temperature pyrolysis, resulting in agglomeration of metal ions. Therefore, the SiO2 layer improves the dispersibility between contrast agent particles and prevents their agglomeration from affecting the relaxation performance. On the other hand, the SiO2 layer outside the coated nitrogen-doped carbon layer serves as a hydrophilic layer, which can improve the water solubility and biocompatibility of the contrast agent particles, thereby reducing chemical toxicity.

[0045] (3) The particle size of the single-atom metal magnetic resonance contrast agent provided in the present application is 65 - 200 nm, and it has strong permeability to tumor tissues. Due to its special hollow bowl-like structure and double-layer structure with an inner layer of nitrogen-doped carbon loaded with single-atom metal and an outer layer of SiO2, this contrast agent has more excellent kinetic inertness and thermal stability than the commercial Gd-DTPA contrast agent, and can achieve passive targeting of tumor sites. At the same time, it has excellent T1 / T2 dual-mode MRI capabilities, can avoid false positive signals, and provide accurate and complementary diagnostic information. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1Schematic diagram of the structural changes during the etching process for preparing the Gd single-atom magnetic resonance contrast agent in Example 1 of this application;

[0047] Figure 2 Transmission electron microscopy image of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 of this application at low magnification;

[0048] Figure 3 Aberration-corrected electron microscopy image of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 of this application;

[0049] Figure 4 X-ray absorption fine structure spectrum of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 of this application;

[0050] Figure 5 Curves of T1 and T2 magnetic resonance relaxation rate measurements of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 of this application at different magnetic field strengths;

[0051] Figure 6 Comparison images of T1 and T2 magnetic resonance imaging in the tube of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 of this application and a commercial Gd contrast agent at a 7T magnetic field strength;

[0052] Figure 7 Comparison images of T1 and T2 brightness at the tumor site of mice before and after tail vein injection of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 of this application;

[0053] Figure 8 Transmission electron microscopy images of Gd single-atom magnetic resonance contrast agents with different etching degrees in Example 1, Comparative Example 1, and Comparative Example 2 of this application;

[0054] Figure 9 Curves of T1 and T2 magnetic resonance relaxation rate measurements of the hollow-sphere structure Gd single-atom magnetic resonance contrast agent prepared in Comparative Example 1 of this application at a 3T magnetic field strength;

[0055] Figure 10 Curve of T1 magnetic resonance relaxation rate measurement of the over-etched Gd single-atom magnetic resonance contrast agent prepared in Comparative Example 2 of this application at a 7T magnetic field strength;

[0056] Figure 11 Transmission electron microscopy image of the Gd single-atom magnetic resonance contrast agent without silica coating prepared in Comparative Example 3 of this application;

[0057] Figure 12 Transmission electron microscopy image of the bowl-shaped Fe single-atom magnetic resonance contrast agent prepared in Example 2 of this application;

[0058] Figure 13 This is the aberration-corrected electron microscopy image of the bowl-shaped Fe single-atom magnetic resonance contrast agent prepared in Example 2 of this application;

[0059] Figure 14 This is the measurement curve graph of the T1 and T2 magnetic resonance relaxation rates of the bowl-shaped Fe single-atom magnetic resonance contrast agent prepared in Example 2 of this application under a magnetic field strength of 7T. Detailed implementation manners

[0060] To further elaborate on the technical means and effects adopted by this application to achieve the intended application purpose, the following combines the accompanying drawings and preferred embodiments to describe in detail the specific implementation manners, structures, features, and their effects according to this application as follows.

[0061] It should be noted that different "one embodiment" or "embodiments" in this application do not necessarily refer to the same embodiment. In addition, the specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form. It should be understood that the embodiments of this application are explanations of the application solutions and do not limit the protection scope of this application.

[0062] Although the transitional term "including" will be used to disclose various features, elements, or steps of a specific implementation manner, it should be understood that this implies alternative implementation manners that can be described by transitional terms such as "consisting of...", "substantially consisting of...". Therefore, for example, the implied alternative implementation manners of a method including A + B + C include the implementation manner where the method consists of A + B + C and the implementation manner where the method is substantially composed of A + B + C.

[0063] Example 1

[0064] This example provides a preparation method for a bowl-shaped Gd single-atom magnetic resonance contrast agent, including the following steps:

[0065] (1) First, in 60 mL of Tris-HCl buffer solution, add 100 mg of polystyrene nanospheres with an average particle size of 165 nm and 0.025 mmol of gadolinium nitrate. Subsequently, slowly inject 5 mL of a dopamine monomer solution with a concentration of 10 mg / mL. After stirring at room temperature for 24 h, centrifuge and purify to remove the unreacted raw materials to obtain polymer nanospheres coated with Gd ions and an ultrathin polydopamine layer, denoted as the first nanospheres. Then disperse the first nanospheres in 4 mL of ultrapure water for standby;

[0066] (2) Disperse 4 mL of the dispersion of the first nanospheres in 25 mL of ethanol. After stirring at room temperature for 5 min, sequentially add 0.4 mL of tetraethoxysilane and 0.65 mL of NH₃·H₂O through a syringe. After continuing to stir for 1 h, centrifuge and wash three times to obtain the first nanospheres coated with SiO₂, denoted as the second nanospheres (Gd-SA@SiO₂), and dry them overnight in a vacuum at 60 °C;

[0067] (3) In an atmosphere of N₂ gas, calcine the second nanospheres at 500 °C for 2 h to obtain hollow SiO₂-coated Gd-loaded nitrogen-doped carbon spheres; then add them to 15 mL of an aqueous NaOH solution with a concentration of 0.01 M. After ultrasonic dispersion, etch them at 50 °C for 1 h respectively to obtain bowl-shaped Gd single-atom magnetic resonance contrast agents (Gd-SA CAs).

[0068] Comparative Example 1

[0069] This comparative example provides a method for preparing hollow-sphere-structured Gd single-atom magnetic resonance contrast agents (Gd-SA CAs);

[0070] Compared with Example 1, the difference is that the etching time in step (3) is 20 min, and the rest are the same as in Example 1.

[0071] Comparative Example 2

[0072] This comparative example provides a method for preparing over-etched Gd single-atom magnetic resonance contrast agents;

[0073] Compared with Example 1, the difference is that the etching time in step (3) is 4 h, and the rest are the same as in Example 1.

[0074] Comparative Example 3

[0075] This comparative example provides a method for preparing Gd single-atom magnetic resonance contrast agents without SiO₂ coating, including the following steps:

[0076] (1) First, add 100 mg of polystyrene nanospheres with an average particle size of 165 nm and 0.025 mmol of gadolinium nitrate to 60 mL of Tris-HCl buffer solution. Then slowly inject 5 mL of a dopamine monomer solution with a concentration of 10 mg / mL. After stirring at room temperature for 24 h, centrifuge and purify to remove unreacted raw materials, and dry to obtain polymer nanospheres coated with Gd ions and an ultrathin polydopamine layer, denoted as the first nanospheres;

[0077] (2) In an atmosphere of N₂ gas, calcine the first nanospheres at 500 °C for 2 h to obtain hollow Gd-loaded nitrogen-doped carbon spheres, which are the Gd single-atom magnetic resonance contrast agents without SiO₂ coating.

[0078] Example 2

[0079] This embodiment provides a method for preparing a bowl-shaped Fe single-atom magnetic resonance contrast agent, comprising the following steps:

[0080] (1) First, in 60 mL of Tris-HCl buffer solution, add 100 mg of polystyrene nanospheres with an average particle size of 165 nm and 0.038 mmol of iron nitrate. Subsequently, slowly inject 10 mL of a dopamine monomer solution with a concentration of 10 mg / mL. After stirring at room temperature for 12 h, centrifuge and purify to remove unreacted raw materials, obtaining polymer nanospheres coated with Fe ions and an ultrathin polydopamine layer, denoted as the first nanospheres. Then disperse the first nanospheres in 6 mL of ultrapure water for later use;

[0081] (2) Disperse 6 mL of the dispersion of the first nanospheres in 25 mL of ethanol. After stirring at room temperature for 5 min, sequentially add 0.4 mL of tetraethoxysilane and 0.65 mL of NH₃·H₂O through a syringe. Continue stirring for 0.5 h, then centrifuge and wash 3 times to obtain SiO₂-coated first nanospheres, denoted as the second nanospheres, and dry them overnight in a vacuum at 60 °C;

[0082] (3) In an atmosphere of N₂ gas, calcine the second nanospheres at 700 °C for 2 h to obtain hollow SiO₂-coated Fe-loaded nitrogen-doped carbon spheres; then add them to 15 mL of an aqueous NaOH solution with a concentration of 0.2 M. After ultrasonic dispersion, etch at 70 °C for 0.5 h respectively to obtain a bowl-shaped Fe single-atom magnetic resonance contrast agent (Fe-SA CAs).

[0083] Characterize and perform performance tests on the magnetic resonance contrast agents in the examples and comparative examples, and the results are as follows:

[0084] (1) Characterize the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 to obtain Figure 2 the transmission electron microscope image at low magnification as shown, Figure 3 the spherical aberration-corrected electron microscope image as shown, Figure 4 the X-ray absorption fine structure spectrum as shown, where Figure 4 a is the absorption spectrum of the L3 edge of Gd, Figure 4 b is the Fourier transform spectrum of the L3 edge absorption of Gd, Figure 4 c is the experimental and fitting results of the Fourier transform spectrum of the L3 edge absorption of Gd in the R space.

[0085] It can be seen from Figure 2 that the Gd single-atom magnetic resonance contrast agent has dispersed particles, uniform sizes, and all are in a bowl-shaped structure, with an average particle size of about 180 nm, and this particle size is beneficial for its penetration into tumor tissues;

[0086] From Figure 3 It can be seen that compared with elements such as C, H, O, and Si, Gd atoms are relatively heavy. The bright spots in the figure are Gd atoms, and these bright spots are in a dispersed state, indicating that the metal Gd in the contrast agent is in a single-atom dispersion;

[0087] From Figure 4 a, it can be seen that Gd exists in the form of an oxidation state; from Figure 4 b, it can be seen that there are no Gd-Gd bonds, Gd-O bonds, and Gd-O-Gd bonds in the figure. Gd is only coordinated with N, and from Figure 4 c, it can be seen that a Gd-N4 structure is formed, proving that Gd is loaded on nitrogen-doped carbon in the structure of Gd-N-C and exists in a single-dispersed atomic state.

[0088] (2) Evaluate the performance of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 as a T1 / T2 dual-mode contrast agent.

[0089] First, measure the T1 and T2 magnetic resonance relaxation rates at different magnetic field strengths to obtain the measurement curve shown in Figure 5 the figure. According to the measured T1 and T2 relaxation times at different gadolinium ion concentrations, the longitudinal relaxation rates T1 of the Gd single-atom magnetic resonance contrast agent at different magnetic field strengths are respectively: 65.5 mM -1 s -1 at 0.5 T, 34.2 mM -1 s -1 at 3 T, and 12.0 mM -1 s -1 at 7 T. And the transverse relaxation rates r2 at different magnetic field strengths are respectively: 74.9 mM -1 s -1 at 0.5 T, 80.42 mM -1 s -1 at 3 T, and 112.8 mM -1 s -1 at 7 T. The above data show that the Gd single-atom magnetic resonance contrast agent prepared in Example 1 has excellent nuclear magnetic imaging ability.

[0090] Secondly, measure the T1 and T2 magnetic resonance imaging maps in the tube of the bowl-shaped Gd single-atom magnetic resonance contrast agent prepared in Example 1 and the commercial Gd contrast agent at a 7 T magnetic field strength. As Figure 6 shown in a, compared with the Gd-DTPA complex, the Gd single-atom magnetic resonance contrast agent prepared in Example 1 shows a brighter T1-weighted image and has a concentration-dependent enhancement effect, indicating that the Gd single-atom magnetic resonance contrast agent prepared in Example 1 can be used as a T1-enhanced contrast agent. At the same time, from Figure 6It can be seen that as the concentration increases, the T2-weighted images of the Gd single-atom magnetic resonance contrast agent gradually darken, showing typical T2 contrast imaging. The above proves that the Gd single-atom magnetic resonance contrast agent prepared in Example 1 can be used as a T1 / T2 dual-mode contrast agent.

[0091] Finally, in vivo experiments were carried out: Under a 7T nuclear magnetic imaging system, the Gd single-atom magnetic resonance contrast agent (the injection dose was 5 μmol Gd per kg of mouse body weight) was intravenously injected into female BALB / c mice carrying 4T1 tumors, and then in vivo magnetic resonance imaging was performed to obtain Figure 7 the T1 and T2 brightness contrast images of the mouse tumor before and after the tail vein injection of the Gd single-atom magnetic resonance contrast agent as shown. From Figure 7 it can be seen that on the T1-weighted image after injection, the tumor (circled) can be clearly observed to become significantly brighter. At the same time, the T2-weighted imaging image also darkens after injection, indicating that the Gd single-atom magnetic resonance contrast agent prepared in Example 1 can be used as an effective T1 / T2 dual-modal magnetic resonance contrast agent. It also shows that the Gd single-atom magnetic resonance contrast agent has excellent tumor passive targeting ability.

[0092] (3) The magnetic resonance contrast agents with different etching degrees in Example 1, Comparative Example 1, and Comparative Example 2 were characterized respectively, and the transmission electron microscope images as shown Figure 8 were obtained. Among them Figure 8 a represents the hollow SiO2-coated Gd-loaded nitrogen-doped carbon spheres without etching in Example 1, Figure 8 b represents the Gd single-atom magnetic resonance contrast agent with a hollow sphere structure obtained by etching for 20 min in Comparative Example 1; Figure 8 c represents the Gd single-atom magnetic resonance contrast agent with a bowl-shaped structure obtained by etching for 1 h in Example 1; Figure 8 d represents the Gd single-atom magnetic resonance contrast agent obtained by etching for 4 h in Comparative Example 2.

[0093] From Figure 8 it can be seen that Figure 8 a to Figure 8d shows the structures of the hollow SiO2-coated Gd-loaded nitrogen-doped carbon spheres after 0 min, 20 min, 1 h, and 4 h of etching, respectively. As the etching degree increases, the silicon layer on the surface of the nitrogen-doped carbon spheres becomes thinner from thick. When etched for 20 min, only the silicon layer on the surface of the nitrogen-doped carbon spheres becomes thinner, and the overall structure remains a complete hollow sphere. When etched for 1 h, the hollow sphere structure becomes a bowl-like structure, and the dispersibility is also improved. When etched for 4 h, the structure of the contrast agent becomes irregular and serious aggregation occurs. It can be seen that during the preparation of the contrast agent, when the hollow SiO2-coated Gd-loaded nitrogen-doped carbon spheres are not etched or slightly etched, a contrast agent with a bowl-like structure cannot be obtained. When over-etched, the structure of the contrast agent is not only irregular, but the aggregation between particles will seriously affect the dispersibility and relaxivity of the contrast agent.

[0094] In addition, the T1 and T2 magnetic resonance relaxation rates of the Gd single-atom magnetic resonance contrast agent with a hollow sphere structure in Comparative Example 1 were measured at a magnetic field strength of 3 T, and the measured curve graph is as Figure 9 shown. As can be seen from Figure 9 it, the longitudinal relaxation rate r1 and the transverse relaxation rate r2 at a magnetic field strength of 3 T are 15.5 mM -1 s -1 and 55.2 mM -1 s -1 respectively. Its relaxation performance is far inferior to that of the bowl-like structure Gd single-atom magnetic resonance contrast agent in Example 1. The reason is that in the Gd single-atom magnetic resonance contrast agent with a hollow sphere structure, the Gd atoms loaded on the nitrogen-doped carbon spheres are coated by SiO2 on the outside and cannot come into full contact with water molecules, so their relaxation ability cannot be fully exerted.

[0095] The T1 magnetic resonance relaxation rate of the over-etched Gd single-atom magnetic resonance contrast agent in Comparative Example 2 was measured at a magnetic field strength of 7 T, and the measured curve graph is as Figure 10 shown. As can be seen from Figure 10 it, the longitudinal relaxation rate r1 at a magnetic field strength of 7 T is 9.57 mM -1 s -1 which is significantly lower than that of the bowl-like structure Gd single-atom magnetic resonance contrast agent in Example 1 under the same conditions.

[0096] (4) The Gd single-atom magnetic resonance contrast agent without SiO2 coating prepared in Comparative Example 3 was characterized, and the transmission electron microscope image is as Figure 11 shown; as can be seen from Figure 11 it, when the surface of the nitrogen-doped carbon spheres loaded with Gd is not coated with silicon dioxide, after calcination, serious aggregation occurs between the carbon spheres, and it is difficult for the carbon spheres to maintain a complete spherical morphology. This shows that the SiO2 coated on the outside of the nitrogen-doped carbon loaded with Gd can play a role in supporting the stable bowl-like morphology of the contrast agent particles and can also play a good dispersing role.

[0097] (5) Characterize the bowl-shaped Fe single-atom magnetic resonance contrast agent prepared in Example 2 to obtain Figure 12 the transmission electron microscope images shown and Figure 13 the spherical aberration corrected electron microscope images shown; It can be seen from Figure 12 that the Fe single-atom magnetic resonance contrast agent has the same bowl-shaped structure and dispersibility as the Gd single-atom magnetic resonance contrast agent in Example 1; It can be seen from Figure 13 that the bright spots in the circle are Fe atoms, and these bright spots are in a dispersed state, indicating that the metallic Fe in the contrast agent is in single-atom dispersion.

[0098] Test the T1 and T2 magnetic resonance relaxation rates of the bowl-shaped Fe single-atom magnetic resonance contrast agent prepared in Example 2 at a magnetic field strength of 7T to obtain Figure 14 the measured curve shown; It can be seen from this figure that the longitudinal relaxation rate r1 and the transverse relaxation rate r2 of the Fe single-atom magnetic resonance contrast agent at a magnetic field strength of 7T are 4.7 mM -1 s -1 and 21.5 mM -1 s -1 , respectively, and its relaxation performance is better than that of commercial Gd contrast agents, and it can be used as a T1 / T2 dual-modal magnetic resonance contrast agent.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application and not to limit them. Although the present application has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present application, and they should all be covered by the scope of the claims of the present application.

Claims

1. A single-atom metal magnetic resonance contrast agent, characterized in that, The contrast agent has a hollow bowl-shaped structure with a nitrogen-doped carbon layer loaded with single-atom metal on the inner layer and a SiO2 layer on the outer layer; The contrast agent is prepared by first coating the surface of the template agent nanospheres with metal ions and nitrogen-containing polymers, then coating with SiO2, and then successively undergoing high-temperature pyrolysis and chemical etching; The temperature of the high-temperature pyrolysis is 500-900 °C; The metal is selected from one of Gd, Dy, Ho, Mn, Fe, and Co.

2. The single-atom metal magnetic resonance contrast agent according to claim 1, characterized in that, The weight content of the metal in the contrast agent is 0.1% - 5.4%.

3. The single-atom metal magnetic resonance contrast agent according to claim 1, characterized in that, The particle size of the single-atom metal magnetic resonance contrast agent is 65-200 nm.

4. A preparation method of the single-atom metal magnetic resonance contrast agent according to any one of claims 1-3, characterized in that, It includes the following steps: (1) In a buffer solution, add template agent nanospheres, metal salts, and nitrogen-containing organic substances, and react to obtain template agent nanospheres with metal ions and nitrogen-containing polymers coated on the outer layer, denoted as the first nanospheres; the nitrogen-containing polymer is polymerized from nitrogen-containing organic substances; (2) Disperse the first nanospheres obtained in step (1) in a solvent, and add a silica precursor and ammonia water, and react to obtain the first nanospheres with SiO2 coated on the outer layer, denoted as the second nanospheres; (3) Under a protective atmosphere, perform high-temperature pyrolysis on the second nanospheres obtained in step (2), then disperse them in an alkaline solution for chemical etching to form a hollow bowl-shaped structure, and finally perform centrifugation, washing, and drying to obtain the single-atom metal magnetic resonance contrast agent.

5. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The metal salt is selected from the water-soluble salts of at least one of the following metals: Gd, Dy, Ho, Mn, Fe, Co.

6. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The nitrogen-containing organic substance is dopamine.

7. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The template agent nanospheres are polymer nanospheres.

8. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The size of the template agent nanospheres is 50-180 nm.

9. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The mass ratio of the template agent nanospheres to the nitrogen-containing organic substance is 100∶(30-100); the molar ratio of the metal salt to the nitrogen-containing organic substance is (0.5-10)∶100.

10. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The mass ratio of the silica precursor to the template agent nanospheres is (15-75)∶10.

11. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The pH of the alkaline solution > 11; when the temperature of the alkaline solution is 40-60 °C, the etching time is 50-200 min; when the temperature of the alkaline solution is 60-100 °C, the etching time is 20-100 min.

12. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The pyrolysis time is 2-4 h, and the heating rate during pyrolysis is 2-10 °C / min.

13. The preparation method of the single-atom metal magnetic resonance contrast agent according to claim 4, characterized in that, The solvent in step (2) is a mixture of methanol and water or a mixture of ethanol and water, and the volume ratio of methanol or ethanol to water is 25∶(3-6).

14. Application of the single-atom metal magnetic resonance contrast agent according to any one of claims 1-3 in the preparation of disease diagnosis materials.

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