Iron-based nanoparticles and their gel thermal preparation method and application in magnetic resonance imaging
Monodisperse iron-based nanoparticles were prepared by gel thermal method, which solved the polydispersity problem in the existing technology, achieved the preparation of uniform nanoparticles and efficient magnetic resonance imaging effect, and is suitable for biomedical applications.
Patent Information
- Application Number
- CN202310277896.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-03-21
- Publication Date
- 2025-09-16
- Estimated Expiration
- 2043-03-21
Smart Images
Figure CN116333330B_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of functional nanomaterials, and in particular to an iron-based nanoparticle and a gel-thermal preparation method thereof, and an application thereof in magnetic resonance imaging. Background Art
[0002] Iron-based materials are gaining increasing attention in the field of biological research due to their unique advantages. However, as nanomedicines, they require shrinking from polydisperse crystals to monodisperse nanocrystals to achieve the target performance. Currently, a variety of synthetic strategies have been reported to synthesize nanoscale iron-based materials, such as using technology-assisted methods (microwaves, ultrasound, spray drying, microfluidics, etc.) to control nucleation and using microemulsion methods (surfactants and capping agents) to limit nanoparticle nucleation and growth. However, the particles synthesized by these methods do not meet the requirements due to poor monodispersity or the presence of surfactants in the pores. Therefore, there is an urgent need to develop new synthetic strategies to improve the shortcomings of the above methods.
[0003] Hydrothermal or solvothermal reactions are commonly used methods for constructing nanoscale iron-based materials. These methods primarily follow the LaMer and Ostwald ripening laws, with the solvent acting as a medium for precursor diffusion, nucleation, and growth. However, secondary nucleation and growth induced by diffusion inevitably produce polydisperse nanoparticles. Therefore, a new synthetic strategy is urgently needed to prepare uniform, monodisperse iron-based nanoparticles. Summary of the Invention
[0004] The purpose of the present invention is to provide an iron-based nanoparticle and a gel thermal preparation method thereof and application in magnetic resonance imaging in order to overcome at least one of the above-mentioned defects of the prior art.
[0005] The purpose of the present invention can be achieved by the following technical solutions:
[0006] The inventors believe that the sol-gel method is a classic wet chemical method that has been used to prepare various materials. Among them, coordination polymer gel (CPG) involves the formation of a sol and the subsequent formation of a gel driven by metal-ligand interactions, in which solvent molecules are fixed. CPG can achieve structural transformation by adjusting the coordination geometry at the molecular level, while providing a restricted gel domain for nucleation and growth. Therefore, utilizing the soft constraint environment of CPG and the need to cross the energy barrier for nucleation, the present invention proposes a method of first gelling and then heating to prepare monodisperse iron-based nanoparticles. Uniformly monodispersed nanoparticles can maximize entry into cells through endocytosis to optimize the imaging effect, while achieving efficient delivery of drugs, genes and proteins, and have potential application value in the biomedical and pharmaceutical fields. The specific scheme is as follows:
[0007] The invention discloses an iron-based nanoparticle, wherein the raw material of the nanoparticle contains a 6-linked trinuclear metal cluster and an organic carboxylic acid ligand.
[0008] Furthermore, the molecular formula of the 6-linked trinuclear metal cluster is [Fe3O(COO)6(H2O)3], and the organic carboxylic acid ligand is one of fumaric acid, terephthalic acid, hydroxyterephthalic acid, aminoterephthalic acid or trimesic acid.
[0009] Furthermore, the organic carboxylic acid ligand is fumaric acid or hydroxyterephthalic acid.
[0010] Furthermore, the nanoparticles have a shape of an octahedron or a double-pointed quadrangular prism, and a size of 50-300 nm.
[0011] In order to realize the application in magnetic resonance imaging, the preferred composition is constructed from all-iron-based 6-linked trinuclear clusters and fumaric acid or hydroxyterephthalic acid, with a preferred size of 50-300 nm, more preferably 50-150 nm.
[0012] Furthermore, the metal iron ion in the molecular formula [Fe3O(COO)6(H2O)3] is partially replaced by metal manganese, cobalt, nickel, copper, zinc or magnesium ions.
[0013] A gel-thermal method for preparing the iron-based nanoparticles as described above comprises the following steps:
[0014] The organic carboxylic acid ligand and the metal iron salt are respectively prepared into solutions and then mixed to obtain a coordination polymer gel based on the metal iron salt and the organic carboxylic acid ligand;
[0015] The polymer gel is aged and then heated to crystallize to obtain iron-based nanoparticles.
[0016] Iron-based nanoparticles prepared by the gel-thermal method have octahedral or bi-pointed quadrangular prism morphologies. Their size can be adjusted from 50 to 1000 nanometers by varying parameters such as solvent, ratio, and crystallization temperature. The particle size uniformity achieved during this process is shown to be less than 0.05. The gel-thermal method utilizes the quasi-solid state of the gel to create a spatially confined environment. This hinders the diffusion of solvent molecules, limiting secondary nucleation and growth, and enabling the preparation of uniformly sized nanoparticles.
[0017] Furthermore, the metallic iron salt is one of nitrate, chloride, perchlorate or acetate;
[0018] The molar ratio of the metal iron salt to the organic carboxylic acid ligand is (0.5-2.5):1;
[0019] The solvent in the solution is one of ethylene glycol, diethylene glycol, polyethylene glycol, water, dimethylformamide, or dimethyl sulfoxide;
[0020] The aging time is 30 minutes to 3 days, and the temperature of heating for crystallization is 50-120° C., and the time is 8-24 hours.
[0021] Furthermore, the metallic iron salt is nitrate or acetate;
[0022] The molar ratio of the metal iron salt to the organic carboxylic acid ligand is (1-1.5):1;
[0023] The solvent of the metal iron salt solution is one of ethylene glycol, diethylene glycol or polyethylene glycol, and the solvent of the organic carboxylic acid ligand is one of water, dimethylformamide or dimethyl sulfoxide;
[0024] The aging time is 2 hours to 1 day, and the heating temperature for crystallization is 90-120°C for 8-12 hours.
[0025] The organic carboxylic acid ligand and the metal iron salt are dissolved in a solvent and treated by ultrasound, heating, and a gelling agent to form a coordination polymer gel, preferably ultrasound. That is, polymer gelation can be achieved by any of ultrasound, standing, heating, and a gelling agent.
[0026] The size of the resulting iron-based nanoparticles can be adjusted from nanometer to micrometer scale by adjusting the type or ratio of solvent during the gelation process. When preparing the solution, the solvent and solute are prepared in a volume ratio of (1-20):1, preferably (1-5):1 when preparing nanosized particles. The solution concentration is 0.2-1.5 mol / L (calculated as iron salt), preferably 0.5-1.0 mol / L.
[0027] The aging treatment of the polymer gel is to heat or leave the gel to stand for a period of time. The heating crystallization of the polymer gel includes placing the gel in a closed environment.
[0028] A method for using the iron-based nanoparticles in magnetic resonance imaging, wherein the r1 relaxation rate is 2-10 mg·mL at 0.5T and room temperature to 37°C. -1 ·s -1 Range, r2 relaxation rate is 3-30 mg·mL -1 ·s -1 range, meeting the requirements of T1 contrast agents or T1-T2 dual-mode contrast agents.
[0029] The iron ions in the iron trinuclear cluster are located in a hexacoordinated octahedral field, with each iron ion coordinated by a single molecule of water. Under the influence of an external field, the hydrogen in the coordinated water molecules readily polarizes, allowing them to relax with ambient water through water exchange, generating an imaging signal. Furthermore, because this structure is easily regulated by ligands, size, and metal doping, these iron-based nanoparticles can be used as excellent T1 imaging agents or T1-T2 dual-mode imaging agents, possessing broad clinical imaging research potential.
[0030] Furthermore, the r2 / r1 value is less than or equal to 2, and the r1 value is between 2-10 mg·mL -1 ·s -1 As a T1 imaging contrast agent, the r2 / r1 value is selected to be between 2 and 10, and the r2 value is between 3 and 30 mg·mL. -1 ·s -1 As a T1-T2 dual-mode contrast agent.
[0031] Compared with the prior art, the present invention has the following advantages:
[0032] (1) The present invention provides a gel thermal method for the preparation of monodisperse iron-based MOFs for the first time, achieving regulation from 170 nm to 1200 nm, and a particle size uniformity PDI parameter of less than 0.05; the experimental method uses simple and readily available raw materials, the experimental process is quasi-solid, and no high temperature or high pressure is required, the operation is simple and safe, and it is suitable for large-scale production; the prepared monodisperse and uniform iron-based nanoparticles have more stable imaging effects than polydisperse nanoparticles prepared by other methods;
[0033] (2) The uniform nanoparticles prepared by the present invention are easy to be taken up by cells, and the cell uptake rate can reach 55% within 6 hours; the iron-based nanoparticles have good biosafety, and the cell uptake rate can reach 100 μg mL -1 The cell viability was still above 80% after incubation with the nanoparticles at the same concentration for 24 hours. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 The scanning electron microscope image and dynamic light scattering image of the monodisperse iron-based nanoparticles prepared in Example 1;
[0035] Figure 2 Transmission electron microscopy and dynamic light scattering images of the monodisperse iron-based nanoparticles prepared in Example 2;
[0036] Figure 3 The scanning electron microscope image and dynamic light scattering image of the monodisperse iron-based nanoparticles prepared in Example 3;
[0037] Figure 4 The in vitro nuclear magnetic resonance imaging images and the relaxivity r1 and r2 values of the monodisperse iron-based nanoparticles prepared in Example 1 are shown;
[0038] Figure 5 This is a scanning electron micrograph of monodisperse iron-based nanoparticles doped with different metals prepared in Example 4;
[0039] Figure 6 The in vitro nuclear magnetic resonance imaging images and the relaxivity r1 and r2 values of the monodisperse iron-based nanoparticles prepared in Example 4 are shown;
[0040] Figure 7 The cytotoxicity of monodisperse iron-based nanoparticles of different concentrations in Example 1 after co-incubation with melanoma cells for 24 hours. The drug loading content meets the requirements of clinical treatment and research.
[0041] Figure 8 These are laser confocal microscopy images of monodisperse MIL-88A nanoparticles loaded with calcein in Example 1 co-incubated with melanoma cells for different time periods;
[0042] Figure 9 The intracellular iron ion concentrations at different times were measured by co-incubating the gel-thermal MIL-88A nanoparticles in melanoma cells with the nanoparticles in Example 1. DETAILED DESCRIPTION
[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.
[0044] A gel thermal method for preparing iron-based nanoparticles and its application in magnetic resonance imaging. The gel thermal method comprises the following steps: (1) preparing a coordination polymer gel based on a metal iron salt and an organic carboxylic acid ligand; and (2) aging the polymer gel and then heating the gel to crystallize the iron-based nanoparticles.
[0045] The metal iron salt is one of nitrate, chloride, perchlorate, and acetate, preferably nitrate or acetate; the organic carboxylic acid ligand is one of fumaric acid, terephthalic acid, hydroxyterephthalic acid, aminoterephthalic acid, and trimesic acid, preferably fumaric acid or hydroxyterephthalic acid. The process for preparing a coordination polymer gel based on the metal iron salt and the organic carboxylic acid ligand comprises: dissolving the metal iron salt and the organic carboxylic acid ligand in a solvent at a molar ratio of (0.5-2.5):1, preferably (1-1.5):1, and forming the coordination polymer gel by ultrasonication, heating, and treating with a gelling agent, preferably ultrasonication.
[0046] The solvent is prepared from one of ethylene glycol, diethylene glycol, and polyethylene glycol and one of water, dimethylformamide, and dimethyl sulfoxide in a volume ratio of (1-20):1. When preparing nanosized particles, the ratio is preferably (1-5):1. The solution concentration is 0.2-1.5 mol / L (calculated as iron salt), preferably 0.5-1.0 mol / L.
[0047] The aging treatment of the polymer gel involves heating or allowing the gel to stand for a period of time ranging from 30 minutes to 3 days, preferably from 2 hours to 1 day. The thermal crystallization of the polymer gel involves placing the gel in a sealed environment and heating it at a temperature range of 50-120°C for 8 to 24 hours, preferably at 90-120°C, for 8 to 12 hours.
[0048] A gel-thermal method is used to prepare iron-based nanoparticles and their application in magnetic resonance imaging. The iron-based nanoparticles contain a 6-linked trinuclear metal cluster and an organic carboxylic acid ligand. The 6-linked trinuclear metal cluster has the general molecular formula [Fe₃O(COO)₆(H₂O)₃]. Specifically, the metallic iron ions can be partially replaced by metallic manganese, cobalt, nickel, copper, zinc, or magnesium ions. The iron-based nanoparticles prepared by the gel-thermal method have an octahedral or bicuspid prism morphology. Their size can be adjusted from 50 nanometers to 1000 nanometers by varying parameters such as solvent, ratio, and crystallization temperature. The particle size uniformity (PDI) parameter during this process is less than 0.05.
[0049] A gel thermal method for preparing iron-based nanoparticles and their application in magnetic resonance imaging. In order to achieve the application in magnetic resonance imaging, the preferred composition is constructed from all-iron-based 6-linked trinuclear clusters and fumaric acid or hydroxyterephthalic acid, with a preferred size of 50-300nm, more preferably 50-150nm.
[0050] The r1 relaxation rate is 2-10 mg·mL in the range of 0.5T to 37℃. -1 ·s -1 Range, r2 relaxation rate is 3-30 mg·mL -1 ·s -1 Range, meeting the requirements of T1 contrast agent or T1-T2 dual-mode contrast agent, preferably the r2 / r1 value is less than or equal to 2, and the r1 value is large as a T1 imaging contrast agent, preferably the r2 / r1 value is between 2-10, and the r2 value is large as a T1-T2 dual-mode contrast agent.
[0051] Example 1
[0052] An iron-based nanoparticle and its gel thermal preparation method and its application in magnetic resonance imaging are as follows:
[0053] Preparation of iron-based nanoparticles: 116 mg of fumaric acid was dissolved in 1 mL of DMF and sonicated for 15 minutes. 405 mg of Fe(NO₃)₃·9H₂O was dissolved in 1 mL of diethylene glycol and sonicated for 15 minutes. After complete dissolution, the DMF was poured into the diethylene glycol and sonicated for another 30 minutes to form a uniform red gel.
[0054] After aging the gel at room temperature for 2-3 days, it was placed in an oven and heated at 90°C for 24 hours. After cooling to room temperature, the resulting sediment was collected by repeated centrifugation washing with DMF and ethanol, and dried at 60°C to obtain a red dry solid powder containing uniform iron-based nanoparticles. Figure 1 The size is around 170 nm and the polydispersity index (PDI) is 0.0152.
[0055] Test Case 0
[0056] The relaxation time and T1 and T2 weighted imaging images of nuclear magnetic resonance imaging were obtained on a 0.5T MRI (MicroMRI. Shanghai Newmai Electronic Technology Co., Ltd.) at a test temperature of 32°C. The gel-thermal iron-based nanoparticles obtained in Example 1 were prepared into aqueous solutions of different mass concentrations and placed in a nuclear magnetic resonance analysis and imaging system to obtain T1 and T2 weighted imaging images and r1 and r2 relaxation rates. The results are shown in Figure 4 The relaxation rates r1 and r2 are 3.27 and 7.13 mg respectively. -1 mL -1 S -1 , which can meet the requirements of medical magnetic resonance imaging diagnosis and research.
[0057] Test Example 1
[0058] The cytotoxicity test of the sample of Example 1 adopts the CCK-8 method, and the method is as follows: B16 cells are digested with trypsin, inoculated in a 96-well plate, and incubated at 37°C, 5% CO2 for 12 hours. Different concentrations of the sample of Example 1 are added respectively, and the incubation is continued for 24 hours. Then 10 μL of CCK-8 culture medium dilution is added to each well, and the well plate is returned to the 37°C incubator and cultured for about 2 hours. The living cells will be stained by CCK-8, and the OD value of each well at 450nm is measured using a microplate reader (TECAN, Infinite M200, Germany), and the cell survival rate is calculated according to the following formula: Cell survival rate (%) = (average absorbance value of the experimental group / average absorbance value of the control group) × 100%. That is, Figure 7 As shown, when the sample concentration is 120 μg·mL -1 When the cells were cultured, the cell survival rate was still as high as over 80%, proving that the sample had good biosafety.
[0059] Test Example 2
[0060] (1) Preparation of gel-thermal iron-based nanoparticles loaded with calcein: 5 mg of the iron-based nanoparticles of Example 1 were ultrasonically dispersed in 5 mL of 4 mg mL -1 Calcein was added to deionized water and stirred at room temperature overnight. The resulting sediment was collected by repeated centrifugation washing with DMF and ethanol, and dried at 60°C to obtain calcein-loaded iron-based nanoparticles.
[0061] (2) The drug loading capacity was observed using a laser confocal microscope with the aid of the green fluorescence of calcein and the blue fluorescence of DAPI. The method was as follows: a certain amount of calcein-loaded iron-based nanoparticles was dispersed into DMEM culture medium at a concentration of 100 μg mL –1 . Then B16 cells were co-incubated with the materials at 37°C for different periods of time, and then the culture medium was removed and the residual nanomaterials and solution were washed out with PBS. After fixing the cells with 4% paraformaldehyde for 15 minutes, the cells were washed three times with PBS, and then DAPI was added to stain the nuclei at 37°C for 20 minutes. After washing twice with PBS, the cells were observed under a microscope. The excitation wavelengths of calcein and DAPI were 497nm and 405nm, respectively, and the emission wavelengths were collected in the range of 518 and 550-650nm, i.e. Figure 8 As shown, as the incubation time gradually increased, the fluorescence intensity of calcein gradually increased, proving that the sample could be gradually taken up by cells.
[0062] Test Example 3
[0063] B16 cells were digested with trypsin, seeded in 6-well plates, and incubated at 37°C, 5% CO2 for 12 h. 100 μg·mL –1 The sample of Example 1 was dispersed into DMEM culture medium, and then B16 cells were incubated with the material at 37°C for different times, and then collected with trypsin without EDTA-Na. After centrifugation (1000rpm, 5min), the cells were washed with PBS. Finally, cell lysis solution (containing 1% SDS, 1% Triton X-100 and 40mM tris acetate) was added to lyse the cells, and the resulting solution was sonicated to ensure complete cell disintegration. Finally, they were suspended in deionized water and analyzed using ICP-MS (Agilent 7800, America), that is, Figure 9 As shown, when the incubation time was only 6 hours, the cell uptake rate was greater than 50%, proving that the sample was easily taken up by cells.
[0064] Example 2
[0065] An iron-based nanoparticle and its gel thermal preparation method and its application in magnetic resonance imaging are as follows:
[0066] Preparation of iron-based nanoparticles: 116 mg of fumaric acid was dissolved in 1 mL of DMF and ultrasonicated for 15 minutes. 403 mg of Fe(NO3)3·9H2O was dissolved in 1 mL of diethylene glycol and ultrasonicated for 15 minutes. After complete dissolution, DMF was poured into diethylene glycol and ultrasonicated for 30 minutes to form a uniform red gel. The gel was aged at room temperature for 2-3 days and then placed in an oven and heated at 70°C for 24 hours. After cooling to room temperature, the resulting sediment was collected by repeated centrifugation and washing with DMF and ethanol, and dried at 60°C to obtain a red dry solid powder, which is uniform iron-based nanoparticles. Figure 2 The size is around 290 nm and the polydispersity index (PDI) is 0.0214.
[0067] Example 3
[0068] An iron-based nanoparticle and its gel thermal preparation method and its application in magnetic resonance imaging are as follows:
[0069] Preparation of iron-based nanoparticles: 116 mg of fumaric acid was dissolved in 1 mL of DMF and ultrasonicated for 15 minutes. 405 mg of Fe(NO3)3·9H2O was dissolved in 1 mL of diethylene glycol and ultrasonicated for 15 minutes. After complete dissolution, DMF was poured into triethylene glycol and ultrasonicated for 30 minutes to form a uniform red gel. The gel was aged at room temperature for 2-3 days and then placed in an oven and heated at 90°C for 24 hours. After cooling to room temperature, the resulting sediment was collected by repeated centrifugation and washing with DMF and ethanol, and dried at 60°C to obtain a red dry solid powder, which is uniform iron-based nanoparticles. Figure 3 As shown in Figure 2, the size is around 144 nm and the polydispersity index (PDI) is 0.0156.
[0070] Example 4
[0071] An iron-based nanoparticle and its gel thermal preparation method and its application in magnetic resonance imaging are as follows:
[0072] Preparation of iron-based nanoparticles doped with different metals: 116 mg of fumaric acid was dissolved in 1 mL of DMF and ultrasonicated for 15 minutes. 242 mg of Fe(NO3)3·9H2O and 0.3 mmol of nitrate (magnesium nitrate, manganese nitrate, zinc nitrate, copper nitrate, cobalt nitrate, nickel nitrate) were dissolved in 1 mL of diethylene glycol and ultrasonicated for 15 minutes. After complete dissolution, DMF was poured into diethylene glycol alcohol and ultrasonicated for 30 minutes to form a uniform red gel. The gel was aged at room temperature for 2-3 days and then placed in an oven for heating at 90°C for 24 hours. After cooling to room temperature, the resulting sediment was collected by repeated centrifugation and washing with DMF and ethanol, and dried at 60°C to obtain a red dry solid powder, which is monodispersed and uniform nanoparticles, i.e. Figure 5As shown, since the coordination forms and atomic sizes of the selected metal salts are similar to those of ferric nitrate, certain metal cluster sites of iron in the MIL-88A structure can be replaced in situ without changing its morphology.
[0073] The relaxation time and T1 and T2 weighted imaging images of nuclear magnetic resonance imaging were obtained on a 0.5T MRI (MicroMRI. Shanghai Newmai Electronic Technology Co., Ltd.) at a test temperature of 32°C. The gel-thermal iron-based nanoparticles obtained in this example were prepared into aqueous solutions of different mass concentrations and placed in a nuclear magnetic resonance analysis and imaging system to obtain T1 and T2 weighted imaging images and r1 and r2 relaxation rates. The results are shown in Figure 6 As the number of uncoordinated electrons in metal ions increases, the MRI relaxation rate increases, with manganese-doped ions exhibiting the best relaxation rate. This result is due to the fact that the MRI signal detection mechanism primarily relies on the relaxation rate of water protons. Unpaired electron spins in metals have a long relaxation time, significantly accelerating the relaxation rate of water protons in tissues and shortening the T1 of hydrogen nuclei.
[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.
Claims
1. An iron-based nanoparticle, characterized in that The nanoparticles are nanoparticles whose raw materials contain 6-linked trinuclear metal clusters and organic carboxylic acid ligands; The molecular formula of the 6-linked trinuclear metal cluster is [Fe3O(COO)6(H2O)3]; The organic carboxylic acid ligand is fumaric acid or hydroxyterephthalic acid; The nanoparticles have an octahedral or bicuspid prism shape and a size of 50-300 nm. The gel thermal method for preparing the iron-based nanoparticles comprises the following steps: The organic carboxylic acid ligand and the metal iron salt are respectively prepared into solutions and then mixed to obtain a coordination polymer gel based on the metal iron salt and the organic carboxylic acid ligand; After aging the polymer gel, heating and crystallizing the polymer gel to obtain iron-based nanoparticles; The metallic iron salt is nitrate or acetate; The molar ratio of the metal iron salt to the organic carboxylic acid ligand is (1-1.5): 1; The solvent of the metal iron salt solution is one of ethylene glycol, diethylene glycol or polyethylene glycol, and the solvent of the organic carboxylic acid ligand is one of water, dimethylformamide or dimethyl sulfoxide; The aging time is 2 hours to 1 day, and the heating temperature for crystallization is 90-120°C for 8-12 hours.
2. The iron-based nanoparticle according to claim 1, characterized in that: The metal iron ion in the molecular formula [Fe3O(COO)6(H2O)3] is partially replaced by metal manganese, cobalt, nickel, copper, zinc or magnesium ions.