Amorphous manganese salts for use as magnetic resonance contrast agents and methods for their preparation

Abstract

The application discloses an amorphous manganese salt used as a magnetic resonance (MRI) contrast agent and a preparation method thereof, the amorphous manganese salt is modified by polycarboxylic acid, meanwhile, the amorphous manganese salt can also be doped by a non-paramagnetic metal to improve the performance of the amorphous manganese salt. A one-step coprecipitation method is provided for preparing the amorphous manganese salt, the preparation method is simple in operation and can realize industrialized mass production. The amorphous manganese salt is stable, high in safety and excellent in MRI after being modified by the polycarboxylic acid, and is particularly suitable for liver-specific and blood vessel-enhanced imaging.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostic technology, specifically relating to an amorphous manganese salt used as a magnetic resonance contrast agent and its preparation method. Background Technology

[0002] Contrast agents can effectively improve the diagnostic sensitivity and specificity of magnetic resonance imaging (MRI). However, clinically used gadolinium-based contrast agents (such as gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) and gadoxetate disodium (Gd-EOB-DTPA)) generally exhibit low longitudinal relaxation efficiency. r 1) This leads to higher clinical dosages, posing certain potential risks. Therefore, the development of products with superior... r 1. Novel contrast agents with high biocompatibility and simple preparation process are very necessary.

[0003] According to SBM theory and Freed equations, the contrast agent... r 1. The relaxation of the inner sphere, the second sphere, and the outer sphere jointly regulates the increase of the water coordination number of the inner sphere. q ), increase the coordination number of hydrogen-bonded water in the second sphere ( q ') and extending the rotation-related time (τ) R ) etc. are improvements r 1. The most effective strategy. Based on these theories, numerous methods have been used to develop high-performance magnetic resonance contrast agents, such as: (1) those with tunable components, gadolinium ions (Gd) 3+ ), manganese ions (Mn) 2+ ), iron ions (Fe 3+ (1) Other lanthanide metal ions and even non-paramagnetic metal systems; (2) Diverse structural forms, chelates or nanomaterials; (3) Innovative structural designs, ultra-small structures, core-shell structures or hollow mesoporous structures; (4) Engineered surface modifications, etc. Among them, ultra-small nanoparticles with advantages such as large specific surface area, adjustable composition and easy surface functionalization are particularly outstanding and are expected to become the next contrast agent that can be applied clinically. However, there are still two problems that have not been significantly solved: (1) Traditional crystalline manganese-based and iron-based nanocontrast agents r 1 is still relatively low, mostly around 20mm. 1 s 1 The following; (2) These contrast agents often require complex synthesis processes, which are not conducive to large-scale industrial production. It is precisely for this reason that, even though the development of magnetic resonance contrast agents has made many improvements, no mature contrast agent has yet entered clinical application. Therefore, it is evident that constructing a contrast agent that can simultaneously optimize... q , q 'and τ R To achieve excellence rMaterials with specific properties are essential for use as MRI contrast agents. Furthermore, such materials should possess characteristics such as simple synthesis processes and high biocompatibility, facilitating large-scale production. Summary of the Invention The purpose of this invention is to provide an amorphous manganese salt for use as a magnetic resonance contrast agent and its preparation method.

[0004] To achieve the objectives of this invention, the following implementation scheme is provided: In one embodiment, the present invention provides an amorphous manganese salt for use as a magnetic resonance contrast agent, wherein the manganese salt is an inorganic acid salt modified with polycarboxylic acid. In some embodiments, the manganese salt is an inorganic acid salt, which is a carbonate, phosphate, silicate, or sulfite.

[0005] In a preferred embodiment, the amorphous manganese salt of the present invention is manganese carbonate (MnCO3).

[0006] In a preferred embodiment, the amorphous manganese salt of the present invention is polyacrylic acid (PAA), more preferably polyacrylic acid (PAA-1800) with an average molecular weight of 1800.

[0007] In some embodiments, the amorphous manganese salt of the present invention further comprises doped with nonparamagnetic metal ions selected from calcium, zinc and magnesium ions.

[0008] In some embodiments, the present invention also provides a method for preparing the amorphous manganese salt of the present invention described above, comprising the following steps: 1) Dissolve polycarboxylic acid and MnCl2 in deionized water and stir, then add sodium inorganic acid solution to react; 2) After the reaction is complete, add ethanol and centrifuge to remove the precipitate; 3) Resuspend the precipitate from the previous step in water, remove free polycarboxylic acid by ultrafiltration, and concentrate to obtain a concentrated solution; 4) Freeze-dry the concentrate to obtain the target sample.

[0009] In some embodiments, the method of the present invention further includes adding a dopant nonmagnetic metal ion in step 1), wherein the nonparamagnetic metal ion is selected from calcium ions, zinc ions and magnesium ions.

[0010] In some embodiments, the amorphous manganese salt in the method of the present invention described above is selected from carbonates, phosphates, silicates, and sulfites, preferably carbonates, i.e., amorphous manganese carbonate. The inorganic sodium acid in step 1) is sodium carbonate.

[0011] In a preferred embodiment, in the method of the present invention described above, the inorganic sodium acid is sodium carbonate, the polycarboxylic acid is PAA-1800, the molar ratio of sodium carbonate to manganese chloride is (1-2):(0.01-1), preferably 1:0.24; the molar mass ratio of sodium carbonate to PAA-1800 is (1-2) mmol:200 mg, preferably 1 mmol:200 mg.

[0012] In some embodiments, the use of the amorphous manganese salt of the present invention in the manufacture of a magnetic resonance contrast agent is also provided.

[0013] In a preferred embodiment, the contrast agent is used as a liver-specific and vascular-enhancing contrast agent.

[0014] The beneficial effects of the amorphous manganese salt of the present invention: The amorphous manganese salt of the present invention, after being modified with polycarboxylic acid such as PAA, can improve the stability and safety of the manganese salt and has the potential for clinical application. In particular, the relaxation efficiency is significantly improved after doping with non-magnetic metal ions.

[0015] This invention successfully prepared a series of PAA-stable ultrasmall amorphous paramagnetic nanoclusters using a one-step coprecipitation method. These nanoclusters exhibit consistently high... r The optimal MnCO3 concentration can reach 41.27 mM. 1 s 1 It has shown superiority in the development of excellent MRI contrast agents, and its synthesis process is simple and easy to commercialize.

[0016] The results of this invention demonstrate that MnCO3 has great potential in hepatocyte-specific MRI and contrast-enhanced magnetic resonance angiography (CE-MRA). Attached Figure Description

[0017] Figure 1 The images shown are the appearance and characterization diagrams of the MnCO3 product prepared in Example 6. In the images, a is the product appearance, b is the appearance of MnCO3 powder resuspended in deionized water and the Tyndall effect test, c is a transmission electron microscope (TEM) image, and d is a particle size distribution diagram under an electron microscope.

[0018] Figure 2 The images shown are conventional characterization diagrams of the MnCO3 product prepared in Example 6. In the diagrams, a is the hydration particle size distribution measured by dynamic light scattering (DLS), b is the zeta potential diagram, c is the Fourier transform infrared (FT-IR) spectrum, and d is the X-ray diffraction (XRD) pattern.

[0019] Figure 3The image shows the X-ray photoelectron spectroscopy (XPS) spectrum of the MnCO3 product prepared in Example 6. In the image, a is the full spectrum, and b is the Mn2 spectrum. p High-resolution spectrum, c represents Mn3 s High-resolution spectrum, d = O 1 s High-resolution spectrum.

[0020] Figure 4 Characterization images of MnCO3 and other manganese salts in Example 7 are shown. In the image, a represents different weighted MRI images of MnCO3, and b represents the longitudinal relaxation efficiency of MnCO3. r 1) The fitted curve (the slope is the corresponding curve) r 1), c is Mn 2+ After simple mixing with different substances r 1. Statistical graph, where d represents the concentrations of MnSiO3, MnSO3, and Mn3(PO4)2. r 1. Fitting curves, e is the XRD pattern of MnSiO3, MnSO3 and Mn3(PO4)2.

[0021] Figure 5 The TGA results for MnCO3 and other manganese salts in Example 7 are shown in Figure a. In Figure a, MnCO3 and the reference standard are TGA images, and MnSiO3, MnSO3 and Mn3(PO4)2 are TGA images.

[0022] Figure 6 The images show the characterization of MnCO3 modified with different polycarboxylic acids in Example 8. In the image, a is the XRD pattern, and b is... r 1. Fitted curve.

[0023] Figure 7 For different Mn in Example 8 2+ Characterization diagram of MnCO3 synthesized by dosage. Where a is... r 1. Fitted curve, b represents the transverse relaxation efficiency ( r 2) Fitted curves, c is the statistical graph of Mn content of some samples, and d is the TGA graph of some samples.

[0024] Figure 8 Characterization diagrams of MnCO3 modified with different amounts of PAA-1800 in Example 8. Where a is... r Figure 1 shows the fitted curve, and b is the appearance of MnCO3 synthesized with 50 mg of PAA.

[0025] Figure 9 Characterization diagrams of MnCO3 prepared with different amounts of Na2CO3 in Example 8. Where a represents... r 1. Fitted curves, b is a statistical graph of Mn content in some samples, and c is a TGA graph of some samples.

[0026] Figure 10 The reaction time and optimized synthesis conditions of MnCO3 obtained in Example 8 are used as a reference. r 1. Fitting curve. Where a represents the samples obtained at different reaction times, and b represents the samples obtained under optimized synthesis conditions.

[0027] Figure 11 This is a flowchart and characterization diagram of the large-scale synthesis of MnCO3 samples in Example 8. In the diagram, a is a detailed flowchart, b is MRI images with different weights, and c is... r 1. Fitted curve.

[0028] Figure 12 This is a fitting curve of the relaxation efficiency of the magnesium-doped MnCO3 (Mn-MgCO3) prepared in Example 9. Where a is... r 1, b is r 2.

[0029] Figure 13 Characterization diagrams of doped MnCO3 samples synthesized with different metal ions and different salts in Example 9 are shown. Wherein, a represents Mn-CaCO3, Mn-MgCO3, and Mn-MgCO3. r 1. Fitted curves, b represents the values ​​of Mn-CaSiO3, Mn-CaSO3, and Mn-Ca3(PO4)2. r 1. Fitting curve, c is the XRD pattern of the corresponding sample, and d is the TGA pattern.

[0030] Figure 14 The images show the MRI results of MnCO3 in mice in Example 10. A) shows whole-body coronal images acquired at different time points after injection of different doses of MnCO3 and Gd-EOB-DTPA; b) shows the dynamic contrast-to-noise ratio (CNR) analysis of the liver; and c) shows the maximum density projection (MIP) reconstruction of blood vessels in mice treated with a 25 μmol Mn / kg dose.

[0031] Figure 15 The image shows the MRI results of MnSiO3, MnSO3 and Mn3(PO4)2 in mice in Example 10.

[0032] Figure 16 The images show MRI images of the liver and corresponding H&E-stained pathological sections of mice with hepatocellular carcinoma after injection of MnCO3 and Gd-EOB-DTPA in Example 10.

[0033] Figure 17 The graphs show the stability evaluation of the MnCO3 powder and solution from Example 11 after fresh synthesis, 6 months of storage, and 15 months of storage. In the graphs, a is the XRD pattern, and b is... r 1. Fitted curve.

[0034] Figure 18 MnCO3 in different solutions and at different times in Example 11 r 1. Fitting curve. Where a represents fresh synthesis, b represents after 1 day of preparation, and c represents after 3 days of preparation.

[0035] Figure 19 The corresponding safety assessment graphs in Example 12 are shown. Among them, a and b are statistical graphs of the effect of MnCO3 on the survival rate of AML-12 cells and Hepa1-6 cells, respectively; c is a statistical graph of hemolysis rate; and d is a statistical graph of manganese content in various major organs measured 3 days after mice were injected with MnCO3.

[0036] Figure 20 The corresponding safety assessment diagram in Example 12 is shown. Among them, a is a statistical graph of blood routine and blood biochemistry test results of mice at different time points after injection of MnCO3, and b is a pathological section of H&E staining of various major organs of mice 14 days after injection of MnCO3. Detailed Implementation

[0037] The following embodiments are provided to describe the present invention in more detail. However, these embodiments are provided only to help further understand the present invention and are not intended to limit the present invention. Those skilled in the art should understand that equivalent substitutions or corresponding improvements made to the content of the present invention still fall within the protection scope of the present invention.

[0038] The characterization methods and property evaluation of the prepared amorphous manganese salts in the following examples were carried out as follows.

[0039] Product structure characterization Transmission electron microscopy (TEM) images were acquired using a Hitachi HT-7700 TEM at an accelerating voltage of 100 kV. High-angle annular dark-field scanning TEM (HAADF-STEM) images and corresponding energy-dispersive spectroscopy (EDS) spectra were acquired using a FEI TalosF200X high-resolution TEM. X-ray powder diffraction (XRD) patterns were recorded on a Bruker D8 advanced X-ray diffractometer. Hydrodynamic particle size and zeta potential were determined using a Malvern Zetasizer nano-ZS90 instrument. Magnetization curves (from -2 to 2 T at room temperature) were measured on a Lake Shore 8604 vibrating sample magnetometer. X-ray photoelectron spectroscopy (XPS) was performed on a Thermo escalab 250Xi photoelectron spectrometer. Fourier transform infrared spectroscopy (FT-IR) was recorded at room temperature on a Bruker Alpha II spectrometer. Thermogravimetric analysis (TGA) was performed using a Mettler Toledo TGA2 thermogravimetric analyzer under a nitrogen atmosphere at a heating rate of 10°C / min. Inductively coupled plasma optical emission spectroscopy (ICP-OES) and inductively coupled plasma mass spectrometry (ICP-MS) were performed on a Thermo iCAP 7200 ICP-OES instrument and a PerkinElmer NexION 300D ICP-MS instrument, respectively.

[0040] In vitro MRI and relaxation efficiency measurement In vitro magnetic resonance imaging was performed using a clinical 3.0 T MRI scanner (GE Signa HDx3.0T, USA) equipped with an 8-channel head coil. A simplified procedure was as follows: After ICP determination of the manganese concentration in the sample, a series of gradient concentration solutions (0.0125, 0.025, 0.05, 0.1, and 0.2 mM) were prepared with deionized water and placed in 2 mL centrifuge tubes. The centrifuge tubes were then fixed to a 48-well centrifuge tube rack and immersed in a plastic container filled with deionized water.

[0041] Imaging uses the following standard sequences: (1) Fast spin echo T1 weighted imaging (FSE T1WI): repetition time (TR) = 450 ms, echo time (TE) = 8.5 ms, region of interest (FOV) = 160×160 mm², matrix = 320×192, slice thickness / spacing = 1.0 mm / 0.3 mm, number of excitations (NEX) = 4; (2) T1 mapping: TE = 9ms, TR = 150, 300, 600, 900, 1200ms, FOV = 160×160mm², matrix = 256×256, layer thickness / spacing = 1.0 mm / 0.3 mm, NEX = 1; (3) FSE T2WI: TR = 2000 ms, TE = 50 ms, FOV = 160×160 mm², matrix = 192×160, layer thickness / spacing = 1.0 mm / 0.3 mm, NEX = 4; (4) T2mapping: TR = 1500 ms, TE = 8.8, 17.6, 26.3, 35.1, 43.9, 52.7, 61.4, 70.2ms, FOV = 160×160mm², matrix = 160×128, layer thickness / spacing = 1.0 mm / 0.3 mm, NEX = 1.

[0042] The T1 and T2 values ​​are calculated using the system's built-in post-processing software.

[0043] Cell culture and cell viability assay AML-12 cell line was purchased from Guangzhou Yuanjing Biotechnology Co., Ltd., while Hepa 1-6 cell line, Hepa 1-6 specific culture medium, and AML-12 complete culture medium were purchased from Wuhan Sewell Biotechnology Co., Ltd. All cells were routinely cultured in a humidified incubator at 37°C with 5% carbon dioxide using the appropriate culture medium.

[0044] The cytotoxicity of AMCs was assessed using the Standard Cell Counting Kit-8 (CCK-8) assay. The specific steps were as follows: First, AML-12 and Hepa 1-6 cells were counted at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured overnight. The medium was then replaced with a series of fresh medium containing AMC (manganese concentrations of 0, 0.03125, 0.0625, 0.125, 0.25, 0.5, and 1 mM) and cultured for another 24 hours. Each concentration was used in triplicate. The medium was then replaced with 100 μL of fresh medium containing 10 μL of CCK-8 solution (Beyotime, China) and cultured for another 1.5 hours. The absorbance was measured at 450 nm using a microplate reader (Synergy H1, USA). Cell viability was calculated using the following formula:

[0045] animal All animal experiments were conducted in accordance with the guidelines approved by the Laboratory Animal Welfare and Ethics Committee of Army Medical University (ethics number: AMUWEC20245286). Male C57BL / 6J mice weighing 16-20 grams and aged 6-8 weeks were purchased from Chongqing Tengxin Laboratory Animal Sales Co., Ltd. and housed under standard conditions at the Laboratory Animal Center of Xinqiao Hospital.

[0046] hemolysis test Fresh mouse blood was collected into anticoagulant tubes and washed repeatedly with PBS until the supernatant was clear. Red blood cell (RBC) suspension (1 × 10⁻⁶ per tube) was then prepared. 7 (Number of cells) were co-incubated with AMCs at gradient manganese concentrations (0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 mM) at 37°C for 4 hours. Deionized water and PBS were used as positive controls (100% hemolysis) and negative controls (0% hemolysis), respectively. After incubation, the samples were centrifuged at 1000 rpm for 5 minutes, and the absorbance of the supernatant was measured at 540 nm using a microplate reader. The hemolysis rate was calculated using the following formula:

[0047] Hepatocellular carcinoma-bearing mouse model This study used the Hepa 1-6 cell line and C57BL / 6J mice to construct a hepatocellular carcinoma model. The specific procedures were as follows: Freshly cultured Hepa 1-6 cells were collected and adjusted to a concentration of 1×10⁻⁶ cells. 7 After achieving a concentration of [number] cells / mL, the cells were stored on ice for later use. Mice were continuously anesthetized with isoflurane, fixed in a supine position, and the abdominal hair was shaved and disinfected with povidone-iodine. A small incision was made on the right side of the xiphoid process, parallel to the midline of the abdomen, to expose the liver lobe tissue without damaging surrounding organs. 50 μL of Hepa 1-6 cell suspension was slowly injected into the liver parenchyma using a microsyringe, leaving the needle in place for at least 30 seconds after injection to prevent cell leakage. Finally, the abdominal and skin incisions were sutured and disinfected again. Mice were fed normally, and tumor growth was monitored weekly by MRI.

[0048] In vivo MRI Mouse hepatocyte-specific MRI and CE-MRA were performed using a clinical 3.0 T MRI scanner (Philips Ingenia CX3.0 T, Netherlands), equipped with a dedicated 8-channel rat coil (MS80-3T; Suzhou Zhongzhi Medical Technology Co., Ltd., China). Mice were anesthetized with isoflurane. Whole-body coronal images were acquired using a three-dimensional fast field echo (3D FFE) sequence, while transverse liver images were acquired before and after tail vein injection using enhanced T1 high-resolution isotropic volume excitation (e... THRIVE sequence acquisition. Scanned samples included AMC (25, 10 and 5 μmmol Mn / kg), Gd-EOB-DTPA (25 μmmol Gd / kg) and other manganese-based UAPNCs (25 μmmol Mn / kg).

[0049] The specific sequence parameters are as follows: 3D FFE: TE = 3.9 ms, TR = 8.2 ms, FOV = 80 (FH) × 40 (RL) × 25 (AP) mm 3Voxel = 0.3 × 0.3 × 0.5 mm 3 Matrix = 268×136×100, layer thickness = -0.25 mm, average signal strength (NSA) = 2, flip angle = 20°; e THRIVE: TE=4 ms, TR=10.7 ms, FOV =30 (RL) × 30 (AP) × 30 (FH) mm 3 Voxel = 0.25 × 0.3 × 1 mm 3 The matrix was 120×100×60, the slice thickness was -0.5 mm, the NSA was 5, and the flip angle was 30°. The vascular images were reconstructed using maximum intensity projection (MIP) through the system's built-in post-processing software.

[0050] The contrast-to-noise ratio (CNR) is calculated using the following formula: CNR = (SI 肝脏 - SI 肌肉 ) / δ 噪声 It should be noted that almost all MRI images in this study were displayed using the same window level (1000) and window width (2000).

[0051] In vivo safety evaluation In the in vivo safety assessment, mice in the experimental group were intravenously injected with AMC (0.1 mmol Mn / kg), while the control group received saline. Mice were sacrificed on days 1, 7, 14, and 28 post-injection, and blood samples were collected for routine blood tests and biochemical analyses, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), and urea (UREA). Simultaneously, on day 14, the major organs (heart, liver, spleen, lungs, kidneys, and brain) of the mice were removed and observed using hematoxylin and eosin (H&E) staining. To clarify the metabolism of MnCO3, after injection of MnCO3 (25 μmmol Mn / kg), the major organs were removed, and manganese content was analyzed by ICP-MS.

[0052] Example 1: Preparation of amorphous MnCO3 PAA-1800 (200 mg) and MnCl2•4H2O (47.5 mg, 0.24 mmol) were dissolved in 10 mL of deionized water and placed in a 100 mL beaker. The mixture was magnetically stirred (500 rpm) for 3 minutes. Then, 5 mL of sodium carbonate (Na2CO3) solution (0.2 M) was rapidly added, and magnetic stirring was continued for 1 minute. The reaction was terminated by adding 20 mL of ethanol, and the mixture was immediately centrifuged at 12000 rpm for 15 minutes. The colorless, transparent gelatinous precipitate was resuspended in deionized water by sonication, followed by ultrafiltration (5 kDa) to remove free PAA. Finally, the concentrated solution obtained by ultrafiltration was lyophilized to obtain the target sample.

[0053] Example 2: Preparation of amorphous MnSiO3 The preparation method was the same as in Example 1, except that Na2CO3 was replaced with Na2SiO3 to obtain amorphous MnSiO3 nanoclusters.

[0054] Example 3: Preparation of amorphous MnSO3.

[0055] The preparation method was the same as in Example 1, except that Na2CO3 was replaced with Na2SO3 to obtain amorphous MnSO3 nanoclusters.

[0056] Example 4 Preparation of amorphous Mn3(PO4)2 The amorphous Mn3(PO4)2 nanoclusters were prepared by replacing Na2CO3 with Na3PO4, as in Example 1.

[0057] Example 5: Preparation of nonparamagnetic metal-doped amorphous MnCO3 Prepared according to the method of Example 1, except that MnCl2 is replaced with MnCl2 + CaCl2, or MnCl2 + MgCl2, or MnCl2 + ZnCl2, provided that the total amount of metal ions remains constant compared to that in Example 1 (e.g., Mn...). 2+ and Mg 2+ The total amount of the substance was 0.24 mmol), and PAA-modified Mn-CaCO3, Mn-MgCO3, or Mn-ZnCO3 nanoclusters were prepared respectively.

[0058] Example 6 Characterization of conventional physicochemical properties of MnCO3 MnCO3 was freeze-dried to obtain a white powder. Figure 1 a) It facilitates transportation and storage. When resuspended in deionized water, it appears colorless and transparent, exhibiting a pronounced Tyndall effect (a). Figure 1 b). TEM image ( Figure 1 c) and the corresponding particle size statistical distribution diagram ( Figure 1d) indicates that MnCO3 has a narrow particle size distribution and good uniformity, with an average diameter of 2.0 ± 0.32 nm.

[0059] The hydrodynamic particle size of MnCO3, measured by dynamic light scattering (DLS), was 3.6 ± 0.51 nm. Figure 2 a). Furthermore, due to PAA surface modification, MnCO3 exhibits a significant negative Zeta potential (-35 mV). Figure 2 b) indicates that it has excellent colloidal stability.

[0060] Furthermore, Fourier transform infrared (FT-IR) spectroscopy indicates that 1712 cm⁻¹ in PAA... -1 The peak at 0.05 cm⁻¹ (corresponding to the stretching vibration of the carboxyl group) is redshifted to 1560 cm⁻¹ in MnCO₃. -1 Attributable to the antisymmetric stretching vibration of the carboxylate group ( Figure 2 c) confirms the success of PAA modification in MnCO3. For example... Figure 2 As shown in Figure d, X-ray powder diffraction (XRD) analysis revealed no obvious characteristic peaks in MnCO3, confirming its amorphous structure. Therefore, it can be concluded that, with the assistance of PAA, MnCO3 exists in a stable, ultra-small amorphous structure.

[0061] X-ray photoelectron spectroscopy (XPS) analysis of MnCO3 showed that its characteristic peaks corresponded to oxygen (O 1) and oxygen (O 1) respectively. s ), carbon (C1) s ) and manganese (Mn 2 p () Figure 3 a). In Mn 2 p In the core energy spectrum ( Figure 3 (b) Two peaks appear at 641.6 and 653.7 eV, corresponding to Mn2, respectively. p 3 / 2 and Mn 2 p 1 / 2 Energy level. Mn3 s 3 / 2 With Mn 3 s 1 / 2 The splitting energy between them is 6 eV ( Figure 3 c), indicating that the main form of manganese in MnCO3 is Mn. 2+ The O 1s core energy spectrum only shows three distinct peaks at 536, 532.6, and 531.6 eV, corresponding to adsorbed water, single-bonded oxygen, and double-bonded oxygen, respectively. Figure 3 d). Due to the structural similarity between carboxylate and carbonate, their characteristic O 1s signals may overlap.

[0062] Example 7: Relaxation efficiency of MnCO3 To evaluate the relaxation efficiency of MnCO3, nanoclusters were dispersed in deionized water at different concentrations and imaged using a 3.0T clinical MRI scanner, employing typical T1WI, T2WI, T1mapping, and T2mapping sequences. Results are as follows: Figure 4 As shown in Figure a, with increasing MnCO3 concentration, the T1WI image gradually brightens, while the T2WI image slightly darkens. Linear fitting analysis ( Figure 4 b) Discovery of MnCO3 r 1 is 26.71 mM 1 s 1 The corresponding lateral relaxation efficiency ( r 2) 74.08 mM 1 s 1 Meanwhile, MnCO3's r 2 / r The ratio is as low as 2.8. Overall, the ratio is relatively high. r 1 value and lower r 2 / r The 1-to-1 ratio makes MnCO3 a potential candidate material for MRI contrast agents.

[0063] Furthermore, in order to elucidate the excellent properties of ultrasmall amorphous structures for MnCO3 r The contribution of the 1 value led to the preparation of a series of control samples. For example... Figure 4 As shown in c, pure Mn 2+ of r The value is only 5.44 mM. 1 s 1 Even after directly adding PAA, it remained almost unchanged (5.79 mM). 1 s 1 ), indicating that PAA and Mn 2+ Simple mixing does not significantly improve r 1 value. Surprisingly, when Mn 2+ When combined with amorphous calcium carbonate (CaCO3), r The value surged to 31.08 mM. 1 s 1 It even surpasses MnCO3. r 1 value. In contrast, when Mn2+, PAA and crystalline CaCO3 are mixed, r1 increased only slightly (9.77 mM) 1 s 1 This further proves the role of ultra-small amorphous structures in improving... r 1 is crucial. Furthermore, the prepared MnSiO3, MnSO3, and Mn3(PO4)2 nanoclusters... r The values ​​of 1 were all close to those of MnCO3, at 28.37, 23.38, and 25.77 mM, respectively. 1 s 1 ( Figure 4 d), and the XRD test results show that they are all amorphous structures ( Figure 4 e). These results demonstrate that ultrasmall amorphous structures can improve... r The advantages and universality of aspect 1.

[0064] Thermogravimetric analysis (TGA) was used to further investigate the detailed enhancement mechanisms of ultrasmall amorphous structures. For example... Figure 5 As shown in Figure a, pure PAA begins to decompose at approximately 180°C, while the slight weight loss observed before this temperature is likely attributed to water evaporation, confirming PAA's hydration capacity. The decomposition of carbonates, however, begins at around 550°C. Clearly, MnCO3 and CaCO3 contain approximately 20% water and about 35% PAA, while crystalline CaCO3 contains neither water nor PAA. Similarly, MnSiO3, MnSO3, and Mn3(PO4)2 also exhibit high water content (…). Figure 5 b). Therefore, it can be concluded that there is a relationship between amorphous and crystalline nanoclusters. r The significant difference in the 1 value is mainly due to their different hydration capacities.

[0065] Example 8: Effects of Synthesis Parameters and Conditions on MnCO3 (1) The effect of different polycarboxylic acids Following the method of Example 1, PAA-1800 was replaced with polyaspartic acid (PASP) with a molecular weight of 8000, polymaleic acid (PMA) with a molecular weight of approximately 1000, and PAA-3000 and PAA-5000, respectively, to prepare MnCO3-PASP, MnCO3-PMA, MnCO3-PAA 3000, and MnCO3-PAA 5000 samples. First, XRD confirmed that all samples had an amorphous structure. Figure 6 a) indicates that, besides PAA, other hydrophilic polycarboxylic acids can also help MnCO3 maintain its amorphous structure. For example... Figure 6 As shown in b, the corresponding sample obtained r The values ​​were 18.99, 20.31, 27.91, and 25.45 mM, respectively. 1 s 1 Therefore, it can be seen that polycarboxylic acids can give MnCO3 excellent properties. r 1. However, due to differences in molecular weight, its r 1. There is still a slight difference, and it seems that its r The value of 1 increases first and then decreases as the molecular weight increases, exhibiting a parabolic change.

[0066] (2) Mn 2+ Effect of dosage Referring to the method in Example 1, while keeping other conditions unchanged, the amount of MnCl2 was varied to 0.01-0.96 mmol. Figure 7 As shown in a, with Mn 2+ The dosage was increased from 0.01 to 0.96 mmol, and the MnCO3 concentration... r 1 decreased from 36.47 to 17.4 mM 1 s 1 ,at the same time r 2. First, it decreased from 93.15 to 62.35 mM 1 s 1 It then rose slightly. Figure 7 b). This is because with Mn 2+ With increased dosage, PAA deficiency occurred, leading to a decrease in the τ of MnCO3. R Reduce, and thus its r The concentration of Mn will gradually decrease. This is consistent with the gradually increasing Mn content. Figure 7 c), while no significant difference was observed in water content ( Figure 7 d). This indicates that, with the PAA dosage remaining constant, Mn 2+ Increasing the dosage will cause MnCO3 to... r 1. Decrease.

[0067] (3) Effect of PAA dosage Following the method of Example 1, and with other conditions remaining unchanged, the dosage of PAA-1800 was adjusted to 50-800 mg to obtain a series of MnCO3 samples with different PAA dosages. Figure 8 As shown in a, with the change of Mn 2+ With varying dosages, as the amount of PAA increases, the amount of MnCO3 decreases. r 1. First, it increased from 14.11 to 26.31 mM 1 s 1The concentration then decreased slightly. Among them, the MnCO3 solution modified with 50 mg PAA (MnCO3-50) turned significantly darker (8b). This is because insufficient PAA resulted in an alkaline reaction solution, leading to a decrease in the concentration of MnCO3. 2+ This is due to oxidation. Therefore, increasing the amount of PAA can indeed increase the concentration of MnCO3 to some extent. r The value is 1, but it is not positively correlated. Furthermore, sufficient PAA can act as a protective agent against Mn. 2+ Oxidation is also a key factor in maintaining the stability of MnCO3.

[0068] (4) Effect of Na2CO3 dosage Following the method in Example 1, and keeping other conditions unchanged, a series of MnCO3 samples with different Na2CO3 concentrations were prepared by varying the amount of Na2CO3 used to 0.25, 0.5, 1, 1.5, and 2 mmol. The results showed that as the amount of Na2CO3 increased, the concentration of MnCO3 decreased. r The value of 1 gradually increases ( Figure 9 a), while the manganese content decreased ( Figure 9 b) while the water content increases ( Figure 9 c). This is because, despite the total PAA dose remaining constant, the actual effective PAA content in MnCO3 increases, as evidenced by the significantly increased degradation observed in MnCO3 synthesized from 0.5 mmol Na2CO3. This is because the PAA is not sufficiently deprotonated, resulting in a decrease in the amount of PAA on the nanoparticle surface. Therefore, it can be seen that with increasing Na2CO3 dosage, the effective PAA content in MnCO3... r 1 will gradually increase.

[0069] (5) Effect of reaction time Following the method of Example 1, and with other conditions remaining unchanged, the reaction time was adjusted to 1, 10, 30, and 120 minutes to obtain a series of MnCO3 samples with different reaction times. Figure 10 As shown in Figure a, no abnormalities were observed in the MnCO3 samples prepared at different reaction times. r The difference is obvious. It is evident that, for this reaction system, reaction time has a significant impact on the MnCO3 content. r 1. No significant impact.

[0070] (6) Optimization and large-scale synthesis Based on the above conclusions, the optimal synthesis conditions for MnCO3 were summarized. r 1 reached 41.27 mM 1 s 1 ( Figure 10 b), corresponding to r2 is 106.58 mM 1 s 1 The product consists of 0.01 mmol Mn 2+ It was prepared using 200 mg PAA, 2 mmol Na2CO3, and a reaction time of 1 minute. However, considering yield and cost, this study preferentially recommends the following typical synthesis conditions for MnCO3: 0.24 mmol Mn... 2+ Prepared with 200 mg PAA, 1 mmol Na2CO3, and a reaction time of 1 minute.

[0071] Finally, large-scale synthesis of MnCO3 was also attempted, such as... Figure 11 As shown in a, it uses 10 mmol Mn 2+ A detailed flowchart of the synthesis of MnCO3 from 4 g PAA and 20 mmol Na2CO3 is provided. Its in vitro MRI imaging results are consistent with those of classically synthesized MnCO3 samples. Figure 11 b) r 1 and r The values ​​were 24.73 and 53.53 mM, respectively. 1 s 1 This further clarifies the feasibility of large-scale synthesis and lays a solid foundation for large-scale synthesis in later clinical translation.

[0072] In summary, several polycarboxylic acids used in this reaction system can serve as stabilizers to achieve stable synthesis of MnCO3. The molecular weight of these polycarboxylic acids has a significant impact on the synthesis of MnCO3. r 1. This will have some impact. Adjusting Mn... 2+ The nature of PAA and Na2CO3 actually affects the effective amount of PAA in the reaction system, which will adjust the τ of MnCO3. R This affects its r 1. Furthermore, the optimization of synthesis parameters provides theoretical guidance for subsequent large-scale synthesis.

[0073] Example 9: Effect of doping with nonparamagnetic metals on MnCO3 The introduction of magnesium ions (Mg) into MnCO3 was further explored. 2+ ), calcium ions (Ca 2+ ) and zinc ions (Zn 2+ The effects of common nonparamagnetic metal ions, such as... Figure 12 As shown in a and 12b, in Mn 2+ and Mg 2+ When the total amount (0.24 mmol) remains constant, as Mn... 2+The dosage was increased from 0.01 to 0.22 mmol, and the Mn-MgCO3 concentration... r 1 decreased from 39.56 to 28.24 mM 1 s 1 ,and r The value decreased from 108.54 to 64.42 mM. 1 s 1 Obviously, Mn-MgCO3... r 1 and r 2 were almost all higher than typical MnCO3 (26.71 and 74.08 mM). 1 s 1 ), indicating the addition of Mg 2+ It will significantly improve its relaxation efficiency, and Mg 2+ The larger the proportion, r 1 and r The higher the value of 2, the better.

[0074] To further clarify the effect of introducing different nonparamagnetic metal ions on improving the concentration of MnCO3... r To ensure the universality of 1, we prepared a series of control samples. For example... Figure 13 As shown in a, using the same amount of Mn 2+ Mn-CaCO3, Mn-MgCO3, and Mn-ZnCO3 were prepared from (0.04 mmol) and nonparamagnetic metal ions (0.2 mmol). r The concentrations were 37.13, 36.74, and 36.37 mM, respectively. 1 s 1 And their r The values ​​were 102.89, 100.66, and 107.56 mM, respectively. 1 s 1 Compared to MnCO3, they r 1 and r The increase was nearly 40%, but there was no significant difference between them. This indicates that the type of non-paramagnetic metal ion does not affect this enhancement effect.

[0075] Furthermore, the variations in inorganic manganese salt samples doped with nonparamagnetic metal ions were investigated. For example... Figure 13 As shown in b, the obtained Mn-CaSiO3, Mn-CaSO3, and Mn-Ca3(PO4)2 r1. Although there are slight differences, compared with the corresponding undoped sample, its r 1 also showed significant enhancement. XRD confirmed that they were all amorphous structures. Figure 13 c), and the moisture content of all of them exceeded 20% ( Figure 13 d). This indicates that anions do not interfere with the enhancement effect brought about by doping non-paramagnetic metal ions.

[0076] Example 10: Mouse Hepatocyte-Specific MRI and CE-MRA To verify the applicability of MnCO3 in hepatocyte-specific MRI and CE-MRA in vivo, contrast-enhanced MRI was performed on mice using a clinical 3.0T scanner. Whole-body coronal images were acquired using three-dimensional fast field echo (3D FFE) sequences after tail vein injection of MnCO3 at doses of 25, 10, and 5 µmol Mn / kg. Figure 14 As shown in Figure a, the liver showed significant enhancement after administration, exhibiting a clear time- and dose-dependent effect, demonstrating the excellent applicability of MnCO3 in hepatocyte-specific imaging. Dynamic contrast-to-noise ratio (CNR) analysis revealed that the liver CNR rapidly increased after MnCO3 injection, peaking at 12 minutes, and then gradually decreased. Figure 14 (b) Furthermore, the liver CNR in the MnCO3 group was not only higher than that in the group receiving the same dose of Gd-EOB-DTPA, but also remained at a higher level 2 hours after injection. Unexpectedly, unlike Gd-EOB-DTPA, no significant vascular enhancement was observed in mice injected with 25 µmol Mn / kg MnCO3 (14c), indicating that MnCO3 is also a potential candidate contrast agent for CE-MRA. Simultaneously, significant enhancement was observed in the bladder and gallbladder of the mice, suggesting that MnCO3, similar to Gd-EOB-DTPA, can be metabolized simultaneously via both hepatic and renal pathways, significantly improving its biocompatibility.

[0077] In addition, such as Figure 15 As shown, MnSiO3, MnSO3, and Mn3(PO4)2 all exhibited the same imaging enhancement effect, demonstrating the universality of this ultrasmall amorphous manganese salt as a magnetic resonance contrast agent. Subsequently, a mouse model of hepatocellular carcinoma was constructed, and after tail vein injection of 25 and 5 µmol Mn / kg doses of MnCO3, enhanced T1 high-resolution isotropic volumetric excitation (e... Imaging is performed using THRIVE sequences. Figure 16 As shown, normal liver tissue in mice exhibits significant enhancement, while tumors show markedly low signal intensity. This demonstrates that MnCO3 can indeed be used for the diagnosis of liver cancer and holds promise as an excellent liver-specific MRI contrast agent.

[0078] Example 11 Stability Evaluation The prepared MnCO3 sample powder was stored at room temperature, and samples were taken at 6 and 15 months respectively. XRD analysis was performed, and the results are as follows: Figure 17 As shown in a, the amorphous structure was maintained 6 months and 15 months after synthesis.

[0079] The synthesized MnCO3 samples were evaluated before and after lyophilization, as well as after storage at room temperature for a certain period of time. r The changes in 1. For example... Figure 17 As shown in b (where N represents the solution before freeze-drying and F represents the powder after freeze-drying), regardless of whether it is stored in solution or powder form, the freeze-drying process before and after freeze-drying... r 1. No significant difference was found, indicating that freeze-drying has no effect on the properties of MnCO3. However, regardless of whether it is a solution or powder, its... r A slight decrease was observed at both 6 and 15 months. This may be due to the fact that Mn at room temperature... 2+ This is caused by slow oxidation, and can be solved by sealing and storing.

[0080] MnCO3 was dispersed in deionized water (DI), normal saline (NS), phosphate buffer (PBS), acetate buffer (pH 5.0), and fetal bovine serum (FBS) to assess its stability in different solutions. Figure 18 As shown, regardless of the dispersion medium, MnCO3... r 1. It remains essentially unchanged over 3 days, indicating that MnCO3 has extremely high stability. And its stability in different solutions... r There is a slight change in τ, which may be related to the viscosity coefficient of the solution, affecting the exchange rate of water molecules. In contrast, there is a significant decrease in FBS, possibly due to protein adsorption, which increases its molecular weight and consequently leads to a decrease in τ. R reduce.

[0081] Example 12 Safety Evaluation The cytotoxicity of MnCO3 was assessed by detecting cell viability using the CCK-8 assay. Figure 19 As shown in a and 19b, the cell viability of both cell lines remained above 80% at all tested concentrations, indicating that MnCO3 has good cell biocompatibility.

[0082] Hemolysis was performed by co-incubating fresh mouse red blood cells with MnCO3. For example... Figure 19 As shown in c, at concentrations below 2 mM, the hemolysis rate is less than 2%, which is well within the acceptable safety threshold of 5%.

[0083] Finally, the in vivo safety of MnCO3 was assessed through biodistribution analysis, routine blood tests, blood biochemistry tests, and histological observation. Considering the rapid metabolism of MnCO3, its in vivo distribution was examined on day 3 after administration of a dose of 25 µmol / kg Mn. Figure 19 As shown in d, compared with the control group, the manganese content in most organs of mice injected with MnCO3 was reduced, with only a slight increase in the liver. This phenomenon may reflect the compensatory excretion mechanism of the body to maintain manganese homeostasis. Meanwhile, routine blood tests and biochemical tests showed that even at a dose (100 µmol Mn) four times the imaging dose, MnCO3 remained safe. Figure 20 a). The light blue area represents the normal range. Furthermore, 14 days after injection of MnCO3 (100 µmol / Kg Mn), major organs of mice were harvested for H&E staining and observation. (See image below.) Figure 20 As shown in b, no obvious histological abnormalities, inflammatory infiltration, necrosis, or pathological damage were observed in any of the organs, indicating that at this dose, MnCO3 does not cause significant organ damage or toxic reactions.

[0084] In summary, this study systematically explored the mechanism, advantages, and versatility of ultrasmall amorphous manganese salts as MRI contrast agents. Furthermore, the feasibility and safety of using MnCO3 for hepatocyte-specific MRI and CE-MRA were evaluated. Based on its excellent stability and safety, the clinical application potential of polycarboxylic acid-stabilized ultrasmall amorphous manganese salts in MRI contrast agents is undeniable.

Claims

1. An amorphous manganese salt for use as a magnetic resonance contrast agent, said manganese salt being an inorganic acid salt, characterized in that, The inorganic acid salt is modified with polycarboxylic acid.

2. The amorphous manganese salt according to claim 1, wherein the inorganic acid salt is a carbonate, phosphate, silicate, or sulfite.

3. The amorphous manganese salt according to claim 1, wherein the polycarboxylic acid is polyacrylic acid, polymaleic acid, or polyaspartic acid.

4. The amorphous manganese salt according to claim 2, wherein the inorganic acid salt is manganese carbonate, manganese phosphate, manganese silicate, or manganese sulfite.

5. The amorphous manganese salt of claim 1, further comprising doped with nonparamagnetic metal ions.

6. The amorphous manganese salt according to claim 5, wherein the nonparamagnetic metal ion is selected from calcium ions, zinc ions, and magnesium ions.

7. A method for preparing the amorphous manganese salt according to any one of claims 1-6, comprising the following steps: 1) Dissolve polycarboxylic acid and manganese chloride (MnCl2) in deionized water and stir, then add sodium inorganic acid solution to react; 2) After the reaction is complete, add ethanol and centrifuge to remove the precipitate; 3) Resuspend the precipitate from the previous step in water, remove free polycarboxylic acid by ultrafiltration, and concentrate to obtain a concentrated solution; 4) Freeze-dry the concentrate to obtain the target sample.

8. The method according to claim 7, wherein the inorganic sodium acid is sodium carbonate, the polycarboxylic acid is PAA-1800, the molar ratio of sodium carbonate to manganese chloride is (1-2):(0.01-1), preferably 1:0.24; and the molar / mass ratio of sodium carbonate to PAA-1800 is (1-2) mmol:200 mg, preferably 1 mmol:200 mg.

9. The method of claim 7, further comprising adding a non-magnetic metal ion in step 1), wherein the non-magnetic metal ion is selected from calcium ions, zinc ions and magnesium ions.

10. The use of the amorphous manganese salt according to any one of claims 1-6 in the manufacture of a magnetic resonance contrast agent, preferably, the magnetic resonance contrast agent being used as a liver-specific or vascular-enhancing contrast agent.

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