Magnetic resonance imaging contrast agent based on gadolinium-ellagic acid as well as preparation method and application of magnetic resonance imaging contrast agent

Abstract

The invention relates to the technical field of functional materials, in particular to a magnetic resonance imaging contrast agent based on gadolinium-ellagic acid as well as a preparation method and application of the magnetic resonance imaging contrast agent. The magnetic resonance imaging contrast agent comprises ellagic acid, gadolinium ions, polyvinylpyrrolidone, NaOH and water. The molar ratio of ellagic acid to gadolinium ions is (1: 1)-(1: 0.3); the mass ratio of PVP to ellagic acid is (20: 1)-(25: 1); and the concentration of the NaOH is 0.02 to 0.025 M. According to the gadolinium-based contrast agent, gadolinium ions can be firmly locked in a physiological environment to prevent leakage through ellagic acid polyphenol hydroxyl and Gd oriented coordination, so that green synthesis, rapid preparation, high stability and biological safety of the gadolinium-based contrast agent are realized; the method is a key direction for promoting sustainable application of the method in the field of medical image diagnosis, reducing clinical risks and expanding new scenes.

Description

Technical Field

[0001] This invention relates to the field of functional materials technology, and in particular to a gadolinium-ellagic acid-based magnetic resonance imaging contrast agent, its preparation method, and its application. Background Technology

[0002] Magnetic resonance imaging (MRI) has irreplaceable value in tumor diagnosis. It can improve the contrast imaging effect between tumor tissue and physiological tissue, improve imaging quality, and reduce misdiagnosis. However, its imaging contrast is significantly dependent on the enhancement efficacy of contrast agents.

[0003] Gadolinium-based contrast agents have become a research hotspot in the field of medical imaging due to their excellent imaging effects and low toxicity. Clinically, low molecular weight gadolinium (Gd) chelate contrast agents are generally used to enhance magnetic resonance signals, but these contrast agents have disadvantages such as short in vivo circulation time, low relaxation rate, and large dosage. In recent years, nanotechnology has been widely used in the research of MRI contrast agents. Studies have shown that nanocontrast agents prepared by incorporating Gd into polymer nanocarriers have good relaxation properties, reducing the total dosage and thus reducing toxic side effects. Gadolinium contrast agent nanoformulations mainly include liposomes, dendritic molecules, micelles, nanoparticles, carbon nanotubes, and fullerenes. However, the synthesis of polymer nanoparticles is complicated, purification is difficult, and stability is insufficient, resulting in the following obvious defects in the current synthesis process of gadolinium-based nanocontrast agents: the construction of nanocarriers requires high-temperature annealing (>200℃) or organic solvent phases (such as chloroform / dimethyl sulfoxide), the purification process must use toxic cross-linking agents (such as carbodiimide), the reaction is time-consuming, generally exceeding 12 hours, and generates a large amount of harmful waste liquid. More seriously, the product structure is not stable enough. For example, in the liposome-loaded gadolinium system, the structural disintegration rate is >30% within 72 hours due to phospholipid hydrolysis, which poses a serious clinical safety risk.

[0004] Patent application 2015108225915 discloses a method for preparing gadolinium phosphate nanomaterials, which requires high-temperature calcination at 900℃ for 4 hours. Patent 2010106075785 discloses a highly efficient gadolinium-loaded liposome formulation and its preparation method, which uses a lipid gel method to prepare gadolinium-loaded liposomes, increasing the gadolinium loading and improving stability. However, the organic solvent used in its liposome preparation is chloroform, and the reaction time is at least 9 hours. The literature "Synthesis and In Vivo Magnetic Resonance Imaging Study of Novel Polymer Nanocontrast Agents" (Journal of Hefei University of Technology, Vol. 46, No. 2) uses a reversible addition-fragmentation chain transfer polymerization method to polymerize amphiphilic polymer nanoparticles with hydrophobic copolymer segments as the core and hydrophilic copolymer segments as branches. After chelation reaction with gadolinium(III) hexahydrate, a novel branched polymer magnetic resonance imaging nanocontrast agent P(DO3A-Gd) is synthesized. 3+ The preparation process is complicated, involves many types of solvents, and takes more than 24 hours to complete.

[0005] Therefore, there is an urgent need to develop novel gadolinium-based nanocontrast agents that are easy to prepare and synthesize and have high performance. Summary of the Invention

[0006] This invention provides a gadolinium-ellagic acid-based magnetic resonance imaging contrast agent, its preparation method, and its application, which solves the problems of existing gadolinium-based nanocontrast agents, such as cumbersome preparation steps, use of toxic cross-linking agents in the purification process, long reaction time and easy generation of large amounts of waste liquid, poor stability of the contrast agent in the physiological environment, and potential clinical safety hazards.

[0007] One of the technical solutions adopted in this invention is: A gadolinium-ellagic acid-based magnetic resonance imaging contrast agent is provided, comprising ellagic acid, gadolinium ions, polyvinylpyrrolidone (PVP), NaOH solution, and water; wherein the molar ratio of ellagic acid to gadolinium ions is 1:1 to 1:0.3; the mass ratio of PVP to ellagic acid is 20:1 to 25:1; and the concentration of the NaOH solution is 0.02 to 0.025 M.

[0008] Furthermore, the molar ratio of ellagic acid to gadolinium ions is 1:0.5; the mass ratio of the total amount of PVP to ellagic acid is 23:1; and the concentration of NaOH is 0.0225 M.

[0009] Furthermore, the gadolinium ions are derived from gadolinium chloride, specifically GdCl3·6H2O.

[0010] Furthermore, the water is deionized water.

[0011] The second technical solution adopted in this invention is: A method for preparing the above-mentioned gadolinium-ellagic acid-based magnetic resonance imaging contrast agent is provided, including the following steps: S1. Dissolve ellagic acid in NaOH solution to form sodium ellagic acid solution; S2. Dissolve GdCl3·6H2O together with the first PVP in water; S3. While stirring, add the sodium ellagic acid solution from S1 dropwise to S2; after the solution turns yellow, add the remaining PVP and react at 50~80℃ for 0.5~3 h. S4. After the reaction, the mixture was centrifuged at 12,000 rpm for 10 min. The precipitate was washed several times with ethanol / water and dried to obtain Gd-EA nanoparticles.

[0012] Furthermore, the volume of NaOH solution in step S1 is 10 mL; the volume of water in step S2 is 20 mL.

[0013] Furthermore, the reaction temperature in step S3 is preferably 60°C, and the reaction time is 1 hour; the drying temperature in step S4 is 60°C.

[0014] Furthermore, the ratio of PVP usage in steps S2 to S3 is 2:5.

[0015] Furthermore, in step S4, the patient is washed three times with ethanol / water.

[0016] In the chelation process described above, ellagic acid phenolic anions and Gd³⁺ form a nanostructure through an octetary coordination bond.

[0017] The third technical solution adopted in this invention is: The application of the aforementioned magnetic resonance imaging contrast agent as a contrast agent for breast tumors is provided.

[0018] The beneficial effects of this invention are: 1. This invention relates to a magnetic resonance imaging contrast agent based on gadolinium-ellagic acid chelate (Gd-EA), successfully constructing a synergistic system of "green synthesis-rapid preparation-high stability-biosafety": Through an aqueous reaction system (deionized water as the sole solvent), one-step chelation of ellagic acid and gadolinium ions is achieved under mild conditions of 60℃, completely eliminating organic reagents; the reaction time is only 60 minutes, 12-24 times shorter than conventional methods, and energy consumption is reduced by more than 95%; the coordination chelation significantly improves the material's high stability. Ellagic acid, as an FDA-recognized safe substance (GRAS), forms a nanostructure through directional chelation of polyphenolic hydroxyl groups. As a stable chelating ligand, it can firmly lock gadolinium ions in the physiological environment to prevent leakage, ensuring biosafety. Simultaneously, its antioxidant and anti-inflammatory properties can reduce the potential damage of the contrast agent to the body. PVP, as a surfactant, helps stabilize nanoparticles to prevent aggregation, regulate particle size and morphology, and improve the dispersibility and interfacial compatibility of nanomaterials. Cytotoxicity experiments confirmed that the complex had no significant toxic effect on normal cells, and the degradation products were safely excreted through the body's metabolic pathways.

[0019] 2. This invention breaks through the industrial technology bottleneck by using aqueous low-temperature synthesis, and simultaneously achieves zero-pollution production, efficient preparation, structural stability and biocompatibility, providing an innovative solution for the field of contrast agents and having significant clinical application value. Attached Figure Description

[0020] Figure 1 This is a TEM image of Gd-EA obtained by the preparation method in Example 1 of the present invention; Figure 2 These are TEM images of Gd-EA obtained by the preparation methods in Examples 2-7 of this invention; Figure 3 The XRD pattern of Gd-EA obtained in Example 1 of this invention; Figure 4 The cell viability of Gd-EA obtained in Example 1 of this invention is shown in the cytotoxicity test. Figure 5 The results of tumor magnetic resonance imaging using Gd-EA contrast agent are shown in an application example of this invention.

[0021] in, Figure 5 In the image, a and b are MRI images of mouse tumors without Gd-EA contrast agent; c and d are MRI images of mouse tumors after injection of the contrast agent. Detailed Implementation

[0022] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings.

[0023] In the following specific embodiments of the present invention, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are permissible with acceptable deviations due to instrument testing accuracy or operational precision. The equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0024] Example 1 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 18.5 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 60 °C for 1 h. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0025] Example 2 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 37 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 60 °C for 1 h. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0026] Example 3 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 11.1 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 60 °C for 1 h. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0027] Example 4 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 18.5 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 50 °C for 1 h. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0028] Example 5 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 18.5 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 80 °C for 1 h. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0029] Example 6 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 18.5 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 60 °C for 30 min. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0030] Example 7 Prepare 10 mL of 0.0225 M NaOH solution containing 30.2 mg ellagic acid (solution A) and 20 mL of aqueous solution containing 18.5 mg GdCl3·6H2O and 0.2 g PVP (solution B). Add solution A dropwise to solution B while stirring. After the solution turns yellow, add 0.5 g PVP and react at 60 °C for 30 min. Centrifuge at 12,000 rpm for 10 min. Wash the precipitate three times with ethanol / water and dry at 60 °C to obtain Gd-EA nanoparticles.

[0031] An appropriate amount of the prepared Gd-EA nanomaterials was dispersed in water and sonicated to achieve complete dispersion, resulting in a Gd-EA aqueous dispersion. A small amount of the Gd-EA aqueous dispersion was dropped onto a copper grid, and the morphology and size of the Gd-EA were characterized by transmission electron microscopy (TEM). The obtained TEM image is shown below. Figure 1 , Figure 2 As shown. Figure 1 Corresponding to the Gd-EA prepared in Example 1, Figure 2 The figures, from left to right and top to bottom, correspond to the Gd-EA prepared in Examples 2 through 7. As can be seen from the figures, the particle size of Gd-EA is approximately 250-300 nm.

[0032] I. Taking the Gd-EA nanoparticles prepared in Example 1 as an example, an appropriate amount was ground and compacted to form a flat sample. The phase analysis was performed on the sample using an X-ray diffractometer (XRD). The scanning range was 5°~80° (2θ), the scanning step size was set to 0.02°, and the scanning rate was 5° / min. The obtained XRD pattern is shown in Figure 3.

[0033] II. To investigate the stability of the Gd-EA nanoparticles, the product described in section I above was placed in PBS buffer solutions at pH values ​​of 7.2, 6.5, and 5.0, respectively, and immersed at 37°C for 72 hours before XRD analysis. Figure 3 The results showed that the intensity of the characteristic diffraction peaks of Gd-EA nanoparticles did not decrease significantly after treatment with buffer solutions of different pH values, indicating that their crystal structure could be maintained intact and could meet the stability requirements of smart contrast agents.

[0034] III. Cytotoxicity Test CCK-8, chemically named 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt, produces formazan in a reaction with substances in living cells in a direct proportionality to the number of living cells. Its absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) scanner can indirectly reflect the number of living cells.

[0035] The cytotoxicity of Gd-EA in "I" against human normal hepatocytes (LO2) and mouse breast cancer cells (4T1 cells) was studied using CCK-8 assay. All cell experiments were performed in a clean bench after UV irradiation and alcohol wiping, and cells were cultured in a sterilized incubator. Specifically, cells were incubated in complete culture medium (containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and DMEM). When the cells reached 70% confluence in the culture dish, they were digested with trypsin and seeded into 96-well cell culture plates, with approximately 10,000 cells per well. After 4 hours of incubation, the cells were washed twice with PBS, and 10 μL of CCK-8 solution and 90 μL of complete culture medium were added. The cells were incubated for at least 20 minutes, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated according to Equation 1.

[0036] Cell viability (%) = ( A test - A 0 / A control - A 0) × 100%. In the formula, "A" test A0, A control "The absorbance represents the experimental wells containing cells and nanoparticles, the blank wells without nanoparticles and cells, and the control wells containing cells but without nanoparticles, respectively."

[0037] Cell survival rate in the Gd-EA group was as follows Figure 4 As shown; by Figure 4 It can be seen that the survival rates of LO2 cells and 4T1 cells co-incubated with Gd-EA are relatively high, both above 95%, meeting the safety requirements for medical contrast agents.

[0038] IV. This embodiment uses Gd-EA contrast agent for tumor magnetic resonance imaging. Mice bearing a 4T1 breast tumor (subcutaneously transplanted into the right thigh) were selected and injected intratumorally with the Gd-EA contrast agent prepared in Example 1 at a dose of 0.1 mmol Gd / kg. The tumor sites in the mice were imaged using a 1.5T magnetic resonance imaging (MRI) system, and the results are shown in Figure 5. Figures 5a and 5b show MRI images of the mouse tumors without Gd-EA injection, while figures 5c and 5d show MRI images of the mouse tumors after Gd-EA injection. As can be seen from the figures, the tumor tissue outline was clearly visualized after Gd-EA injection. Further research showed that the Gd-EA contrast agent has excellent stability in the physiological environment and can effectively inhibit gadolinium ion leakage. Systematic biosafety evaluation confirmed that it has no significant toxicity and meets the application requirements of magnetic resonance imaging, providing reliable imaging support for the accurate diagnosis of tumors.

[0039] The foregoing provides a detailed description of a gadolinium-ellagic acid-based magnetic resonance imaging contrast agent, its preparation method, and its applications. The specific embodiments described above should not be construed as limiting the scope of protection of this invention. Any alternative modifications or variations made to the embodiments of this invention by those skilled in the art will fall within the scope of protection of this invention.

[0040] Any aspects of this invention not described in detail are well-known to those skilled in the art.

Claims

1. A gadolinium-ellagic acid-based magnetic resonance imaging contrast agent, characterized in that, The mixture includes ellagic acid, gadolinium ions, polyvinylpyrrolidone (PVP), NaOH solution, and water; the molar ratio of ellagic acid to gadolinium ions is 1:1 to 1:0.3; the mass ratio of PVP to ellagic acid is 20:1 to 25:1; and the concentration of the NaOH solution is 0.02 to 0.025 M.

2. The gadolinium-ellagic acid-based magnetic resonance imaging contrast agent according to claim 1, characterized in that, The molar ratio of ellagic acid to gadolinium ions is 1:0.5; the mass ratio of PVP to ellagic acid is 23:1; and the concentration of the NaOH solution is 0.0225 M.

3. The gadolinium-ellagic acid-based magnetic resonance imaging contrast agent according to claim 1, characterized in that, The source of the gadolinium ions is gadolinium chloride.

4. The method for preparing the magnetic resonance imaging contrast agent according to any one of claims 1-3, characterized in that, The following steps are included: S1. Dissolve ellagic acid in NaOH solution to form sodium ellagic acid solution; S2. Dissolve GdCl3·6H2O together with the first PVP in water; S3. While stirring, add the sodium ellagic acid solution from S1 dropwise to S2; after the solution turns yellow, add the remaining PVP and react at 50~80℃ for 0.5~3 h. S4. After the reaction, the mixture was centrifuged at 12,000 rpm for 10 min. The precipitate was washed several times with ethanol / water and dried to obtain Gd-EA nanoparticles.

5. The preparation method according to claim 4, characterized in that, The preferred reaction temperature for step S3 is 60°C, and the reaction time is 1 hour; the drying temperature for step S4 is 60°C.

6. The preparation method according to claim 4, characterized in that, The ratio of PVP usage in steps S2 and S3 is 2:

5.

7. The use of the magnetic resonance imaging contrast agent as described in any one of claims 1-3 as a contrast agent for magnetic resonance imaging of breast tumors.

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