Surface modified hepatocyte targeting molecule-based ferroferric oxide nanoparticle as nuclear magnetic resonance imaging contrast agent as well as preparation method and application of surface modified hepatocyte targeting molecule-based ferroferric oxide nanoparticle as nuclear magnetic resonance imaging contrast agent

Abstract

The invention discloses a preparation method of a nuclear magnetic resonance imaging contrast agent based on surface hepatocyte targeting molecule modification ferroferric oxide nanoparticles, which comprises the following steps: synthesizing ferroferric oxide nanoparticles (Fe3O4 NPs) by a thermal decomposition method, and connecting the Fe3O4 NPs with a ligand dopamine-PEG2000-EOB carrying hepatocyte targeting molecules by a ligand exchange method to obtain the nuclear magnetic resonance imaging contrast agent. The nanoparticles Fe3O4-EOB-PEG capable of carrying out selective T2 nuclear magnetic resonance imaging on the hepatocytes are formed. The method is simple and easy to operate, the controllability of the preparation process is high, the biocompatibility of the obtained product is good, T2 signals of hepatocytes can be remarkably improved, the good early diagnosis effect on liver tumors is achieved, and wide application prospects are achieved.

Description

TECHNICAL FIELD

[0001] The present application relates to the multi-disciplinary field of material science, hepatoma, medical imaging, and the like, and in particular to a kind of Fe3O4-EOB-PEG based on surface modification hepatocyte targeting molecule Fe3O4 Nanoparticle used as magnetic resonance imaging contrast agent and its preparation method and application. BACKGROUND

[0002] Magnetic resonance imaging (MRI) is a non-invasive, non-invasive medical imaging technology, widely used in the diagnosis of liver tumors, especially the early diagnosis of primary liver tumors and liver metastases. MRI can provide high-resolution images to help doctors assess tumor size, shape and its relationship with surrounding tissues. The use of contrast agents significantly improves the signal-to-noise ratio of MRI, which is an important strategy for early diagnosis.

[0003] Currently, clinically approved liver-specific contrast agents mainly include two types: T1 contrast agent Primescan (Gd-EOB-DTPA) targeting hepatocytes and T2 contrast agent ferroferric oxide nanoparticles targeting Kupffer cells of liver macrophages. Primescan is a T1 contrast agent that targets hepatocytes through an ethoxybenzyl (EOB) group. Its targeting group binds to specific receptors on the surface of hepatocytes, significantly enhancing the T1 signal in normal liver tissue. However, the high T1 signal of Primescan may mask the differences between tumors and surrounding normal tissues, making the liver tumor boundary indistinct and affecting accurate diagnosis. In addition, this gadolinium-based contrast agent may cause potential safety problems such as nephrogenic systemic fibrosis in some patients, limiting its application.

[0004] Ferroferric oxide nanoparticles, as a classic T2 contrast agent, are efficiently taken up by Kupffer cells, resulting in significant distribution in liver tissue. Due to the low content of macrophages in liver tumor tissue, ferroferric oxide nanoparticles have less enrichment in tumor areas, thereby forming a significant negative contrast, which theoretically helps to improve the imaging contrast between tumors and surrounding normal tissues. However, ferroferric oxide nanoparticles still have many problems in use, including insufficient targeting, non-specific enhancement of macrophage uptake, metabolic burden caused by macrophage uptake, and the like, which limit their application in liver imaging.

[0005] Although T1 contrast agents dominate in the diagnosis of liver tumors, they still have problems such as blurred tumor boundaries and insufficient identification of small lesions. T2 contrast agents, on the other hand, can significantly improve the contrast between tumors and normal tissues by reducing the signal of tumor tissue, appearing as a "dark area" in the image, making them suitable for early diagnosis of small lesions. Therefore, developing a T2 contrast agent with strong targeting, high safety and significant imaging contrast is an important research direction for precise diagnosis of liver cancer.

[0006] In view of this, the present application uses dopamine-PEG 2000 The EOB modifies the surface of the ferroferric oxide nanoparticles to prepare an MRI contrast agent capable of targeting liver cells and enhancing T2 signals, which significantly improves the signal-to-noise ratio of liver tumor diagnosis by combining the excellent distribution of ferroferric oxide nanoparticles in the liver and the EOB molecule-mediated hepatocyte targeted uptake, and provides a new means for early and accurate diagnosis of liver tumors, which has great significance for early diagnosis of liver tumors. SUMMARY

[0007] The present application aims to utilize the blood flow distribution and strong T2 imaging performance of ferroferric oxide nanoparticles and the characteristics of EOB molecules targeting liver cells to provide a nuclear magnetic resonance imaging contrast agent Fe3O4-EOB-PEG, its preparation method and application. The contrast agent is surface-modified by EOB molecules on ferroferric oxide nanoparticles to achieve efficient uptake by liver cells, thereby improving the effect of liver tumor nuclear magnetic imaging and providing a new idea for clinical liver tumor nuclear magnetic resonance imaging.

[0008] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In a first aspect of the present application, a nuclear magnetic resonance imaging contrast agent Fe3O4-EOB-PEG is provided, wherein the dopamine-PEG 2000 The structural formula of EOB and the structure of Fe3O4-EOB-PEG are shown as follows:

[0009] In a second aspect of the present application, a preparation method of the above-mentioned nuclear magnetic resonance imaging contrast agent Fe3O4-EOB-PEG is provided, and a schematic diagram of the preparation method is as follows, which specifically comprises: (1) After vacuum degassing of the iron oleate, oleic acid and 1-octadecene mixture, heat reflux and thermal decomposition reaction to obtain Fe3O4 NPs.

[0010] (2) Disperse Fe3O4 NPs in tetrahydrofuran, then add ligand molecules dopamine-PEG 2000 -EOB for ligand exchange to obtain the nuclear magnetic resonance contrast agent Fe3O4-EOB-PEG.

[0011]

[0012] In step (1), the vacuum degassing step is preferably carried out under vacuum conditions, the temperature is preferably 90℃, and the time is preferably 30 minutes.

[0013] In step (1), the heating reflux reaction is preferably carried out under a nitrogen atmosphere, the heating rate is preferably 3.3 ℃ / min, the reaction temperature is preferably 320 ℃, and the reaction time is preferably 1 hour.

[0014] In step (1), after the reaction is completed, a nano precipitation and washing process is further carried out, the precipitation process preferably uses isopropyl alcohol, the centrifugal speed is preferably 11000 rpm, the centrifugal time is preferably 15 minutes, the washing process reagent is preferably anhydrous ethanol, and the storage condition of the product is preferably storage in trichloromethane at 4 ℃.

[0015] In step (1), the amounts of iron oleate, oleic acid and 1-octadecene are preferably 0.93 g, 160 μL and 15 mL, respectively.

[0016] In step (2), the ligand exchange process is preferably a heating reflux reaction under nitrogen protection and continuous stirring, the temperature is preferably 60 ℃, and the reaction time is preferably 5 hours.

[0017] In step (2), after the reaction is completed, a product precipitation and washing process is carried out, and the reagent used is preferably ice anhydrous ether.

[0018] In step (2), the amounts of Fe3O4 nanoparticles, dopamine-PEG 2000 The amounts of EOB and tetrahydrofuran are preferably 30 mg, 150 mg and 10 mL, respectively.

[0019] In a third aspect of the present application, the application of the above-mentioned nuclear magnetic resonance contrast agent Fe3O4-EOB-PEG is provided, and the targeting performance of Fe3O4-EOB-PEG is verified in vitro. In order to intuitively evaluate the targeted uptake effect of Fe3O4-EOB-PEG in liver cells, a fluorescent molecule fluorescein isothiocyanate (FITC) labeled Fe3O4-EOB-PEG, namely Fe3O4-EOB-PEG-FITC, is prepared. The specific method is as follows: in the step of synthesizing Fe3O4-EOB-PEG in S22, dopamine-PEG 2000 is replaced by FITC labeled dopamine-PEG 2000 is replaced by FITC labeled dopamine-PEG

[0020] In order to verify the liver cell targeting of Fe3O4-EOB-PEG in vitro, human normal liver cell line (LO2 cells) and human hepatoma cell line (Huh-7 cells) were selected as research objects in the in vitro experiment. The specific steps are as follows: (1) LO2 cells and Huh-7 cells were inoculated in laser confocal dishes respectively, and the culture medium was removed after the cells adhered; (2) the culture medium was replaced with serum-free medium, and then Fe3O4-EOB-PEG-FITC solution was added to the confocal dish; (3) the confocal dish added with the material was incubated in a cell incubator at 37°C, 5% CO2 for different time (such as 1 h, 2 h, 4 h); (4) after incubation, the culture medium was discarded, and the cells were washed with pre-cooled PBS three times to remove the un-uptaken material and ensure the accuracy of the observation results; (5) finally, the intensity and distribution of the fluorescence signal inside the cells were observed by using a laser confocal microscope to analyze the uptake and targeting performance of Fe3O4-EOB-PEG-FITC.

[0021] The fourth aspect of the present application provides the application of the above-mentioned nuclear magnetic resonance contrast agent Fe3O4-EOB-PEG, which verifies the in vitro and in vivo Fe3O4-EOB-PEG biological safety evaluation. In order to evaluate the in vitro cytotoxicity of Fe3O4-EOB-PEG, human normal liver cells (LO2 cells) were inoculated in a 96-well plate, and after the cells adhered, different concentrations of Fe3O4-EOB-PEG solution were added to the culture medium. After 24 hours of incubation of the cells in a cell incubator at 37°C, 5% CO2, the cytotoxicity was detected by MTT method.

[0022] In order to evaluate the in vivo biological safety of Fe3O4-EOB-PEG, Fe3O4-EOB-PEG was injected into healthy Balb / c mice through the tail vein. (1) At different time points (7 days, 14 days), the blood samples of the mice were collected, and the blood samples of the mice were detected by routine blood test. At the same time, Fe3O4-EOB-PEG was injected into healthy Balb / c mice through the tail vein. (2) At different time points (7 days, 14 days), the blood samples of the mice were collected, and the serum was separated and detected for liver and kidney function related indexes. (3) After collecting the blood samples, the main organs (heart, liver, spleen, lung, kidney) of the mice were taken and preserved in paraformaldehyde fixing solution. Then the tissues were paraffin-embedded, sectioned and H&E stained to evaluate the safety of the material.

[0023] In a fifth aspect of the present application, the application of the above-mentioned nuclear magnetic resonance contrast agent Fe3O4-EOB-PEG is provided, and the liver cell uptake capacity of Fe3O4-EOB-PEG is verified in vivo. In order to evaluate the liver targeting property of Fe3O4-EOB-PEG in mice and the effect of nuclear magnetic resonance T2-weighted imaging, the following experimental steps are designed: (1) After the healthy Balb / c mice are anesthetized and fixed, the Fe3O4-EOB-PEG solution is injected into the Balb / c mice through the tail vein. Immediately after the injection of the material, liver nuclear magnetic imaging is performed at different time points using a 3.0T nuclear magnetic resonance instrument to detect the change of T2 signal at the liver site, so as to evaluate the liver imaging effect of Fe3O4-EOB-PEG.

[0024] In a sixth aspect of the present application, the application of the above-mentioned nuclear magnetic resonance contrast agent Fe3O4-EOB-PEG is provided, and the liver tumor imaging effect evaluation of Fe3O4-EOB-PEG is verified in vivo. 1. In vivo imaging effect evaluation of liver orthotopic tumor: Hepa1-6-luc cell line is used to construct a liver orthotopic tumor model in C57BL / 6 male mice. After the modeling is successful, the mice are anesthetized and fixed, and Fe3O4-EOB-PEG is injected into the liver orthotopic tumor model mice through the tail vein. Then, a 3.0T nuclear magnetic resonance instrument is used to scan the liver site of the mice immediately after the injection of the Fe3O4-EOB-PEG contrast agent, and the signal change at the liver site at different time points after the injection of the Fe3O4-EOB-PEG contrast agent is recorded to diagnose the liver orthotopic tumor of the mice. The imaging effect is evaluated.

[0025] 2. In vivo imaging effect evaluation of liver metastasis tumor: CT26-luc cell line is used to construct a colorectal cancer liver metastasis tumor model in Balb / c female mice. After the modeling is successful, the mice are anesthetized and fixed, and Fe3O4-EOB-PEG is injected into the liver orthotopic tumor model mice through the tail vein. Then, a 3.0T nuclear magnetic resonance instrument is used to scan the liver site of the mice immediately after the injection of the Fe3O4-EOB-PEG contrast agent, and the signal change at the liver site at different time points after the injection of the Fe3O4-EOB-PEG contrast agent is recorded to diagnose the liver metastasis tumor of the mice. The imaging effect is evaluated.

[0026] The present application has the following technical effects or advantages: (1) The present application combines ferroferric oxide nanoparticles and liver cell targeting molecule EOB, fully utilizes the advantages and characteristics of both, realizes specific imaging of liver tumors, and can significantly improve the tumor diagnosis effect; (2) The ferroferric oxide nanoparticles and EOB molecules used in the present application have been fully verified for safety and effectiveness; (3) The method of the present application is simple, low in production cost, and easy to scale up; (4) The synthesis route, modification route and method of the ferric oxide in the application are mature, and the conditions are easy to control. (5) The required reaction conditions and reagents are conventional and easy-to-buy reagents. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a TEM image of Fe3O4-EOB-PEG NPs; Figure 2 is the hydrodynamic particle size of Fe3O4-EOB-PEG NPs; Figure 3 is a T2-weighted MRI image of Fe3O4-EOB-PEG NPs; Figure 4 is the T2 relaxation value of Fe3O4-EOB-PEG NPs; Figure 5 is the confocal microscope evaluation of the hepatocyte targeting of Fe3O4-EOB-PEG; Figure 6 is the cytotoxicity evaluation of Fe3O4-EOB-PEG at different concentrations; Figure 7 is a T2-weighted image of the liver of a normal mouse after injection of Fe3O4-EOB-PEG; Figure 8 is a T2-weighted image of the liver of a liver orthotopic tumor and colorectal cancer liver metastasis mouse model after injection of Fe3O4-EOB-PEG; Figure 9 is a schematic diagram of the preparation method steps of the magnetic resonance imaging contrast agent Fe3O4-EOB-PEG. DETAILED DESCRIPTION

[0028] The advantages and various effects of the present application will be more clearly presented from the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0029] Throughout the specification, unless otherwise specifically indicated, the terms used herein are to be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs. If there is a contradiction, the present specification takes precedence.

[0030] Unless otherwise specifically stated, the various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or obtained by existing methods.

[0031] As an optional embodiment of the present invention, the present invention provides a method for preparing the nuclear magnetic resonance imaging contrast agent Fe3O4-EOB-PEG, comprising the following steps:

[0032] (1) Add ferric oleate, oleic acid and 1-octadecene to a dry three-necked flask. After vacuum degassing, react for a period of time. After precipitation, centrifugation and washing, Fe3O4 NPs are obtained. Finally, disperse them in chloroform and store them at 4°C for later use.

[0033] (2) Fe3O4 NPs were dispersed in tetrahydrofuran and dopamine-PEG was added. 2000 After heating and refluxing for a period of time, Fe3O4-EOB-PEG was finally obtained by precipitation, washing, and freeze-drying.

[0034] As one of the preferred solutions in the specific implementation plan, in step (1), the degassing step is carried out under vacuum conditions, at a temperature of 90 ℃, for a time of 30 minutes.

[0035] As one of the preferred embodiments of the specific implementation plan, in step (1), the reaction is carried out in a nitrogen atmosphere under continuous stirring, with a heating rate of 3.3 ℃ / min, a reaction temperature of 320℃, and a time of 1 hour.

[0036] As one of the preferred embodiments of the specific implementation plan, in step (1), the ratio of ferric oleate, oleic acid and 1-octadecene is 0.93 g: 160 μL: 15 mL.

[0037] As one of the preferred options for the specific implementation plan, in step (1), the precipitating agent is isopropanol, the washing agent is anhydrous ethanol, and the centrifugation conditions are 11000 rpm for 15 minutes.

[0038] As one of the preferred embodiments of the specific implementation plan, in step (2), the reaction is carried out in a nitrogen atmosphere under continuous stirring, the reaction temperature is 60°C, and the time is 5 hours.

[0039] As one of the preferred embodiments of the specific implementation plan, in step (2), the Fe3O4 nanoparticles and dopamine-PEG are... 2000- The dosage ratio of EOB to tetrahydrofuran is 30 mg: 150 mg: 10 mL.

[0040] As one of the preferred options for the specific implementation plan, the precipitating reagent and the washing reagent in step (2) are both ice-cold anhydrous diethyl ether.

[0041] Example 1, a liver-specific T2 magnetic resonance imaging contrast agent based on ethoxybenzyl molecular surface modification and a preparation method thereof 1. First, the oleic acid-capped Fe3O4 nanoparticles with uniform particle size were prepared by thermal decomposition method. The preparation method of the Fe3O4 NPs was as follows: a dry three-necked flask was added with 0.93 g of iron oleate, 160 μL of oleic acid and 15 mL of 1-octadecene, respectively. The mixed solution was first degassed at 90 ℃ for 30 minutes under vacuum. After cooling, the mixed solution was heated to 320 ℃ at a rate of 3.3 ℃ / min under nitrogen protection and continuous stirring for 1 hour. After the reaction was completed, the temperature was lowered, isopropanol was added to precipitate the nanoparticles, and the nanoparticles were washed with anhydrous ethanol for several times to obtain the Fe3O4 NPs, which were finally dispersed in chloroform and stored at 4 ℃.

[0042] 2. Then, the Fe3O4 nanoparticles surface-modified with ethoxybenzyl molecules were synthesized by ligand exchange method. The preparation method of the Fe3O4-EOB-PEG was as follows: under nitrogen protection, 30 mg of Fe3O4 nanoparticles dispersed in 10 mL of tetrahydrofuran were added with dopamine-PEG 2000 -EOB ligand molecules, and the reaction was carried out at 60 ℃ for 5 hours under continuous stirring and heating reflux. After the reaction was completed, the temperature was lowered, and the nanoparticles were precipitated and washed with ice anhydrous ether, and finally the contrast agent Fe3O4-EOB-PEG powder was obtained by freeze-drying.

[0043] Experimental Example 1, characterization of Fe3O4-EOB-PEG The contrast agent Fe3O4-EOB-PEG in the implementation example 1 was characterized, as shown in Figure 1 , spherical Fe3O4-EOB-PEG nanoparticles with a particle size of about 20 nm were obtained. Figure 2 showed that the fluid was 45 nm. And Figure 3 and 4 The results showed that the Fe3O4-EOB-PEG nanoparticles had strong T2 imaging performance, and the r2 value was about 20.04 mM-1 s-1. This laid a foundation for further imaging.

[0044] Experimental Example 2, in vitro verification of the targeting of Fe3O4-EOB-PEG In order to intuitively evaluate the targeting uptake effect of Fe3O4-EOB-PEG in liver cells, the fluorescent molecule fluorescein isothiocyanate (FITC) labeled FFe3O4-EOB-PEG, i.e. Fe3O4-EOB-PEG-FITC, was prepared. The specific method was as follows: the dopamine-PEG 2000 -EOB in S22 was replaced by fluorescein-labeled dopamine-PEG2000 - EOB-FITC, and synthesized according to the same ligand exchange method steps. The obtained Fe3O4-EOB-PEG-FITC has fluorescence properties and can be used for visual research.

[0045] In vitro experiments, the human normal liver cell line (LO2 cells) and human hepatoma cell line (Huh-7 cells) were selected as the research object. The specific steps are as follows: (1) LO2 cells and Huh-7 cells were inoculated in laser confocal dishes, and the culture medium was removed after the cells adhered; (2) replace with serum-free medium, then add Fe3O4-EOB-PEG-FITC solution to the confocal dish; (3) the confocal dish added with the material was incubated in a cell incubator at 37 ℃, 5% CO2 for different time (such as 1 h, 2 h, 4 h); (4) after incubation, discard the culture medium, wash the cells with pre-cooled PBS three times, remove the unabsorbed material, and ensure the accuracy of the observation results; (5) Finally, the intensity and distribution of the fluorescence signal inside the cells were observed using a laser confocal microscope to analyze the uptake and targeting performance of Fe3O4-EOB-PEG-FITC.

[0046] The results are shown in Figure 5 As the incubation time increases, the green fluorescence intensity in LO2 and Huh7 cells increases, representing the increase in the uptake of Fe3O4-EOB-PEG-FITC over time. At the same time, whether incubated for several hours, the uptake of LO2 is always higher than that of Huh7 cells, indicating that Fe3O4-EOB-PEG-FITC has a certain targeting property for liver cells.

[0047] Experimental Example 3, Fe3O4-EOB-PEG biological safety evaluation In order to evaluate the in vitro cytotoxicity of Fe3O4-EOB-PEG, human normal liver cells (LO2 cells) were inoculated in a 96-well plate, and after the cells adhered, different concentrations of Fe3O4-EOB-PEG solution were added to the culture medium. After 24 hours of incubation at 37℃, 5% CO2 in a cell incubator, the cytotoxicity was detected by MTT method. In order to evaluate its in vivo toxicity, Fe3O4-EOB-PEG was injected into healthy Balb / c mice through the tail vein. At different time points (7 days, 14 days), the blood samples of mice were collected, and the blood samples of mice were detected by blood routine test. The results are shown in Figure 6 and Table 1, Fe3O4-EOB-PEG NPs have low cytotoxicity to human normal liver cells LO2, and have little effect on mouse blood routine.

[0048] Experimental Example 4, in vivo evaluation of liver cell uptake of Fe3O4-EOB-PEG To evaluate the liver targeting of Fe3O4-EOB-PEG in mice and its effect on T2-weighted magnetic resonance imaging, the following experimental steps were designed: (1) After the healthy Balb / c mice were anesthetized and fixed, Fe3O4-EOB-PEG solution was injected into the Balb / c mice through the tail vein. Immediately after the injection of the material, liver magnetic resonance imaging was performed at different time points using a 3.0T nuclear magnetic resonance instrument to detect the changes in T2 signal in the liver region, thereby evaluating the liver imaging effect of Fe3O4-EOB-PEG. The results are shown in Figure 7 As shown in Figure 1, after injecting Fe3O4-EOB-PEG into the tail vein of normal mice, the liver region in the MRI scan image of the mice became significantly darker, indicating that Fe3O4-EOB-PEG significantly enhanced the local T2 signal and had strong liver targeting ability.

[0049] Table 1 is the blood routine test results of mice at different times after Fe3O4-EOB-PEG injection

[0050] Experimental Example 5: Evaluation of Liver Tumor Imaging Effect of Fe3O4-EOB-PEG Evaluation of in vivo imaging effect of liver orthotopic tumor: Hepa1-6-luc cell line was used to construct liver orthotopic tumor model in C57BL / 6 male mice. After the model was successfully constructed, the mice were anesthetized and fixed, and Fe3O4-EOB-PEG was injected into the liver orthotopic tumor model mice through the tail vein. Then, a 3.0T nuclear magnetic resonance instrument was used to scan the liver region of the mice, and the signal changes at different time points after the injection of Fe3O4-EOB-PEG contrast agent were recorded to diagnose the liver orthotopic tumor of the mice. The imaging effect was also evaluated.

[0051] Evaluation of in vivo imaging effect of liver metastasis tumor: CT26-luc cell line was used to construct colorectal cancer liver metastasis tumor model in Balb / c female mice. After the model was successfully constructed, the mice were anesthetized and fixed, and Fe3O4-EOB-PEG was injected into the liver orthotopic tumor model mice through the tail vein. Then, a 3.0T nuclear magnetic resonance instrument was used to scan the liver region of the mice, and the signal changes at different time points after the injection of Fe3O4-EOB-PEG contrast agent were recorded to diagnose the liver metastasis tumor of the mice. The imaging effect was also evaluated. The results are shown in Figure 8 As shown in Figure 2, in the liver cancer and colorectal cancer liver metastasis mouse models, the liver of the mice became significantly darker after the injection of Fe3O4-EOB-PEG, but the tumor did not show obvious changes, indicating that Fe3O4-EOB-PEG had good liver imaging effect.

[0052] Therefore, the method of using the liver cell surface targeting molecule to modify the ferroferric oxide nanoparticles for magnetic resonance imaging is feasible, and is expected to achieve the goal of noninvasive imaging diagnosis of early liver tumor, and to provide a powerful imaging tool for the diagnosis of liver tumor.

[0053] Finally, it should be noted that the terms "comprising", "including", or any other variation thereof are intended to cover a non-exclusive inclusion, such that processes, methods, articles, or apparatuses that comprise a list of elements are not limited to those elements but can include other elements not expressly listed or inherent to such processes, methods, articles, or apparatuses.

[0054] Although preferred embodiments of the application have been described, those skilled in the art will recognize that additional modifications and variations can be made thereto without departing from the spirit and scope of the application. It is therefore intended that the appended claims cover all such modifications and variations as fall within the scope of the application.

[0055] Obviously, various modifications and changes can be made to the present application by those skilled in the art without departing from the spirit and scope of the present application. Thus, it is intended that the present application encompass all such modifications and changes as fall within the scope of the appended claims and their equivalents.

Claims

1. A ferroferric oxide nanoparticle based on a surface-modified hepatocyte targeting molecule for use as a magnetic resonance imaging contrast agent Fe3O4-EOB-PEG, characterized in that, The dopamine-PEG 2000 The structure of EOB and the structure of the contrast agent are shown below: 。 2. A process for the preparation of a ferroferric oxide nanoparticle based on surface modified hepatocyte targeting molecule as a magnetic resonance imaging contrast agent (Fe3O4-EOB-PEG) as claimed in claim 1, characterized in that, The method comprises the following steps: S1: preparing oleic acid-capped Fe3O4 NPs with uniform particle size by thermal decomposition method; S2: The prepared Fe3O4 nanoparticles were exchanged with ligands by the method of dopamine-PEG 2000 -EOB coupling, Fe3O4-EOB-PEG preparation process is shown as follows: 。 3. A method for preparing a ferroferric oxide nanoparticle nuclear magnetic resonance imaging contrast agent (Fe3C>4-EOB-PEG) based on a surface-modified hepatocyte targeting molecule according to claim 2, characterized in that, The method for obtaining Fe3O4 NPs in step S1 comprises: S11: first, 0.93 g of iron oleate and 160 μL of oleic acid are dispersed into 15 mL of 1-octadecene, the mixture is degassed at 90°C in vacuum for 30 minutes, and then is cooled to room temperature; S12: the above mixed solution is transferred to a dry three-neck flask, a condensation reflux device is added, stirring is continued, and heating is performed to 320°C at a heating rate of 3.3 ℃ / min under nitrogen protection and for 1 hour, and it is observed that the solution changes from reddish brown to black, and then the heating device is removed and the temperature is slowly lowered; S13: after the obtained reaction solution is cooled to room temperature, isopropanol is used to precipitate the nanoparticles, centrifugal separation (11000 rpm, 15 minutes) is performed, and ethanol is used for washing several times, the obtained nanoparticles are dispersed in chloroform, and are stored at 4°C for later use.

4. The method for preparing the Fe3O4-EOB-PEG, a ferroferric oxide nanoparticle nuclear magnetic resonance imaging contrast agent based on a surface-modified hepatocyte targeting molecule according to claim 2, characterized by, The method for obtaining Fe3O4-EOB-PEG in step S1 comprises: S21: first, 2 mL of Fe3O4 NPs dispersed in chloroform (containing 30 mg of nanoparticles) is taken at room temperature, ethanol is added thereto, and centrifugal separation (11000 rpm, 15 minutes) is performed, and the material at the bottom of the centrifugal tube is fully dispersed with 10 mL of tetrahydrofuran to obtain a transparent reaction solution; S22: 150 mg dopamine-PEG 2000 - EOB was dispersed in 10 mL of tetrahydrofuran containing 30 mg of ferroferric oxide nanoparticles, a condensation reflux device was added, and the mixture was heated to 60°C under nitrogen atmosphere and maintained at this temperature for 5 hours under constant stirring; S23: after the reaction is completed, the temperature is lowered to room temperature, ethyl ether is used to precipitate the nanoparticles, and the nanoparticles are washed several times with ethyl ether, and are dispersed in ultrapure water and then freeze-dried to obtain a black powder for subsequent use.

5. The method of claim 2, wherein: Nanoparticles with a suitable particle size are prepared to achieve T2 enhancement effect, and the particle size can be further controlled to achieve T1WI or T2WI enhancement effect and the T2 imaging enhancement effect under the above raw material ratio and synthesis conditions.

6. The preparation method according to claim 2, characterized in that, The step S2 ligand coupled on the surface of the ferroferric oxide nanoparticles includes but is not limited to dopamine-PEG 2000 -EOB, which can be further coupled with other molecules targeting hepatocytes and corresponding ligands targeting tumor cells or cells of different tissues according to different applicable scenarios.

7. The preparation method according to claim 2, characterized in that, The steps S1 and S2 complete the function of liver cell targeting molecular T2 imaging, and further comprise surface multi-ligand modification to realize multi-modal imaging in different scenarios.

8. The preparation method according to claim 2, characterized in that, The ferriferrous oxide nanoparticles are used as T2 enhanced contrast agent, and the dopamine-PEG is coupled by expanding the application of the properties of the nanoparticles 2000 The EOB is used for liver cell targeted contrast T2 enhancement, and the nanoparticles are applied to drug loading, tumor treatment, complete diagnosis and treatment integration design and expanded application.

9. A nuclear magnetic resonance contrast agent Fe3O4-EOB-PEG obtained by the preparation method in any one of claims 2-8, which is used for extended application of Fe3O4 NPs as a nuclear magnetic resonance imaging contrast agent.

10. Extended application of the nuclear magnetic resonance imaging contrast agent Fe3O4-EOB-PEG in claim 1 in liver tumors.

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