KR100974082B1 - A contrast agent for diagnosis of liver diseases and preparation method thereof - Google Patents

Abstract

The present invention relates to a diagnosing agent for diagnosing liver disease and a method for preparing the same, and more specifically, the liver is characterized by a magnetic resonance image represented by the following Chemical Formula 1 including a superparamagnetic iron oxide nanoparticle, a linker, and a ligand targeting a liver parenchymal cell. The present invention relates to a diagnosing agent for diagnosing liver disease, and further comprising a radionuclide labeling agent, injecting a contrast agent to contrast not only magnetic resonance images but also gamma images, so that liver disease can be diagnosed quickly and easily.

<Formula 1>

(In the above formula, SPIO is a superparamagnetic iron oxide nanoparticle.)

Liver disease diagnosis, contrast agent, gamma imaging, magnetic resonance imaging, iron oxide nanoparticles, hepatic parenchymal cells

Description

 A contrast agent for diagnosis of liver diseases and preparation method

The present invention relates to a contrast agent for diagnosing liver disease and a preparation method thereof.

Liver disease is one of the late-substantiated diseases, and has been found to be difficult to treat since it has already progressed considerably, or after complications including other chronic liver diseases and cirrhosis. Therefore, early diagnosis of liver disease is known as a very important factor in the treatment of liver disease.

Representative methods of liver disease include blood tests, biopsies, molecular imaging. Blood tests include serums such as bilirubin, aminotransferases, alkaline phosphatase (ALP), albumin (globuin), and globulin, whose concentrations change in the blood depending on the function of the liver. There is a method of diagnosing liver disease by measuring ammonia, drug clearance, etc., but there is a problem in that it is not detected in the early stage of liver disease because it is not directly measured. Next, the biopsy is a method of directly removing a tissue and observing it directly under a microscope, but it is an essential step for accurate diagnosis, but there is a problem in that the examination is not easy.

Molecular imaging is a technique that analyzes and evaluates various molecular-level changes in a cell, such as gene expression, biochemical changes, and biological changes, by combining molecular biology and advanced imaging techniques. It is an integrated technology of biology, cytology, chemistry, pharmacy, physics, computer science, medical engineering, radiology and nuclear medicine.

Recently, with the rapid development of molecular imaging technology, it is possible to diagnose diseases and detect lesions. In particular, nuclear medicine imaging and magnetic resonance imaging technology has attracted attention as a very useful test system for the diagnosis of liver disease. In the late 1980s, iron oxide nanoparticles prepared by coprecipitation method have been used in clinical practice, and these are used as negative contrast agents by applying the feature of reducing the signal intensity by dispersing iron oxide nanoparticles in tissue in magnetic resonance imaging. . These nanoparticles are generally phenotyped by Cooper cells, which, when injected in vivo, account for most of the reticuloendothelial cell system, primarily reducing liver signals. Liver tumors, on the other hand, lack Cooper cells and show contrasting signals. This phenomenon is currently used in the diagnosis of liver lesions in clinical practice.

The purpose of the contrast agent is to artificially create a difference in contrast between the lesion site and normal tissue to detect and diagnose a more accurate disease. Most of the contrast agents used in the clinic are dextran-coated superparamagnetic iron oxide contrast agents, which are mainly targeted to liver cells of the liver and are released as some products. Conventional contrast agent has a particle size of 50 ~ 200 nm and is fast phagocytized by liver cells of the liver, short blood half-life, but can obtain a higher image image than gadolinium (Gd) preparations, is less toxic and than in the blood It has the advantage of having a long half life.

Since the late 1990s, technologies for targeting specific lesions have been developed by introducing antibodies, functional proteins, peptides, and functional small molecules into the nanoparticles. Molecular and cellular images are now attracting attention in magnetic resonance imaging. In addition, research on multi-image preparations that can utilize various imaging systems such as magnetic resonance images and optical images has been actively conducted.

 At present, a contrast agent for contrasting the liver using co-precipitation has been developed and reported. Recently, a patent (Korea Patent No. 0541282) has been registered as a liver contrast agent using superparamagnetic iron oxide-based nanoparticles and a method of manufacturing the same. The method using the coprecipitation method is difficult to control the size of the magnetic nanoparticles, there is a problem that the dispersibility, crystallinity, paramagnetic properties of the particles are not as good as compared to the thermal decomposition method. Therefore, various attempts have been made to develop contrast agents for diagnosing specific lesions based on a technique of manufacturing magnetic nanoparticles having high crystallinity, superparamagnetism and monodispersity using pyrolysis and molecular biology.

Accordingly, the present inventors have developed a contrast agent capable of imaging not only magnetic resonance images but also gamma images, and completed the present invention.

An object of the present invention is to provide a contrast agent for diagnosing liver disease.

Another object of the present invention is to provide a method for preparing a contrast agent for diagnosing liver disease.

In order to achieve the above object, the present invention provides a contrast agent for diagnosing liver disease in which ultra-fine superparamagnetic iron oxide nanoparticles and beta-galactose which can specifically interact with hepatic parenchymal cells are combined, and the liver can be contrasted with magnetic resonance imaging. to provide.

Furthermore, the present invention provides a method for preparing the contrast agent for diagnosing liver disease.

In addition, the present invention provides a contrast agent for diagnosing liver disease capable of imaging the liver as a gamma image in combination with DTPA in addition to the contrast agent capable of contrast imaging the liver in the magnetic resonance image.

Contrast agent according to the present invention comprises a superparamagnetic iron oxide-based nanoparticles, a linker and a ligand targeting the liver parenchymal cells, magnetic resonance imaging is possible, and further comprises a radionuclide labeling material to inject one contrast agent gamma Imaging can be performed to diagnose liver disease quickly and easily.

Hereinafter, the present invention will be described in detail.

The present invention provides a contrast agent for diagnosing liver disease capable of contrasting the liver with a magnetic resonance image represented by Formula 1 below.

(In the above formula, SPIO is a superparamagnetic iron oxide nanoparticle.)

The contrast agent according to the present invention comprises a superparamagnetic iron oxide nanoparticles (SPIO), ligands targeting liver parenchymal cells on the surface of the nanoparticles and a linker that serves to connect them.

The size of the superparamagnetic iron oxide nanoparticles is not particularly limited, but is preferably 4 to 200 nm. In this case, when the superparamagnetic iron oxide nanoparticle size exceeds 200 nm, there is a problem that is easily recognized by macrophages in vivo, and when it is less than 4 nm, there is a problem that its preparation is not easy.

In the contrast agent according to the present invention, the linker preferably contains a hydroxyl group and an amine group at the same time. The hydroxyl group binds to SPIO, and the amine group on the other side binds to a ligand that targets liver parenchymal cells. Ligands targeting liver parenchymal cells bound to the amine groups may be strongly bound to the SPIO surface and remain on the SPIO surface even after being injected into the body. Preferably, dopamine represented by the following formula (2) may be used as the linker.

In the contrast agent according to the present invention, the ligand targeting the hepatic parenchymal cells can specifically interact with hepatic parenchymal cells, and can perform magnetic resonance imaging without depending on Cooper cells present in hepatic parenchymal cells. have. As the ligand, Lactobionic acid (LA) represented by the following Chemical Formula 3 is preferable. Beta-galactose contained in the lactobionic acid (β-galactose) may specifically interact with sialic acid glycoprotein receptors (asialoglycoprotein receptors) present on the surface of the liver parenchyma.

In another aspect, the present invention provides a contrast agent for diagnosing liver disease represented by the following formula (4) further comprising a radionuclide marker in the contrast agent of Formula 1.

(Wherein SPIO is a superparamagnetic iron oxide nanoparticle and DTPA is diethylene triamine penta acetic acid.)

In the contrast agent according to the present invention, the radionuclide labeling substance is a chelating agent capable of labeling radionuclides in the body, and serves to additionally provide gamma imaging ability to the contrast agent. The radionuclide labeling substance is a material containing a thiocyanate group capable of bonding with an extra amine group of the linker, and is represented by the following formula (5): Diethylene triamine penta acetic acid (DTPA) Is preferred.

Furthermore, the present invention comprises the steps of synthesizing and growing iron oxide nanoparticles (step 1); Modifying the grown iron oxide nanoparticles of step 1 with a linker (step 2); And a step (step 3) of modifying the iron oxide nanoparticles modified with the linker of step 2 with a ligand targeting liver parenchymal cells.

Hereinafter, the method for preparing the contrast medium according to the present invention will be described in more detail step by step.

Step 1 according to the present invention is a step of synthesizing and growing superparamagnetic iron oxide nanoparticles.

The superparamagnetic iron oxide nanoparticles can be prepared using a common co-precipitation method, sol-gel method, pyrolysis method or emulsion method without limitation, but preferably pyrolysis method can be used. The thermal decomposition method is easier to control the size of the superparamagnetic iron oxide nanoparticles than the coprecipitation method, it is possible to produce nanoparticles excellent in dispersibility, crystallinity and paramagnetic properties. In addition, the growth of the superparamagnetic iron oxide nanoparticles may be manufactured to a desired size by applying a seed mediated growth method again.

Step 2 according to the present invention is a step of modifying the linker with the iron oxide nanoparticles grown in step 1, as shown in Scheme 1 below.

Modification of this step can be carried out by reacting the dopamine and the SPIO grown in step 1 as a linker in a mixed solvent of toluene and DMSO. In this case, it is preferable to add the linker material in excess to SPIO, and more preferably 3 to 7 molar times. In this case, when the linker is added in less than 3 mol compared to the superparamagnetic iron oxide nanoparticles, there is a problem that it does not dissolve in the aqueous solution, and when the linker is added in excess of 7 mol, there is a problem that the modification of the linker is not efficient compared to the amount. .

Step 3 according to the present invention is a step of modifying the iron oxide nanoparticles modified with a linker in step 2 with ligands targeting liver parenchymal cells, as shown in Scheme 2 below.

This step is performed by adding SPIO modified with the linker to the buffer, dispersing it by sonication, and dissolving the ligand by adding 0.3 to 0.8 molar ratio of dopamine to the nanoparticle surface in step 2. Can be. It is preferable to use lactobionic acid as said ligand. 1-ethyl-3- (3-dimethylamino) -propyl) (1-ethyl-3- (3- (dimethylamino) -propyl, EDC), N Hydroxysuccinimide (N-Hydroxysuccinimide, NHS) is added and reacted for at least 48 hours, followed by washing and freeze drying to obtain a contrast agent for diagnosing liver disease according to the present invention.

The nanoparticles prepared as described above have beta-galactose, which is contained in the lactobionic acid on the surface thereof, and thus can specifically bind to sialoglycoprotein receptors present on the surface of the liver parenchyma cells. As a result, it can be specifically recognized by liver parenchymal cells and can be used as a contrast agent for diagnosing liver disease capable of magnetic resonance imaging by the characteristics of the bound nanoparticles.

In addition, the present invention, after dispersing the iron oxide nanoparticles modified with a ligand prepared in step 1 to step 3 in a buffer solution, the radionuclide labeling material is added in a 0.2 to 0.5 molar ratio with respect to the linker introduced in step 2 It provides a method of manufacturing a contrast agent for diagnosing liver disease, further comprising the step of modifying.

As the radionuclide labeling material, it is preferable to use diethylene triamine penta acetic acid (DTPA). In order to introduce the DTPA on the surface of SPIO, DTPA dissolved in DMSO was added to the reaction and reacted for 12 hours, followed by washing and freeze drying to obtain a contrast agent for diagnosing liver disease according to the present invention.

The nanoparticles prepared in step 4 may be gamma imaged by placing a radionuclide on the surface of the nanoparticles.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the examples.

Example 1 Preparation of Contrast Agent for Diagnosing Liver Disease

Step 1. Synthesis and Growth of Iron Oxide Nanoparticles

2 mmol of Fe (acac) ₃ , 6 mmol of oleic acid, 6 mmol of oleylamine and 10 mmol of hexadecanoic acid are placed in a round flask and the organic solvent phenylether (phenyl) 20 ml of ether) is added and purged with nitrogen to remove air from the flask. Next, the flask was held at 100 ° C. for 30 minutes, then heated to 260 ° C. and reacted for 30 minutes. At this time, when the reaction proceeds, the color of the solution changes to black, and after completion of the reaction, particles were collected using a magnet. Thereafter, the collected particles were washed and dried to synthesize superparamagnetic iron oxide nanoparticles of 4 nm.

The seed growth method was repeated twice on the synthesized superparamagnetic iron oxide nanoparticles to grow superparamagnetic iron oxide nanoparticles of 12 nm.

Step 2. Introduction of the linker

In order to remove oleic acid on the surface of the superparamagnetic iron oxide nanoparticles prepared in step 1, 50 mg of the superparamagnetic iron oxide nanoparticles were placed in a 20 ml vial and dispersed in a butanol solution containing 1 mol of ammonium hydroxide. And sonicated for 2 hours. After washing with toluene, acetone, and ethanol, the iron oxide nanoparticles were dispersed in 5 ml of toluene, and 2 ml of dopamine-dissolved methanol was added at 6-fold molar concentration of nanoparticles, followed by sonication for 30 minutes, and for 24 hours. By reacting with stirring, dopamine was introduced into the superparamagnetic iron oxide nanoparticles.

Step 3. Remove Liver Parenchymal Cells Targeting Ligand  Introduction

50 mg of superparamagnetic iron oxide-based nanoparticles dopamine introduced in step 2 were added to 5 ml of a buffer solution (0.1 M MES (2- [N-morpholino] ethane sulfonic acid), dispersed, and then lactobionic acid. ) Was dissolved by adding 0.6 mole ratio of modified dopamine to the surface of the nanoparticles, followed by hydroxysuccinimide (NHS) and 1-ethyl-3 (3-dimethylaminopropyl-carbodimide). (1-ethyl-3 (3-dimethylaminopropyl) -carbodimide (EDC) was added at 3 times the number of moles of lactobionic acid and reacted for 48 hours to prepare particles into which beta-galactose was introduced. After washing, the particles were collected and frozen. Dried.

Step 4. Introduction of the chelator for radionuclide labeling

50 mg of the nanoparticles into which lactobionic acid was introduced in step 3 was added to 10 ml of a buffer solution (0.1 M MES (2- [N-morpholino] ethane sulfonic acid), and sonicated to disperse. The nanoparticles were dispersed To the solution was added 1 ml of DMSO in which DTPA was dissolved at a ratio of 0.2 to 0.5 mole of dopamine introduced on the surface of nanoparticles, and then stirred for 12 hours to react, washed and lyophilized to prepare nanoparticles introduced with DTPA. .

<Analysis>

1. Transmission electron microscope and particle size analysis

The size of the iron oxide nanoparticles (a) prepared in step 1 and Example 1 were measured by a particle size analyzer (Dynamic Light Scattering) and a transmission electron microscope and are shown in FIG. 2.

As shown in FIG. 2, the prepared iron oxide nanoparticles not only have a diameter of 12 nm, but also showed a uniform distribution.

2. X-ray Diffraction Analysis

In order to confirm the crystal structure of the nanoparticles prepared in step 1, Example 1 is shown in Figure 3 by X-ray diffraction analysis.

As shown in FIG. 3, Example 1 confirmed the crystal directions of 220, 311, 400, 511, and 440 representing the magnetite structure to confirm that the prepared nanoparticles were superparamagnetic iron oxide nanoparticles.

3. Fourier-Transform Infrared Spectroscopy

In order to confirm that dopamine and hepatic parenchymal cell labeled ligand were introduced into the iron oxide nanoparticles according to the present invention, the iron oxide nanoparticles prepared in step 1 (a) and the iron oxide nanoparticles of step 1 from which oleic acid was removed (b) , Nanoparticles (c) modified with lactobionic acid and Example 1 (d) is shown in Figure 4 by infrared spectroscopy.

As shown in Figure 4, the dopamine-modified nanoparticles (c) was confirmed that the dopamine-modified by the vibration of the amine group and CH group was observed, the nanoparticles (d) introduced with lactobionic acid is C = O and Stretching vibrations of COC were observed to confirm that lactobionic acid was introduced.

Experimental Example 1 Interaction with Liver Parenchymal Cells

In order to evaluate the interaction between the superparamagnetic nanoparticles in which hepatocytes and lactobionic acid were introduced, nanoparticles ingested by HepG2 human hepatocytes were identified through Prussian blue staining and are shown in FIG. 5.

As shown in FIG. 5, nanoparticles (a) without ligands showed less visible intake into cells, whereas nanoparticles (b) with lactobionic acid were relatively high in cells by ASGP-R of hepatocytes. Confirmed. This indicates that the nanoparticles into which lactobionic acid is introduced can be easily recognized by ASGP-R in hepatocytes, and hepatocyte target imaging is possible.

Experimental Example 2 Gamma Image Shooting

 6 shows the results obtained by gamma imaging the radionuclide labeled hepato parietal cell target iron oxide nanoparticles. Example 1 was labeled with techninium (Tc-99m) and injected into the tail vein of a mouse (balb / c mice femal, 20 g), and 30 minutes after, 1 hour after each image was obtained.

As shown in FIG. 6, the labeling efficiency was 95% or more, and after 1 hour of injection of Example 1, it was confirmed that most of Example 1 was distributed in the liver, and the gamma image using Example 1 was obtained through the nanoparticles. The biodistribution of can be evaluated as a whole, and the liver parenchymal cells reduced by cirrhosis of the liver reduced the interaction with the nanoparticles and confirmed this through imaging to confirm that the disease can be quickly and accurately diagnosed.

Experimental Example 3 MR Image Shooting

To quantitatively evaluate the effect of T ₂ attenuation, the signal intensity in the liver tissue (SI) is selected before and after nanoparticle injection to measure the ROI (regions of interest) and the signal strength of the back muscles close to the liver tissue. The liver T ₂ attenuation effect was calculated by Equation 1 below.

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In addition, according to Table 1 and Equation 1, it was confirmed that the liver disease site was reduced by about 38% compared to the signal strength before injection of Example 1 to confirm the liver disease site.

Example 1 SI before injection SI after injection Liver 4011 (± 130) 2210 (± 77) back muscles 1882 (± 97) 1889 (± 57)

Experimental Example 4 Detection of Contrast Contrast Agent

Example 1 was injected to confirm the presence of iron oxide nanoparticles injected into the body, liver tissue was extracted, treated with a fixative solution, and then a block was prepared. The photograph of the tissue section observed with a transmission electron microscope is shown in FIG. 9. .

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As shown in Figure 9, it was confirmed that Example 1 is present in the parenchymal intracellular cytoplasm and mitochondria.

1 is a schematic view illustrating a method of manufacturing an embodiment of the present invention and a process of recognizing liver parenchymal cells and binding to hepatic parenchyma membranes ((a) lactobionic acid, (b) technium, (c) ) ASGP receptor);

FIG. 2 is a transmission electron microscope (TEM) photograph and a particle size analysis graph of superparamagnetic iron oxide nanoparticles (a) and one embodiment (b) of the present invention; FIG.

3 is an X-ray diffraction analysis (XRD) graph of one embodiment of the present invention;

Figure 4 is a graph of Fourier Transform Infrared Spectroscopy (FT-IR) according to an embodiment of the present invention ((a) iron oxide nanoparticles prepared in step 1, (b) oleic acid is removed after the step 1 Iron oxide nanoparticles, (c) nanoparticles modified with lactobionic acid, and (d) Example 1);

5 is a photograph observing uptake into human HepG2 cells expressing ASGP receptors ((a) is cell uptake of nanoparticles without surface modification (b) cell uptake of Example 1);

FIG. 6 is a gamma image photograph taken after injection by labeling technicium in one embodiment according to the present invention ((a) heart, (b) liver, (c) spleen, (d) bladder);

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Figure 9 is a photograph of a parenchymal cell in liver tissue before (a) and after injection (b) of one embodiment of the present invention observed with a transmission electron microscope.

Claims (11)

Contrast agent for diagnosing liver disease for contrasting the liver with a magnetic resonance image represented by the following Formula 1 comprising a superparamagnetic iron oxide nanoparticle, a linker, and a ligand targeting liver parenchymal cells. <Formula 1>

(In the above formula, SPIO is a superparamagnetic iron oxide nanoparticle.) The contrast agent for diagnosing liver disease according to claim 1, wherein the size of the superparamagnetic iron oxide nanoparticles is 4 to 200 nm. [Claim 2] The contrast agent for diagnosing liver disease according to claim 1, wherein the linker is dopamine. The method of claim 1, wherein the ligand is a contrast agent for diagnosing liver disease, characterized in that the lactobionic acid containing beta-glucose. The contrast agent for diagnosing liver disease for contrasting the liver with a magnetic resonance image and a gamma image represented by the following Chemical Formula 4, further comprising a radionuclide labeling substance in the contrast agent. <Formula 4>

(Wherein SPIO is a superparamagnetic iron oxide nanoparticle and DTPA is diethylene triamine penta acetic acid.) The contrast agent for diagnosing liver disease according to claim 5, wherein the radionuclide labeling substance is diethylene triamine penta acetic acid (DTPA). Synthesizing and growing iron oxide nanoparticles (step 1); Modifying the grown iron oxide nanoparticles of step 1 with dopamine (step 2); And Liver to contrast the liver with the magnetic resonance image of claim 1, characterized in that it comprises the step of modifying the dopamine-modified iron oxide nanoparticles of step 2 with lactobionic acid targeting liver parenchymal cells (step 3). Method for preparing a contrast agent for diagnosis of disease. The method of claim 7, wherein the iron oxide nanoparticles of step 1 are prepared by pyrolysis, and grown by a seed mediated growth method. The method of claim 7, wherein the modifying of Step 2 is performed by adding iron oxide nanoparticles and dopamine of 3-7 mole times to the nanoparticles to the mixed solvent of toluene and DMSO. . The method of claim 7, wherein the modification of step 3 is carried out by dispersing the iron oxide nanoparticles modified in step 2 in a buffer solution, and then adding the lactobionic acid 0.3 to 0.8 molar times with respect to the dopamine introduced in step 2 Method for producing a contrast agent for diagnosing liver disease. After performing steps 1 to 3 of claim 7, additionally dispersing the iron oxide nanoparticles modified with the lactobionic acid prepared in step 3 in a buffer solution, the radionuclide labeling material is 0.2 to about the dopamine introduced in step 2 Method of preparing a contrast agent for diagnosing liver disease in which the liver is contrasted with the magnetic resonance image and the gamma image of claim 5, further comprising the step of modifying by adding 0.5 mole times (step 4).

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