KR101042399B1 - Multifunctional iron oxide nanoparticles and a diagnostic agent using the same - Google Patents

Abstract

The present invention relates to multifunctional water-soluble iron oxide nanoparticles, a method for producing the same, and a use thereof for cancer diagnosis. More specifically, the present invention provides iron oxide nanoparticles comprising iron oxide-based core particles, a shell coated with a water-soluble substance having a phosphate group and an amine group bondable to the target ligand, and a water-soluble monosaccharide bonded to the shell surface. A method and diagnostic use of the nanoparticles for cancer.

Therefore, the iron oxide-based nanoparticles according to the present invention can be used as a contrast agent useful in diagnosing specific tissues or diseases since the water dispersibility is increased by coating a water-soluble material and can provide a functional site capable of binding a target ligand. Surface modification with water-soluble monosaccharides can increase blood circulation time. In addition, by combining the fluorescent probe can provide a dual contrast medium that can also be used for optical imaging.

Iron Oxide Nanoparticles, ATP, NAD, Magnetic Resonance Imaging, Contrast Agent

Description

Multifunctional iron oxide nanoparticles and a diagnostic agent using the same

The present invention relates to multifunctional water-soluble iron oxide nanoparticles, a method for producing the same, and a use thereof for cancer diagnosis. More specifically, iron oxide-based nanoparticles comprising a shell coated with a water-soluble substance having iron oxide-based core particles, a phosphate group and an amine group bondable to the target ligand, and a water-soluble monosaccharide bonded to the shell surface, and a method for manufacturing the same; It relates to the cancer diagnostic use of the nanoparticles.

Iron oxide nanoparticles have a magnetizing property and thus have a very high biomedical value. In particular, superparamagnetic iron oxide nanoparticles (SPION) have recently been in the spotlight in the biomedical field such as magnetic resonance imaging (MRI) diagnosis, drug delivery and treatment. Because of its superparamagnetic properties, SPION can exhibit higher contrast in MRI than conventional paramagnetic Gd-based contrast agents.

In order to use the iron oxide nanoparticles as a contrast agent in vivo, it must be well dispersed in water and have a uniform and small particle size. However, even if these prerequisites are met, most of the iron oxide nanoparticles injected in vivo are rapidly eroded by reticular endothelial cells and removed from the blood, and mainly by cooper cells, which are distributed by the liver and thus do not function as a contrast agent.

Therefore, in order to reduce the extent to which iron oxide nanoparticles are phagocytized and removed by macrophages, not only the control of the nanoparticles but also the modification of the particle surface may be an important factor. Currently, many studies have been reported to increase blood circulation time by coating iron oxide nanoparticles with water-soluble polymers. Water-soluble iron oxide nanoparticles coated with polyvinylpyrrolidone (Korea Patent Registration No. 10-0746312), contrast agent using polysuccinimide-based polymer (Korea Patent Registration No. 10-0635026), iron oxide nanoparticles coated with polyethylene glycol, In addition to silicon-coated iron oxide nanoparticles, dextran-coated iron oxide nanoparticles, and many other studies have been reported.

However, most of these water-soluble iron oxide nanoparticles have a disadvantage in that they have to go through a step of chemically modifying a polymer to present a ligand introduction site in order to diagnose a specific organ or disease in vivo.

Accordingly, the present inventors have completed the present invention by coating iron oxide nanoparticles with ATP or NAD to increase water dispersibility and introducing a target ligand as a contrast agent for diagnosing a specific disease.

An object of the present invention is an iron oxide nanoparticle comprising a shell coated with a water-soluble substance having iron oxide-based core particles, a phosphate group and an amine group bondable with a target ligand, and a water-soluble monosaccharide bonded to the shell surface. To provide.

Another object of the present invention is to provide a cancer diagnostic nanoparticle or a contrast agent comprising the same, characterized in that the antibody or peptide for cancer cell target is further bound to the shell surface of the nanoparticle.

In addition, another object of the present invention to provide a method for producing the nanoparticles.

In order to achieve the above object, the present invention comprises a shell coated with a water-soluble material having iron oxide-based core particles, a phosphate group and an amine group bondable to the target ligand, and a water-soluble monosaccharide bonded to the shell surface It provides an iron oxide-based nanoparticles.

The present invention also provides a nanoparticle for cancer diagnosis, characterized in that the antibody or peptide for cancer cell target is further bound to the shell surface of the nanoparticle.

The present invention also provides a contrast agent for cancer diagnosis comprising the nanoparticles.

In addition, the present invention comprises the step of forming a shell by coating the surface of the iron oxide core particles with a water-soluble material having a phosphate group and an amine group (a); And (b) modifying the surface by bonding a water-soluble monosaccharide to the surface of the shell.

Hereinafter, the present invention will be described in detail.

According to the first aspect of the present invention, the present invention includes a shell coated with a water-soluble substance having an iron oxide-based core particle, a phosphate group and an amine group bondable with a target ligand, and a water-soluble monosaccharide bonded to the shell surface. Provided are iron oxide nanoparticles.

The water-soluble material for coating the iron oxide-based core particles can bind to the iron oxide-based core particles by having a phosphate group, and can bind to antibodies and the like by having an amine group.

In the present invention, the water-soluble material may be selected from the group consisting of ATP, NAD, ADP, AMP and NADP. In one embodiment of the present invention, by selecting ATP or NAD having the formula, ATP or NAD-iron oxide nanoparticles were prepared.

Chemical Formula of ATP

Chemical Formula of NAD

In the present invention, the water-soluble monosaccharide can be selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid, by increasing the size of the iron oxide nanoparticles by the surface coating of the nanoparticles by such a water-soluble monosaccharide Improve blood circulation reduction time by macrophages. In one embodiment of the present invention, gluconic acid having the following formula was selected.

Gluconic acid improves the blood circulation time decrease by macrophages to reduce the intake by Cooper cells to increase the blood circulation time, if too much treatment can not be combined or very small amounts of fluorescent probes or antibodies Since there may be, preferably the gluconic acid and the surface-coated nanoparticles are combined in a weight ratio of 1: 1 to 15, most preferably 10 times more effective.

In addition, in the present invention, the fluorescent probe can be further bonded to the shell surface of the iron oxide nanoparticles. The fluorescent dye used as the fluorescent probe is IRDye, Cy2 (trade name), Cy3 (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 (trade name), Cy-chrome, phycoerythrin, PerCP (Perridinine Chlorophyll-a Protein), PerCP-Cy5.5, Alexa Fluor (trade name) 350, Alexa Floor (trade name) 430, Alexa floor (trade name) 488, Alexa floor (trade name) 532, Alexa floor (trade name) ) 546, Alexa floor (trade name) 568, Alexa floor (trade name) 594, Alexa floor (trade name) 633, Alexa floor (trade name) 647, Alexa floor (trade name) 660, Alexa floor (trade name) 680. In one embodiment of the present invention, chlorotoxin Cy5.5 ^™ was used as the fluorescent probe.

In addition, in the present invention, the average diameter of the nanoparticles is preferably 5 to 500 nm.

According to the second aspect of the present invention,

The present invention provides a cancer diagnostic nanoparticles, wherein the antibody or peptide for cancer cell targeting is further bound to the shell surface of the nanoparticles. Preferably, a linker capable of binding an antibody may be further bound to the shell surface.

According to the third aspect of the present invention,

The present invention provides a contrast agent comprising the nanoparticles for diagnosing cancer. Since the contrast agent of the present invention includes a fluorescent probe, it has a dual function that can be used for both optical and magnetic resonance images.

According to the fourth aspect of the present invention,

The present invention includes a step of forming a shell by coating the surface of the iron oxide-based core particles with a water-soluble material having a phosphate group and an amine group and (b) modifying the surface by bonding a water-soluble monosaccharide to the shell surface It provides a method for producing iron oxide-based nanoparticles.

Preferably, the water-soluble material of step (a) may be selected from the group consisting of ATP, NAD, ADP, AMP and NADP.

In addition, iron oxide-based nanoparticles can be prepared using a commonly known co-precipitation method, sol-gel method, pyrolysis method or emulsion method without limitation, but must be prepared including ATP or NAD, ATP or NAD relatively unstable to heat In consideration of the coprecipitation method it is preferable to select and manufacture. In one embodiment of the present invention, after dissolving FeCl ₃ and FeCl ₂ in distilled water purged with nitrogen in a nitrogen environment, ATP or NAD is added to dissolve uniformly, and after adding ammonium solution, separated by a magnet and washed with distilled water Drying yielded ATP or NAD-iron oxide nanoparticles.

In the present invention, the water-soluble monosaccharide of step (b) is preferably selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid.

In addition, in the present invention, in order to enable binding of the fluorescent probe or antibody, it is preferable to bind the gluconic acid and the surface-coated nanoparticles in a weight ratio of 1: 1 to 15, and bind in a weight ratio of 1:10. Most preferably.

In addition, in the present invention, the step (c) of introducing a fluorescent probe on the surface-modified shell surface of step (b) may be further combined. At this time, the fluorescent probe is preferably introduced into the nanoparticles by reacting by adding a molar ratio of 1: 1 to 2 to the nanoparticles modified with a water-soluble monosaccharide.

The fluorescent dye used as the fluorescent probe is IRDye, Cy2 (trade name), Cy3 (trade name), Cy3.5 (trade name), Cy5 (trade name), Cy5.5 (trade name), Cy-chrome, phycoerythrin, PerCP (Perridinine Chlorophyll-a Protein), PerCP-Cy5.5, Alexa Fluor (trade name) 350, Alexa Floor (trade name) 430, Alexa floor (trade name) 488, Alexa floor (trade name) 532, Alexa floor (trade name) 546, Alexa floor (trade name) 568, Alexa floor (trade name) 594, Alexa floor (trade name) 633, Alexa floor (trade name) 647, Alexa floor (trade name) 660, Alexa floor (trade name) 680.

In one embodiment of the present invention, after adding ATP-iron oxide nanoparticles modified with gluconic acid to a borate buffer solution, Cy5.5 NHS ester was added to react for 12 hours to introduce Cy5.5, a fluorescent probe.

The present invention also relates to a method for producing iron oxide-based nanoparticles for cancer diagnosis, further comprising the step of (d) binding the antibody or peptide for cancer cell target to the surface-modified shell surface.

In one embodiment of the present invention, ATP or NAD-iron oxide nano modified with gluconic acid and bonded to Cy5.5 using sulfosuccinimidyl 6- [3 '(2-pyridyldithio) -propionamido] (SPDP) having two functional groups. The particles were added to the buffer for SPDP, followed by reaction for 6 hours after the addition of SPDP, followed by washing with buffer and reaction for 12 hours with the addition of the antibody to bind the antibodies.

Iron oxide-based nanoparticles according to the present invention increases the water dispersibility by coating a water-soluble material, can provide a functional site that can bind the target ligand, is coated with water-soluble monosaccharides of gluconic acid is well dispersed in water It can also increase circulation time in the blood. In addition, since the fluorescent probe, the antibody for diagnosing cancer or a peptide can be bound to the surface of the nanoparticles by a water-soluble substance bound to the surface of the nanoparticles, it can be usefully used for diagnosing cancer effectively. Can also be used.

Hereinafter, although this invention is demonstrated in detail, this invention is not limited to this.

< Example  1> Preparation of Contrast Agents

1-1 ATP  or NAD Synthesis of Coated Iron Oxide Magnetite Nanoparticles

First, the iron oxide magnetite nanoparticles can be prepared using a common coprecipitation method, sol-gel method, pyrolysis method or emulsion method without limitation, but preferably coprecipitation method can be used. Since it must be prepared including ATP or NAD, the coprecipitation method is selected in consideration of ATP or NAD which is relatively unstable to heat.

By coprecipitation, iron oxide nanoparticles coated with ATP or NAD can be prepared as shown in the following preparation method. It is prepared by dissolving FeCl ₃ and FeCl ₂ in distilled water purged with nitrogen in a nitrogen environment and then uniformly dissolving by adding ATP or NAD and adding ammonium solution. The prepared ATP or NAD-iron oxide nanoparticles are separated by a magnet and washed with distilled water.

Synthesis Method 1

1-2 Gluconic  Modification of Iron Oxide Nanoparticles by Bonding

This example is a step of modifying the iron oxide nanoparticles synthesized in Example 1-1 with gluconic acid, as shown in Scheme 1 below.

The modification of this step is performed by converting gluconic acid and the synthesized ATP or NAD-iron oxide nanoparticles into 1-ethyl-3- (3-dimethylamino) -propyl) (1-ethyl-3- (3- (dimethylamino) -propyl, EDC) and N-hydroxysuccinimide (N-Hydroxysuccinimide, NHS) were added and reacted for at least 24 hours, followed by washing.

In this case, it is preferable to add gluconic acid in excess with respect to ATP or NAD-iron oxide nanoparticles, and it is more preferable to add it by 1 to 15 times (weight ratio). Too much treatment can lead to the inability to bind the next level of fluorescence probes or antibodies, or even very small amounts of binding.

Gluconic acid binding process to iron oxide nanoparticles coated with ATP (top) or NAD (bottom)

1-3 Binding of Fluorescent Probes

This example is to bind the fluorescent probe Cy5.5 to the iron oxide nanoparticles modified with gluconic acid in Example 1-2, as shown in Schemes 2 and 3.

In this embodiment, the ATP-iron oxide nanoparticles modified with gluconic acid were added to a borate buffer, followed by reaction for 12 hours by adding Cy5.5 NHS ester. The fluorescent probe is preferably added in a 1 to 2 molar ratio to the iron oxide nanoparticles.

 Cy5.5 Binding Process to ATP-Gluconate Iron Oxide Nanoparticles

 Cy5.5 Bonding Process to NAD-Gluconate Oxide Nanoparticles

1-4 Binding of Cancer Target Antibodies

This embodiment is a step of binding an antibody for cancer targeting using sulfosuccinimidyl 6- [3 '(2-pyridyldithio) -propionamido] (SPDP) having two functional groups having the following formula.

In this embodiment, the ATP or NAD-iron oxide nanoparticles modified with gluconic acid and Cy5.5 are added to the buffer solution for SPDP, followed by reaction for 6 hours after the addition of SPDP, followed by washing with buffer solution. The antibody was added and reacted for 12 hours.

< Experimental Example  1> Cancer diagnosis Dual  Preparation of Contrast Agents

1-1. ATP Of Oxide Coated Iron Oxide Nanoparticles

760 mg and 180 mg of FeCl ₃ · 6H ₂ O and FeCl ₂ · 4H ₂ O, respectively, were put in a round flask, and distilled water purged with nitrogen was added, followed by adding 500 mg of ATP or NAD, followed by purging with nitrogen. Remove the air in the flask. After complete dissolution, 7. ml of ammonium solution was added and allowed to react 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 iron oxide nanoparticles were washed with distilled water and nanoparticles were obtained by drying.

1-2. Gluconic  Introduction

To introduce gluconic acid into the ATP or NAD-iron oxide nanoparticles prepared in step 1, 50 mg of iron oxide nanoparticles were placed in a 20 ml vial, and 10 times of gluconic acid was added to 0.1 M MES (2- [N-morpholino). dissolved in ethane sulfonic acid, and then hydroxysuccinimide (NHS) and 1-ethyl-3 (3-dimethylaminopropyl-carbodimide). , EDC) was added at a 3-fold molar number of gluconic acid and reacted for 24 hours to prepare iron oxide nanoparticles containing gluconic acid.

1-3. Neon Of probe  Combination

In step 2, 50 mg of ATP or NAD-iron oxide nanoparticles to which gluconic acid was introduced were added to 5 ml of borate buffer (pH 8.3), dispersed, and then dissolved by adding 10 μg of Cy5.5 NHS ester in DMSO. The reaction was carried out for a time.

1-4. Antibody Introduction for Cancer Targets

In order to introduce the antibody into the iron oxide nanoparticles synthesized in Step 3, SPDP was first bound and then antibody was bound. After SPDP was dissolved in DMSO and added to the PBS buffer solution for SPDP in which iron oxide nanoparticles were dispersed, it was reacted for 6 hours, washed, and then reacted for 12 hours by adding 500 μg of antibody. After washing, the main body of the refrigerator was used for the next experiment.

< Experimental Example  2> for cancer diagnosis Dual  Diagnosis and Analysis Using Contrast Agents

2-1 Transmission Electron Microscope

ATP or NAD-coated iron oxide nanoparticles prepared in Experiment 1-1 were analyzed by transmission electron microscopy (TEM, Transmittane electron microscopy, H-7650, Hitachi-Ltd, JAPAN).

As shown in FIG. 2, it was confirmed that the prepared ATP or NAD-iron oxide nanoparticles had a diameter of 3-5 nm on average and their distribution was uniform.

2-2 Optical  Obtain

The iron oxide nanoparticles synthesized in Experimental Example 1 were injected into an animal mouse model (U87MG tumor model) to obtain an optical image. After injecting the synthesized iron oxide nanoparticles into the mouse tail vein, images were obtained for 10 minutes, 3 hours, 16 hours, 24 hours, and 48 hours, and the organs were extracted to obtain optical images.

As shown in FIG. 3, it was confirmed that the nanoparticles were distributed in the cancer site three hours after the injection. It was confirmed that iron oxide nanoparticles ingested into cancer increase with time.

2-3 magnetic resonance imaging

In order to take a cancer target MR image, the middle cancer image of the mouse before (a) and after (b) injection of the iron oxide nanoparticles synthesized in Experimental Example 1 was taken.

In addition, in order to quantitatively evaluate the effect of T ₂ attenuation, signal intensity (SI) in liver tissue was selected before and after nanoparticle injection, and then ROI (regions of interest) and signal strength of the back muscle were measured. The cancer tissue T ₂ attenuation effect was calculated by Equation 1.

As shown in Figure 4, before the injection of Experimental Example 1, the cancer tissue looks somewhat bright, but after the nanoparticle injection, the image of the cancer tissue can be seen to turn significantly darker than before injection.

In addition, according to Table 1,2 and Equation 1, it was confirmed that the liver disease site was reduced by about 21.4% compared to the signal strength before injection of Experimental Example 1 to confirm the liver disease site.

Antibody-Gluconic Acid-ATP-Coated Iron Oxide Nanoparticles Experimental Example 1 SI before injection SI after injection Cancer tissue 11777 9480 back muscles 2211 2265

Iron Oxide Nanoparticles Coated with Antibody-Gluconic Acid-NAD Experimental Example 1 SI before injection SI after injection Cancer tissue 11260 7690 back muscles 2406 2493

2-4 cancer Within the organization  Contrast Agent Detection

In order to confirm the presence of iron oxide nanoparticles injected into the body, the contrast agent prepared in Experimental Example 1 was injected, cancer tissues were extracted, treated with a fixative solution, a block was prepared, and tissue sections were prussian blue stained under a microscope. Observed.

As shown in FIG. 5, the contrast agent of Experimental Example 1 stained blue in the cancer tissue was observed, and it was confirmed that the contrast agent was present in the cancer tissue.

As described above, the iron oxide-based nanoparticles of the present invention may be usefully used to effectively diagnose cancer, and may also be used as an optical imaging contrast agent.

1A is a schematic diagram showing a manufacturing process of an embodiment of the present invention.

Figure 1b is the formula (left) of ATP and iron oxide nanoparticles (right) coated with ATP, Figure 1c is the formula (left) of NAD and iron oxide nanoparticles (right) coated with NAD, Figure 1d is a The chemical formula is shown.

2 is a transmission electron microscope (TEM) photograph of iron oxide nanoparticles coated with ATP (a) or NAD (b) of the present invention.

3 is an optical image obtained by intravenous injection of an antibody-gluconic acid-ATP-iron oxide nanoparticle (a) and an antibody-gluconic acid-NAD-iron oxide nanoparticle (b) of an embodiment of the present invention into a U87MG mouse animal model .

4 is a magnetic resonance image obtained by intravenous injection of an antibody-gluconic acid-ATP-iron oxide nanoparticle (a) and an antibody-gluconic acid-NAD-iron oxide nanoparticle (b) of an embodiment of the present invention into a U87MG mouse animal model to be.

Figure 5 shows cancer tissue after intravenous injection of antibody-gluconic acid-ATP-iron oxide nanoparticles (a) and antibody-gluconic acid-NAD-iron oxide nanoparticles (b) of an embodiment of the present invention into a U87MG mouse animal model. It is a micrograph taken by extraction and stained with Prussian blue.

Claims (16)

A shell coated with a water-soluble material having an iron oxide core particle, a phosphate group and an amine group bondable to a target ligand, and a water-soluble monosaccharide bonded to the shell surface, and a fluorescent probe further bound to the shell surface. Iron oxide nanoparticles. The iron oxide nanoparticles of claim 1, wherein the water-soluble material is selected from the group consisting of ATP, NAD, ADP, AMP, and NADP. The iron oxide nanoparticles of claim 1, wherein the water-soluble monosaccharide is selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid. delete The iron oxide nanoparticles of claim 1, wherein the fluorescent probe is chlorotoxin. The iron oxide nanoparticles of claim 1, wherein the nanoparticles have a diameter of 5-500 nm. The nanoparticle for cancer diagnosis, characterized in that the antibody or peptide for cancer cell targeting is further bound to the shell surface of the nanoparticle according to any one of claims 1 to 3 and 5 to 6. Cancer diagnostic contrast agent comprising the nanoparticles according to claim 7. (A) coating the surface of the iron oxide core particle with a water-soluble material having a phosphate group and an amine group to form a shell; (B) modifying the surface by binding a water-soluble monosaccharide to the shell surface; And (c) introducing a fluorescent probe to the surface-modified shell surface of step (b). The method of claim 9, wherein the water-soluble substance of step (a) is selected from the group consisting of ATP, NAD, ADP, AMP and NADP. The method of claim 9, wherein the water-soluble monosaccharide of step (b) is selected from the group consisting of gluconic acid, citric acid, propionic acid, butyric acid and oleic acid. The method of claim 11, wherein the gluconic acid of step (b) and the surface-coated nanoparticles of step (a) are combined in a weight ratio of 1: 1 to 15. delete 10. The method of claim 9, wherein the fluorescent probe of step (c) is chlorotoxin. The method of claim 14, wherein the fluorescent probe of step (c) is introduced into the nanoparticles by adding and reacting the surface-modified nanoparticles of step (b) in a molar ratio of 1: 1 to 2. The method of any one of claims 9 to 12 and 14 to 15, further comprising the step of (d) binding an antibody or peptide for cancer cell targeting to the surface-modified shell surface. Method for producing iron oxide-based nanoparticles for cancer diagnosis.

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