KR101018221B1 - Gold-deposited iron oxide/glycol chitosan composite, preparation method thereof and MRI contrast agent comprising the same - Google Patents

Abstract

The present invention relates to a gold-deposited iron oxide / glycol chitosan complex, a preparation method thereof, and an MRI contrast agent comprising the same. Gold-deposited iron oxide / glycol chitosan composite according to the present invention adsorbs glycol chitosan to gold-deposited iron oxide nanoparticles to form a complex, and then surface modified with heparin and impregnated it in an aqueous solution of triblock copolymer, followed by lyophilization. It features.

Description

Gold-deposited iron oxide / glycol chitosan composite, preparation method thereof, and MRI contrast agent comprising the same {Gold-deposited iron oxide / glycol chitosan composite, preparation method about and MRI contrast agent comprising the same}

The present invention relates to a gold-deposited iron oxide / glycol chitosan complex, a preparation method thereof, and an MRI contrast agent comprising the same.

Among the methods that can diagnose the disease of the human body by internal observation, MRI is the safest recently developed technology compared to other imaging techniques. The advantage of MRI is that it is sensitive to tissue and is not exposed to radiation unlike other imaging techniques. Recently, the application and utilization of MRI has been rapidly increasing, and much researches on MRI contrast agents have been conducted.

MRI contrast agents are classified into paramagnetic and super-paramagnetic agents according to their effect on the magnetic field. Paramagnetic MRI contrast agents dominate the T1 attenuation effect and appear bright on T1-weighted images, while superparamagnetic MRI contrast agents predominately darken the signal on T2-weighted images.

Paramagnetic MRI contrast agents include gadolinium (Gd) and manganese (Mn) preparations that are currently clinically widely used. Since gadolinium (Gd) and manganese (Mn) itself are highly toxic, the current formulations are in the form of chelates bound to ligands, preventing the release of toxicity and improving stability. Injecting an MRI contrast agent made of gadolinium and manganese into the body requires dilution with physiological saline or 5% glucose solution and intravenously. However, during dilution, the organic matter in the chelates can fall, exposing the toxic metals. In addition, MRI contrast agents made of gadolinium or manganese metal have a very short half-life of about 14 minutes and are rapidly expelled into the urine after administration (Hiroki Yoshikwa et al, Gazpshindan, 6, 959-969, 1986). It is difficult to diagnose distribution, blood flow distribution, distribution amount, permeation, and the like. Therefore, in order to obtain a clear image contrast of the MRI between the normal and damaged areas, the length of stay in the body is preferred as an MRI contrast agent.

Iron paramagnetic particles are typical of superparamagnetic MRI contrast agents. MRI contrast agents made of iron oxide are non-toxic contrast agents that have passed the stability test results and can stay in the body for about 8 hours. In particular, among the magnetic nanoparticles, iron oxide exhibits high magnetic resonance efficiency even at nanomolar concentrations, and thus, many studies have been conducted with MRI contrast agents. These superparamagnetic iron oxide particles have a longer blood half-life than gadolinium (Gd) and manganese (Mn), and the magnetic resonance efficiency is about 10 to 100 times higher than that of the paramagnetic composite, so that a smaller amount can be used.

The most widely used contrast agent among the superparamagnetic iron oxide MRI contrast agents described above is dextran-coated superparamagnetic iron oxide contrast agent. Dextran-coated superparamagnetic iron oxide contrast agents target Kupffer cells in hepatocytes and were commercialized in 1988 under the trade name ferumixde, aka 'feridex'. However, the dextran-coated superparamagnetic iron oxide contrast agent has a short blood half-life since the average diameter of the particles is 100 nm or more. The superparamagnetic iron oxide contrast agent called 'resovist', coated with carboxydextran, which compensates for the shortcomings of the dextran-coated superparamagnetic iron oxide contrast agent, has an average diameter of 60 nm, Similar principle and image are the same, but rapid infusion does not have side effects such as hypotension and can obtain dynamic image.

Recently, methods using pyrolysis of various iron oxide precursors such as FeCup ₃ , Fe (acac) ₃ , and Fe (CO) ₅ have been developed. Regarding these methods, Korean Patent No. 10-482278 discloses pyrolysis of low-grade Fe (CO) ₅ in an easy and convenient way in a low-cost, low-cost surfactant and a solvent, and proceeds with oxidation by residual oxygen. A method of preparing a mixed trivalent mixed iron oxide nanopowder or injecting air into this powder solution to produce a gamma -Fe ₂ O ₃ nanopowder having further advanced oxidation reaction is described. However, the method has a problem that the manufacturing temperature is too high and the manufacturing time is too long, such as iron oxide is synthesized at 286 ℃ or more and oxidized for 1-7 days. In addition, Fe (CO) ₅ which is used as a raw material for producing iron oxide is very expensive and has difficulty in storage.

Korean Patent No. 10-550194 describes a synthesis of magnetite nanoparticles and a method for producing a Fe-based nanomaterial by mixing iron salts with alcohols, carboxylic acids and amines in an organic solvent and heating the mixture to 200 to 360 ° C. have.

However, these methods are high in manufacturing temperature, long manufacturing time, complicated process, expensive raw materials, and can cause toxicity in the body by using organic solvent. In addition, iron oxides produced by the above methods are well soluble in organic solvents, which leads to limitations in their application to medicine.

Superparamagnetic iron oxide nanoparticles can cause toxicity in vivo such as chemical bonding, viscosity and immunity due to aggregation of contrast agent, and prohibition of enzymatic action such as lysozyme, and thus cannot be used on their own. Therefore, the use of superparamagnetic iron oxide nanoparticles as an MRI contrast medium requires high imaging images, low toxicity, high efficiency, long half-life in blood, dispersion in tissue specificity, good solubility and stability in physiological fluids. It should be coated with a biocompatible polymer.

Therefore, there is an urgent need for the development of a complex that can be easily absorbed into cancer cells and tissues by simply and effectively coating superparamagnetic iron oxide nanoparticles with a biocompatible polymer without using an organic solvent.

The present inventors studied the preparation of a biocompatible composite using iron oxide without using an organic solvent, adsorbing glycol chitosan to gold-deposited iron oxide nanoparticles to form a complex, and then modifying the surface with heparin and applying the triblock air. After the impregnation in the aqueous solution was lyophilized to prepare a complex, the complex was confirmed to exhibit an excellent contrast effect on the MRI image, can be selectively absorbed into cancer cells and tissues, and completed the present invention.

The present invention seeks to provide gold-deposited iron oxide / glycol chitosan complexes with tumor specific target properties and methods for their preparation.

The present invention also provides an MRI contrast agent comprising the gold-deposited iron oxide / glycol chitosan complex.

According to the present invention, gold-deposited iron oxide / glycol chitosan is characterized by adsorbing glycol chitosan on gold-deposited iron oxide nanoparticles to form a complex, and then surface modifying it with heparin and impregnating it in an aqueous triblock copolymer solution. To provide a complex.

In addition,

1) adding a basic solution to an aqueous solution of FeCl ₂ · 4H ₂ O and FeCl ₃ · 6H ₂ O in distilled water and then preparing the iron oxide nanoparticle seeds dispersed in distilled water by ultrasonic grinding;

2) depositing gold by adding gold salt (HAuCl ₄ ) to the iron oxide nanoparticle seed dispersed in the distilled water and reducing the gold salt with sodium citrate (Na ₃ C ₆ H ₅ O ₇ );

3) preparing a complex by adsorbing glycol chitosan of the following Chemical Formula 1 on the surface of the iron oxide nanoparticles on which gold is deposited;

4) stabilizing the surface of the formed complex with heparin of Formula 2; And

5) It provides a method for producing a gold-deposited iron oxide / glycol chitosan complex comprising the step of immersing the stabilized complex in an aqueous solution of triblock copolymer of Formula 3 and then lyophilized.

In Formula 1, n is an integer of 1000 to 5000.

In Formula 3, Y is> 10 and 2X is a number such that these terminal portions comprise 5 to 95% by weight, preferably 20 to 90% by weight of the polymer.

The present invention also provides an MRI contrast agent comprising the gold-deposited iron oxide / glycol chitosan complex.

Hereinafter, the step of the gold-deposited iron oxide / glycol chitosan composite manufacturing method according to the present invention in detail as follows.

Step 1) in the preparation method of the present invention is to prepare the iron oxide nanoparticle seed, adding a basic solution to an aqueous solution of FeCl ₂ · 4H ₂ O and FeCl ₃ · 6H ₂ O mixed in distilled water in a 1: 2 molar ratio To adjust the pH to 9-12. In this case, the basic solution may use NH ₄ OH or NaOH. When the reaction starts, the first brown mixed solution forms a dark brown colloid, and a precipitate occurs. After the reaction, the iron oxide is dispersed using an ultrasonic mill. The iron oxide nanoparticle seed thus prepared was put in a dialysis membrane, and distilled water was exchanged every 3 hours in tertiary distilled water and dialyzed for 24 hours to remove unreacted components and solvent after the reaction.

Step 2) is a step of preparing the iron oxide nanoparticles deposited with gold, gold salt (HAuCl ₄ ) is added to the dispersed iron oxide nanoparticle seeds and then sodium citrate (Na ₃ C ₆ H ₅ O ₇ ) as a reducing agent The gold salt is reduced to deposit gold. The zeta potential of the particle surface charge of the gold-deposited metal nanoparticles of the present invention is about −45 to −55 mV, indicating an anionic charge. Accordingly, by depositing gold on the surface of the iron oxide nanoparticle seed, it is possible to induce an ionic reaction between the iron oxide and the biocompatible polymer to firmly fix the iron oxide and the biocompatible polymer.

Step 3) is a step of preparing a composite, the composite is prepared by adsorbing the glycol chitosan of the formula (1) on the surface of the gold oxide iron oxide nanoparticles are deposited. The mixing ratio of the gold-deposited metal nanoparticles to glycol chitosan is in a volume ratio of 2: 8 to 99: 1, preferably in a volume ratio of 1: 4 to 5: 5. If the ratio of gold-deposited metal nanoparticles to glycol chitosan is out of the above range, there is a disadvantage in that the yield in manufacturing the composite is decreased or the size of the composite is rapidly increased.

The glycol chitosan of Chemical Formula 1 is soluble in chitosan and dissolved in water. Chitosan is a basic polysaccharide prepared by N-deacetylation by treating chitin with a high concentration of alkali, and is known to be superior to other synthetic polymers in terms of cell adsorption capacity, biocompatibility, biodegradability and moldability. The glycol chitosan used in the present invention preferably has a molecular weight of 400,000, but is not limited thereto.

Step 4) is to stabilize the surface of the complex with heparin. The mixing ratio of glycol chitosan to heparin contained in the complex prepared above is in a volume ratio of 2: 8 to 99: 1, preferably in a volume ratio of 5: 5 to 9: 1. If the mixing ratio of glycol chitosan to heparin contained in the complex is out of the above range, there is a disadvantage in that the yield in preparing the heparin-modified complex is markedly lowered or the size of the complex is rapidly increased.

Heparin of Formula 2 consists of sulfated linear polysaccharides, mainly composed of uronic acid (uronic acid). Heparin is a mixture of various structures and has various properties such as antithrombogenicity, inflammation inhibition, angiogenic cancer growth inhibition and immunosuppression. Heparin used in the present invention preferably has a molecular weight of about 20,000, but is not limited thereto. Heparin is a solid at room temperature and soluble in water. Heparin, which is used as an anticoagulant, is also used to prevent the thrombosis that can occur in cancer patients. In addition, it is possible to inhibit the formation of fibrin by interfering with the process of the coagulation promoting enzyme around the tumor cells, and to prevent abnormal hemostatic mechanisms. In this regard, fibrin formation around cancer cells is generally difficult to be affected by the immune system by forming a thick shell, but when heparin inhibits fibrin formation, tumor cells become more easily accessible from natural killer cells. , Cytokines, and the like, or by controlling their activity, can induce the destruction or death of tumor cells by immune system action.

The time required for steps 3) and 4) is generally about 10 to 30 minutes at room temperature, and varies depending on the amount of the mixture.

In step 5), the stabilized complex is impregnated in the triblock copolymer aqueous solution and then lyophilized to obtain a complex in powder form. The triblock copolymer of Chemical Formula 3 is a triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene and dissolved in water. The triblock copolymer is preferably a poloxamer. Poloxamers may be prepared by the methods described in the literature or may use commercially available products. Poloxamers used in the present invention have a molecular weight of 1000 to 16000, and their properties depend on the ratio of the polyoxypropylene block and the polyoxyethylene block, that is, the ratio of Y and 2X in the formula (3). Poloxamers are solid at room temperature, soluble in water and ethanol, and examples of poloxamers are poloxamers 68, 127, 188, 237, 338, 407, and the like. For example, poloxamer 188 means a poloxamer having a molecular weight of about 8350 as a compound of Formula 3 wherein Y is 30 and 2X is about 75.

The average size of the composite prepared by the above method is 100 nm to 10 μm, preferably 50 nm to 5 μm.

Since the method of manufacturing the complex according to the present invention does not use an organic solvent, there is no residual material, and thus, stability is excellent, and an excellent contrast effect is shown in MRI images, and it can be selectively absorbed into cancer cells and tissues, thereby real-time tissue imaging and noninvasive. It can be used as a sensor for early diagnosis of disease. Therefore, the complex according to the present invention can be usefully used as an MRI contrast agent with tumor specific target properties.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1 Preparation of Composites According to the Invention

1. Preparation of Iron Oxide Nanoparticle Seeds

450 ml of distilled water was placed in a reaction vessel, heated to 80 ° C., and 0.235 g (1.18 mmol) of FeCl ₂ · 4H ₂ O dissolved in 25 ml of distilled water was added thereto. After maintaining at this temperature for 20 minutes, 0.639 g (2.36 mmol) of FeCl ₃ · 6H ₂ O dissolved in 25 ml of distilled water so that the molar ratio of FeCl ₂ · 4H ₂ O to FeCl ₃ · 6H ₂ O is 1: 2. Was added and held again at this temperature for 20 minutes. Then 2.6 ml of 7% (v / v) NH ₄ OH was slowly added to the mixed solution using a syringe. When the reaction began, it was confirmed that the first brown mixed solution formed a dark brown colloid and precipitated. After the reaction was completed, the reaction was terminated by cooling to room temperature. Iron oxide nano seeds formed were dispersed in an ultrasonic mill at 25 kHz, 80 W. In order to remove the unreacted components and the solvent after the reaction, the prepared iron oxide nanoparticle seeds were placed in a dialysis membrane (Spectra / PorMembrane, Spectrum Laboratories, Inc., USA) having a molecular weight cut-off (MWCO) 50,000. Distilled water was exchanged every 3 hours in tea distilled water and dialyzed for 24 hours.

The iron oxide nanoparticle seeds thus obtained were observed by transmission electron microscopy (TEM). The results are shown in Fig.

As shown in Figure 2, it was confirmed that the size of the iron oxide nanoparticle seed of the present invention is about 5 ~ 8nm.

In addition, by varying the reaction temperature from 40 ℃ to 80 ℃ in 10 ℃ unit to obtain the yield (%) of the iron oxide nanoparticle seed according to the temperature change Inductively Coupled Plasma Atomic emission spectroscopy, ICP-AES Was observed. The results are shown in FIG.

As shown in FIG. 3, the yield of iron oxide nanoparticle seeds was about 25% at 40 ° C., about 50% at 50 ° C., about 58% at 60 ° C., about 88% at 70 ° C., and about 90% at 80 ° C. . Therefore, it can be seen that the yield of the iron oxide nanoparticle seed is the best when reacted at 80 ℃.

2. Preparation of Metal Nanoparticles Deposited with Gold

200 ml of the solution in which the iron oxide nanoparticle seeds prepared in 1 were dispersed was placed in a reaction vessel and heated to 80 ° C. Then 10 ml of 1 mM gold salt (HAuCl ₄ ) was added and maintained at this temperature for 20 minutes. 1 wt% sodium citrate (Na ₃ C ₆ H ₅ O ₇ ) was added in the same volume as the gold salt to the solution in which the gold salt and the iron oxide nanoparticle seed were mixed. Over time, gold deposited on the iron oxide nanoparticle seeds.

The particle surface charge of the gold-deposited metal nanoparticles according to the volume change of the gold salt aqueous solution was analyzed by zeta potential, and the results are shown in FIG. 4.

In addition, in order to confirm the deposition of gold on the surface of the iron oxide nanoparticles, the spectroscopic characteristics of the gold-deposited metal nanoparticles according to the volume change of the gold salt aqueous solution were measured by UV-visible spectroscopy. , Results are shown in FIG. 5.

As shown in FIG. 4, the zeta potential of the particle surface charge of the gold-deposited metal nanoparticles of the present invention was about −45 to −55 mV, indicating anion charge.

In addition, as shown in FIG. 5, there was no specific absorption band of the iron oxide nanoparticles between 400 and 600 nm, and the maximum absorption band of the metal nanoparticles on which gold was deposited was found at 540 nm. Accordingly, it can be seen that gold was deposited on the iron oxide nanoparticle surface.

3. Preparation of the composite

0.15 g of Tween 80 was mixed with 5 ml of an aqueous solution containing the gold-deposited metal nanoparticles (0.19 mg / ml) prepared in 2 above, followed by 0.5 ml of a 0.05 wt% glycol chitosan solution dissolved in distilled water at once. Add quickly to form a complex. While maintaining the agitation of the aqueous solution in which the complex was formed, 0.5 ml of 0.0625% by weight aqueous solution of heparin dissolved in distilled water was quickly added at a time to stabilize the complex. The formed complex was impregnated in 5 wt% aqueous solution of Poloxamer 188 (Pluronic F-68) and then lyophilized to obtain a composite in powder form.

The particle size in the aqueous solution of the prepared composite was measured by a particle size analyzer, the measurement results are shown in FIG. As shown in Figure 6, it was confirmed that the particle size in the aqueous solution of the composite of the present invention is about 150nm.

In addition, the prepared complex was observed with a field emission scanning electron microscope (FE-SEM), and the measurement results are shown in FIG. 7.

Experimental Example 1 MRI image according to the iron concentration change of the complex according to the present invention (in vitro)

In order to determine the in vitro diagnostic potential of the complex according to the present invention, the following experiment was performed using 3.0 T MRI (Magnetic Resonance Imaging).

Pentom imaging samples were prepared by suspending the complex prepared in Example 1 in 50 ml of 1% agarose (BioRad, Hercules, CA). The suspension was loaded into a pre-prepared 24-well agarose sample vessel and solidified at 48 ° C. MRI images according to the iron concentration change (9.75μM ~ 1250μM) of the complex was confirmed. As a control, resovist, a commercially available superparamagnetic iron oxide contrast medium, was used.

The MRI image according to the iron concentration change of the complex of the present invention is shown in Figure 8, the data of quantitative analysis of the MRI image signal intensity of Figure 8 is shown in Figure 9.

As shown in Figures 8 and 9, the complex according to the present invention showed an improved T2 negative image compared to the control resovist. Therefore, it can be seen that the complex according to the present invention shows excellent contrast effect on the MRI image.

Experimental Example 2 MRI image (in vivo) of the complex according to the present invention

In order to determine the possibility of in vivo diagnosis of the complex according to the present invention, the following experiment was performed using a 4.7 T Bruker Biospin imager (Bruker Medical Systems, Karlsruhe, Germany).

Squamous cell carcinoma (SCC cells; ATCC, Rockville, MD, USA) was incubated in RPMI medium 1640 supplemented with 10% FBS at 37 ° C. in a humidified 5% CO ₂ incubator. A suspension of 1 × 10 ⁶ cells in physiological saline was inoculated into the subcutaneous right calf of C3H / HeN mice (7 weeks old, 20-25 g). When the tumor size was 1 cm in diameter, 0.75 mg of the complex of Example 1 mixed with 0.1 ml saline was administered through the tail vein of the tumor-bearing mouse. MRI images were checked at 1 hour, 3 hours and 6 hours after injection.

The results are shown in FIG.

As shown in FIG. 10, MRI image intensity was increased at the tumor location 1 hour after injection. Significant improvement in MRI imaging contrast was observed at the tumor location up to 6 hours after injection. Therefore, the complex according to the present invention can be usefully used as an MRI contrast agent with tumor-specific target properties.

Since the preparation method of the complex according to the present invention does not use an organic solvent, there is no residual material, excellent stability, excellent contrast effect on MRI images, and selective absorption into cancer cells and tissues. It can be used as a sensor for early diagnosis of disease. Therefore, the complex according to the present invention can be usefully used as an MRI contrast agent with tumor specific target properties.

1 is a schematic diagram of a gold-deposited iron oxide / glycol chitosan complex according to the present invention.

FIG. 2 is a diagram of an iron oxide nanoparticle seed according to the present invention observed with a transmission electron microscope (TEM).

3 is a diagram illustrating the yield (%) of the iron oxide nanoparticle seed of the present invention according to the temperature change by inductively coupled plasma atomic emission spectroscopy (ICP-AES).

FIG. 4 is a diagram illustrating particle surface charges of gold-deposited metal nanoparticles of the present invention according to volume change of an aqueous gold salt solution as zeta potential.

5 is a diagram measuring the spectroscopic characteristics of the gold-deposited metal nanoparticles of the present invention according to the volume change of the gold salt aqueous solution by UV-Vis spectrophotometer.

Figure 6 is a measure of the particle size in the aqueous solution of the composite according to the present invention with a particle size analyzer.

7 is a view of the complex according to the present invention with a field emission scanning electron microscope (FE-SEM).

Figure 8 is a diagram showing the MRI image of the in vitro experiment according to the iron concentration change (9.75μM ~ 1250μM) of the complex of the present invention.

FIG. 9 is data quantitatively analyzing the MRI image signal intensity of FIG. 8.

10 is a diagram showing an MRI image of the in vivo experiment of the complex according to the present invention.

Claims (8)

1) adding a basic solution to an aqueous solution of FeCl ₂ · 4H ₂ O and FeCl ₃ · 6H ₂ O in distilled water and then preparing the iron oxide nanoparticle seeds dispersed in distilled water by ultrasonic grinding; 2) depositing gold by adding gold salt (HAuCl ₄ ) to the iron oxide nanoparticle seed dispersed in the distilled water and reducing the gold salt with sodium citrate (Na ₃ C ₆ H ₅ O ₇ ); 3) preparing a complex by adsorbing glycol chitosan of the following Chemical Formula 1 on the surface of the iron oxide nanoparticles on which gold is deposited; 4) stabilizing the surface of the formed complex with heparin of Formula 2; And 5) A method for preparing a gold-deposited iron oxide / glycol chitosan complex comprising the step of immersing the stabilized complex in an aqueous triblock copolymer of Formula 3 and then lyophilizing. <Formula 1>

In Formula 1, n is an integer of 1000 to 5000. <Formula 2>

<Formula 3>

In Formula 3, Y is> 10 and 2X is a number such that these terminal portions comprise 5 to 95% by weight of the polymer. The method of claim 1, wherein FeCl ₂ · 4H ₂ O and FeCl ₃ · 6H ₂ O in step 1) is mixed with distilled water in a 1: 2 molar ratio. The method of claim 1, wherein the basic solution in step 1) is a NH ₄ OH aqueous solution or a NaOH aqueous solution. The method of claim 1, wherein the mixing ratio of the metal nanoparticles to which the gold is deposited in the glycol chitosan in the step 3) is 2: 8 to 99: 1 by volume. The method of claim 1, wherein the mixing ratio of the glycol chitosan to heparin contained in the complex in step 4) is 2: 8 to 99: 1 by volume. The method of claim 1, wherein the triblock copolymer in step 5) is a poloxamer. A gold-deposited iron oxide / glycol chitosan complex prepared by the method of any one of claims 1 to 6, characterized in that the average size of the complex is from 100 nm to 10 μm. An MRI contrast agent comprising the gold-deposited iron oxide / glycol chitosan complex of claim 7.

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