KR100974083B1 - Iron oxide nano particles coated with PVLA, preparation method thereof and contrast agent for diagnosing liver diseases - Google Patents

Abstract

The present invention relates to a superparamagnetic iron oxide nanoparticles coated with PVLA, a method for preparing the same, and a contrast agent for diagnosing liver disease, including the same, wherein the superparamagnetic iron oxide nanoparticles coated with PVLA are stable in a superparamagnetic iron oxide solution (ferrofluid). It can be usefully used as a stable contrast agent to accurately diagnose liver disease by securing and selectively delivering to the liver.

Contrast Agent, Superparamagnetic, Iron Oxide Nanoparticles

Description

Superparamagnetic iron oxide nanoparticles coated with PPLA, preparation method thereof, and contrast agent for diagnosing liver disease, including the same

The present invention relates to a superparamagnetic iron oxide nanoparticles coated with PVLA, a preparation method thereof, and a contrast agent for diagnosing liver disease, including the same.

As the average life expectancy increases, the elderly population has increased, and patients with various cancer diseases and cerebrovascular diseases have increased. As the eating habits and the living environment have changed, the age of patients with adult disease has been decreasing. Therefore, early detection of various diseases and increasing treatment rates are very important for improving the health of people's lives.

Various diagnostic methods are being developed for the early detection of diseases, and the use of nuclear magnetic resonance imaging (MRI), a state-of-the-art method for visually early detection before subjective symptoms appear, is increasing.

MRI is a measurement of a signal from a proton and is diagnosed by non-invasive methods in humans.However, the use of contrast agents for MRI can increase the sensitivity and specificity of the diagnosis. From MRI angiography to perfusion imaging, the area of application of MRI contrast agents is expanding.

Contrast agent affects magnetic field and X-ray passage of MRI, and it is administered to internal organs such as tissues, blood vessels, stomach, and intestines that cannot be identified by general MRI or X-ray imaging. It is a substance used to observe a target site or to evaluate a function, and is a substance that forms a clearer image. The contrast agents are classified into paramagnetism and superparamagnetism agents according to their effect on the magnetic field, and are also called positive contrast agents and negative contrast agents, respectively.

A positive contrast agent is predominantly T ₁ due to the attenuation effect of the proton longitudinal relaxation time (T ₁ ) in the tissue part to which the contrast agent is administered. Highlight image (T ₁ In the weighted image, the negative contrast agent, T ₂ transeverse relaxation time (T ₂ transeverse relaxation time) attenuation effect of the tissue predominantly T ₂ Highlight image (T ₂ weighted image) [R. Weissleder et. al. (2000) Nat. Med. 6: 351-355.

The superparamagnetic material used for the negative contrast agent may be representative of superparamagnetic iron oxide (SPIO) such as magnetite (Fe ₃ O ₄ ) or maghemite (maghemite, Fe ₂ O ₃ ). They are made into uniform particles having a size of several tens of nanometers or less and made of ferrofluid, which is composed of a stable colloidal form, and administered into the body.

On the other hand, in order to use the superparamagnetic nanoparticles as an effective contrast agent, a small, uniform and stable magnetic iron oxide solution (ferrofluid) having high saturation magnetization should be prepared. The ferrofluid is Fe ₃ O ₄ It is a colloidal dispersion solution of magnetic nanoparticles such as Fe ₂ O ₃ and must be able to maintain liquid state even under very strong magnetic field. However, pure superparamagnetic iron oxide particles are 1) hydrophobic and have a high volume-to-surface area ratio, resulting in a strong hydrophobic interaction between the particles, leading to agglomeration and forming clusters. The original structure can be changed, the magnetic properties can be changed, 3) it can be rapidly biodegradable when it comes into contact with the living environment, 4) pure iron oxide itself is toxic and harmful to the human body, there is a problem that there is a limit to use as a contrast agent.

Thus, surface modification of the particles is required to ameliorate these problems and maintain the stability of the iron oxide solution comprising the superparamagnetic nanoparticles. In general, a method of coating the nanoparticles with a stabilizer such as a surfactant or a polymer has been developed. Conventional polymers for coating nanoparticles include synthetic polymers such as polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), polyvinyl alcohol (polyvinylalcohol, PVA), polyethylene glycol (Polyethylene, PEG), etc. MH Liao et. al. (2002) J. Mater. Chem. 12: 3654-3659; M. Chastellain et. al. (2004) J. Colloid Interface Sci. 278: 353-360] and natural polymers of gelatin, dextran, chitosan and pullulan [Y.C. Chang et. al. (2005) J. Collide Interface Sci. 283: 446-451); M. C. Bautista et. al. (2005) J. Magn. Magn. Mater. 293: 20-27.

As a coating method using such a polymer, 1) an in situ coating method for coating a polymer in a process of manufacturing iron oxide nanoparticles, and 2) a post-synthetic coating method for coating a polymer after the production of iron oxide nanoparticles is completed. However, when the coating method is used, if the coated iron oxide nanoparticles exceed 50 nm, due to the phagocytosis of macrophages present in the liver and spleen, it may be possible to rely on the reticuloendotherial system after injection. Removal in seconds or minutes makes it difficult to obtain images of other tissue sites, with an optimal size of 20 nm [L. Illum et. al. (1982) Int. J. Pharm. 12: 135-146.

On the other hand, the superparamagnetic iron oxide nanoparticles coated using the above-mentioned polymers are difficult to be used as contrast agents that target specific tissues or organs due to nonspecific accumulation in tissues or organs. Accordingly, there is a need for a polymer that is specific to tissue and can remain in tissue for a long time and can penetrate into cells to improve imaging efficiency of local lesions and to reduce side effects and toxicity.

PVLA (poly (vinylbenzyl- O -β-D-galactopyranosyl-D-gluconamide)) is a hydrophobic polystyrene (polystyrene, O- β-D-galactopyranosyl-D-gluconamide) PS) Amphiphilic polymer consisting of a backbone and a side chain, which is a hydrophilic carbohydrate, contains galactose, which is an aminoal glycoprotein present on the surface of liver cells. It is recognized by receptors (asaloglycoprotein receptors (ASGP-R)) to form a ligand-receptor complex. This complex leads to specific influx into hepatocytes [ Biochemistry , vol. 21, no. 24, pp. 6292-6298, 1982 .; CS Cho et al., Biomaterials , vol. 27, no. 4, pp. 576585, 2006]. Furthermore, the positive nature of PVLA acts as an emulsifier to disperse hydrophobic superparamagnetic nanoparticles in aqueous solution.

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Therefore, the inventors of the present invention, while studying a polymer that meets the above requirements, hepatocyte-specific delivery of contrast agent through the coating of superparamagnetic iron oxide nanoparticles using PVLA, a kind of galactose-carrying polymer It was confirmed that the stability in the physiological environment was greatly improved and the present invention was completed.

An object of the present invention is to provide a PVLA-coated superparamagnetic iron oxide nanoparticles and a method of preparing the same, which functions as a specific delivery to the hepatocytes and an emulsifier in order to prevent cohesion due to hydrophobic attraction between superparamagnetic iron oxide nanoparticles and instability in aqueous solution. To provide.

In addition, the present invention provides a contrast agent for diagnosing liver disease using superparamagnetic iron oxide nanoparticles coated with PVLA, which shows excellent liver target directivity.

In order to achieve the above object, the present invention provides a superparamagnetic iron oxide nanoparticles coated with PVLA.

Hereinafter, the present invention will be described in detail.

The superparamagnetic iron oxide nanoparticles according to the present invention have a structure in which PVLA is coated on the iron oxide nanoparticle surface.

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The PVLA is a positive polymer composed of a hydrophobic polystyrene main chain and a hydrophilic carbohydrate as a side chain, and serves as an emulsifier capable of dispersing hydrophobic superparamagnetic nanoparticles in an aqueous solution. Furthermore, galactose contained in the PVLA is recognized by the asialo glycoprotein receptor (ASGP-R) present on the surface of hepatocytes to form a ligand-receptor complex, thereby coating the PVLA according to the present invention. Specific iron oxide nanoparticles can be introduced into hepatocytes.

The superparamagnetic iron oxide nanoparticles coated with PVLA according to the present invention have a maghemite (Fe ₂ O ₃ ) structure and have an average particle size of 10 to 20 nm. For rapid inflow into hepatocytes, the size of the particles is preferably 50 nm or less. The superparamagnetic nanoparticles according to the present invention are fluorescently treated and examined for inflow into hepatocytes 1 hour after injection into hepatocytes. About 75% of the observed cells Introduces iron oxide nanoparticles coated with PVLA.

In another aspect, the present invention provides a contrast agent for diagnosing liver disease comprising a superparamagnetic iron oxide nanoparticles coated with PVLA.

After injecting the iron oxide nanoparticles coated with PVLA according to the present invention through the tail vein of the rat (rat), the MR image was taken, the signal strength in the liver after the injection is significantly reduced, T ₂ attenuation effect compared to the control group Significantly increased results in much darker images. From this, iron oxide nanoparticles coated with PVLA according to the present invention can be used as a contrast agent for diagnosing liver disease by showing excellent liver target orientation.

Furthermore, the present invention comprises the steps of precipitating iron oxide nanoparticles by adding a basic solution to the aqueous iron chloride solution (step 1);

Oxidizing and dialysis the precipitated iron oxide nanoparticles of step 1 to prepare a stock solution (step 2); And

Provided is a method for producing superparamagnetic iron oxide nanoparticles coated with PVLA comprising the step (step 3) of mixing and stirring the PVLA solution to the stock solution of step 2.

Step 1 for preparing PVLA-coated nanoparticles according to the present invention is a step of precipitating iron oxide nanoparticles by adding a basic solution to an aqueous solution of iron chloride.

The aqueous iron chloride solution of step 1 may use a mixed aqueous solution of iron (II) chloride hydrate (FeCl ₂ · 4H ₂ O) and iron (III) chloride hydrate (FeCl ₃ · 6H ₂ O) in distilled water. Precipitation of the iron oxide nanoparticles in this step is coprecipitation method of divalent iron ions (Fe ^{2 +} ) and trivalent iron ions (Fe ³⁺ ) [M. Liao et. al. (2002) J. Mater. Chem. 12: 3654-3659; DB Shieh et. al. (2005) Biomaterials 26: 7183-7191. When the iron oxide nanoparticles are combined by the following reaction scheme, the iron oxide nanoparticles made of magnetite (Fe ₃ O ₄ ) may be produced by meeting with hydroxide ions (OH ⁻¹ ).

Fe ^{2 +} + ² Fe ^{3 +} + 8 OH ^-1- > Fe ₃ O ₄ + 4H ₂ O

As the basic solution, sodium hydroxide, potassium hydroxide, aqueous ammonia solution or the like can be used.

When the basic solution is added with vigorous stirring of the mixed ferric chloride aqueous solution, the reaction proceeds as in Scheme 1, whereby black iron oxide nanoparticles are obtained by precipitation. Only sediments are collected separately using permanent magnets for the next step.

Next, step 2 according to the present invention is a step of preparing a stock solution by oxidizing and dialysis the iron oxide nanoparticles precipitated in step 1.

In step 2, nitric acid (HNO ₃ ) solution may be used to oxidize the precipitated iron oxide nanoparticles, and ferric nitrate (Fe (NO ₃ ) ₂ ) solution may be used together. Iron oxide is dialyzed with dilute nitric acid solution after oxidation of the nanoparticles to prepare a stock solution.

Next, the iron oxide nanoparticles can be simply coated by mixing and stirring the PVLA solution in the stock solution of Step 2 by Step 3 according to the present invention.

PVLA superparamagnetic iron oxide nanoparticles prepared according to the present invention is coated PVLA to secure the stability of the magnetic iron oxide solution (ferrofluid) and can be selectively delivered to the liver can be used as an accurate and stable contrast agent for the diagnosis of liver disease.

Example 1 Preparation of Superparamagnetic Iron Oxide Nanoparticles Coated with PVLA

1) Preparation of Iron Oxide Nanoparticles

    In order to prepare iron oxide nanoparticles, coprecipitation was performed in which Fe (II) and Fe (III) ions were reduced as basic solutions to precipitate iron oxide nanoparticles [M. Liao et. al. (2002) J. Mater. Chem. 12: 3654-3659; D.B. Shieh et. al. (2005) Biomaterials 26: 7183-7191.

A mixed aqueous solution is prepared by adding 3.255 g of commercially available iron (III) chloride (FeCl ₃ .6H ₂ O) and 1.197 g of iron (II) chloride (FeCl ₂ .4H ₂ O) to 70 ml of distilled water. 7 ml of aqueous ammonia solution is added while vigorously stirring the mixed aqueous solution. At this time, a black product was produced, which precipitated and collected only the precipitate using a permanent magnet having a magnetic force of 6000G.

2) Preparation of Stock Solution of Iron Oxide Nanoparticles

The precipitate was oxidized using nitric acid solution (HNO ₃ ) and ferric nitrate (Fe (NO ₃ ) ₃ ) solution and dialyzed in a dilute nitric acid solution to prepare a stock solution.

The precipitate of step 1 was oxidized with 20 ml of 2 M nitric acid solution and 30 ml of 0.35 M ferric nitrate aqueous solution, and then dialyzed with 0.01 M dilute nitric acid to prepare a stock solution of iron oxide nanoparticles [GA van Ewiji]. et al., "Convenient preparation methods for magnetic colloids", Journal of Magnetism and Magnetic Materials , 201 (1-3), 31-33. 1999, M. Chastellain et al., "Particle size investigations of a multistep synthesis of PVA coated superparamagnetic nanoparticles", Journal of Colloid and Interface Science , 278 (2), 353-360, 2004.].

3) PVLA Coating of Iron Oxide Nanoparticles

To the iron oxide nanoparticles stock solution, 1 ml of an aqueous solution of PLVA prepared by adding 0.32 g of PVLA to 4 ml of distilled water was added and stirred for 30 minutes to prepare coated iron oxide nanoparticles.

Comparative Example 1

As a conventionally used contrast agent, iron oxide nanoparticles whose surface is modified with 2-pyrrolidone were prepared and used [Z. Li et. al. (2004) "One-pot reaction to synthesize water-soluble magnetite nanocrystals" Chem. Mater. 16: 1391-1393.

<Analysis>

1) Fourier Transform Infrared Spectroscopy (FT-IR) Analysis

In order to confirm that the surface of the iron oxide nanoparticles prepared according to the present invention is coated with PVLA, PVLA, iron oxide nanoparticles and Example 1 were Fourier transform infrared spectrometers (Nicolet Magna 550 series II spectrometer, Midac, Atlanta, GA, USA), and the results are shown in FIG. 1. In order to proceed with the analysis, PVLA, uncoated iron oxide nanoparticles and each of Example 1 was coated on a silicon wafer surface to prepare a film form.

As it is shown in Fig. 1, in the spectrum of the non-coated iron oxide nanoparticles ((a) of Fig. 1) 631 cm ^- was a strong absorption band at ¹ and 564 cm ^-1. The peak is at 570 cm ⁻¹ by FeO bonding of magnetite This is caused by the split of ν ₁ . In addition, an absorption peak was observed at 440 cm ⁻¹ , indicating that ν ₂ of the FeO bond appearing at 375 cm ⁻¹ was shifted to a higher frequency [M. Yamaura et al., Journal of Magnetism and Magnetic Materials , vol. 279, no. 2-3, pp. 210-217, 2004.].

The spectrum of pure PVLA (FIG. 1 (b)) shows strong absorption peaks at 1078 cm ⁻¹ due to the stretching vibrations of the CO groups contained in the oligosaccharides, which are included in the main chain of polystyrene. The absorption peaks corresponding to the ring stretching vibrations of the C = C bonds of the -CH ₂ and benzene groups appear in pairs at 2926 cm ^-1 and 1542 cm ^{- 1} and 1423 cm ^{- 1} , respectively, and the bending of the -NH group ) And absorption peaks due to the stretching vibration of the C = O group overlapped at 1647 cm ^-1 to confirm the intrinsic peak of PVLA [CV Luyen et al., Chitin and derivatives, in: JC Salomone (Ed.), Polymeric Materials Encyclopedia, vol. 2, CRC Press, Boca Raton, 1996, pp. 1208-1217, P. Galgali et al., Carbohydrate Polymers , vol. 67, no. 4, pp. 576-585, 2007.), YC Chang et al., Reactive & Functional Polymers , vol. 66, no. 3, pp. 335-341, 2006].

In the spectrum of Example 1 (FIG. 1 (c)), absorption peaks at 1078 cm ⁻¹ and 2926 cm ⁻¹ , indicating CH ₂ of oligosaccharides and polystyrene main chain of PVLA, respectively, were coated with PVLA. It can be seen. The absorption peaks at 631 cm ^-1 and 564 cm ^-1 , which are the characteristic peaks of the iron oxide nanoparticles, also shifted to higher frequencies, 636 cm ^-1 and 588 cm ^-1 , due to the hydrogen bonding with PVLA. Above, it was confirmed that the iron oxide nanoparticle surface is coated by PVLA, it can be seen that the coating by the hydrogen bond between the oxide (oxide) surface of the iron oxide nanoparticles and the OH group of PVLA.

2) X-ray Diffraction Analysis

The crystal structures of Example 1 and Comparative Example 1 were analyzed using an X-ray diffractometer (XRD) (GADDS; Bruker-AXS D5005, Karlsruhe, Germany) and shown in FIG. 2.

As shown in FIG. 2, six characteristic peaks unique to iron oxide in the form of magnetite (Fe ₃ O ₄ ) appeared in the diffraction analysis graph (FIG. 3 (a)) of Comparative Example 1. FIG. The peak of the graph of Comparative Example 1 is consistent with the database of JCPDS files (PCPDFWIN v.2.02, PDF No. 85-1436) related to magnetite, confirming that pure magnetite iron oxide nanoparticles of spinel structure were prepared. . On the other hand, the graph of Example 1 (Fig. 3 (b)) showed a peak characteristic for the iron oxide in the form of maghemite (Fe ₂ O ₃ ). It was confirmed that the conversion from magnetite to maghemite occurred during the second step of the iron oxide nanoparticle manufacturing process [JP Jolivet et al., Journal of Colloid and Interface Science , vol. 125, no. 2, pp. 688-701, 1988), TH Hyeon et al., Journal of the American Chemical Soiety , vol. 123, no. 51, pp. 12798-12801, 2001].

3) Transmission electron microscopy analysis

The size and shape of Comparative Example 1 and Example 1 were measured using a transmission electron microscopy (TEM) (JEM 1010, JEOL, Japan) and shown in FIG. 3. Samples for transmission electron microscopy measurements were placed in distilled water, and the ultrasonic dispersion was dropped in small amounts onto a copper grid and dried in air for 15 minutes.

As shown in FIG. 3, Comparative Example 1 (FIG. 3A) shows that particles having a very fine and even distribution having a size of 10 nm or less were produced. In addition, it was confirmed that the form of Example 1 coated with PVLA was spherical and had particles having a fine and uniform size distribution of 20 nm or less.

4) Total Light Scattering Meter (ELS) Analysis

In order to measure the size and distribution of Example 1 and Comparative Example 1 prepared according to the present invention, a total light scattering spectrometer (electrophoretic light scattering spectrophotometer, ELS 8000, Otsuka Electronics, Osaka, Japan) adjusted to a scattering angle of 90 ° was used. It was measured at 25 ℃ using it is shown in FIG. Samples for the analysis of the total light scattering meter were prepared by sonicating each of Example 1 and Comparative Example 1 in distilled water.

As shown in FIG. 4, the size of Comparative Example 1 (FIG. 4A) was 20.8 ± 4.4 nm in number average, and the size of Example 1 (FIG. 4B) was 25.8 ± 6.1 nm in number average. It was found that the size of the nanoparticles did not increase significantly after the PVLA coating. It can be seen that the nanoparticles were prepared to meet the conditions that the size of the particles should be less than 50 nm in order to quickly enter the hepatocytes. However, the particle size was analyzed to be larger than that measured by TEM. In the case of TEM, the size of one particle is measurable, whereas the ELS is regarded as one particle up to 2-3 aggregated particles in an aqueous solution. It is thought to be a difference according to KMK Selim et al., Biomaterials , vol. 28, no. 4, pp. 710-716, 2007.]. In addition, Comparative Example 1 and Example 1 showed that the size distribution of the nanoparticles is very even, especially since the iron oxide nanoparticles coated with PVLA show a distribution tending to be monodisperse, PVLA plays a role as an emulsifier. It can be seen.

Experimental Example 1 Influx of PVLA Coated Iron Oxide Nanoparticles into Hepatocytes

Confocal scanning microscopy over time was measured to confirm that Example 1 according to the present invention is introduced into hepatocytes. Samples required for this analysis were prepared by the following method.

1) Isolation and Culture of Hepatacyte

Hepatocytes were isolated from the liver of ICR mice using the two-step collagenase perfusion technique proposed by Seglen group [P.O. Seglen et. al. (1976), Methods Cell Biol. 13: 29-83). ICR mice (Charles River Japan, Inc. Kanagaea, Japan) used females 5 to 7 weeks old.

First, the liver was treated with 0.5 mM ethylene glycol-bis- [beta-amino ethyl ether] -N, N, N ', N' -tetraacetic acid / HBSS (ethylene glycol-bis [β-amino ethyl ether] -N, After perfusion with N, N ', N'- tetraacetic acid (EGTA) / Hank's balanced salt solution (HBSS) solution, it was perfused with 5 x 10 ^-3 wt% collagenase / HBSS solution. The liver perfused with the collagenase was cut and placed in HBSS, filtered using a membrane of 100 μm, and densified for 10 minutes at 50 g using 45% Percoll solution (Pharmacia, Piscataway, NJ, USA) at 4 ° C. Pure hepatocytes were isolated by gradient-gradient.

Next, the isolated hepatocytes were suspended in William's medium E (WE) containing only 50 μg / ml penicillin and 50 μg / ml streptomycin without fetal bovine serum (FBS). Collagen type I-coated 12-well cell culture plates were dispensed 1 x 10 ⁵ cells per well incubated for 2 hours at 37 ℃ temperature and 5% carbon dioxide atmosphere. After incubation, the medium of the cell culture plate was removed and washed three times with PBS.

2) Injection of Example 1

For fluorescence microscopy observation of iron oxide nanoparticles present in hepatocytes, fluorescein isothiocyanate (FITC) -bound PVLA (FITC-PVLA) was synthesized and used as Example 1.

Example 1 was dispersed in a WE medium containing 1.0 wt% Bovine Serum Albumin (BSA) to prepare a dispersion having a concentration of 1 mg / ml. 1 ml per well was added to the prepared hepatocytes, and cultured at 37 ° C. and 5% carbon dioxide atmosphere for 15, 30, and 60 minutes, respectively.

<Analysis>

1) Confocal Laser Scanning Microscope Analysis

In order to confirm Example 1 injected into the hepatocytes, the sample after a predetermined incubation time was washed three times with PBS to remove Example 1 remaining outside the cells, and then confocal laser scanning microscopy (Micro) Systems CLSM 410, Carl Zeiss, Germany) and observed the fluorescence and retardation image of the iron oxide nanoparticles present in the cells are shown in Figure 5 (a) and 5 (b).

At this time, if Example 1 is labeled using FITC, it may be visually confirmed whether nanoparticles are introduced into hepatocytes. Since hepatocytes can also fluoresce themselves, untreated hepatocytes were used as a control to confirm whether the fluorescence image obtained by the experiment resulted from FITC labeled on the iron oxide nanoparticles.

As can be seen in Figure 5 (a) there is almost no fluorescence appears in the control group, while in the sample added to Example 1 it can be observed that the fluorescence is increasingly vivid as the treatment time elapses, Example 1 is added After 1 hour, hepatocytes were observed to fluoresce in about 75% or more of cells compared to FIG. 5 (b), and it was confirmed that PVLA-coated nanoparticles were introduced into hepatocytes.

In addition, Example 1 is shown in Figure 6 using the gallery mode of the optical cross-section to confirm whether it is internalized inside the cell to ensure that it is located in the cytoplasm or only attached to the cell membrane.

As shown in Figure 6, the fluorescence was observed to be uniformly expressed in the cytoplasm of hepatocytes, it was confirmed that Example 1 is introduced into the cytoplasm.

Experimental Example 2 Magnetic Resonance Imaging Using Iron Oxide Nanoparticles Coated with PVLA

In order to investigate the applicability of Example 1 as a contrast agent for diagnosing liver disease, Example 1 was injected through a tail vein into a 6-week-old rat, followed by an animal coil (4.3 cm Quadrature volume coil, Nova Medical). Magnetic reasonance was taken with a 1.5 T MR scanner (GE Signa Exite Twin-speed, GE Health Care, Milwaukee, WI) using System, Wilmington, DE. At this time, it is shown in Figure 7 using Comparative Example 1 as a control. In addition, for the effect of T2 attenuation, the signal intensity (SI) of Example 1 and Comparative Example 1 was changed to ROI (regions of the pre-liver) and the post-injection (SI post of liver) in the same liver region. ROI was measured in the same dorsal muscle before injection (SI pre of BM) and after injection (SI post of BM) of the back muscles adjacent to the liver. The relative signal intensity (SI) increase in the liver after injection of the example was calculated by the following equation.

Example 1 SI before injection SI after injection Liver 3,600 760 back muscles 2,600 2,200

Comparative Example 1 SI before injection SI after injection Liver 3,000 2,700 back muscles 2,250 3,200

As shown in Fig. 7, Table 1 and Table 2, after the injection of Example 1 (Fig. 7 (b)) than the magnetic resonance photograph (Fig. 7 (a)) before the injection of Example 1, SI dropped significantly, resulting in a relatively dark image. In addition, although the magnetic resonance photograph (FIG. 7 (d)) was darker than the magnetic resonance photograph (FIG. 7 (c)) before the comparative example 1 was injected, the image was darker. After injection of 1, a brighter image was observed than the magnetic resonance image. As a result of calculating the SI increase and decrease of Example 1 and Comparative Example 1 by the above equation, after the injection of Comparative Example 1 showed a T ₂ attenuation effect of 36%, after the injection of Example 1 of 75.4% By exhibiting a T ₂ attenuation effect, it was confirmed that conventional 2-pyrrolidone exhibited better target orientation than coated iron oxide nanoparticles.

Therefore, it was confirmed that the PVLA-coated superparamagnetic nanoparticles prepared according to the present invention can be used as a hepatocyte-specific contrast agent such as mangafodipir trisodium known as Mn-DPDP.

1 is an FT-IR graph of one embodiment made in accordance with the present invention; (iron oxide nanoparticles before PVLA coating (a), PVLA (b), Example 1 (c))

2 is an XRD graph of one embodiment prepared according to the present invention; (Comparative Example 1 (a), Example 1 (b))

3 is a TEM photograph of one embodiment prepared according to the present invention; (Comparative Example 1 (a), Example 1 (b))

4 is an ELS graph of one embodiment prepared according to the present invention; (Comparative Example 1 (a), Example 1 (b))

5 is a confocal laser scanning micrograph of one embodiment prepared according to the present invention present in hepatocytes; (Fluorescence micrograph (a), phase contrast micrograph (b))

6 is a gallery mode photograph of a confocal laser scanning microscope of one embodiment made according to the invention present in hepatocytes;

7 is a magnetic resonance image of one embodiment prepared according to the present invention present in hepatocytes. (Example 1 before (a), after Example 1, after 1 hour (b), Comparative Example 1 Before (c), after the introduction of Comparative Example 1 (d))

Claims (8)

PVLA-coated superparamagnetic iron oxide nanoparticles containing PVLA-coated superparamagnetic maghemite (Fe ₂ O ₃ ). delete The superparamagnetic iron oxide nanoparticles coated with PVLA according to claim 1, wherein the average particle size of the maghemite (Fe ₂ O ₃ ) nanoparticles is 10-20 nm. The contrast agent for diagnosing liver disease according to claim 1, comprising PVLA-coated superparamagnetic iron oxide nanoparticles in which PVLA-coated superparamagnetic maghemite (Fe ₂ O ₃ ) is mixed. Adding a basic solution to the aqueous iron chloride solution to precipitate the iron oxide nanoparticles (step 1); Oxidizing and dialysis the precipitated iron oxide nanoparticles of step 1 to prepare a stock solution (step 2); And The method for preparing PVLA-coated superparamagnetic iron oxide nanoparticles of claim 1, comprising the step (3) of coating the PVLA solution by mixing and stirring the stock solution of step 2. The superparamagnetic coating of claim 1, wherein the aqueous iron chloride solution of step 1 is a mixed aqueous solution of iron (II) chloride (FeCl ₂ ) hydrate and iron (III) chloride (FeCl ₃ ) hydrate. Method for producing iron oxide nanoparticles. The method of claim 5, wherein the basic solution of step 1 is an aqueous solution of sodium hydroxide, potassium hydroxide or ammonia, wherein the PVLA-coated superparamagnetic iron oxide nanoparticles of claim 1 are used. The superparamagnetic coated PVLA of claim 1, wherein the oxidation of step 2 is performed by using a nitric acid (HNO ₃ ) solution and a ferric nitrate (Fe (NO ₃ ) ₂ ) solution. Method for producing iron oxide nanoparticles.

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