KR100949465B1 - Superparamagnetic iron oxide nanoparticles coated with mannan, 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 mannan, a method for preparing the same, and a contrast agent for diagnosing liver disease, including the same, wherein the superparamagnetic iron oxide nanoparticles of the present invention are coated with a superparamagnetic iron oxide solution (ferrofluid). As a result of securing stability and selectively delivering to liver Kupffer cells, it can be usefully used as a stable contrast agent for accurately diagnosing liver disease.

Contrast agent, superparamagnetic, iron oxide nanoparticles, met, liver disease

Description

Superparamagnetic iron oxide nanoparticles coated with mannan, preparation method approximately and contrast agent for diagnosing liver diseases}

The present invention relates to a superparamagnetic iron oxide nanoparticles coated with mannan, a method for preparing the same, and a contrast agent for diagnosing liver disease, including the same. More specifically, the mannan is coated and specifically introduced into a liver Kupffer cell. The high superparamagnetic iron oxide nanoparticles and the use as a contrast agent.

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.

Positive contrast agents longitudinal axis proton relaxation time (T _1, longitudinal relaxation time) is dominant and T ₁ weighted images damping effect of the systematic part of the contrast agent administration (T ₁ weighted image) appears as a bright signal from the, audio contrast agent to the horizontal axis relaxation time (T ₂ relaxation time transeverse) attenuation of tissue predominantly T ₂ weighted images (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 into a ferrofluid in the form of a stable colloid 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 iron oxide solution (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, and is known to be 20 nm in optimal size [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.

Mannan is the main constituent of some yeasts and consists mainly of D-mannose. Saccharomyces cereviae encounters with α- (1,6)-, α- (1,3)-and α- (1,2)-and partially phosphorylated D-mannose It is a water-soluble molecule composed of residues. The encounter is recognized by mannose receptors on macrophages.

The inventors have focused on the production of coated iron oxide nanoparticles using the met polymer.

Looking at the prior art related to the contrast agent, hepatic contrast agent using superparamagnetic iron oxide-based nanoparticles and its manufacturing method (Korean Patent Registration No. 10-0541282), iron oxide nanopowder used in MRI contrast agent prepared using ultrasound and its preparation Method and preparation method of MRI contrast agent using the same, MRI contrast agent (Korean Patent Registration No. 10-0637275), contrast agent for use in medical and diagnostic procedures and its use (Korean Patent Publication No. 10-2006-0052675) There is this. However, there is no description of a method for producing iron oxide nanoparticles by polymer coating, and the Republic of Korea Patent Publication No. 10-2006-0052675 discloses that manna can be used as an osmotic agent, which delivers the manna into an anatomical part. It was used to promote, the mannan is not coated and bound form, it shows a significant difference in structure with the present invention.

In addition, the nanoparticles were coated with nanoparticles, and none of the prior arts confirmed the inflow of hepatocytes. Therefore, the present inventors endeavored to make the hepatocyte specificity of the contrast agent through the superparamagnetic iron oxide nanoparticle coating using the metnan. The present invention has led to a greatly improved stability in the delivery and physiological environment. The coating component, mannan, is composed of D-mannose and is recognized by the mannose receptor on the macrophages of the liver, thereby enabling specific inflow into hepatocytes.

Accordingly, an object of the present invention is a superparamagnetic iron oxide nanoparticles coated with mannan and specific delivery to hepatocytes and an emulsifier function in order to prevent cohesion due to hydrophobic attraction between the superparamagnetic iron oxide nanoparticles and instability in aqueous solution. It is to provide a manufacturing method.

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

The above object of the present invention is to prepare a superparamagnetic iron oxide nanoparticles coated with mannan, characterize it and confirm its role as a contrast agent for diagnosing liver disease through cytotoxicity, intracellular influx, MR imaging, Prussian blue staining, etc. By achieving this.

In the present invention, the superparamagnetic iron oxide nanoparticles may also be represented as SPIONs (SuperParamagnetic Iron Oxide Nanopaticles), and the manna coated superparamagnetic iron oxide nanoparticles may be represented as met-SPIONs or mannan-SPIONs.

The present invention is characterized by providing superparamagnetic iron oxide nanoparticles coated with mannan. The coating component, mannan, is obtained from yeast, is composed of D-mannose, and is recognized by mannose receptors on the macrophages of the liver, thereby allowing specific inflow into hepatocytes.

The manganese-coated superparamagnetic iron oxide nanoparticles preferably have an average particle size of 10-20 nm for rapid entry into hepatocytes.

In another aspect, the present invention is characterized by providing a contrast agent for diagnosing liver disease comprising superparamagnetic iron oxide nanoparticles coated with metna.

In another aspect, the present invention is a step of precipitating iron oxide nanoparticles by adding a basic solution to the aqueous solution of iron chloride, step 2 of preparing a storage solution by oxidizing and dialysis the precipitated iron oxide nanoparticles, and mixing and stirring the solution met in the storage solution It characterized by providing a manufacturing method of the superparamagnetic iron oxide nanoparticles coated with mannan made comprising a three step of coating.

The aqueous iron chloride solution of step 1 of the manufacturing method 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 the co-precipitation 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 ).

Scheme 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.

In step 2 of the above production method, to oxidize the precipitated iron oxide nanoparticles can be used a nitric acid (HNO ₃ ) solution, it can be used with a ferric nitrate (Fe (NO ₃ ) ₂ ) solution. 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 met solution in the stock solution of Step 2 by Step 3 according to the present invention.

The met-superparamagnetic iron oxide nanoparticles prepared according to the present invention are very useful inventions in the medical field because they can be used as an accurate and stable contrast agent for the diagnosis of liver disease by stabilizing the magnetic ferrofluid and selectively delivering the liver. something to do.

Hereinafter, the present invention will be described in detail by way of examples, but the scope of the present invention is not limited only to these examples.

Example  1: Met ( mannan ) Coated Superparamagnetism  Iron oxide nanoparticles ( SPIONs Manufacturing

Iron precipitation

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.

3.255 g of iron (III) chloride (FeCl ₃ 6H ₂ O) and 1.197 g of iron (II) chloride (FeCl ₂ 4H ₂ O) purchased from Sigma-Aldrich Co .; St. Louis, MO, USA It was added to 70 mL to prepare a mixed aqueous solution. 7 mL of aqueous ammonia solution was added while vigorously stirring the mixed aqueous solution. At this time, a black precipitate is formed, and the precipitate is washed with distilled water several times until the pH drops from 10 to 7 while fixing the precipitate using a permanent magnet. Only precipitates with pH 7 were collected.

Preparation of Iron Oxide and Iron Oxide Nanoparticle Storage Solutions

The precipitate was oxidized using a nitric acid solution (HNO ₃ ) and ferric nitrate (Fe (NO ₃ ) ₃ ) solution, and then dialyzed in a dilute nitric acid solution to prepare an iron oxide nanoparticle storage solution. 20 mL of 2 M nitric acid solution and 30 mL of 0.35 M ferric nitrate (Fe (NO ₃ ) ₃ ) aqueous solution were added to the precipitate for 1 hour to oxidize. The brown suspension formed due to oxidation was dialyzed with 0.01 M nitric acid for 2 days to prepare iron oxide nanoparticle stock solutions and stored at 4 ° C.

Meeting of iron oxide nanoparticles mannan ) coating

To 2 mL of the iron oxide nanoparticle stock solution was added 1 mL of 0.01 M nitric acid solution to 1 mL of 0.14 g of mannan, which was prepared by adding 1 mL of an aqueous mannan solution and stirring at 4 ° C. for 12 hours to prepare coated iron oxide nanoparticles. . The pH of the sample was adjusted to 7 using ammonia water for in vitro and in vivo experiments.

As a control, superparamagnetic iron oxide nanoparticles coated with polyvinyl alcohol were prepared in the same manner as above.

Example  2: Met mannan ) Coated Superparamagnetism  Iron oxide nanoparticles ( mannan Characterization of SPIONs

Check the coating: Fourier transform infrared spectroscopy (FT-IR) analysis

In order to check whether the surface of the iron oxide nanoparticles prepared according to the present invention is coated with mannan-manganese-superparamagnetic iron oxide nanoparticles (mannan-SPIONs) prepared by Example 1 Fourier transform infrared spectroscopy (Fourier transform infrared) spectrometer; Nicolet Magna 550 series II spectrometer (Midac, Atlanta, GA, USA) and analyzed by uncoated iron oxide nanoparticles (SPIONs), mannan and met-superparamagnetic iron oxide nanoparticles ( Each mannan-SPIONs was coated on the surface of a silicon wafer to prepare a film, and the results of analysis are shown in FIG. 1.

As shown in FIG. 1, in the spectrum of the uncoated iron oxide nanoparticles (a), strong absorption peaks appeared at 631 cm ⁻¹ and 578 cm ⁻¹ . The peak is at 570 cm ⁻¹ by FeO bonding of magnetite This is caused by the split of ν ₁ . Absorption peaks were also observed at 440 cm ^-1 , indicating that ν ₂ of FeO bonds appearing at 375 cm ^-1 were shifted to higher frequencies [M. Yamaura et al., Journal of Magnetism and Magnetic Materials , vol. 279, no. 2-3, pp. 210-217, 2004.].

Spectrum of met itself (b) is 1650, 1370 and showed the characteristic absorption peak of the polysaccharide in the 1057 cm ^-1, exhibited a unique peak met at 816cm ^-1.

Comparing the FTIR spectra of the uncoated iron oxide nanoparticles (a) and the coated iron oxide nanoparticles (c), it was found that the characteristic absorption peaks due to the encounter were found at 1653 cm ^-1 and 816 cm ^-1 . As a result, it was confirmed that the met was coated on the surface of the nanoparticles. Also, the absorption peaks at 631 cm ^-1 and 578 cm ^-1 , the characteristic peaks of the uncoated iron oxide nanoparticles, are quite weak, and the higher frequencies 639 cm ^-1 and 586 cm ^{-1 due} to hydrogen bonding with the iron oxide nanoparticles met. It can be seen that each moved to. This shows that hydrogen bonding occurred between the quantized iron oxide surface and the OH group of the meet.

Met- Superparamagnetism  Morphology Measurement of Iron Oxide Nanoparticles

The shape and size of the coated iron oxide nanoparticles (mannan-SPIONs) that were met using a transmission electron microscopy (TEM) (JEM 1010, JEOL, Japan) were measured. The sample for measuring the transmission electron microscope was measured by distilled water in a small amount of ultrasonic dispersion on a copper grid and dried in air for 15 minutes.

As a result of the analysis, as shown in FIG. 2a, the nanoparticles coated with mannan were 10 nm or less in size, and it was confirmed that a well-formed oval was formed.

However, as shown in FIG. 2B, in the case of uncoated iron oxide nanoparticles, they were considerably agglomerated, showing that the encounter contributes to the stability of the nanoparticles.

Met- Superparamagnetism  Measurement of Size Distribution of Iron Oxide Nanoparticles

In order to measure the size and distribution of the met-superparamagnetic iron oxide nanoparticles prepared according to the present invention, an electrophoretic light scattering spectrophotometer (ELS 8000, Otsuka Electronics, Osaka, Japan) adjusted to a scattering angle of 90 ° was used. Measured at 25 ° C. Samples for the total light scattering analyzer analysis were prepared by soaking each in distilled water.

As a result of the analysis, as shown in FIG. 3a, it was found that evenly distributed particles were generated. The average particle size was 28.4 ± 7.2, and 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 ELS is one to two or three aggregated particles in aqueous solution. It is considered that the difference is regarded as the particle of KMK Selim et al., Biomaterials , vol. 28, no. 4, pp. 710-716, 2007.].

On the other hand, as can be seen in Figure 3b for the uncoated iron oxide nanoparticles, the average particle size was 22.9 ± 4.3. As a result of the analysis, the size distribution of the nanoparticles was found to be very uniform. In particular, the iron oxide nanoparticles coated with mannan show a distribution tending to be close to monodispersion, and thus it can be seen that the mannan serves as an emulsifier.

Met- Superparamagnetism  Electrophoretic Mobility of Iron Oxide Nanoparticles electrophoretic  mobility) and hydrodynamic size ( hydrodynamic size ) Measure

In order to investigate the adsorption on the surface of nanoparticles according to the content of mannan, the platinum electrode was changed by varying the weight ratio of met / iron to electrophoretic mobility and hydrodynamic size of mannan-superparamagnetic iron oxide nanoparticles. Measurement was carried out at 25 ° C. using an equipped ELS. All samples were prepared such that the weight ratio of met / iron was in the range of 0-20 (0, 0.5, 1, 2, 3, 10, 20) to a final concentration of iron of 0.06 mg / ml. To prepare a sample having a weight ratio of manna / iron 20, a manna solution (2.17 mg of manna was dissolved in 5 ml of 0.01 M nitric acid) at a concentration of 2.34 mg / ml was prepared first, and then the iron oxide nanoparticle stock solution prepared in Example 1 To 1 mL of a 100-fold dilution (concentration of 0.117 mg / ml of iron) was added 1 mL of the above-mentioned aqueous solution and stirred at 4 ° C. for 12 hours to prepare coated iron oxide nanoparticles. In addition, a sample having a weight ratio of met / iron of 10, 3, 2, 1, 0.5 was prepared in the same manner as above after diluting the met aqueous solution with an appropriate ratio using 0.01 M nitric acid. The platinum electrode was used after washing with 10 ml of distilled water and 5 ml of 0.01 M nitric acid for each measurement.

As a result of the analysis, as shown in Figure 4a, the mobility of the met-SPIONs in the acidic condition of pH 2 sharply decreased until the critical value met / iron weight ratio 2 and remained constant. In addition, as shown in Figure 4b, the hydrodynamic size of the particles also increased up to the same threshold value due to the increase in the adsorption of the mannan was maintained constant above the threshold value.

In the acidic aqueous solution, the surface of the iron oxide nanoparticles before the coating is charged with a positive charge, and when the electric field is applied to the surroundings, the particles move toward the cathode, and the mobility is proportional to the amount of charge on the particle surface. However, when the surface of the iron oxide nanoparticles are coated with a polymer such as met, the positive charge of the surface is shielded by the adsorbed polymer, thereby reducing mobility.

 Therefore, when the met / iron weight ratio was 2 or more, the mobility and the hydrodynamic size of the particles were constantly maintained.

Example  3: met ( mannan ) Coated Superparamagnetism  Iron oxide nanoparticles ( mannan Cytotoxicity of -SPIONs cytotoxicity ) Measure

  MTT analysis is a measurement method for determining the toxicity of biomaterials by measuring the viability of cells. In this example, MTT analysis was performed using Raw 274.7 macrophages by the method described below to measure the cytotoxicity of met-SPIONs.

Cell culture

Rat macrophages of the raw 264.7 cell line were obtained from the cell line bank, and DMEM medium containing 10% fetal bovine serum (FBS), 100 μg / mL of streptomycin and 100 units / mL of penicillin Life Technologies, Paris, France). The cultivation was performed at 37 ° C. in an atmosphere containing 5% carbon dioxide.

MTT analysis

Raw 264.7 macrophages were aliquoted into 24-well plates at a concentration of 1 × 10 ⁵ cells per well and incubated for 24 hours. After incubation, the medium was replaced with fresh medium containing various concentrations of PVA-SPIONs or Mannan-SPIONs. After 24 hours, the control group (SPIONs) and the experimental group (Mann-SPIONs, PVA-SPIONs) were further incubated for 4 hours after the addition of 0.5 mg / mL MTT. The medium was discarded, and the formed formazan salt was dissolved in dimethylsulfoxide (DMSO), and the absorbance was measured at 540 nm. Results are shown as percentages relative to the control.

As shown in FIG. 5, although the cell viability of the met-SPIONs decreased with increasing SPIONs concentration, the met-SPIONs showed better cell viability than the PVA-SPIONs. As a result, the met-SPIONs could replace PVA-SPIONs as a safe contrast medium.

Example  4: met ( mannan ) Coated Superparamagnetism  Iron oxide nanoparticles ( mannan -SPIONs) Intracellular  Inflow measurement

When fluorescein isothiocyanate (FITC) is bound to the nanoparticles, fluorescence flow cytometry can be used to quantitatively analyze whether the nanoparticles have been introduced into cells. The inflow efficiency of the met-SPIONs and the control PVA-SPIONs was compared.

Raw 264.7 cells were dispensed into 6-well plates at 1 × 10 ⁶ cells per well. Cells were washed with 0.1 M PBS and 1 mL of DMEM medium containing 100 μL of FITC-Mann-SPIONs (6.3 Fe mg / mL) was added to each well and incubated at 37 ° C. for 1 hour. The cultured cells were washed three times with PBS and then removed from the plate after trypsin treatment and redissolved in 500 μL of PBS. Cell-related fluorescence screening was performed by Fluorescence-activated cell sorting using FACSCalibur (Beckton Dickinson, Mansfield, Mass., USA). The data were analyzed using CELLQuest software.

 The results of the analysis are shown in FIG. 6, (a) of FIG. 6 is a macrophage of Raw 264.7 cultured only in a medium containing no sample as a control, and (b) is incubated in a medium containing FITC-PVA-SPIONs. Phagocytes, (c) are the results of fluorescent screening of macrophages cultured in a medium containing FITC-Men-SPIONs. The graph of FIG. 5 shows the degree of fluorescence by FITC of the iron oxide nanoparticles introduced into the macrophages, and the larger the graph area entering the M1 gate, the greater the degree of fluorescence by the FITC, which means more iron oxide nanoparticles. Means introduced into the cell. As can be seen in Figure 6, when raw 264.7 cells, which are mannose receptor-positive macrophages, are cultured in the presence of FITC-mannan-SPIONs with mannose, compared to FITC-PVA-SPIONs without mannose It can be seen that the inflow of FITC is high. This shows that the endocytosis of SPIONs through the mannose-receptors into macrophages is efficient.

Example  5: met ( mannan ) Coated Superparamagnetism  Iron oxide nanoparticles ( mannan Magnetic resonance imaging using SPIONs

In order to investigate the applicability of Mannan-SPIONs as a contrast agent for diagnosing liver disease, rats with Mann-SPIONs were injected and magnetic resonance imaging was performed.

Six-week old rats were anesthetized with a mixed gas containing 1.5% isoflurane in a 1: 2 ratio of oxygen: nitrogen and injected with SPIONs through the tail vein. PVA-SPIONs were injected as a control. Then, magnetic reasonance with a 1.5 T MR scanner (GE Signa Exite Twin-speed, GE Health Care, Milwaukee, WI) using an animal coil (4.3 cm Quadrature volume coil, Nova Medical System, Wilmington, DE) ) Was taken. Fast-spin echo (FSE) T2-weighted (repetition time ms / echo time ms of 4,200 / 102, flip angle 90 °, echo train lenth of 10, 5 cm field of view, 2mm section thickness, 0.2- mm intersection gap, 256 × 160 matrix) MR images were performed.

The experimental results are shown in FIG. 7, (a) before the injection of PVA-SPIONs as a control group, (b) after the injection, (c) before injection of the met-SPIONs as the experimental group, (d ) Means after injection.

As a result of the analysis, as shown in FIG. 7, the magnetic resonance image (FIG. 7B) was darker than the magnetic resonance image (FIG. 7A) before the control PVA-SPIONs were injected, and the met-SPIONs were also injected. The picture was darker after the injection (FIG. 7D) than before the magnetic resonance picture (FIG. 7C). When comparing the control group and the experimental group, the photograph after the injection of the experimental group, Mannan-SPIONs, was darker than after the injection of the control group, PVA-SPIONs. Through this, met-SPIONs was found to be a more effective contrast agent.

In addition, for the quantitative analysis of T2 attenuation effects, the signal intensity (SI) of met-SPIONs and PVA-SPIONs was measured in the same liver region before and after injection of the contrast agent (SI pre of liver). ROI (regions of interest) was measured. ROI was also measured in the same dorsal muscle before and after injection (SI pre of BM) and after injection (SI post of BM). The measurement result was substituted into Equation 1 to calculate the degree of decrease in signal strength.

[Equation 1]

Signal intensity drop (%)

Signal strength drop by met-SPIONs Met-SPIONs SI before injection SI after injection Liver 1552 441 back muscles 1153 1339 % Decrease in signal intensity = 75.6 (after 1.5 hours)

Signal strength drop by PVA-SPIONs PVA-SPIONs SI before injection SI after injection Liver 1552 441 back muscles 1153 1339 % Decrease in signal intensity = 75.6 (after 1.5 hours)

As shown in Table 1 and Table 2, after 1.5 hours of injection of PVA-SPIONs, a 65% T ₂ attenuation effect was observed, whereas after 1.5 hours of infusion of mannan-SPIONs, a 75.6% T ₂ attenuation effect was observed. It was.

Through the above results, it was confirmed that the met-SPIONs are better absorbed into the liver than the PVA-SPIONs, and the influx of the met-SPIONs is achieved through the mannose receptor-mediated uptake.

Example  6: met ( mannan ) Coated Superparamagnetism  Iron oxide nanoparticles ( mannan Liver of -SPIONs In macrophages  Check for existence

Prussian blue staining was performed to determine whether the met-SPIONs were present in the macrophages present in the liver. Prussian blue dyeing is an application of iron ions reacting with ferrocyanide compounds to produce iron ferrocyanide compounds. After injection of the met-SPIONs and PVA-SPIONs, liver tissues were fixed with formaldehyde and cut for staining. The tissue was also treated with eosin for counter-staining of the cytoplasm.

As shown in FIG. 8, as compared with PVA-SPIONs (FIG. 8A), mainly the influx of met-SPIONs (FIG. 8B) occurs near the hepatic vasculature (cardiovascular and vasculature) and optionally Kupffer cells. It was confirmed that it is distributed in (Fig. 8b).

Therefore, the mannan-coated superparamagnetic nanoparticles prepared according to the present invention may be usefully used as a hepatocyte-specific contrast agent.

FIG. 1 shows FT-IR graphs of (a) iron oxide nanoparticles before coating (SPIONs), (b) met, and (c) met-SPIONs.

2A is a TEM picture of met-SPIONs.

2B is a TEM picture of SPIONs.

3A shows an ELS graph of met-SPIONs.

3B shows an ELS graph of SPIONs.

Figure 4a shows the electrophoretic mobility of the met-SPIONs according to the weight ratio of met / iron.

4B shows the hydrodynamic size of mannan-SPIONs according to the weight ratio of met / iron.

FIG. 5 is a graph showing cytotoxicity of Raw 264.7 macrophages of PVA-SPIONs and met-SPIONs by MTT assay.

6 is a graph showing the cellular influx of met-SPIONs.

7 is a resonance photograph before (a), after (b) and before (a) and after (b) of injection of PVA-SPIONs.

Figure 8 is a photograph showing the influx of SPIONs in Cooper cells between rats by Prussian blue staining after the addition of PVA-SPIONs (a) and Mannan-SPIONs (b).

Claims (4)

Contrast agent for diagnosing liver disease, wherein mannan contains superparamagnetic iron oxide nanoparticles coated with hydrogen bonds. delete delete Precipitating iron oxide nanoparticles by adding a basic solution to an aqueous solution of iron chloride; Oxidizing and dialysis the precipitated iron oxide nanoparticles to prepare a stock solution; And Coating the surface of the iron oxide nanoparticles with met by mixing and stirring the met solution into the stock solution through hydrogen bonding; Method of producing a superparamagnetic iron oxide nanoparticles coated with metnan, characterized in that comprises a.

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