KR101417869B1 - Contrast agent coated with phosphate-polymer and method for preparing the same - Google Patents

Abstract

The present invention relates to a contrast agent comprising iron oxide nanoparticles coated with a phosphate-bonded polymer and a method for producing the same. The iron oxide nanoparticles coated with the phosphate-bonded polymer of the present invention are excellent in stability in the body and biocompatibility, can stably display all organs, and particularly can display lymph nodes which have been difficult to visualize in the meantime.

Description

TECHNICAL FIELD The present invention relates to a contrast agent coated with a phosphate-coated polymer and a method for preparing the same,

The present invention relates to a contrast agent comprising iron oxide nanoparticles coated with a phosphate-bonded polymer and a method for producing the same.

Magnetic resonance imaging (MRI), which is a typical CT imaging technique, is a non-invasive method for acquiring 3D images, and is widely used for diagnosing diseases because of its excellent contrast and spatial resolution.

MRI is a measure of the signal from a proton that has been diagnosed by a noninvasive method in the human body. However, since it has been shown that using a contrast agent for MRI can increase the sensitivity and specificity of the diagnosis, MRI angiography and perfusion imaging have been expanding the scope of application of MRI contrast agents.

MRI contrast agents enhance the contrast between normal and abnormal tissues by sensing the difference in the changes in T1 and T2 relaxation time caused by strong external magnetic field and high frequency energy after in vivo injection, (MRI) is a chemical that allows imaging of anatomical or functional areas of the brain, usually a paramagnetic contrast agent and a superficial contrast agent (Eur. Radiol. 11: 2319, 2001).

Paramagnetic contrast agents are also referred to as positive contrast agents because they exhibit enhanced (bright) signals on T1-weighted images and are made using paramagnetic metal ions such as gadolinium (Gd) and manganese (Mn). These paramagnetic contrast agents are low toxic, such as DTPA (diethylene triamine pentaacetic acid), DOTA (1,4,7,10-tetraazacyclododecane N, N ', N' ', N' '' - tetraacetic acid) And a biocompatible polymer having a functional group (for example, a carboxyl group) capable of being immobilized. 6, 2427, 2006; Acad. Radiol. 9: S20, 2002) can be used as both T1 and T2 contrast agents because they can reduce T1 relaxation time and T2 relaxation time. The paramagnetic contrast agent is mainly used as a T1 contrast agent and its enhancement effect is mainly caused by the contribution of the inner sphere which shortens the T1 mitigation time by hydration with water molecules and partly because the water molecule diffuses itself by the contrast agent And may be caused by the contribution of the out-sphere, which is affected by the magnetic field generated by the magnetic field, resulting in self relaxation enhancement. However, these paramagnetic contrast agents are highly biotoxic and capable of imaging at a level of mM, so they are more interested in the supernatant contrast agent capable of high sensitivity imaging at the μM level (Nano Lett., 6: 2427, 2006; , 2007).

Superparamagnetic contrast agents widely used in clinical practice are superparamagnetic nanoparticles represented by superparamagnetic iron oxide (SPIO) such as magnetite (Fe ₃ O ₄ ) or maghemite (Fe ₂ O ₃ ) And is classified as a negative contrast agent. They are made of uniform particles with a size of several tens of nanometers or less and made into a ferrofluid in a stable colloid form and put into the body. Tissue containing the supernatant contrast agent is distorted by the local magnetic field at the proton position in the water molecule caused by the susceptibility difference with the peripheral part according to the external environment, so that the proton is dephasing more rapidly, An enhancement (reduction in T2 relaxation time) image can be obtained.

The superficial magnetic contrast agents are commercially available that are surface coated with dextran or derivatives of dextran, which are natural polymers. The super-magnetic contrast agents coated with dextran and the like are generally used as a co-precipitation method (CPM ) (IEEET Magn. 17: 1247, 1981). Briefly, in the presence of Fe ³⁺ / Fe ²⁺ ions and low molecular weight dextran, a basic solution is added momentarily to precipitate iron oxide nanoparticles, followed by centrifugation and gel filtration (Magnet. Reson. Med. 29: 599, 1993). The manufacturing method based on this coprecipitation method has a problem that super-magnetic nanoparticles, which are a core part of the contrast agent, are synthesized at room temperature and are generally low in crystallinity and distributed in various sizes. On the other hand, the thermal decomposition method (TDM) developed in the early 1990s is a method for synthesizing highly crystalline nanoparticles without size selection process after synthesis (Angew. Chem. Int. 2007; J. Am. Chem. Soc. 126: 273, 2004). In this method, super-magnetic nano particles are synthesized by heating at a high temperature of 200 ° C. or higher using a precursor containing Fe ^{3 +} ions in general. The kind and amount of the solvent, precursor and ligand, (Nat. Mater. 3: 891, 2004; J. Am. Chem. Soc. 124: 8204, 2002), the desired size can be precisely controlled according to process parameters such as temperature change.

Dispersion in the aqueous phase is very important in order to be suitable as a contrast agent to be injected into a living body by highly sensitive superparamagnetic nanoparticles synthesized by a pyrolysis method. That is, in order to use the super-magnetic nanoparticles as an effective contrast agent, the magnetic nanoparticles should be made of a small and uniform stable magnetic iron oxide solution having a high saturation magnetism. This iron oxide solution is a colloidal dispersion solution of magnetic nanoparticles such as Fe ₃ O ₄ or Fe ₂ O ₃ and should be able to maintain a liquid state under a very strong magnetic field. However, since pure super magnetic iron oxide particles are hydrophobic and have a large ratio of volume to surface area, hydrophobic interaction is strong between the particles, which results in agglomeration and formation of clusters. If the particles are not sufficiently stable, And the biodegradation may occur rapidly. When pure iron oxide itself is toxic by itself, it is harmful to the human body, so there is a problem that it is limited to use as a contrast agent.

Therefore, in order to solve such a problem and maintain the stability of the iron oxide solution containing the super-magnetic nanoparticles, surface modification of the particles is required. The surface ligand exchange method and the surface ligand addition method are widely used for such surface modification (J. Am. Chem. Soc. 127: 12387, 2005; Science 307: 538, 2005; 19: 3870, 2007).

The surface ligand substitution method is a method of replacing the hydrophobic part of the surface of the superpowder nanoparticles with a hydrophilic ligand. At this time, if the hydrophilic ligand usable includes a carboxyl group or a dihydroxyphenyl group, it can maintain stable binding with the magnetic material after ligand exchange. The magnetic nanoparticles can be stably dispersed in the receiving phase through the exchange of surface ligands.

The surface ligand addition method, which is another method for stably dispersing the supernatant magnetic nanoparticles in a water phase, is carried out by using an amphipathic polymer capable of coating the hydrophobic nanoparticles. The polymer which can be used herein is not only poly (vinyl alcohol), Pluonic series commercial surfactants but also synthetic amphipathic polymers (Chem. Mat. 19: 3870, 2007; 317: 34, 2007, Advanced Functional Materials 18 (2007), J. Mol. Inter. Sci. 319: 429, 2008, Biomaterials 29: 2548, 2008, J. Magn. : 258, 2008, Angew Chem. Int. Edit. 46: 8836, 2007). When the organic solvent is evaporated by forcibly emulsifying the water phase (including the amphipathic polymer) and the organic phase (including the hydrophobic super-magnetic nanoparticles) and reducing the size of the droplet to the nano level, A super-magnetic nano-contrast agent is made.

To date, several tissue-specific contrast agents have been commercialized. Examples include contrast media (MS325 from Epix, USA), liver and pancreas contrast media (Ferridex and Combidex from Advanced Magnetics, USA), gastrointestinal contrast agents (Gastromark from Advanced Magnetics of the US and Gadolite from Pharmacyclic) (Teslascan, Nycomed Amersham, Norway). However, the development of MRI contrast agents that can identify lymph nodes effectively is not yet clear. However, considering that most cancers undergo lymph node metastasis, the development of lymph node MRI contrast agents that are effective in the diagnosis of cancer metastasis is very important.

Therefore, the present inventors continued to search for a new contrast agent capable of imaging lymph nodes, and as a result, confirmed that the contrast agent containing iron oxide nanoparticles coated with a phosphate-linked polymer can effectively enhance the lymph nodes, thereby completing the present invention Respectively.

Domestic patent application number: 10-2009-7009975

It is an object of the present invention to provide a contrast agent comprising iron oxide nanoparticles coated with a phosphate-bonded polymer.

It is still another object of the present invention to provide a method for producing the contrast agent.

In order to achieve the above object, the present invention provides a contrast agent comprising iron oxide nanoparticles coated with a phosphate-bonded polymer.

The present invention also provides a method of producing the contrast agent.

The iron oxide nanoparticles coated with the phosphate-bonded polymer of the present invention are excellent in stability in the body and biocompatibility, can stably display all organs, and particularly can display lymph nodes which have been difficult to visualize in the meantime.

Brief Description of the Drawings Fig. 1 is a diagram showing a process for producing phosphate-adhered polyethylene glycol (PEG).
FIG. 2 is a view illustrating a process for preparing iron oxide nanoparticles coated with a phosphate-adhered polymer.
FIG. 3 is a graph showing XPS measurement results of iron oxide nanoparticles coated with a phosphate-adhered polymer ((a) C1s, (b) O1s, (c) P2p, and (d) Fe2p.
4 is a graph showing the EDX measurement results of the iron oxide nanoparticles coated with the phosphate-adhered polymer.
FIG. 5 is a graph showing FT-IR measurement results of iron oxide nanoparticles coated with phosphate-adhered polymer (before and after (a) and (b) before ligand substitution with a phosphate-attached polymer).
FIG. 6 is a TEM photograph showing the iron oxide nanoparticles coated with the phosphate-adhered polymer (before and after (a) and (b) before the ligand substitution with the phosphate-attached polymer).
FIG. 7 is a graph showing cytotoxicity of iron oxide nanoparticles coated with a phosphate-adhered polymer. FIG.
FIG. 8 shows intracellular MR images using iron oxide nanoparticles coated with phosphate-adhered polymer ((a) T1-weighted, (b) T2-weighted).
FIG. 9 is an MR photograph of a lymph node of a mouse using iron oxide nanoparticles coated with a phosphate-adhered polymer. FIG.

Hereinafter, the present invention will be described in more detail.

As used herein, the term "magnetic resonance imaging" refers to a medical imaging method for detecting a magnetic resonance imaging signal from a water molecule after irradiating a sample with low electromagnetic energy, which is based on hemodynamics, It differs from positron emission tomography (PET), which uses radioisotopes based on molecular sciences, in terms of its use.

In the present invention, the term "contrast agent" refers to an agent used for visualizing blood vessels and / or tissues by making a difference in artificial contrast so that blood vessels and / or tissues can be seen more easily. Contrast agents can determine the presence and degree of disease and / or damage by increasing the visibility and contrast of the surface under study.

The contrast agent is characterized by enhancing the contrast effect by reducing T1 or T2 relaxation time near the tissue in the body at the time of organ diagnosis for a living body.

In one aspect, the present invention provides a contrast agent comprising iron oxide nanoparticles coated with a phosphate-linked polymer.

Wherein the contrast agent is a supernatant contrast agent.

The contrast agent of the present invention is capable of imaging all parts of the living body and cells (for example, blood vessels, liver, spleen, pancreas, digestive organs, cancer cells, macrophages, etc.), and particularly lymph nodes can be visualized.

The nanoparticles have a diameter of 1 nm to 1,000 nm, preferably 1 nm to 200 nm, and most preferably 1 nm to 20 nm. When the size of the nanoparticles exceeds 200 nm, the functionality as a contrast agent drops.

Various polymers such as hydrophilic polyamino acid, hydrophilic vinyl-based polymer and polyetherimide may be used, and examples thereof include polyethyleneglycol (PEG), polyacrylic acid (PAA), polyvinylpyrrolidone Polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), gelatin, dextran, chitosan, and pullulan, and preferably polyethylene glycol (Polyethyleneglycol, PEG).

The average molecular weight of the polymer is not limited thereto, but may be 100 Da to 50,000 Da, and preferably 200 to 20000 Da. When the molecular weight is less than 100 Da, the chain length is not sufficient enough to coat iron oxide, so that it is difficult to obtain uniform particles due to agglomeration. When the molecular weight exceeds 50,000 Da, agglomeration of polymers occurs, which is not preferable .

The magnetic material of the nanoparticles is not limited thereto, but may be selected from the group consisting of MnO, MnFe ₂ O ₄ , Fe-Pt alloy, Co-Pt alloy, (Co), and the like, and preferably superparamagnetic iron oxide (SPIO) such as magnetite (Fe ₃ O ₄ ) or maghemite (Fe ₂ O ₃ ).

The contrast agent of the present invention may contain the nanoparticles in a pharmaceutically acceptable carrier. Examples of the carrier that can be used in the contrast agent composition include a carrier and a vehicle commonly used in the medical field, including, but not limited to, alumina, sorbitol, dextran, salt, water, electrolyte and the like. In addition, the contrast agent of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, a preservative, etc. in addition to the above components.

The contrast agent according to the present invention can be prepared as an aqueous solution for parenteral administration. Preferably, a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline can be used. Aqueous injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Other preferred embodiments of contrast agents of the present invention may be in the form of sterile injectable preparations of aqueous or oily suspensions. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (for example, a solution in 1,3-butanediol). Vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. Non-volatile oils with low irritation, including synthetic mono- or diglycerides, can be used for this purpose.

The contrast agent composition according to the present invention may be administered to a living body or a sample, and an image may be obtained by sensing a signal emitted from the living body or the sample. Thus, the present invention can be used for biomedical applications applicable to diagnosis of cancer or specific disease of a desired target site, study of mechanism of bio-signal, or study of differentiation of stem cells.

The term "administering" as used herein means introducing a given substance into a patient in any suitable manner, with useful dosages and the particular mode of administration being dependent upon the age, body weight and the particular site to be treated, Will vary depending on factors such as the nature of the drug, the diagnostic use and the form of the agent (e.g., suspension, emulsion, microsphere, liposome, etc.), as will be apparent to those skilled in the art. Typically, doses are administered at low levels and increased until the desired diagnostic effect is achieved. Imaging is performed using techniques known to those skilled in the art. Generally, a sterile aqueous solution of contrast agent can be administered to a patient in a dosage range of from about 0.01 to about 1.0 mmole per kg (body weight), including all combinations and subcombinations of the dosage range, and the particular dose included therein have.

The step of injecting the contrast medium into a living body or a sample can be administered through a route commonly used in the medical field, and parenteral administration is preferred, and for example, intravenous, intraperitoneal, intramuscular, subcutaneous or local route Lt; / RTI >

In another aspect, the present invention provides a method for preparing a polymer, comprising: (a) binding a phosphate to a terminal of a polymer; And (b) coating the iron oxide nanoparticles with the phosphate-bound polymer through the ligand replacement method of step (a).

In step (a), the ginseng salt is bound to the end of the polymer. In one embodiment of the present invention, the polymer is first added to an organic solvent, t-butoxide and ethyl bromoacetate are added, -Hydroxyl-omega-carboxyl polymer. Next, the? -Hydroxyl-? -Carboxyl polymer is reacted with POCl ₃ to prepare a polymer-phosphate compound.

The step (b) is a step of preparing iron oxide nanoparticles from a phosphate-bound polymer, which is prepared using a surface ligand substitution method. In one embodiment of the present invention, the iron oxide nanoparticles are dissolved in an organic solvent and reacted with the polymer-phosphate prepared in the step (a) to prepare iron oxide nanoparticles coated with the phosphate-bonded polymer. The iron oxide nanoparticles coated with the phosphate-bonded polymer prepared by the above method are stably dispersed in an aqueous solution.

The organic solvent in the steps (a) and (b) is not limited to the above, but includes, but is not limited to, acetone, dichloromethane, dichloroethane, chloroform, ether, ketone, benzene, ethyl acetate, methanol, ethanol, hexane, carbon tetrachloride, toluene, , Tetrahydrofuran (THF), dimethylformamide (DMF), N-methyl-2-pyrrolidone (NMP), dimethyl sulfoxide (DMSO), and dimethylether (DME).

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

Example  1. Phosphate Combined  Synthesis of polymer-coated iron oxide nanoparticles

1-1. Phosphate Combined  Manufacture of Polymers

The synthesis process of the phosphate-bonded polymer is shown in FIG.

(a). α- Hydroxyl -ω- Carboxyl PEG Manufacturing

Polyethylene glycol at 25 ℃ glycol _{(PEG, Polyethylene glycol, M n} = 2000, 25 g, 12.5 mmol) was dissolved in 150ml of toluene and potassium tert- butoxide (potassium tert -butoxide, 1.7 g, 15 mmol) was added to the reaction Respectively. The reaction solution was distilled while refluxing until 40 ml of the solution remained. The remaining solution was cooled at room temperature with stirring, ethyl bromoacetate (3.4 mL, 16 mmol) was added, and the reaction was allowed to proceed overnight. The precipitate was then removed through a filter, and the filtrate was precipitated with cold ether. The material obtained through the above procedure was dissolved in 50 ml of 1 M NaOH, and then 10 g of NaCl was added. The mixture was stirred for 1 hour, then acidified to pH 3.0 and extracted with dichloromethane (CH ₂ Cl ₂ , 3 x 100 mL). After drying the organic layer over anhydrous magnesium sulfate (MgSO ₄₎ and the filter, it precipitated with cold ether, and dried in vacuo (in vacuo). Pure α-hydroxyl-ω-carboxyl polyethylene glycol (PEG) was isolated by ion exchange chromatography using DEAE-Sephadex A-25 and confirmed by NMR

^{[1 H NMR (500 MHz,} CDCl 3): δ 4.12 (s, -CH 2 - COOH, 2H), 3.63 (s, PEG backbone).]

[ ¹³ C NMR (125 MHz, CDCl 3):? 171.72, 72.78, 70.51, 69.22, 61.72.]

(b). PEG - Preparation of Phosphate Compounds

5 mmol of [alpha] -Hydroxyl- [omega] -carboxyl PEG was treated under vacuum for 1 hour and dissolved in 15 ml of anhydrous THF. While vigorously stirring this, POCl ₃ (0.6 mL, 6.35 mmol) was added dropwise. The transparent material obtained through the reaction was stirred at room temperature overnight. The reaction was quenched by addition of water and the material was extracted with chloroform (3 x 20 mL). After drying the organic layer over anhydrous magnesium sulfate (MgSO ₄₎ and the filter, it precipitated with cold ether, and dried in vacuo (in vacuo). The finally obtained PEG-phosphate molecule was confirmed by NMR.

^{[1 H NMR (500 MHz,} CDCl 3): δ 4.12 (s, -C H 2 -COOH, 2H), 3.63 (s, PEG backbone).]

^{[13 C NMR (125 MHz,} CDCl 3): δ 172.20, 70.59, 68.77, 66.17, 63.80.]

1-2. surface Ligand  Phosphates via displacement Combined  Preparation of polymer-coated iron oxide nanoparticles

A process for producing iron oxide nanoparticles coated with phosphate-bound polymer through surface ligand replacement is shown in FIG. To prepare iron oxide nanoparticles coated with a phosphate-bound polymer, a surface ligand exchange method was used. More specifically, Fe ₃ O _{4 having a} size of 6 nm To prepare iron oxide nanoparticles, pyrolysis of previously known Fe (acac) ₃ and oleic acid as a ligand were used (Weissleder, R. et al. Adv . Drug Delivery Rev. 1995, 16 , 321., Kircher, MF et al. L. Cancer Res . 2003, 63 , 8122., Rosler, A .; Vandermeulen, GWM et al. Drug Delivery Rev. 2001, 53 , 95. Huh, YM et al. J. Am . Chem . Soc . 2005, 127 , 12387.) To convert nanoparticles from hydrophobic to hydrophilic, first 10 mg of Fe ₃ O ₄ The nanoparticles were dissolved in 2 mL of THF and 25 mg of PEG-phosphate molecules were added. The reaction was heated at 60 ° C for 12 hours, then the THF was removed, 5 mL of water was added, and this was filtered through a 200 nm syringe filter. The product was purified through dialysis in demineralized water for 2 days (MW cutoff of 12 kDa) to remove excess polymer and lyophilized under reduced pressure. The finally obtained iron oxide nanoparticles coated with the phosphate-bound polymer were stably dispersed in an aqueous solution.

Experimental Example  1. Phosphate Combined  Characterization of polymer-coated iron oxide nanoparticles

1-1. XPS  analysis

The surface of the iron oxide nanoparticles coated with the phosphate-bonded polymer prepared in Example 1 was analyzed using an X-ray photoelectron spectroscopy (XPS) analyzer. The results are shown in Fig.

As shown in Fig. 3, a typical spectrum of iron oxide nanoparticles coated with a phosphate-bound polymer having a size of 6 nm was confirmed. Specifically, the C 1s spectrum (FIG. 3 (a)) showed a broad shoulder at the three peaks of 285.2, 286.8 and 289.1 eV, due to the CC, CO and COOH groups, respectively. The O 1s spectrum (FIG. 3 (b)) also showed peaks at 532.7, 531.2, and 529.8 eV, due to the presence of oxygen in CO, PO and metal oxides. Furthermore, it was P 2p spectra (Fig. (C) a 3) a peak is observed in the results, 132.5 and 133.5 eV, which is caused by the P 2p3 / 2 and 2p1 P / 2. In addition, as a result of the Fe 2p spectrum (Fig. 3 (d)), peaks were observed at 710 and 724 eV, which is due to the Fe-O bond and is typical of the spectrum of Fe ₃ O ₄ .

1-2. EDX  analysis

To analyze the surface of the iron oxide nanoparticles coated with the phosphate-bound polymer prepared in Example 1, EDX (energy dispersive X-ray spectroscopy) was analyzed using a Hitachi FE-SEM equipped with an EDX detector (Horiba) Respectively. The results are shown in Fig.

As shown in FIG. 4, it was confirmed that the phosphate was coated on the surface of the iron oxide nanoparticles.

1-3. FT - IR  analysis

The surface of the iron oxide nanoparticles coated with the phosphate-bound polymer prepared in Example 1 was analyzed using FT-IR (Shimadzu IRPrestige-21). The results are shown in Fig.

As shown in Fig. 5, it was confirmed that there was a band difference before (Fig. 5 (a)) and after (Fig. 5 (b)) of the ligand substitution. In particular, the iron oxide nanoparticles coated with oleic acid before ligand replacement showed a strong absorption band at 2852 and 2923 cm ^{-1 due} to the symmetrical and asymmetric CH of the oleyl chain, but were coated with a phosphate-linked polymer by ligand substitution It was confirmed that the intensity of the band was weakened in the iron oxide nanoparticles. Also, 1735 cm? A new absorption band at ^{? 1} (C = O) indicates that the carboxylic acid group is present and that the ligand substitution can be made at the nanoparticle surface.

1-4. TEM  analysis

In order to analyze the morphology of the iron oxide nanoparticles coated with the phosphate-bonded polymer prepared in Example 1, a TEM (transmission electron microscopy) photograph was taken using a Hitachi H7650 TEM at 100 kV. The results are shown in Fig.

As shown in Fig. 6, the iron oxide nanoparticles coated with the phosphate-bound polymer had no significant change in core size before (Fig. 6 (a)) and after (Fig. 6 Were dispersed stably without any aggregation.

From the above experimental results, it was confirmed that the phosphate-bonded polymer was efficiently coated on the iron oxide nanoparticles by the manufacturing method of Example 1, and that the iron oxide nanoparticles were uniformly dispersed in the aqueous solution with a fine and uniform size of 6 nm Respectively.

Experimental Example  2. Cytotoxicity investigation

To confirm the cytotoxicity of the iron oxide nanoparticles coated with the phosphate-bound polymer prepared in Example 1, an MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) Respectively. More specifically, mouse macrophages (RAW 264.7) were cultured in 96-well plates at 37 ° C and 5% CO ₂ until the cell concentration reached 1 × 10 ⁴ cells / well. Iron oxide nanoparticles coated with a phosphate-bound polymer having a final concentration of 50, 100, 150 and 200 μg ml ^-1 were added to the cells and cultured for 48 hours. The cells were washed twice with PBS, replenished with fresh medium, and then cytotoxicity was confirmed using MTT reagent, which is shown in FIG.

As shown in FIG. 7, the iron oxide nanoparticles coated with the phosphate-bound polymer did not show cytotoxicity even at a high concentration of 200 μg ml ^-1 . The concentration is in excess of the concentration of iron conventionally used as a mouse MRI contrast agent. Accordingly, it was confirmed from the above results that the iron oxide nanoparticles coated with the phosphate-bonded polymer of the present invention are not cytotoxic and can be used as a contrast agent.

Experimental Example  3. MR imaging

The MR system is designed to minimize motion artifacts and maintain respiratory anesthesia while scanning MR imaging of the abdomen of the mouse. Fourier transform (Fourier transform) of a multi-slice spin echo T ₁ -weighted image (TR), 361.5 ms; echo time [TE], 11 ms; (TR, 3500 ms; TE, 36 ms; 25.6 x 25.6 mm; matrix size, 256 x 256; slice thickness, 0.5 mm; interslice distance, 0.5 mm; acquisition time, 10 minutes 20 seconds) and a refocused echo T ₂ -weighted image FOV, 25.6 x 25.6 mm; matrix size, 256 x 256; slice thickness, 0.5 mm; interslice distance, 0.5 mm; acquisition time, 11 minutes 12 seconds) MR images were obtained.

3-1. Intracellular ( in vitro ) In MR  shooting

In order to confirm the applicability of the iron oxide nanoparticles coated with the phosphate-bound polymer prepared in Example 1 as an MRI contrast agent, the iron concentration was adjusted so that cervical cancer cells (Hela cells) or Raw264.7 cells were used MR imaging was performed. Feridex, a commercially available iron oxide MR contrast agent, was used as a control. The results are shown in Fig.

As shown in FIG. 8, the iron oxide nanoparticles coated with the phosphate-bonded polymer of the present invention showed the possibility of size-and concentration-dependent T 1 -weighted and T 2 -weighted magnetic resonance contrast agents. In particular, iron oxide nanoparticles coated with a phosphate-bonded polymer exhibited high T 1 -weighted and low T 2 -weighted signal intensities at high iron concentrations, indicating that T1 and T2 contrast enhancers As a result.

3-2. In vivo in vivo ) In MR  shooting

Superparamagnetic iron oxide nanoparticles of size less than 10 nm can be injected intravenously, which is considered to be a potential material for detecting lymph nodes, and MR imaging was performed in vivo.

Three Balb / C mice were anesthetized with isoflurane (1% in 100% oxygen) using a gas evacuation system. MR images were taken and iron oxide nanoparticles (6 nm or 12 nm in size) coated with a phosphate-bonded polymer of 0.2 mmol of iron (Kg body weight) ^-1 ) were injected through the tail vein. Feridex, a commercially available iron oxide MR contrast agent, was used as a control. T 2 -weighted MR images of lymph nodes were obtained using a 4.7-T animal MRI system (Pharmascan; Bruker, Ettlingen, Germany) 24 hours and 48 hours after injection. The results are shown in Fig.

As shown in Fig. 9, it was confirmed that the iron oxide nanoparticles coated with the phosphate-bonded polymer having a size of 6 nm or 12 nm were accumulated in the lymph nodes and showed a significant decrease in signal intensity after 24 hours of injection, as compared with the control group. From the above results, it was confirmed that the iron oxide nanoparticles coated with the phosphate-bonded polymer of the present invention have excellent biocompatibility and can circulate in the blood stream for a long time.

Claims (8)

A lymphadenogram involving iron oxide nanoparticles coated with a phosphate-linked polymer, obtained by reacting an? -Hydroxyl-? -Carboxyl polymer prepared by reacting a polymer with t-butoxide and ethyl bromoacetate with a phosphate, Possible superparamagnetic T1 and T2 contrast agents.
delete delete The method of claim 1, wherein the polymer is selected from the group consisting of polyethyleneglycol (PEG), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), gelatin, Dextran, Chitosan, and Pullulan. [Claim 10] The method according to claim 1, wherein the contrast agent is at least one selected from the group consisting of Dextran, Chitosan, and Pullulan.
2. The hyperlucent T1 and T2 contrast agent according to claim 1, wherein the polymer has an average molecular weight of 100 to 50000 Da.
The method of claim 1, wherein the iron oxide is magnetite (magnetite, Fe ₃ O ₄₎ or MAG H. mite (maghemite, Fe ₂ O ₃₎ is capable of portrait lymph node imaging, characterized in that the magnetic T1 and T2 contrast agent.
2. The hyperlucent T1 and T2 contrast agent according to claim 1, wherein the nanoparticles have a size of 1 to 20 nm.
(a) reacting a polymer with t-butoxide and ethyl bromoacetate to prepare an? -hydroxyl-? -carboxyl polymer;
(b) reacting the -hydroxyl-carboxyl polymer with a phosphate to produce a phosphate-bound polymer-phosphate compound at the end of the polymer; And
(c) coating the iron oxide nanoparticles with the polymer-phosphate compound of step (b) through a ligand replacement method. < Desc / Clms Page number 19 >

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