KR101510835B1 - Mussel adhesive protein derived chitooligosaccharide ligand and contrast agent comprising the same - Google Patents

Abstract

The present invention relates to a chitosan oligosaccharide ligand derived from mussel adhesive protein, and a contrast medium including the same. More specifically, the chiotsan oligosaccharide ligand is obtained by attaching catechol, used when mussel adheres to an object, and methoxy polyethylene glycol (mPEG), which is a non-toxic polymer, to chitosan oligosaccharide, which is a biocompatible substance. The chitosan oligosaccharide ligand derived from mussel adhesive protein has excellent ability to be bound with iron oxide, is not cytotoxic, has excellent biocompatibility, can be circulated through blood stream for a long time, and thus can be advantageously used as a contrast medium.

Description

A mussel adhesive protein-derived chitosan oligosaccharide ligand and a contrast agent comprising the same,

The present invention relates to a mussel adhesive protein-derived chitosan oligosaccharide ligand and a contrast agent containing the same, and more particularly, to a mugnet oligosaccharide ligand and a mucopolysaccharide conjugate, which are useful as a biocompatible chitosan oligosaccharide A chitosan oligosaccharide ligand and a contrast agent comprising the chitosan oligosaccharide ligand.

Magnetic resonance imaging (MRI) uses a strong magnetic field and applies a high-frequency magnetic field to a human body placed inside a device capable of applying a strong magnetic field. The applied magnetic field resonates the hydrogen nucleus of each tissue of the body, The difference in the relaxation time of the signal from each tissue occurs, which is a technique of imaging through a computer.

MRI can be divided into T1 (long) and T2 (transverse) emphasized images. Iron oxide nanoparticles are mainly used as a contrast agent for a typical T2-weighted image.

Among them, magnetic nanoparticles have been used as magnetic resonance image (MRI) contrast agents for several years. It is known that magnetic nanoparticles exhibit superparamagnetic characteristics at a size of less than 10 nm, and their applicability in the biomedical field In fact, magnetic nanoparticles have been functionalized to track specific cells, reaching levels of clinical trials beyond animal testing in areas such as cancer tracking and high-temperature therapeutic drug delivery.

However, since it is a very toxic substance in the human body, a polymer material suitable for a living body should be used for coating and there should be no side effects as a drug.

Polymers suitable for the living body mainly used for coating include poly-ε-caprolactone (PCL), PLA (ploylactide) and their copolymers and dextran, silane, chitosan, . Among these, dextrane and chitosan are known to have various physiological functions such as antibacterial action, anticancer action, immunity strengthening action, and lactic acid bacterin promoting action in the human body. Chitosan derivatives are non-toxic, non-pollutant, biodegradable and have been applied in various fields such as drug delivery system, blood coagulant, food additives.

However, the conventional polymeric material as described above has a disadvantage in that it has a weak binding force with iron oxide and is easily peeled off, thus exhibiting toxicity, and has a disadvantage in that the solubility is lowered in an aqueous solution.

PEGylation, a technique using biopolymers, involves the modification of proteins, peptides, and non-peptide molecules by the binding of one or more polyethylene glycol (PEG) chains. PEG is a non-toxic , Non-immunogenic, non-immunogenic, and highly water-soluble, and FDA-approved. In addition, the binding of PEG to active ingredients such as proteins and peptides has the advantage that the degradation is reduced by reduction or elimination of the metabolic enzyme and protein immunity, and the efficacy in the body can be maintained for a long time.

Korean Patent Laid-Open Publication No. 10-2012-0013519 Korean Patent No. 10-0974082

In order to solve the problems of the prior art as described above, it is an object of the present invention to provide a chitosan oligosaccharide ligand derived from a mussel adhesive protein which has no toxicity due to its excellent binding force with iron oxide.

It is another object of the present invention to provide a contrast agent comprising iron oxide nanoparticles coated with the chitosan oligosaccharide ligand derived from the mussel adhesive protein.

In order to achieve the above object, the present invention provides a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by the following formula (1).

[Chemical Formula 1]

In Formula 1, x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80.

The present invention also relates to (S1) reacting methoxypolyethylene glycol with acrylonitrile and sulfuric acid to prepare mPEG-COOH; (S2) preparing mPEG-g-COS (PEGylated COS) by reacting the mPEG-COOH with a chitosan oligosaccharide solution; COS-DOPA (3,4-Dihydroxyphenylalanine) by reacting a mPEG-g-COS (PEGylated COS) and a (S3) And a method for producing a mussel adhesive protein-derived chitosan oligosaccharide ligand.

In particular, mPEG-COOH in step (S2) is preferably reacted at 10 to 70% with respect to the number of -NH ₂ groups in the chitosan oligosaccharide.

The hydrocapphosphoric acid of step (S3) is preferably reacted at 10 to 70% with respect to the number of -NH ₂ .

The present invention also provides a contrast agent comprising iron oxide nanoparticles coated with a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by Formula 2 below.

(2)

In Formula 2, x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80.

The size of the iron oxide nanoparticles is preferably 10 to 200 nm.

The iron oxide nanoparticles and the mussel adhesive protein-derived chitosan oligosaccharide ligands are preferably mixed in a weight ratio of 1: 3 to 10.

The chitosan oligosaccharide ligand derived from mussel adhesive protein according to the present invention has excellent toxicity and excellent stability with iron oxide and is excellent in stability and is suitable for use as a contrast agent because it can display all organs of human body and is well dissolved in an aqueous solution, Stability is excellent.

1 is a graph showing the results of ¹ H-NMR spectrum analysis of mPEG-COOH formed in the step of producing mPEG-COS-DOPA according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of ¹ H-NMR spectrum analysis of chitosan oligosaccharides used in the step of producing mPEG-COS-DOPA according to an embodiment of the present invention.
3 is a graph showing the results of ¹ H-NMR spectrum analysis of mPEG-g-COS (PEGylated COS) formed in the step of producing mPEG-COS-DOPA according to an embodiment of the present invention.
4 is a graph showing the results of ¹ H-NMR spectrum analysis of mPEG-COS-DOPA prepared according to an embodiment of the present invention.
FIG. 5 is a graph showing XPS analysis results of mPEG-COS-DOPA coated iron oxide nanoparticles prepared according to an embodiment of the present invention. FIG. 5A shows C1s, FIG. 5B shows O1s, 5c shows N1s, and FIG. 5D shows spectral results of Fe2p.
FIG. 6 is a graph showing FT-IR analysis results of mPEG-COS-DOPA coated iron oxide nanoparticles prepared according to an embodiment of the present invention. 6a shows iron oxide nanoparticles coated with oleic acid before ligand replacement, 6b shows iron oxide nanoparticles coated with a mussel adhesive protein-derived chitosan oligosaccharide ligand, and 6c shows a mussel adhesive protein-derived chitosan oligosaccharide ligand.
FIG. 7 is a graph showing the results of thermogravvimetric analysis (TGA) analysis of mPEG-COS-DOPA coated iron oxide nanoparticles according to an embodiment of the present invention. 7a shows iron oxide nanoparticles coated with oleic acid before ligand replacement, and 7b shows iron oxide nanoparticles coated with a mussel adhesive protein-derived chitosan oligosaccharide ligand.
FIG. 8 is a graph showing the effect of mPEG-COS-DOPA coated iron oxide nanoparticles prepared according to an embodiment of the present invention on cytotoxicity by MTT analysis.
FIG. 9 is a graph showing the relationship between the concentration of R 2 (= 1 / T 2) and R 1 (= 1 / T 1) of iron oxide nanoparticles coated with mPEG-COS-DOPA prepared according to an embodiment of the present invention Fig. 9A shows the T2 horizontal axis relaxation rate, 9b the T2 map image, and 9c the T1 vertical axis relaxation rate.
10 is an MR photograph of a mouse lymph node measured using mPEG-COS-DOPA coated iron oxide nanoparticles prepared according to an embodiment of the present invention. FIG. 10A shows the results before injection of contrast agent, 10B immediately after injection, 10C after 10 hours, and 10D after 24 hours of injection.

Hereinafter, the present invention will be described in detail.

The term "magnetic resonance imaging" as used herein refers to a medical imaging method for detecting a magnetic resonance imaging signal emitted from a water molecule after irradiating a sample with low electromagnetic energy, which is based on hemodynamics and uses a change in magnetic resonance signal , Is different from positron emission tomography (PET), which is based on molecular science and uses radioactive isotopes.

In the present invention, the term "contrast agent" refers to a preparation used for visualizing blood vessels and / or tissues by making a difference in artificial contrast so that the blood vessels and / or tissues are more easily seen. Contrast agents can determine the presence and extent 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 in the vicinity of tissue in the organ during organ diagnosis for a long time.

The present invention provides a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by the following formula (1).

[Chemical Formula 1]

In Formula 1,

x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80. [

That is, the mussel adhesive protein-derived chitosan oligosaccharide ligand represented by Formula 1 of the present invention is prepared by conjugating catechol and non-toxic mPEG (methoxy polyethylene glycols) used in attachment of a mussel to a biocompatible chitosan oligosaccharide .

More specifically, the process for producing a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by Formula 1 of the present invention can be represented by the following Reaction Scheme 1. It is to be understood that the process of the present invention is not limited to the following Reaction Scheme 1.

[Reaction Scheme 1]

In the above Reaction Scheme 1, a and b are each independently the same as or different from each other and are an integer of 30 to 80, and x, y, m, z and n are as defined in the above formula (1).

Hereinafter, a method for preparing a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by Formula 1 of the present invention will be described in detail with reference to the above Reaction Scheme 1.

(S1) Production of mPEG-COOH

First, mPEG-COOH is prepared by reacting methoxy polyethylene glycoll mPEG with acrylonitrile and sulfuric acid (H ₂ SO ₄ ) to prepare mPEG-COOH.

Specifically, mPEG-OH solution and potassium hydroxide are added to distilled water to completely dissolve the solution. Acrylonitrile is slowly added to this solution and stirred at 0 to 5 ° C for 1 to 10 hours. At this time, since the acrylonitrile generates heat upon dropwise addition, it is slowly added to the solution. Again, the concentrated sulfuric acid is added to the solution, heated at 95 to 100 ° C for 0.5 to 10 hours to react, cooled to room temperature, and the reaction product is extracted to obtain mPEG-COOH.

(S2) Production of mPEG-g-COS (PEGylated COS)

Then, mPEG-g-COS (PEGylated COS) is prepared by reacting the mPEG-COOH with a chitosan oligosaccharide solution.

That is, the above-prepared mPEG-COOH and N-hydroxysuccinimide (NHS) were added to a solution prepared by dissolving chitosan oligosaccharide in deionized water, and then carbodiimide hydrochloride (EDC) was added And the solution is stirred at room temperature for 1 to 10 hours to prepare mPEG, that is, mPEG-g-COS (PEGylated COS) grafted with chitosan oligosaccharide.

In particular, it is preferable that the mPEG-COOH is reacted at 10 to 70%, preferably 30%, with respect to the number of -NH ₂ of the chitosan oligosaccharide. When the mPEG-COOH is reacted with the number of -NH ₂ of the chitosan oligosaccharide within the above-mentioned range, it can be dissolved well in water and dissolved well in CHCl ₃ .

(S3) Preparation of mPEG-COS-DOPA

For the production of mPEG-COS-DOPA which is a chitosan oligosaccharide ligand derived from the mussel adhesive protein of the present invention, the carboxylic acid group of the hydrocaffeic acid and the amine group of the chitosan oligosaccharide are coupled through carbodiimide chemistry do.

Specifically, hydrocapphosphoric acid is dissolved in anhydrous DMF, and a solution in which carbodiimide hydrochloride (EDC) is dissolved in methanol is added to this solution to activate the hydrocapphosphoric acid. Then, the activated hydrocaiophosphoric acid is added to a solution of mPEG-g-COS (PEGylated COS) dissolved in methanol in a nitrogen atmosphere and reacted to prepare mPEG-COS-DOPA.

The hydrocafphosphoric acid is preferably reacted in an amount of 10 to 70%, preferably 30%, based on the number of -NH ₂ in the chitosan oligosaccharide. When the hydrocafphosphoric acid is reacted with the number of -NH ₂ of the chitosan oligosaccharide within the above-mentioned range, it can be dissolved well in water and dissolved well in CHCl ₃ .

The hydrocafphosphoric acid then forms dopamine for iron oxide (Fe ₃ O ₄ ) and a -OH of the catechol group for ligand exchange reaction.

The chitosan oligosaccharide ligand derived from the mussel adhesive protein represented by the formula 1 of the present invention has excellent binding force with iron oxide nanoparticles and is free from toxicity due to the peeling off of the coating and is excellent in stability when applied to human body, .

The present invention also provides a contrast agent comprising iron oxide nanoparticles coated with a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by Formula 2 below.

(2)

In Formula 2, x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80.

The size of the iron oxide nanoparticles is preferably 10 to 200 nm, more preferably 12 nm. When the size of the iron oxide nanoparticles is within the above-mentioned range, it is better because the life time in the body is long and the lymph nodes can be visualized.

The compound of Formula 2 can be prepared by reacting iron oxide nanoparticles with a chitosan oligosaccharide ligand derived from mussel adhesive protein represented by Formula 1 in a solvent.

For example, chloroform, methylene chloride, ethyl acetate and the like can be used. In particular, chloroform has a high solubility, so that it is possible to dissolve the polar manufacturing materials Since it is easy to use, chloroform is more preferably used.

The iron oxide and the mussel adhesive protein-derived chitosan oligosaccharide ligands are preferably mixed in a weight ratio of 1: 3 to 10, more preferably 1: 5. If the mixing ratio is out of the above range, the ligand may not be sufficiently coated on the iron oxide surface.

The iron oxide nanoparticles coated with the mussel adhesive protein-derived chitosan oligosaccharide ligand of the present invention have no cytotoxicity, have excellent biocompatibility, and can be circulated in the blood stream for a long time, so that they can be usefully used as a contrast agent.

The contrast agent of the present invention may be contained in a pharmaceutically acceptable carrier as needed. Examples of usable carriers include carriers and vehicles conventionally used in the medical field, such as, but not limited to, alumina, sorbitol, dextran, salts, water, electrolytes, 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 of 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. Water-soluble injection suspensions may be added with a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Contrast agents of the 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 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. In general, sterile aqueous solutions of contrast agents can be administered to a patient in a dosage range of from about 0.01 to about 1.0 mmole per kilogram of body weight, including all combinations and subcombinations of the dosage range, and the particular dose included therein.

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 >

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

Example 1. Preparation of chitosan oligosaccharide ligand (mPEG-COS-DOPA) derived from mussel adhesive protein

(1) Preparation of mPEG-COOH

Put the methoxy polyethylene glycol (mPEG-OH, 6mmol, M n = 2,000) 12g to 15g of distilled water was completely dissolved, then put in a potassium hydroxide 0.4g (7mol) 30 minutes to completely dissolve in the ice bath conditions. 0.5 g (9.42 mmol) of acrylonitrile was slowly added dropwise to the above solution while stirring in an ice bath, followed by stirring at 0 to 5 ° C for 2 hours. Subsequently, 30.0 g of concentrated sulfuric acid was slowly added to the solution and heated to 95 to 100 캜 for 3 hours. Then, the mixture was heated to room temperature, 200 ml of distilled water was added, and the reaction product was extracted with DCM (3 x 30 ml). The extract was dried with anhydrous sodium sulfate to remove the solvent, and pure mPEG-COOH was separated by ion exchange chromatography using DEAE-Sephadex A-25.

_{^{1 H NMR (500MHz, CDCl 3}} ): δ 3.64-3.56 (m, PEG backbone), 3.33 (s, 3H, CH 3 -O-), 2.54 (t, 2H, J = 6.0Hz, -CH 2- C (O) O-).

(2) Preparation of mPEG-g-COS (mPEG-grafted chitosan oligosaccharide)

0.5 g (3 mmol) of chitosan oligosaccharide was dissolved in 30 ml of deionized water, and then 1.87 g (0.9 mmol) of mPEG-COOH prepared in the above (1) and 0.5 g (4.5 g) of N-hydroxysuccinimide mmol) was added and 0.86 g (4.5 mmol) of carbodiimide hydrochloride (EDC) was added. Then, the solution was reacted by stirring at room temperature for 24 hours, dialyzed with deionized water for 2 days (cut-off molecular weight 12 kDa) and lyophilized to prepare mPET-g-COS.

(3) Preparation of mPEG-COS-DOPA

1.24 g of EDC was dissolved in 5 ml of methanol. Subsequently, the EDC solution was slowly added to a solution prepared by dissolving 0.6 g of hydrocapphosphoric acid in 10 ml of anhydrous dimethylformamide (DMF) to activate hydrocaiphosphoric acid. The activated hydrocapphosphoric acid was reacted with a solution of 0.54 g of mPEG-g-COS in 10 ml of methanol under a nitrogen atmosphere for 24 hours. An excess amount of cold ethyl ether was added to the reaction solution to precipitate, and the residual solvent was removed under vacuum. The precipitated polymer was dialyzed (fractional molecular weight 1.2 kDa) in acidified deionized water (pH = 5.5) for 2 days and then lyophilized to prepare mPEG-COS-DOPA.

Example 2. Mussel adhesive protein-derived chitosan oligosaccharide ligand (MAC) -Fe ₃ O ₄  Manufacture of nanoparticles

(1) Production of Fe ₃ O ₄ nanoparticles

Iron oxide nanoparticles are described by Park, J .; An, K .; Hwang, Y .; Park, JG; Noh, HJ; Kim, JY; Park, JH; Hwang, NM; Hyeon, T. Nat . Mater . 2004, 3, 891. The oleic acid was coated on the surface of the substrate.

(2) Preparation of MAC-coated Fe ₃ O ₄ nanoparticles

10 mg of Fe ₃ O ₄ nanoparticles prepared above and 50 mg of mPEG-COS-DOPA prepared in Example 1 were added to 10 mL of chloroform, followed by stirring at room temperature for 1 hour. After stirring, the chloroform was evaporated, and deionized water was added thereto. The resulting aqueous solution was filtered with a 200 nm syringe filter and purified with a rotary filter (fractional molecular weight 100 kDa, 5,000 x g, 10 minutes, Millipore) were removed to prepare a Fe ₃ O ₄ nanoparticles, the MAC is coated.

Experimental Example 1. Characterization of chitosan oligosaccharide ligand derived from mussel adhesive protein

1-1. ^One H-NMR analysis

¹ H-NMR spectra of mPEG-COOH, chitosan oligosaccharide, mPEG-g-COS (PEGylated COS) and mPEG-COS-DOPA formed in the step of preparing mPEG-COS-DOPA in Example 1, The results are shown in Figs.

As shown in FIGS. 1 to 4, mPEG-COOH, chitosan oligosaccharide, mPEG-g-COS (PEGylated COS) and mPEG-COS-DOPA were successfully prepared at each step in the production of mPEG-COS-DOPA according to the present invention .

1-2. XPS analysis

The surface of the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 was analyzed using an X-ray photoelectron spectroscopy (XPS) analyzer. The results are shown in FIG.

As shown in Figure 5, the resulting carbon bond in the C1s spectrum (Figure 5a) was confirmed and the resulting oxygen binding of O1s (Figure 5b) was confirmed, confirming the resulting nitrogen binding of N1s (Figure 5c) was able to verify the result of the Fe binding (Fig. 5d), Fe ₃ O _4, which was typical of the spectrum.

1-3. FT-IR analysis

The surface of the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 was analyzed using FT-IR (Shimadzu IROrestige-21). The results are shown in FIG.

As shown in FIG. 6, the iron oxide nanoparticles coated with oleic acid before ligand replacement (FIG. 6A) showed strong absorption bands at 2852 and 2923 cm ^{-1 due} to the asymmetric CH of the oleyl chain, by at the primary wavelength and 520㎝ Ah due 1664㎝ ^{-1 -1} vicinity of the iron oxide nanoparticles (Fig. 6b) coated with mussel adhesive protein derived from chitosan oligosaccharide ligands according to the invention is well-ligand to iron oxide by checking the Fe-O stretching Coating. Further, iron oxide derived from mussel adhesive protein that is not coated with the nanoparticles of chitosan oligosaccharide ligands (Fig. 6c) Bay ~ 1664㎝ primary amine group of ^-1, the bending vibration and 1107㎝ ^-1 vicinity of the secondary amine group of 1580㎝ ^-1 COC As a result of confirming the presence of stretching, it was confirmed that the protein was a chitosan oligosaccharide ligand.

1-4. TGA analysis

The surface of the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 was analyzed using TGA (Thermogravvimetric Acalysis), and the results are shown in FIG.

As shown in FIG. 7, iron oxide nanoparticles coated with oleic acid before ligand replacement (FIG. 7A) started pyrolysis at about 350 ° C. and finished at about 450 ° C., confirming that it was a typical oleic acid coated iron oxide nanoparticle. In the iron oxide nanoparticles (FIG. 7B) coated with the chitosan oligosaccharide ligand derived from the mussel adhesive protein according to the present invention by the ligand substitution, since the pyrolysis started at about 210 ° C. and finished at 400 ° C., it was confirmed to be a chitooligosaccharide ligand composed of an organic branch there was.

Experimental Example 2: Cytotoxicity test

To confirm the cytotoxicity of the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) Respectively.

First, mouse macrophages (RAW 264.7) and HeLa cells were supplemented with 10% (v / v) fetal bovine serum (BioWhittaker, Walkersville, MD, USA) and 1% penicillin / streptomycin (Gibco BRL, NY, USA) And cultured in Dulbecco's modified eagle's medium (DMEM) at 37 ° C and 5% CO ₂ .

The mouse macrophages and HeLa cells were inoculated in 96-well plates at 37 ° C and 5% CO ₂ until the cell concentration reached 1 × 10 ⁴ cells / well. The MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 were added to the cells at concentrations of 12.5, 25, 50, 100 and 200 μg / ml, and cultured for 48 hours. Cells were washed twice with PBS, replenished with fresh medium, and cytotoxicity was confirmed using MTT reagent. The results are shown in FIG.

As shown in FIG. 8, it was confirmed that the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 according to the present invention did not show cytotoxicity at a concentration of 200 μg / ml. The above concentration exceeds the concentration of iron used as a conventional mouse MRI contrast agent. Accordingly, the iron oxide nanoparticles coated with the chitosan oligosaccharide ligand derived from the mussel adhesive protein of the present invention can be used as a contrast agent because of its cytotoxicity And it was found.

Experimental Example 3. Magnetic Resonance Imaging

3-1. Changes in T2 horizontal axis relaxation rate and T1 vertical axis relaxation rate by concentration

The values of R 2 (= 1 / T 2) and R 1 (= 1 / T 1) according to various concentrations of iron ions contained in the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 The graph is shown in Fig. At this time, the slopes of the graph represented by the concentration and the functional relationship between R2 and R1 are referred to as r2 and r1, respectively. This value is finally a measure of the possibility of the MAC-coated Fe ₃ O ₄ nanoparticles prepared according to the present invention as an MRI contrast agent.

The concentrations of iron ions were 0.01, 0.03, 0.12, 0.25 and 0.5 mM, R1 was 1.6 mM ^-1 s ^-1 as shown in the T2 horizontal axis relaxation rate of Fig. 9 (a) As shown in the relaxation rate, the concentrations of iron ions were 0.1, 0.2, 0.3, 0.55 and 1 mM, and R1 was 1.6 mM ^-1 s ^-1 . FIG. 9 (b) is a T2 map image of a MAC-coated Fe ₃ O ₄ nanoparticle coated with a chitosan oligosaccharide ligand derived from the mussel adhesive protein of the present invention at 12 nm in iron oxide, As shown in FIG. 9 (b), the color of the T2 contrast agent became darker as the mM number of iron increased. Therefore, it was found that the contrast medium according to the present invention can be used not only as T1 but also as a T2 contrast agent.

3-2. MR imaging in vivo

Magnetic resonance imaging of the MAC-coated Fe ₃ O ₄ nanoparticles prepared in Example 2 was performed using a 4.7T-MRI (Bruker BioSpec 47/40) instrument at Ochang Center, Korea Basic Science Research Institute.

First, 1 ml (2 mg / ml) of the MAC-coated Fe ₃ O ₄ nanoparticle solution of Example 2 was mixed with 10 μl of FITC solution (10 mg of FITC dissolved in 1 ml of DMSO) , And then purified using a spin filter to prepare a MAC-coated Fe ₃ O ₄ nanoparticle-FITC.

Animal protocols approved by the Animal Experimental Ethics Committee of the Korea Basic Science Research Institute were used. The MR images of the mice were measured before and after the injection of the above-prepared MAC-coated Fe ₃ O ₄ nanoparticles-FITC (2.5 mg Fe / kg) using a 4.7T MRI (Bruker BioSpec 47/40) T2-weighted MR photographs were obtained and the results are shown in FIG. At this time, the measurement conditions were FA: 90 ㅀ, TR = 3,500 ms, TE = 12 ms, FOV = 50 횞 50 mm, matrix size = 256 x 256 mm, slice thickness = 1 mm and NEX = 2.

As shown in FIG. 10, it was confirmed that the MAC-coated Fe ₃ O ₄ nanoparticles according to the present invention accumulated in the lymph nodes compared to before injection, and the signal intensity decreased significantly after 24 hours of injection. From the above results, the iron oxide nanoparticles coated with the chitosan oligosaccharide ligand derived from the mussel adhesive protein of the present invention have excellent biocompatibility and can be circulated in the blood stream for a long time, so that they can be usefully used as a contrast agent.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (7)

A mussel adhesive protein-derived chitosan oligosaccharide ligand represented by the following Formula 1:
[Chemical Formula 1]

In Formula 1,
x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80. [ (S1) reacting methoxypolyethylene glycol (mPEG) with acrylonitrile and sulfuric acid to prepare mPEG-COOH;
(S2) preparing mPEG-g-COS (PEGylated COS) by reacting the mPEG-COOH with a chitosan oligosaccharide solution; And
(S3) activating the hydrocafphophosphoric acid solution and reacting it with mPEG-g-COS (PEGylated COS) to prepare mPEG-COS-DOPA (3,4-Dihydroxyphenylalanine);
Wherein the mussel adhesive protein-derived chitosan oligosaccharide ligand is represented by the following formula (1)
[Chemical Formula 1]

In Formula 1,
x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80. [ 3. The method of claim 2,
Wherein the mPEG-COOH of step (S2) is reacted at 10 to 70% with respect to the number of -NH ₂ of the chitosan oligosaccharide. 3. The method of claim 2,
Wherein the hydrocafphosphoric acid in step (S3) is reacted in an amount of 10 to 70% based on the number of -NH ₂ groups. A contrast agent comprising iron oxide nanoparticles coated with a mussel adhesive protein-derived chitosan oligosaccharide ligand represented by Formula 2:
(2)

In Formula 2, x, y, m and z are each independently the same or different from 1 to 500 and n is an integer from 30 to 80. 6. The method of claim 5,
Wherein the size of the iron oxide nanoparticles is 10 to 200 nm. 6. The method of claim 5,
Wherein the iron oxide nanoparticles and the mussel adhesive protein-derived chitosan oligosaccharide ligand are mixed at a weight ratio of 1: 3 to 10.

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