[Invention TitleJ

PREPARATION OF EXTREMELY SMALL AND UNIFORM SIZED, IRON OXIDE-BASED PARAMAGNETIC OR PSEUDO-PARAMAGNETIC NANOPARTICLES AND MRI Tl CONTRAST AGENTS USING THE SAME

[Technical Field}

<i> The present invention relates to a preparation method of uniform sized iron oxide-based paramagnetic or pseudo-paramagnetic nanoparticles, iron oxide-based nanoparticles prepared by the same, and an MRI Tl contrast agent including the same. More particularly, the present invention relates to a method for preparation of iron oxide nanoparticles having a extremely small and uniform size of 4nm or less based on thermal decomposition of iron oleate complex, iron oxide nanoparticles prepared by the same, and a Tl contrast agent including paramagnetic or pseudo-paramagnetic nanoparticles.

[Background Art]

<2> A great deal of study involving various kinds of nanoparticles has recently and actively been conducted in biomedical fields such as cell staining, cell separation, in vivo drug delivery, gene delivery, diagnosis and treatment of disease or abnormality, molecular imaging, or the like.

<3> In order to identify substantial significance of medical applications of such nanoparticles, satisfactory results should be achieved both in-vitro and in -vivo.

<4> That is, nanoparticles with beneficial effects primarily proved through cell experiments, are then subjected to secondary animal testings to support that the tested nanoparticles so they may be applicable for medical use.

<5> Magnetic resonance imaging (MRI) is a well known method for provision of anatomic, physiological and/or biochemical information of a human body through images by spin relaxation of hydrogen atoms in a magnetic field and is at present an excellent image diagnostic instrument that enables real time imagination of organs of an animal or human in a non-invasive way.

<6> For precious and various utilizations of MRI in biological science or medical fields, a process of injecting a foreign substance into the body to increase contrast of an (MRI) image is used. In this regard, the foreign substance is often referred to as a contrast agent. Such a contrast agent may be a substance using super-paramagnetic or paramagnetic material that induces contrast of signals on a site to be observed through MRI, thus allowing the site to be clearly distinguished.

<7> On MRI images, contrast between tissues is a phenomenon occurred due to difference in relaxation between tissues, wherein the relaxation refers to recovery of nuclear spin of water molecules in the tissues to an equilibrium state. The contrast agent influences such relaxation and thus may increase the difference in relaxation between tissues and induce variation in MRI signals, thus enabling clearly distinguishable contrast of the tissues. However, the contrast agent may cause differences in utility and precision, depending on the characteristics and functions of the contrast agent, subjects for injection of the contrast agent, or the like.

<8> In addition, when contrast is improved using the contrast agent which helps to regulate image signals of specific organs and/or tissues to be higher or lower than adjacent organs and/or tissues, a more distinctive (sharp) image is created. A contrast agent increasing the level of image signals at a desired site of the body, from which MRI images are obtained, than that of the other site (adjacent to the desired site), may be referred to as a 'positive' contrast agent ( 'Tl contrast agent ^' ). On the other hand, a contrast agent decreasing the level of image signals at a desired site than that of the other side may be referred to as a 'negative' contrast agent ( 'T2 contrast agent' ). More particularly, the MRI contrast agent may be classified into a Tl contrast agent using high spin of a paramagnetic material and a T2 contrast agent using magnetic inhomogeneity around a paramagnetic or super-paramagnetic material. The 'positive' contrast agent relates to Tl relaxation, that is, longitudinal relaxation. Such longitudinal relaxation means that, after a magnetized component ^' Μζ' in Z-axis direction of the spin absorbs RF energy impact applied from X-axis, the magnetized component is aligned along Y-axis on an X-Y plane and emits energy to the outside, in turn returning to the original value (or state) of Mz. The foregoing action is expressed as 'ΊΊ relaxation.' The time taken for returning Mz to 63% of an original value refers to "Tl relaxation time' and, as the Tl relaxation time is decreased, MR I signals are greater, which in turn, decreases a period of time for acquiring images.

<'·>> Likewise, the 'negative' contrast agent relates to T2 relaxation, that is, transversal relaxation. As described above, after the magnetized component 'Mz' in Z-axis direction of the spin absorbs RF energy impact applied from X-axis, the magnetized component is aligned along Y-axis on an X-Y plane and spontaneously decays and/or emits energy to adjacent spins, in turn returning to the original value of Mz. In this regard, another spin component 'My' equally widen on the X-Y plane is decayed by an exponential function and this is expressed as 'T2 relaxation.' A time taken until My is decayed to 37% of an original value refers to 'T2 relaxation time' and a My value measured through a receiving coil mounted on Y-axis by a function of time, wherein the My value is decreased over time, refers to a free induction decay (FID). Tissues with a short T2 relaxation time are shown as a dark region on the MRI .

<io> In MRI contrast agents commercially available on the market, paramagnetic compounds are used as a 'positive' contrast agent while super ^¬ paramagnetic nanopart icles are used as a 'negative' contrast agent.

<u> A current T2 contrast agent includes iron oxide nanoparticles such as

SPIOC superparamagnetic iron oxide). In this case, T2 contrast is a negative contrast, that is, a negative contrast method wherein desired sites are darker than the surrounding part. Therefore, this method does not embody remarkable contrast effects and has a demerit of causing blooming effect to contrast a larger area than an actual size.

<i2> On the other hand, the Tl contrast agent has a merit of offering positive contrast to brightly display a desired site, and comprises a high spin material. Therefore, a gadolinium complex having 7 hole-spins in 4f orbital is usually employed. However, the gadolinium complex has very short in vivo and/or vascular retention time due to a relatively small molecular weight, causing difficulties in precisely diagnosing. Further, the above Tl contrast agent cannot be used to persons having weak kidneys because of a danger to derive nephrogenic systemic fibrosis and has recently received the warning by the U.S. Food and Drug Administration. Accordingly, there is a strong need for development of an improved Tl contrast agent that may solve such disadvantages of the gadolinium complex including, for example, short retention time, severe toxicity to patients with kidney diseases, or the like.

<i3> Among new trends in research on Tl contrast agents, an article regarding the use of manganese oxide nanoparticles having 5 hole-spins at -3d orbital has been disclosed (H. B. Na et al., Angew. Chem. Int. Ed. 2007, 46, 5397).

<i4> A manganese oxide nanoparticle has advantages such as a high Tl relaxation effect, which is a characteristic of manganese ions, and easy bonding to target molecules and easy intracellular injection which are characteristics of the nanoparticle. However, in the case where the manganese oxide nanoparticle is introduced into endosome, manganese ions escape from the nanoparticle due to internal acidic environments. Therefore, if such manganese ions remain in the body, these may cause a calcium channel disturbance problem (L. K. Limbach, et al., Environ. Sci. Techno 1. 2007, 41, 4158).

<15> In order to overcome the above disadvantages, use of iron oxide as a Tl contrast agent, wherein the iron oxide has five hole-spins as well as higher biocompa ibi lity than manganese, may be proposed.

<i6> General iron oxide (especially, magnetite or maghemite) nanoparticles are super-paramagnetic near room temperature. Due to such super-paramagnetic properties, that is, high magnetization, a Γ2 level is increased and susceptibility characteristics may occur, thus causing problems such as signal distortion. Consequently, it has been reported that magnetite is not suitable to be used as a Tl contrast agent (Y. -w. Jun, et al. J. Am. Chem. Soc. 2005, 127, 5732).

<i7> However, the foregoing problems may be overcome by controlling the size of iron oxide nanoparticles. More particularly, as the size of iron oxide particles is decreased, magnetic properties thereof may be reduced, which in turn deteriorates magnetic inhomogeneity. Accordingly, use of the iron oxide nanopart icies as a Tl contrast agent may be expected. For instance, US Patent No. 6,638,494 (inventor: Herbert Pilgrim) disclosed enhancement of Tl relaxivity (rl) by decreasing a particle size of super-paramagnetic iron oxide. According to the patent, iron oxide nanopart icles synthesized by co- precipitation, which have a particle size of 1 to lOnm, and an average size (d50: median) of 2 to 4nm, and hydrophilic surface, show that Tl relaxivity ranges from 2 to 50 L/mmoi.sec and r2/rl is 5 or less. However, although the particle average size (median) is small, a range of the particle size is considerably broad such as 1 to lOnm, to thereby produce irregularity in particle size. If the size of an iron oxide particle is 4nm or greater, the T2 effects may increase rapidly with the particle size. Therefore, even though the average size is small, improvement in Tl relaxivity is not so high when the particles have irregular size. Therefore, these nanopart icles are also not suitable to be used as a Tl contrast agent.

<18> Among recent studies, use of iron oxide nanopart icles with a size of 4 to 6nm as a Tl contrast agent has been reported (E. Taboada et al., Langmuir, 2007, 23, 4583; U. I. Tromsdorf et al . , Nano Lett. 2009, 9, 4434). However, due to a relatively large particle size, T2 effects are still significant, thus the nanopart icles entail limitations in application thereof as a Tl contrast agent.

<i9> Combidex® (AMAG Co.) which is currently under clinical trials in regard to use thereof as a T2 contrast agent for lymph nodes, had also been investigated for Tl contrast performance thereof. However, since an average size of iron oxide nanopart icles was relatively large in the range of 4 to 6nm and a size of constitutional particles is irregular, it was known that T2 effect is predominant over Tl effect (Claire Corot et al . , Advanced Drug Delivery Reviews 58 (2006) 1471).

<2o> Further, there is a method for preparation of iron oxide particles having a uniform size through thermal decomposition. However, strict requirements for the preparation of iron oxide nanopart icles having a size of 4nra or less are needed, in turn not being preferable in commercial applications (Jongnam Park, et al., Nature Mate . , 3(2004), 891).

<2i> Moreover, even if nanoparticles having a size of 4nm or less may be prepared, raw materials are expensive and/or have severe toxic properties, thus having little significance in the aspect of commercial applications (Xiaowei Teng, J. Mater. Chem., 14(2004), 774).

<22> Accordingly, synthesis and mass-production of iron oxide nanoparticles having a extremely small and uniform size of 4nm or less, in a highly reproducible manner at low costs, as well as Tl contrast research using the same, have not yet been reported, which are still required.

<23>

[Disclosure]

[Technical Problem]

<24> As an existing magnetic resonance Tl contrast agent, a gadolinium complex has a considerably small molecular weight, thus showing a too short in vivo and vascular retention time. In addition, the complex may cause severe toxicity problems to patients with kidney diseases. Since an iron oxide nanopart icle is a crystal, it has a relatively particle size, to thereby lead to extend the in vivo and vascular retention time, and it still has minimal toxicity. Based on such advantages, it is intended to develop a new Tl constant agent using the iron oxide nanoparticles. However, iron oxide nanoparticles prepared by conventional methods are so large that they still entails a problem of highly predominant T2 effect over Tl effect and, therefore, may not be suitably used for Tl contrast.

<25> Accordingly, an object of the present invention is to provide a method for manufacturing iron oxide nanoparticles, wherein the nanoparticles are able to be used as a Tl contrast agent, have an extremely small and uniform particle size, are easily produced, and enable mass production thereof.

<26> More particularly, the foregoing object relates to provision of a method for preparation of iron oxide nanoparticles, wherein the nanoparticles have paramagnetic or pseudo-paramagnetic (hereinafter, referred to as (pseudo)paramagnetic' ) properties, size uniformity (average size ± Iran) and a small average size of 4nm or less, as compared to conventional iron oxide nanopart icles with super-paramagnetic properties.

<27> Another object of the present invention is to provide iron oxide nanopart icles which have (pseudo)paramagnet ic properties, size uniformity (average size ± lnm) and a extremely small average size of 4nm or less, and which have not been made in the related art.

<28> Another object of the present invention is to provide an MR I Tl contrast agent including (pseudo)paramagnetic iron oxide nanoparticles described above and, more particularly, an MR I Tl contrast agent including iron oxide nanoparticles, with various advantages, wherein: the contrast agent has improved Tl contrast effects without image distortion while providing bright images; is present in the nanoparticle form to increase intracellular penetration rate and intracellular uptake capacity; imparts target-specific contrast effects; is easily delivered to a target and safely eliminated from the body; minimizes side effects, or the like. Moreover, the present invention provides a Tl contrast agent having desired in vivo and vascular retention time, which is not too short(that is, relatively extended), as compared to conventional Gd-based Tl contrast agents.

[Technical Solution]

<29> In order to overcome the foregoing problems in the related art, the present inventors have implemented intensive and extensive studies and accomplished synthesis of iron oxide nanoparticles having extremely small and uniform size of 4nm or less based on thermal decomposition of iron oleate complex through a simple process. Using the synthesized iron oxide nanoparticles as a Tl contrast agent, the present invention has been com leted.

<30> Accordingly, the present invention provides a method for preparation of iron oxide nanoparticles, which includes (a) reacting: an iron complex having iron as a central atom and a carboxylate group having 4 to 25 carbon atoms ( 'C4 to C25 carboxlylate group' ) that is bonded to the central atom in a ligand form; a C4 to C25 fatty acid: and a C4 to C25 aliphatic alcohol or C4 to C25 aliphatic amine at 150 to 350 ^° C to prepare the iron oxide nanoparticles and, after operation (a), further includes (b) dispersing a preci itate in an organic solvent, wherein the precipitate is obtained by cooling and washing the nanoparticles described above.

<3i> The iron oxide nanoparticles prepared as above may have a size of 4nm or less and (pseudo)parametric properties, thus being useable as an MR I Tl contrast agent.

<32> An iron precursor used in the preparation of iron oxide nanoparticles may comprise an iron atom and a CIO to C22 fatty acid group in a ligand form that is bonded to the iron atom and, more preferably, is an iron oleate complex (hereinafter, 'iron oleate' ).

<33> In addition, the fatty acid and/or aliphatic alcohol (or aliphatic amine) used in the preparation of iron oxide nanoparticles may include CIO to C22 fatty acids and/or aliphatic alcohols (or aliphatic amines). More preferably, the fatty acid and aliphatic alcohol are oleic acid and oleyl alcohol while the aliphatic amine is oleyl amine.

<34> Meanwhile, with regard to practical processing conditions, the preparation of iron oxide nanoparticles described above may be performed by heating reaction materials, that is, an iron complex, fatty acid and aliphatic alcohol (or aliphatic amine), in a mixed state, from room temperature to 200 to 310°C at a temperature elevation rate of 5 ^° C/min or more, and allowing reaction at 200 to 310 ^° C for 5 to 60 minutes.

<35> According to another object, the present invention also provides iron oxide nanoparticles prepared according to the foregoing extremely small and uniform sized preparation method. In this regard, each of the iron oxide nanoparticles may have a size of 4nm or less and exhibit (pseudo) paramagnetic properties. The size of the iron oxide nanoparticle prepared according to the present invention may be controlled by regulating molar ratios of the reaction materials such as C4 to C25 fatty acid, C4 to C25 aliphatic alcohol (or aliphatic amine) introduced during the preparation.

<36> Since the iron oxide nanoparticles have organic materials capped on their surface, which come from reaction materials, they are hydrophobic and well dispersib!e in nonpolar organic solvent such as hexane, toluene, and the like.

<37> Further, the present invention provides hydrophilic iron oxide nanoparticles by modifying the surface of the hydrophobic nanoparticles with hydrophilic materials through ligand exchanging or encapsulating methods.

<38> The hydrophilic iron oxide nanoparticles, according to the present invention, may be obtained by modifying the surface of iron oxide with polyethyleneglycol ( 'PEG' ), phosphol ipid-PEG, PEG-phosphate, monosaccharide phosphate, derivatives of monosaccharide phosphates, betaines or citric acid. More preferably, the surface of iron oxide may be modified with a PEG- phosphate (PO-PEG) molecule that has a PEG bonded to a phosphate group or phosphine oxide group, glucose 6-phosphate, glucose 6-phosphate-ethanol ami e, glucose 6-phosphate-PEG, Betaine or citric acid.

<39> Further, the present invention provides colloidal solution including the hydrophilic iron oxide nanoparticles dispersing the nanoparticles in water .

<40> Further, the present invention provides magnetic resonance imaging Tl contrast agent including the colloidal solution of hydrophilic iron oxide nanoparticles.

<4i> According to the present invention, even where the iron oxide nanoparticle is the one synthesized by any known method, the nanoparticle may be (pseudo)paramagnet ic if it has a size of 4nm or less and, therefore, nanoparticles may exhibit improved Tl contrast effects.

<42> An alternative method for preparation of iron oxide nanoparticles according to the present invention may include reacting: an iron complex having iron as a central atom and a C4 to C25 carboxlylate group that is bonded to the central atom in a ligand form; and a C4 to C25 fatty acid at 290 to 310 °C with a temperature elevation rate of 3 to 3.5 ^° C/min to prepare the iron oxide nanoparticles. Another alternative method for preparation of iron oxide nanoparticles according to the present invention may include primarily reacting: a iron complex having iron as a central atom and a C4 to C25 carboxlylate group that is bonded to the central atom in a ligand form; and a C4 to C25 fatty acid at 265 to 275 ^° C, followed by conducting a secondary reaction thereof at 315 to 325 ^° C, to prepare the iron oxide nanopart icles.

<43> The present invention is not particularly limited to the foregoing objects and, instead, the other objects and advantages of the present invention will be apparent from the following detailed description and clearly understood by preferred embodiments of the present invention.

<44> Moreover, it will be easily understood that other purposes, features and aspects of the present invention may be realized by measures and/or means and combinations thereof described by the appended claims.

[Advantageous Effects 1

<45> According to the present invention, (pseudo)paramagnet ic iron oxide nanoparticles having an extremely small and uniform size of 4nm or less may be reproducibly and massively manufactured using raw materials at low costs by an easier and simple preparation process, compared to existing methods in the related art. Also, a size of the nanoparticles may be easily controlled.

<46> In addition, iron oxide nanoparticles manufactured according to the foregoing method have a uniform size distribution, compared to the existing methods in the related art, thereby imparting constant Tl contrast effects.

<47> Furthermore, the present invention provides a Tl contrast agent including (pseudo)paramagnetic iron oxide nanoparticles, to thereby enable high quality of Tl contrast, which cou'd not be given by the existing methods in the related art .

[Description of Drawings]

<48> The above and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments given in conjunction with the accompanying drawings, in which:

<49> FIG. 1 shows transmission electron microscopy (TEM) images of 3nm-size iron oxide nanopart icle synthesized by a method described in Example 1, more particularly; (a) a TEM image; (b) a TEM image in a wide range; (c) a high resolution-transmission electron microscopy (HR-TEM) image! and (d) a selected area electron diffraction (3AED) pattern, ^"

<50> FIG. 2 shows a X-ray diffraction (XRD) spectrum of 3nm-size nanopart icles synthesized by a method described in Example 1,"

<5i> FIG. 3 shows a TEM image of 2.3nm-size iron oxide nanopart icles synthesized by a method described in Example 2;

<52> FIG. 4 shows a TEM image of l.Snrrrsize iron oxide nanopart icles synthesized by a method described in Example 3;

<53> FIG. 5 shows a TEM image of 3.3nm ^~ size iron oxide nanopart icles synthesized by a method described in Example 4;

<54> FIG. 6 shows a TEM image of 3.5nm-size iron oxide nanoparticles synthesized by a method described in Example 5;

<55> FIG. 7 shows a TEM image of 1.6nm ^~ size iron oxide nanoparticles synthesized by a method described in Example 6;

<56> FIG. 8 shows a TEM image of 2.4nm-size iron oxide nanoparticles synthesized by a method described in Example 7;

<57> FIG. 9 shows a TEM image of 3.5nm-size iron oxide nanoparticles synthesized by a method described in Example 8;

<58> FIG. 10 shows a TEM image of 2.3nm-size iron oxide nanoparticles synthesized by a method described in Example 9;

<59> FIG. 11 of (a) shows a TEM image of 2.7nm-size iron oxide nanoparticles synthesized by a method described in Exam le 10; (b) shows a TEM i.mage of iron oxide nanoparticles synthesized by a method described in Comparative Example 1; and (c) shows a TEM image of iron oxide nanoparticles synthesized by a method described in Comparative Example 2;

<60> FIG. 12 of (a) shows M-H graphs at 5K and 300 , respectively, of 3nm- size nanoparticles synthesized by the method described in Example l; (b) shows variation in M-H graph at 300K of nanoparticles with particle size; (c) shows zero field cooling and field cooling M-T graphs of 2.3nm ^~ size nanoparticles synthesized by the method described in Example 2, respectively; (d) shows an M-T graph of 3nm-size nanoparticles synthesized by a method described in Example 1; (e) shows an M-T graph of 12nm ^~ size nanoparticles synthesized by a method described in Comparative Example 3; (f) shows M-H graphs at 5K and 300K, respectively, of 1.6nnrsize nanoparticles synthesized by the method described in Example 6; (g) shows an M-T graph of the nanopart icle in Example 6; and (h) shows M-H graphs at 5K and 300K, respectively, of 2.3nnrsize nanoparticles synthesized by the method described in Example 9;

<6i> FIG. 13 illustrates a distribution of number-average hydrodynamic diameters (number -aver age of 11.8nm) of 3nm-size nanoparticles which are dispersed in water by using PEG-phosphate (P0-PEG) according to a method described in Example 13;

<62> FIG. 14 shows MRI phantom Tl image of dispersions which are prepared by primarily modifying the surface of iron oxide nanoparticles with PEG- phosphate (P0-PEG) and phospholipid PEG, respectively, with particle size, and then secondarily dispersing the surface-modified nanoparticles in water; in particular, in the case where P0-PEG is used to treat the nanoparticles having a size of 2.3nm, 3nm, 4nm and 7nm, respectively, they are each indicated as 2.3P, 3P and 4P; likewise, in the case where phospnol ipid-PEG is used to treat the foregoing nanoparticles, they are each indicated as 2.3L, 3L, 4L and 7L;

<63> FIG. 15 shows cell phantom MRI results of 3nm-size nanoparticles and

12nm-size nanoparticles, respectively; in particular, (a) shows cell phantom MR image of 3nm-size nanoparticles; and (b) shows cell phantom MR image of 12nm-size nanoparticles;

<64> FIG. 16 shows clear contrast images of jugular veins, carotid arteries and aortic arch, which are obtained using the inventive nanoparticles, as compared to a gadolinium complex, Gadovist® (Bayer Schering Co.) (a) shows in vivo MRI vascular contrast image using 3nm-size nanoparticles; and (b) shows in vivo Wl vascular contrast image using Gadovist®;

<65> FIG. 17 shows a TEM image of 4nm ^~ size iron oxide nanoparticles synthesized by a method described in Example 20;

<66> FIG. 18 illustrates MTT assay results of MCF-7 cells using hydrophi lie- modified iron oxide nanoparticles according to Example 21;

<67> FIG. 19 illustrates molecular weight assay results of iron oxide nanoparticles by means of MALDI-T0F in Example 22;

<68> FIG. 20 shows a TEM image of 12nm-size iron oxide nanoparticles synthesized by a nethod described in Comparative Example 3;

<69> FIG. 21 shows a TEM image of 12nm-size iron oxide nanopar t i c 1 es synthesized by a method described in Comparative Example 4;

<70> FIG. 22 shows a TEM image of 7nm-size iron oxide nanopar t icles synthesized by a method described in Comparative Example 5;

<7i> FIG. 23 shows a TEM image of 4nm-size iron oxide nanopar tides, which were encapsulated in an aggregate form, followed by negative staining, according to the method described in Comparative Example 6; and

<72> FIG. 24 shows a TEM image of 6nm-size iron oxide nanoparticles synthesized by comparative Example 7,

<73> FIG. 25 shows an enhanced MRI vascular contrast images using the 3nm- size nanoparticles capped by glucose 6-phosphate described in example 17. [Best Model

<74> The foregoing objects, features and advantages will become more apparent from the following description of preferred embodiments of the present invention with reference to accompanying drawings, which are set forth hereinafter. Accordingly, those having ordinary knowledge in the related art to which the present invention pertains will easily embody technical ideas or spirit of the present invention. In addition, the terminology used herein includes the meanings commonly understood in the related art by those having ordinary knowledge in the related art, except where otherwise noted. When technical configurations known in the related art are considered to make the contents obscure in the present invention, the detailed description thereof will be omitted.

<75> Hereinafter, preferred embodiments of the present invention will be described in detail with reference the accompanying drawings.

<76> A method for preparation of iron oxide nanoparticles according to the present invention comprises, (a) reacting: an iron complex having iron as a central atom and a carboxylate group having 4 to 25 carbon atoms ( 'C4 to C25 carboxlylate group,' hereinafter the same defined as above) that is bonded to the central atom in a ligand form; a C4 to C25 fatty acid; and a C4 to C25 aliphatic alcohol or C4 to C25 aliphatic amine at 150 to 350 ^° C to prepare the iron oxide nanoparticles and, after the preparation (a), further includes (b) dispersing a precipitate in an organic solvent, wherein the precipitate is obtained by cooling and washing the nanoparticles described above.

<77> An iron precursor used in the preparation (a) of the iron oxide nanoparticles may be one having an iron atom and a CIO to C22 fatty acid group bonded thereto in a ligand form and examples of such ligands may include: stearic acid; oleic acid; linoleic acid; palmitic acid; palmitoleic acid; myristric acid; lauric acid; arakidonic acid; behenic acid, or the like. More preferably, the iron precursor used herein is iron oleate.

<78> Fatty acid and aliphatic alcohol used in the preparation (a) of the iron oxide nanoparticles may include CIO to C22 fatty acid, CIO to C22 aliphatic alcohol and/or CIO to C20 aliphatic amine. Examples of the fatty acid may include: stearic acid; oleic acid; linoleic acid; palmitic acid; palmitoleic acid; myristic acid; lauric acid; arakidonic acid; ricinoleic acid; behenic acid, or the like. Examples of the aliphatic alcohol may include ^' - stearyl alcohol (octadecanoi ) ; oleyl alcohol; linoleyl alcohol; hexadecanol; palmitoleyl alcohol; uetradecanol > ^' dodecanol ; arakidonyl alcohol; eicosanol; docosanol; hexadecanediol , or the like. Also, examples of the aliphatic amine may include- ^' stearylamine (octadecylamine) ; oleylamine; hexadecyl amine; palmitoleylamine! tetradecylamine; dodecyl amine; arakidonyl amine, or the like.

<79> More preferably, the fatty acid and aliphatic alcohol used herein are oleic acid and oleyl alcohol, respectively. Likewise, the aliphatic amine used herein is oleyl amine.

<so> With regard to practical processing conditions, the preparation of the iron oxide nanoparticles may be performed by heating reactive materials, that is, an iron complex, fatty acid and aliphatic alcohol (or aliphatic amine) in a mixed state, from room temperature to 200 to 310 ^° C with a temperature elevation rate of 5°C/min or more, and allowing reaction at 200 to 310 ^° C for 5 to 60 minutes.

<8i> The iron oxide nanoparticles prepared in the present invention may have a size of 4nm or less. Characteristics of the iron oxide nanoparticles obtained according to the preparation method of the present invention are described below.

<82> FIGS. Ka) and (b) show TME images of 3nm-size iron oxide nanopart icles. From the figures, it can be seen that the nanopart icles are extremely small and uniform. FIG. Kb) shows a wide area of image to demonstrate that small particles are not aggregated but uniformly distributed throughout the wide area without large particles mixed therein. Compared to metal nanopart icles, an oxide with a low electron density and, therefore, extremely small oxide particles are not clearly observed by TEM. So, as shown in FIG. 1(c), a lattice structure is not clearly detected by HR-TEM owing to TEM electron-beam energy. But FIG. 1(d) illustrates peaks (311, 400) of typical magnetite or maghemite structure through an electron diffraction (ED) pattern.

<83> FIG. 2 is an XRD spectrum of 3nm-size nanopart icles. From the figure, it can be seen that 3nnrsize nanopart icles have a magnetite or maghemite structure, although they show relatively wide peaks (311, 400, 440) owing to a small size thereof. A clear distinction between magnetite and maghemite is very difficult because XRD patterns of these two structures are very similar. According to the calculation by Debye-Scherrer equation, the nanopart icles have a size of 3nm, which is the same size as TEM images(FIG. 1), thereby demonstrating excellent crystal 1 inity.

<84> A size of the iron oxide nanopart icles prepared according to the present invention may be controlled by regulating a moiar ratio of the reactive materials such as C4 to C25 fatty acid or C4 to C25 aliphatic alcohol (or aliphatic amine) introduced during the preparation.

<85> According to the present invention, the size of the iron oxide nanopar t i c 1 es may be reduced by decreasing a concentration of the iron precursor, so as to control the size of the iron oxide nanopart icles .

<86> When an amount of the aliphatic alcohol (or aliphatic amine), as one of the reaction materials, is increased, nanoparticles having reduced size may be synthesized. However, the size control may depend on types of reaction materials and/or reaction conditions. <87> For instance, in the case where iron oleate is used as the iron precursor, nanoparticles having a smaller size may result by reducing a concentration of the reaction material, that is, the iron oleate. That is, 3nm-size nanoparticles synthesized with an initial precursor concentration of 0.2M; and 2.3nm-size nanoparticles synthesized with an initial precursor concentration of 0.1M. As a result, it can be understood that the size of iron oxide nanoparticles may be controlled by the amount of iron precursor.

<88> With regard to size control based on the ratio between aliphatic alcohol (or aliphatic amine) and fatty acid, the foregoing ratio is not deemed to significantly influence variation in the size of nanoparticles. However, the size of nanoparticles is reduced by increasing an amount of the aliphatic alcohol such as oleyl alcohol.

<89> Even when the aliphatic alcohol used herein is replaced by aliphatic amine as a weak reductive agent, iron oxide nanoparticles may be prepared. The aliphatic amine used herein may be C4 to C25 aliphatic amine and, preferably, oleyl amine.

<90> Even when using alkanediol instead of the aliphatic alcohol, iron oxide nanoparticles may be prepared. However, in this case, iron oxide nanoparticles having a size of 4run or less cannot be produced.

<9i> Considering size and uniformity of iron oxide nanoparticles, temperature elevation may be relatively rapid. A temperature elevation rate may be 5 ^° C/min or higher and, more preferably, 10 ^° C/min or higher.

<92> The reason of the foregoing fact may be presumed that burst nucleation suitably occurs when the temperature is rapidly raised, and such reaction is advantageous for synthesis of iron oxide nanoparticles having an extremely small and uniform size. As described above, the size and uniformity of the iron oxide nanopart icles may be controlled by regulating the temperature elevation rate. More particularly, smaller and more uniform iron oxide nanoparticles may be prepared by increasing the temperature elevation rate. In this regard, the temperature elevation rate may be 200 ^° C/min or less.

<93> For example, with regard to correlation between the size of iron oxide nanoparticles and the temperature elevation rate where oleic acid and oleyl alcohol are used, with the temperature elevation rates of 3.3 ^° C/min, 5 ^° C/min, non-uniform size particles including relatively large nanoparticles with a size of about 4nm to β ^ηη ι are synthesized. On the other hand, with the temperature elevation rates of lCC/min and 20 ^° C/min, uniform particles having a size of 3nm and 2.7nm are synthesized, respectively.

<94> The iron oxide nanoparticles prepared according to the present invention become (pseudo)paramagnetic due to a reduced size thereof, to thus minimize interference with Tl relaxation caused by an increase in T2 relaxation, which in turn, is suitable to be used as an MRI Tl contrast agent. Consequently, the iron oxide nanoparticles of the present invention may be applicable to MRI contrast agents.

<95> The iron oxide nanoparticles prepared according to the present invention are less toxic than other metal oxides, thus are biocompatible. The iron oxide nanoparticles may be used as an MRI Tl contrast agent by modifying the surface thereof with phosphol ipid-PEG, PEG-phosphate(PO-PEG) , monosaccharide phosphates, derivatives of monosaccharide phosphates, betaines or citric acid. More preferentially the surface of iron oxide maybe modified with a PO-PEG, glucose 6-phosphate, glucose 6-phosphate-ethanol amine, glucose 6-phosphate-PEG or citric acid. The capping materials on the nanoparticle affect strongly the hydrodynamic size, stability in water and toxicity. Even when the iron oxide core size is extremely small, hydrophilic composition after ligand exchanging can be large enough to increase T2 effects. So the hydrophilic layer is crucial in a Tl MRI contrast agent.

<96> With regard to preparation of iron oxide nanoparticles, the iron oxide nanoparticles may be synthesized by thermal decomposition of iron oleate as disclosed in Nat. Mater. 2004,4,891. However, since the method disclosed in the foregoing document uses only the oleic acid attached in the iron complex, as a surfactant to synthesize the nanoparticles, the quantity of oleic acid is not enough to prepare iron oxide nanoparticles having a size of 4nm or less. In contrast, the present inventors have successfully synthesized the iron oxide nanoparticles having a size of 4nm or less by varying conditions for generation of nanoparticles, with different temperature elevation conditions from the patented method in the art. Two different methods have been adopted to control the conditions for generation of nanoparticles.

<97> A first method is to generate a number of nuclei during reaction as described in Examples 6 and 7, wherein nanoparticles having a small size are synthesized to reduce the number of iron atoms adhered to each particle during growth of the particle. This method may control formation of nuclei by regulating a reaction temperature. However, compared to a preferred preparation method of the present invention which includes; reacting an iron complex formed of iron as a central atom and a C4 to C25 carboxylate group bonded thereto in a ligand form; a C4 to C25 fatty acid; and a C4 to C25 aliphatic alcohol or C4 to C25 aliphatic amine at 150 to 350 ^° C to prepare iron oxide nanoparticles, the foregoing nucleation method through temperature control entails difficulties in controlling the temperature and thus poor reproducibility.

<98> A second method is to synthesize nanoparticles having a small size by controlling a growth rate of nanopa ticles, as described in Example 8. In particular, this method delays growth of particles to ensure an intermediate reaction stage in growth of particles. That is, the second method may also adopt temperature control. In contrast, compared to a preferred preparation method of the present invention which includes, ^' reacting an iron complex formed of iron as a central atom and a C4 to C25 carboxylate group bonded thereto in a ligand form; a C4 to C25 fatty acid; and a C4 to C25 aliphatic alcohol or C4 to C25 aliphatic amine at 150 to 350 ^° C to prepare iron oxide nanoparticles, the foregoing method has disadvantage that it is difficult to control the reaction temperature accurately and so a growth period of particles is not constant during reaction, which in turn, causes problems such as lower yield and less reproducibility.

<99> The following examples are implemented with sodium oleate, oleyl alcohol and diphenylether , which are purchased from TCI; iron chloride(3) hexahydrate, oleic acid (90%), 1-octadecene (90%) and 1,2-hexadecanediol , which are purchased from Aldrich; and oleyl amine which is purchased from Acros. In addition, ethanol and hexane are purchased from Sam-Chun Chemical for use.

<ioo> TEM is measured by JEOL-2010, XRD is measured by Rigaku Ka,

VSM(Vibrating Sample Magnetometer) is measured by VSM-PPMS, and M-T relationship is measured by VSM while raising a temperature by 5K/min.

<ioi> An iron oleate complex ( 'iron oleate' ) used herein is the one prepared by reacting sodium oleate with

FeCl ₃ accordingtothemethoddisclosedinJ.Parketal . ,Nat .Mater.2004, 4, 891. More particularly, 10.8g of iron chloride hexahydrate and 36.5g of sodium oleate are mixed in 60mL of water, 80mL of ethanol and 140 mL of hexane, followed by reaction of the mixture at 60 ^° C for 4 hours under strong agitation. From a reaction product having two separate phases, the transparent lower phase is removed through a separation funnel. The remaining brown organic phase is mixed with water and then the lower water phase is removed again. The water- soluble salt in the organic phase is eliminated by the washing process. The foregoing washing processes are repeated three times. The organic solution is subjected to evaporation of the hexane, resulting in iron oleate complex.

<i02> With regard to analysis of the products in the following examples and comparative examples, the accompanying drawings are as follows-

<i03> FIG. 1 shows transmission electron microscopy (TEM) images of 3nm-size iron oxide nanoparticle synthesized by a method described in Example 1, more particularly; (a) a TEM image; (b) a TEM image in a wide range; (c) a high resolution-transmission electron microscopy (HR-TEM) image; and (d) a selected area electron diffraction (SAED) pattern. FIG. 2 shows an X-ray diffraction (XRD) spectrum of 3nm-size nanoparticles synthesized by a method described in Example 1. FIG. 3 shows a TEM image of 2.3nm-size iron oxide nanoparticles synthesized by a method described in Example 2. FIG. 4 shows a TEM image of l.Snm-size iron oxide nanoparticles synthesized by a method described in Example 3. FIG. 5 shows a TEM image of 3.3nm-s i ze iron oxide nanoparticles synthesized by a method described in Example 4. FIG. 6 shows a TEM image of 3.5nm-size i on oxide nanoparticles synthesized by a method described in Example 5. FIG. 7 shows a TEM image of 1.6nm-size iron oxide nanoparticles synthesized by a method described in Example 6. FIG. 8 shows a TEM image of 2.4nm-size iron oxide nanoparticles synthesized by a method described in Example 7. FIG. 9 shows a TEM image of 3.5nm-size iron oxide nanoparticles synthesized by a method described in Example 8. FIG. 10 shows a TEM image of 2.3nm-size iron oxide nanoparticles synthesized by a method described, in Example 9. FIG. 11 of (a) shows a TEM image of 2.7nm-size iron oxide nanoparticles synthesized by a method described in Example 10; (b) shows a TEM image of iron oxide nanoparticles synthesized by a method described in Comparative Example 1; and (c) shows a TEM image of iron oxide nanoparticles synthesized by a method described in Comparative Example 2. FIG. 12 of (a) shows M-H graphs at 5K and 300K, respectively, of 3nm-size nanoparticles synthesized by the method described in Example 1; (b) shows variation in M-H graph at 300K of nanoparticles with particle size; (c) shows zero field cooling and field cooling M-T graphs of 2.3nm-size nanoparticles synthesized by the method described in Example 2, respectively; (d) shows an M-T graph of 3nm-size nanoparticles synthesized by a method described in Example 1; (e) shows an M-T graph of 12nm ^~ size nanoparticles synthesized by a method described in Comparative Example 3; (f) shows M-H graphs at 5K and 300K, respectively, of 1.6nm-size nanoparticles synthesized by the method described in Example 6; (g) shows an M-T graph of the nanoparticle in Example 6; and (h) shows M-H graphs at 5K and 300K, respectively, of 2.3nm-size nanoparticles synthesized by the method described in Example 9. FIG. 13 illustrates a distribution of number-average hydrodynamic diameters (number- average of 11.8nm) of 3nm-size nanoparticles, which are dispersed in water by using PEG-phosphate (P0-PEG) according to the method described in Example 13. FIG. 14 shows M I phantom Tl image of dispersions which are prepared by primarily modifying the surface of iron oxide nanoparticles with PEG- phosphate (PO-PEG) and phospholipid PEG, respectively, with particle size, and then secondarily dispersing the surface-modified nanoparticles in water; in particular, in the case where using PO-PEG to treat the nanoparticles having a size of 2.3nm, 3nm and 4nm, respectively, they are each indicated as 2.3P, 3P and 4P; likewise, in the case where using phosphol ipid-PEG to treat the foregoing nanopar icles, they are each indicated as 2.3L, 3L, 4L and 7L. FIG. 15 shows cell phantom MR I results of 3nm-size nanoparticles and 12nm- size nanoparticles, respectively; in particular, (a) shows cell phantom MR image of 3nm-size nanoparticles; and (b) shows cell phantom MR image of 12nra- size nanoparticles. FIG. 16 shows clear contrast images of jugular veins, carotid arteries and aortic arch, which are obtained using the inventive nanoparticles, as compared to a gadolinium complex, Gadovist ® (Bayer Schering Co.); in particular, (a) shows in vivo MRI vascular contrast image, using 3nm-size nanoparticles! and (b) shows in vivo MRI vascular contrast image using Gadovist® . FIG. 17 shows a TEM image of 4nm-size iron oxide nanoparticles synthesized by a method described in Example 20. FIG. 18 illustrates MTT assay results of MCF-7 cells using hydrophi 1 ic-modi f ied iron oxide nanoparticles according to Example 21.

<i04> FIG. 19 illustrates molecular weight assay results of iron oxide nanoparticles by means of MALDI-TOF according to Example 22, FIG. 20 shows a TEM image of 12nni-size iron oxide nanoparticles synthesized by a method described in Comparative Example 3, FIG, 21 shows a TEM image of I2nm-size iron oxide nanoparticles synthesized by a method described in Comparative Example 4, FIG. 22 shows a TEM image of 7nm-size iron oxide nanoparticles synthesized by a method described in Comparative Example 5, FIG. 23 shows a TEM image of 4nm-size iron oxide nanoparticles, which were encapsulated to form an aggregate, followed by negative staining, according to the method described in Comparative Example 6, and FIG. 24 shows a TEM image of 6nm-size iron oxide nanoparticles synthesized by Comparative Example 7, and FIG. 25 shows enhanced MRI vascular contrast images using the 3-nm nanoparticles capped by glucose 6-phosphate, described in Example 17.

<i05> EXAMPLE 1

<106> Synthesis of 3nm ^~ size iron oxide nanoparticles

<i07> 1.8g (2mmol) of iron oleate, 0.57g (2mmol) of oleic acid and 1.61g

(6mmol) of oleyl alcohol were mixed with lOg of diphenylether and placed in a round-bottom flask. Vapor was removed from the flask by vacuuming at 80 ^° C for 1 hour, then, an argon gas was fed into the flask to make an inert environment. After reacting in the flask while raising the temperature to 250 ^° C with HTC/min, the reaction material becomes black during the reaction. After raising the temperature to 250 ^C C, the reaction was continued for 30 minutes, resulting in 3nm ^~ size nanopart icles (FIGS. 1 and 2). After the reaction the product was rapidly cooled and then washed with excess acetone. After washing, the resulting precipitate was dispersed in an organic solvent such as chloroform or hexane.

<io8> EXAMPLE 2

<109> Synthesis of 2.3nm-size iron oxide nanoparticles

<iio> The synthesis of 2.3nm-size nanoparticles was performed by thermal decomposition under the same conditions as described in Example 1, after mixing 0.9g (lmmol) of iron oleate and 3.22g (12mmol) of oleyl alcohol with lOg of diphenyl ether , without addition of oleic acid.

<m> EXAMPLE 3

ii2> Synthesis of 1.8nm-size iron oxide nanoparticles

<!i3> l.Snm-size nanoparticles were synthesized by mixing 0.9g (lmmol) of iron oleate and 3.22g (12mmol) of oleyl alcohol with lOg of di phenyl ether , without addition of oleic acid, followed by raising a temperature to 200 ^° C with a temperature elevation rate of 20 ^° C/min and conducting the reaction at 200 ^° C for 30 minutes. The other conditions are substantially the same as described in Example 1.

<ii4> EXAMPLE 4

<ii5> Synthesis of 3.3nm-size nanoparticles using 1-octadecene

<ii6> Nanoparticles were synthesized by mixing 1.8g of iron oleate, 0.57g of oleic acid and 1.6g of oleyl alcohol with lOg of 1-octadecene, followed by raising a temperature to 250 "C with a temperature elevation rate of 10 ^° C/min and growing particles at 250 ^° C for 30 minutes. The other conditions are substantially the same as described in Example 1.

<ii7> EXAMPLE 5

<H8> Synthesis of 3.5nm-size nanoparticles using oleyl amine

<U9> Nanoparticles were synthesized by mixing 1.8g of iron oleate, 0.57g of oleic acid and 1.6g of oleyl amine with lOg of diphenyl ether , followed by raising a temperature to 250 ^° C with a temperature elevation rate of 10 ^° C/min and growing particles at 250 ^" C for 30 minutes. The other conditions are substantially the same as described in Example 1.

<i20> EXAMPLE 6

<i2i> Preparation of 1.6nm-size iron oxide nanoparticles

<i22> With reference to known synthetic methodCNature Mater. 3(2004), 891), very small iron oxicie nanoparticles were synthesized by preventing the growth rate of particles. The synthesized particles were centrifuged to result in very small iron oxide nanoparticles. For nanoparticle synthesis through thermal decomposition of iron oleate, a synthesis temperature is generally 320 ^° C and particles are rapidly grown at this temperature. On the other hand, energy for growing particles is not sufficiently provided at 300 ^° C, thus the growth rate decreases (that is, the growth of particles). Consequently, nanoparticles having a very small size generated during growth at 300 ^° C could be obtained, although which cannot be yielded during reaction at 3201.

<i23> After mixing 1.8g (2mmol) of iron oleate (Fe-oleate) and 0.57g (2mmol) of oleic acid with lOg of 1-octadecene, the mixture was placed in a round- bottom flask and vapor was removed fron; the flask by vacuuming at 80 ^° C for 1 hour. Then, an argon gas was fed into the flask to make an inert environment. Next, after raising the temperature to 300 ^° C with 3.3 ^° C/min, reaction was conducted at 300 : for 30 minutes, followed by rapidly cooling the reaction product to room temperature. Following this, the cooled product was subjected to precipitation by adding ethanol thereto at room temperature, resulting in 1.6nm-size iron oxide.

<I2 > EXAMPLE 7

<]25> Preparation of 2.4nm-size iron oxide nanoparticles

<i26> 2.4nm-size iron oxide nanoparticles were synthesized by preventing the growth rate of nanoparticles as described in Example 6.

<i27> More particularly, after mixing 1.8g (2mmol) of iron oleate (Fe-oleate) and 0.57g (2mmol) of oleic acid with lOg of 1-octadecene, the mixture was placed in a round-bottomed flask and vapor was removed from the flask by vacuuming at 80°C for 1 hour. Then, an argon gas was fed into the flask to make an inert environment. Next, after raising the temperature to 300 ^° C with 3.3 ^° C/min, reaction was conducted at 300 ^° C for 35 minutes, followed by rapidly cooling the reaction product to room temperature. Following this, the cooled product was subjected to precipitation by adding ethanol thereto at room temperature, resulting in 2.4nm-size iron oxide.

<i28> EXAMPLE 8

<i29> Preparation of 3.5nm-size iron oxide nanoparticles

<i30> With reference to known synthetic method (Nature Mater. 3(2004), 891), iron oxide nanoparticles having a small size were synthesized through lowering the temperature to produce much more nuclei. Since the nucleation temperature during thermal decomposition of Fe-oleate was about 270 ^° C, reaction materials remained longer at the foregoing temperature of 270 ^° C , to thereby facilitate generation of nuclei in large quantities. The reason for this is that, if numerous nuclei are formed, the number of iron atoms adhered to each particle may be reduced, thus the size of each the particle may decreases .

<i3i> After mixing 1.8g (2mmol) of iron oleate (Fe-oleate) and 0.57g (2mmol) of oleic acid with lOg of 1-octadecene, the mixture was placed in a three- neck round-bottom flask, and heated to 270°C under an inert atmosphere and stayed therein for 20 minutes to perform synthesis at the same temperature. After nucleation, the temperature was raised to 318 ^° C as a growth temperature of particles to inhibit further reaction and then stayed at the same temperature, that is, 318 ^° C for 10 minutes to perform synthesis, thereby resulting in 3.5nm-size nanoparticles.

<!32> EXAMPLE 9

<133> Synthesis of 2.3nm-size iron oxide nanoparticles

<i34> A solution comprising 0.64g of oleic acid, 0.59g of 1,2-hexadecanediol and 15.81g of di henyl ether was purified by removing impurities after vacuuming it at about 70 °C for 1 hour. Then, an argon gas was fed thereto until an inert environment is formed and then stopped. 0.3ml of iron pentacarbonyl was injected thereto. By raising the temperature to 250 ^° C with a temperature elevation rate of 3.3 ^° C/min and conducting reaction at 250 ^° C for 30 minutes, nanoparticles having a size of 2.3nm were prepared. The other conditions are substantially the same as described in Example 1.

<i35> EXAMPLE 10

<i36> Particle size and distribution depending upon temperature elevation rate

<i37> After mixing 1.8g of iron oleate, 0.57g of oleic acid and 1.6g of oleyl alcohol with lOg of di phenyl ether , a temperature was raised to 250 ^° C with a temperature elevation rate of 20 ^° C/min and the mixture reacted and nanoparticles was grown at 250 ^° C for 30 minutes. As a result, uniform nanoparticles having a size of 2.7nm were synthesized. The other conditions are substantially the same as described in Example 1.

<138> EXAMPLE 11

<i39> Physico-chemical properties of the prepared iron oxide nanoparticles

<i40> Using a vibrating sample magnetometer (VSM), magnetic property of the nanoparticles was measured. FIG. 12 shows magnetization-magnetic field (M-H) graphs of nanoparticles having different sizes of 1.6, 2.3, 3 and 12nm, which were synthesized in the foregoing examples. More particularly, in FIG. 12, (a) shows M-H graphs at 5K and 300K, respectively, of 3nm-size nanoparticles synthesized by the method described in Example 1; (b) shows variation in M-H graph at 300 , of nanoparticles with particle size, ^' (c) shows zero field cooling and field cooling M-T graphs of 2.3nm-size nanoparticles synthesized by the method described in Example 2, respectively; (d) shows an M-T graph of 3nm-size nanoparticles synthesized by a method described in Example 1; (e) shows an M-T graph of 12nm-size nanoparticles synthesized by a method described in Comparative Example 3; (f) shows M-H graphs at 5K and 300K, respectively, of 1.6nm-size nanoparticles synthesized by the method described in Example 6; (g) shows an M-T graph of 1.6nm nanoparticles synthesized by the method described in Example 6; and (h) shows M-H graphs at 5K and 300K, respectively, of 2.3nm-size nanoparticles synthesized by the method described in Example 9.

<i4i> Referring to FIG. 12, the 12nm-size iron oxide nanoparticles at 5R are ferri magnetic to exhibit coercivity and remanent magnetization. 3nm-size iron oxide nanoparticles are also ferr magnetic to exhibit a little coercivity as well as remanent magnetization. However, the 2.3nm-size nanoparticles neither show such remanent magnetization nor coercivity at the temperature. That is, these nanoparticles remain in a paramagnetic state until the temperature of 5K. This is an unexpected case in magnetic nanoparticles having super ^¬ paramagnetic properties. The foregoing conditions may be clearly identified from a magnetization-temperature (M-T) graph and, for instance, it can be seen that a blocking temperature is 200K for the 12nm-size nanoparticles and 10K for the 3nm-size nanoparticles, however, the blocking temperature of the 2.5nm nanoparticles is not observed even at 5R. The blocking temperature means a transition temperature, at which physical properties such as super ^¬ paramagnetic, ferromagnetic and/or ferrimagnetic properties are exchanged, and may be proportional to the volume of particles. Accordingly, when the particle size is decreased, the blocking temperature may also be lowered. For instance, if the particle size is decreased to 3nm or less, the blocking temperature does not appear even at 5K. As a result, it can be seen that the 3nm-size particle is paramagnetic or has paramagnet ic— 1 ike physical properties. Such characteristic was firstly discovered in iron oxide nanoparticles having a ferrite structure and, since it is similar to paramagnetic property, is referred to as 'pseudo-paramagnetic property' to be distinguishable from super-paramagnetic property. Since the iron oxide nanoparticles having a small size of 3nm or less are mostly super ^¬ paramagnetic nanoparticles and/or have disordered spins on the surface thereof, these particles may look like paramagnetic. More specifically, although the foregoing nanoparticles are not paramagnetic, they show paramagnetic- like behavior, thus being pseudo-paramagnetic.

For assaying, M-H graphs at room temperatures were overlapped (FIG.

12b). It can be seen that magnetization is reduced with decreasing particle size. The reason for this may be presumed that, when the particle is smaller, anisotropic energy is decreased, which in turn, allows high occurrence of so-called Neel relaxation, and overall particles has low magnetization to reduce Zeeman energy, thereby increasing thermal fluctuation. However, it is surprisingly found that a difference in magnetic properties is considerably great between 3nm particle and 2.3nm particle, in spite of a small variation in size. The foregoing result may be interpreted by spin-canting effects (J. M. D. Coey, Phys. Rev. Lett. 1971, 27, 1140). For instance, magnetic nanopart icles generally have lower magnetization than in a bulk state. The reason for this is that: surface atoms are under a different environment from bulk atoms, therefore, a spin direction of the surface atoms has a different angle from bulk atoms, which in turn, lower overall spin angle and thus degrades magnetization. This refers to the spin- canting effects described above. Alternatively, Linderoth has estimated a thickness of the spin-canting surface to about 0.9 nm (S. Lindroth et al., J. Ap l . Phys. 1994, 75, 6583). According to the foregoing, a 2.3nm sized nanoparticle may include about 1.0% of core portion in relation to a total volume of the particle, which is not influenced by spin-canting effects.

<I43> On the other hand, a 3nm-sized nanoparticle may include about 6.4% of core portion in the particle. Consequently, it may be considered that a different in magnetization is relatively great.

<144> According to Lindroth' s calculation, 1.6nm-size nanoparticle, of which the almost entire portion is influenced by a spin-canting surface, exhibits typical paramagnetic properties wherein an M-T relationship is linear at room temperature (FIG. 12f). In addition, it can be seen that a large-scale magnetic field is not saturated up to large magnetic field at 5K.

<i45> Even through the nanopart icles of the present invention are substantially iron oxide nanopart icles having a ferrite structure (FIG. 2), they have paramagnetic or pseudo-paramagnetic property rather than super ^¬ paramagnetic. Accordingly, the foregoing nanopart icles may be highly useful to be used as a MRI Tl contrast agent.

<146> EXAMPLE 12

<147> Hydrophilic modification of iron oxide nanopart icles using phosphol ipid-PEG

<i48> lOmg of the iron oxide nanopart icles having a size of 3nm prepared in

Example 1 was dispersed in 10ml of chloroform, and then, lOmg of phosphol ipid-PEG {l,2-distearyl-sn-glycero-3 ^~ phosphoethanol amine-

N[methoxy(polyethyleneglycol-2000)]} was added to the dispersion solution. After agitating, the chloroform was slowly evaporated, followed by adding water and finally a well dispersed aqueous iron oxide nanoparticle colloidal was resulted. The hydrodynamic diameter of the hydrophilic nanoparticle was 15nm, measured by DLS(dynamic light scattering).

<149> EXAMPLE 13

i50> Hydrophilic modification of iron oxide nanopart icles using PEG- phosphate (PO-PEG)

<i5i> 0.15g of

P0Cl ₃ and6gofpolyethyleneglycolmethylether(Mn:2000)werefedto 7mlofatetrahydrofur an(THF)solutionandagitatedfor4hours. PO-PEG was obtained after removing THF therefrom. lOmg of the 3nm iron oxide nanoparticles having oleate on the surface prepared in Example 1 and lOOmg of PO-PEG were mixed in ethanol, sealed, and agitated at 70 ^° C for 4 hours, to exchange ligands thereof. The resulting product was washed with N-hexane three times and, after evaporating the ethanol portion, water was added to the residue to disperse the same, thus obtaining colloidal particles having a hydrodynamic diameter of 11.8nm(FIG. 13).

<152> EXAMPLE 14

<I53> MR in vitro relaxation of hydrophi 1 ic-modif ied iron oxide nanoparticles

<i54> In order to determine MR contrast ability of the hydrophilic iron oxide nanoparticle colloids, hydrophi lie-modified prepared by the way of Examples 13 and 17, and Comparative Example 6, respectively, several phantoms with concentrations of 0.5, 0.25, 0.13, 0.063, 0.031, 0.016. 0.0078 and 0.0039ing/ml were prepared using the iron oxide nanoparticles having different sizes of 1.6nm (Example 6), 2.4nm (Example 7), 3nm (Example 1), 4nm (Example 20) and 7nm (Comparative Example 5), respectively. 1.5T MR image was obtained using an MR scanner (GE Health Care, Signa Excite) equipped with a head coil. A Tl value was obtained by means of IR-FSE sequence with the following parameters: TR/TE/T1 = 4000 ms/8.4 ms/50 to 4000 ms. A T2 value was obtained by means of CPMG sequence with the following parameters: TR/RE =

<i55> For 4.7T MR image, a BGA12 gradient coil (Biospec 47/40, Bruker Biospin

MRI GmbH) was used to analyze relaxation performance. More particularly, after analyzing iron content of an iron oxide-PLGA nano-capsule powder by ICP-AES, this powder was subjected to measurement at different concentrations of 2, 1, 0.5, 0.25 and 0.125mg/l in 0.01M PBS (phosphate buffer saline, pH7.4). T2 relaxation time was measured by means of multi si ice-mul t i echo (MSME) pulse sequence, wherein the parameters used herein may be as follows:

<I56> TR (repetition time) = 10,000 ms; TEC Echo time) = 8 to 2048 ms (256 times with 8ms intervals); F0V = 60X40 mm; Resolution = 0.234x0.156 mm/pixel; slice thickness = 1 mm; number of acquisition = l; matrixXsize = 128X128.

<i57> The following Table 1 shows rl, r2 and their ratio therebetween of nanopart icles having different sizes at 1.5T and 4.7T. Referring to Table 1, 1.5 Tesla phantom MRI relaxation values depending upon size of nanopart icle may be offered. The greatest rl value is near 7nm but a difference between rl values is not so great. On the contrary, when the particle size is decreased, r2 value is considerably reduced. As a result, r2/rl may also be greatly decreased. For instance, 1.6nm-size nanopart icles may have a very small r2/rl, that is, about 1.47 in a magnetic field of 1.5T. Small r2/rl demonstrates that the corresponding nanopart icles are suitable to be used as Tl MRI contrast agent. Further, referring to Comparative Example 6 in Table 1, it can be seen that, even when a diameter of each iron oxide nanopart icle is 4nm, r2/rl is excessively increased where a plurality of nanopart icles are aggregated (FIG. 23), thus being not preferable for Tl contrast agent. TABLE 1

<I59>

<i60> EXAMPLE 15

<i6i> MR imaging of cell

<I62> In vitro Tl weighted MR images of MCF-7 cells incubated with various concentra ions of the nanoparticles (0, 25, 100 ug Fe/mL) were obtained on a 1.5 T MR scanner. Significant Tl signal enhancement was observed for the cells labeled with 25 and 100 pg Fe/mL of 3nm nanoparticles capped by PO-PEG while non-labeled cells were not brightened (FIG. 15a). Although nanostructured materials are usually clustered in the endosome, 3nm nanoparticles provide Tl contrast effect nor. only in deionized water but also in the cellular environment resulting from their low volume anisotropy. In contrast, in the cell phantom Tl weighted MR image, the cells labeled with 12 nm-sized particles showed much less signal enhancement; even they were darkened at the high concentration(FIG. 15b). The attenuated Tl signal of cell incubated with the 12 nm-sized iron oxide nanoparticles seems to result from the susceptibility effect derived from the strong magnetic moment of aggregates of the large-sized magnetic nanoparticles.

<i63> EXAMPLE 16 <i64> MR in vivo imaging of the 3nm iron oxide nanopart icles POPEG on the surface

<i 5> Dynamic time-resolved MR angiography and 3d ^~ FLASH images of rats were acquired using a wrist coil on a 3 T MRI scanner before and after the injection of 3nm iron oxide nanopart icles capped by PO-PEG, prepared according to example 13(dose: 2.5 mg Fe/kg). The pre-contrast images were subtracted from post-contrast images, and the resulting images were reconstructed using maximum intensity projection (MIP) protocol with OsriX (Version 3.8.1; 32bit; OsiriX foundation, Geneva). Dynamic time-resolved MR angiography was obtained with an interpolated temporal resolution of 1.25 second and the following parameters: flip angle = 20, ETL = 1, TR =3.1 ms, TE

= 1.13 ms, field of view F0V = 75 X 140 mm ² , matrix = 256 X 106, slice thickness/gap= 2.5 mm/Omm, and NEX = 1. The imaging parameters of 3d~FLASH are as follows: flip angle = 25, ETL = 1, TR = 25 ms, TE = 5.1 ms, field of

2

view F0V = 110 X 65 mm , matrix = 256 X 169, slice thickness/gap = 1.0mm /0mm, and NEX = 2.

<i66> FIG. 16a blood vessels were brightened on the Tl weighted MR images, demonstrating that the 3nm nanopart icles can enhance Tl relaxation in the circulating system. The bright signal of blood vessel can be maintained for 1 h on dynamic time-resolved MR angiography (not shown in FIG. 16), showing that the 3nm nanopart icles can be used for Tl enhanced blood pool MRI contrast agent.

<167> Blood pool imaging is important in clinical MR imaging because it can detect the myocardial infarction, renal failure, atherosclerotic plaque, thrombosis, and angiogenesis of tumor cells. Long-term blood pool imaging is beneficial for steady-state imaging, which is critical to obtain high- resolution images. For example, pulmonary artery imaging could clearly be obtained by the steady-state imaging using USPIOCUltra Small Superparamagnetic iron oxide)s. The 3nm nanopart icles can be good Tl contrast agent for steady-state imaging because they have a long blood half-life derived from their optimal particle size.

<i68> The particles should not be so large to avoid uptake by the reticuloendothelial system and should not be so small to keep the particles from being excreted through the kidney. In contrast to the 3nm nanoparticles, gadolinium complex Gadovist (Bayer Schering Pharma), which is a commonly used Tl MRI contrast agent, has a short blood half-life. Immediately after the injection of Gadovist, in vivo MR image exhibited high contrast effect due to its high relaxivity, but the bright signal vanished rapidly in 2 ininutes(FIG. 16b).

<i69> EXAMPLE 17

<i70> Hydrophilic modification of iron oxide nanoparticles using monosaccharide phosphate and MR in vivo imaging

<i7i> Iron oxide nanoparticles capped by oleic acid on the surface were prepared by the method of example 1.

<172> lOOmg of 3nm iron oxide nanoparticles were dispersed in 8ml of

THF(tetrahydrofuran) and mixed with aqueous solution of 200mg of glucose 6- phosphate sodium salt in 2ml of water. The mixed solution was agitated and reacted at 60°C for 4 hr. After cooling, upper phase of THF of the mixture solution was separated and water was added to the lower phase of iron oxide nanoparticles capped by glucose 6-phosphate on the surface to prepare a stable colloid of iron oxide nanoparticles.

<i73> The hydrodynaraic diameter of the nanoparticles having glucose 6- phosphate on the surface was 3.8nm, measured by dynamic light scattering methodCMalvern Zetasizer Nano ZS).

<i74> MR imaging using the 3nm nanoparticles capped by glucose 6-phosphate was performed according to example 16.

<i75> As shown in FIG. 25 blood vessels were brightened on the Tl weighted MR imaging. The bright signal of blood could be maintained for 2 hour, but the bright signal was not shown after 24hours. This means the composition of 3nm nanoparticles capped by glucose 6-phosphate can be a good MR blood pool agent .

<176> EXAMPLE 18

<i77> Hydrophilic modification of iron oxide nanoparticles using citric acid

<i78> Iron oxide nanoparticles capped by oleic acid on the surface were prepared by the method of example 1.

<|79> lOOmg of 3nin iron oxide nanoparticles were dispersed in 8ml of

THF(tetrahydrofuran) and mixed with aqueous solution of 400mg of sodium citrate in 2ml of water. The mixed solution was agitated and reacted at 60 ^° C for 4 hr. After cooling, upper phase of THF of the mixture solution was separated and water was added to the lower phase of iron oxide nanoparticles capped by citric acid on the surface to prepare a stable colloid of iron oxide nanoparticles.

<i8o> The hydrodynamic diameter of the nanoparticles having citric acid on the surface was lOnm, measured by dynamic light scattering method(Malvern Zetasizer Nano ZS) .

<181> EXAMPLE 19

<i82> Hydrophilic modification of iron oxide nanoparticles using betaine.

<183> Iron oxide nanoparticles capped by oleic acid on the surface were prepared by the method of example 1.

<184> 150mg of 3nm iron oxide nanoparti les \fere dispersed in 25ml of n- hexane and mixed with 600ing of betaine (2-(tr imethyl azaniumyl )acetate hydrochloride) in 25ml of ethanol. The mixed solution was agitated and reacted at 50 ^° C for 8 hr. After cooling, the ligand exchanged nanoparticles were separated from the solvent by centri fugation at 3000rpm for lOmin. Water was added to the ligand exchanged nanoparticles Betaine to prepare a stable colloid of iron oxide nanoparticles.

<185> The hydrodynamic diameter of the nanoparticles having betaine on the surface was 7nm, measured by dynamic light scattering methodCMalvern Zetasizer Nano ZS).

<i86> EXAMPLE 20

<I87> Synthesis of 4nm-size iron oxide nanoparticles

<i88> With reference to the method described in Example 6, 4nm-size iron oxide nanoparticles were synthesized through control of particle growth rate. More particularly, 4nm ^~ size nanoparticles were synthesized as follows: after mixing 1.8g of iron oleate and 0.57g of oleic acid with lOg of 1-octadecene, a temperature was raised to 318 ^° C with a temperature elevation rate of 10 ^° C /min, followed by conducting reaction at 318°C for 30 minutes and then rapidly cooling the reaction product to room temperature. Following this, the cooled product was subjected to precipitation by adding ethanol thereto at room temperature, resulting in 4nm-size iron oxide.

<i89> EXAMPLE 21

<i90> MTT assay experiment of hydrophi 1 ic-modi f ied iron oxide nanoparticles

<i9i> Under a wet atmosphere 37°C and with 5% C0 ₂ concentration, human breast cancer cell lines, that is, MCF-7 cells were grown on a Dulbecco' s modified eagle' s mediumCDMEM, Welgene) containing 10% fetal bovine serum(FBS) and 1% penici 11 in/streptomycin(100U/ml and HXV.g/ml, respectively, Gibco).

<i92> For observation of intracellular intake, the MCF-7 cells were incubated on eight (8)-well chamber slide, followed by mixing the cultured cells with each of 3nm-size and 12nm-size iron oxide nanoparticles, which were surface- modified with P0-PEG. After 24 hours, the cells were washed with PBS and then fixed using 4% para-formaldehyde. Fluorescent images were obtained by a confocal laser scanning microscope (LSM 510, Carl Zeiss, Germany).

<i93> In order to assay survival and growth of cells in the presence of nanoparticles, analysis using 3-[4,5-dimethylthialzol-2-yl ]-2,5- diphenyltetrazol ium bromide (MTT, Sigma) was performed. For this purpose, MCF-7 cells were grown on 200 pL medium for 1 day. The grown cells were mixed with each of 3nm-size and 12nm-size nanoparticles capped by P0-PEG on the surface having different concentrations (e.g., 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 /zgFe/mL). After the mixture was cultured overnight, the cultured material was mixed with a medium containing O.lmg/mL of MMT for 1 hour. Following this, the medium was removed and the precipitated formazan was dissolved in DMS0. Using a VerseMaxTM microplate reader (Molecular Devices), absorbance at 540nm was detected to identify cell viability.

<194> Following FIG. 18, both nanoparticles with 4nm and lOnm show about 100% of MCF-7 cell viability up to lOOmg Fe/ml. This means both nanoparticles capped by P0-PEG don' t have cell toxicity up to the concentration.

<1 5>

<196> EXAMPLE 22 i 7> MALDITOF mass spectrometry

i 98> 10 mg/ml of iron oxide nanopart icles and 10 mg/ml of 9-nitroanthracene used as a matrix were dissolved in chloroform. The nanopart icles and 9- nitroanthracene were blended in a relative ratio of 1:100 and only a droplet of the mixture was added to an LDl substrate and then evaporated into the atmosphere. The substrate was placed in an MALDI-T0F spectrometer (Voyager- DETM STR Biospectrometry Workstation, Applied Biosystems Inc.), followed by laser irradiation to measure a mass of the nanopart icles in the range of 500 to 300,000 Da in a linear model and cation detection model, respectively. FIG. 19 shows the result of molecular weight of the nanopart icles by MALDI ^¬ TOF. (a) is a TEM image of 1.6nm-size iron oxide nanopart icles; (b) shows assay results of the 1.6nm-size iron oxide nanoparticles through MALDI-T0F, wherein the nanoparticles have a molecular weight of 9,000 Da! (c) is a TEM image of 2.4nm-size iron oxide nanoparticles; (d) shows assay results of the 2.4nm-size iron oxide nanoparticles through MALDI-TOF, wherein the nanoparticles have a molecular weight of 65,000 Da; and (e) shows mass assay results of a core part of the 1.6nnrsize iron oxide nanoparr icles through thermogravimetric analysis (TGA), wherein the core mass is 35.8% and this means that each 1.6nm-size particle has a core fraction of 35.8%, therefore, a molecular weight of the core is 3,330 Da.

<199> EXAMPLE 23

<200> Hydrophilic modification of iron oxide nanoparticles using glucose 6- phosphat e-ethano1 amine

<2oi> EXAMPLE 23-1

<202> Synthesis of glucose 6-phosphate-ethanolamine

<203> Ig of Glucose 6-phosphate sodium salt, 0.68g of EDC (l-ethyl-3-[3- dimethylaminopropyl lcarbodi imide hydrochloride) and 0.4g of NHS (N- hydroxysuccinimide) were mixed with 10ml of MES(2-(N- morphol ino)ethanesul fonic acid) buffer solution. The mixed solution was agitated and reacted at 30 ^° C for 30min. After reacting, 0.22ml of ethanolamine(2-aminoethanol ) mixed with the solution then reacted at 30 ^° C for 12 hours. Glucose 6-phosphate-ethanolami e was obtained after removing MES buffer solution.

<204> EXAMPLE 23-2

<205> Hydrophilic modification of iron oxide nanopart icles using Glucose 6- phosphat e ^~ e t hano 1 am i ne

<206> Iron oxide nanoparticles capped by oleic acid on the surface were prepared by the method of example 1.

<207> lOOmg of 3nm iron oxide nanoparticles were dispersed in 10ml of

THF(tetrahydrofuran) and mixed with aoueous solution of 300mg of Glucose 6- phosphate-ethanolamine in 2ml of water. The mixed solution was agitated and reacted at 60 ^° C for 4 hr. After cooling, upper phase of THF of the mixture solution was separated and water was added to the lower phase of iron oxide nanoparticles capped by Glucose 6-phosphate-ethanol amine on the surface to prepare a stable colloid of iron oxide nanopartic es.

<208> The hydrodynamic using the 3nm nanoparticles capped by Glucose 6- phosphate-ethanol amine on the surface to prepare a stable colloid of iron nanoparticles. The hydrodynamic diameter of the nanoparticles having Glucose 6-phosphate-ethanol amine on the surface was 8nm, measured by dynamic light scattering method(Malvern Zetasizer Nano ZS) .

<209> EXAMPLE 24

<2!0> Hydrophilic modification of iron oxide nanoparticles using glucose 6- phosphate-PEG

<2ii> EXAMPLE 24-1

<2i2> Synthesis of glucose 6-phosphate-PEG

<2i3> lg of Glucose 6-phosphate sodium salt, 0.68g of EDC (l-ethyl-3-[3- dimethylaminopropyl lcarbodi imide hydrochloride) and 0.4g of NHS (N- hydroxysuccinimide) were mixed with 10ml of MES(2-(N- morphol ino)ethanesulfonic acid) buffer solution. The mixed solution was agitated and reacted at 30 ^° C for 30min. After reacting, 0.23ml of diethyl amine mixed with the solution then reacted at 30 ^° C for 12 hours. After reacting, the solution mixed with EDC activated mPEG-COOH solution which prepared by 17.75g of mPEG-C00H(Methoxy-Polyethylene glycol-carboxyl , Mn:5000), 1.361g of EDC and 0.817g of NHS were mixed with 40ml of MES buffer solution. The mixed solution was agitated and reacted at 30 ^° C for 24 hours. After removing the byproduct by dialysis process, the Glucose 6-phosphate-PEG was obtained after removing DIW.

2i4> EXAMPLE 24-2

2i5> Hydrophilic modification of iron oxide nanopart icles using Glucose 6- phosphate-PEG

<2i6> Iron oxide nanopar icles capped by oleic acid on the surface were prepared by the method of example 1.

<2i7> lOOmg of 3nm iron oxide nanopart icles were dispersed in 10ml of

THF(tetrahydrofurari) and mixed with aqueous solution of lg of Glucose 6- phosphate-PEG in 2ml of water. The mixed solution was agitated and reacted at 60 ^° C for 4 hr. After cooling, upper phase of THF of the mixture solution was separated and water was added to the lower phase of iron oxide nanopart icles capped by Glucose 6-phosphate-PEG on the surface to prepare a stable colloid of iron oxide nanopart icles.

<2i8> The hydrodynam i c using the 3nm nanopart icles capped by Glucose 6- phosphate-PEG on the surface to prepare a stable colloid of iron nanopart icles. The hydrodynam ic diameter of the nanopart icles having Glucose 6-phosphate-PEG on the surface was 14nm, measured by dynamic light scattering method(Malvern Zetasizer Nano ZS).

<2i > COMPARATIVE EXAMPLE 1

<220> Particle size and distribution depending upon temperature elevation rate

<22i> After mixing 1.8g of iron oleate, 0.57g of oleic acid and 1.6g of oleyl alcohol with lOg of diphenylether, a temperature was raised to 250 ^° C with a temperature elevation rate of 3.3 ^° C/min and the mixture was grown at 250 ^° C for 30 minutes. As a result, non-uniform nanoparticles including particles having almost 6nm-size nanoparticles were synthesized. The other conditions are substantially the same as described in Example 1.

<222> COMPARATIVE EXAMPLE 2

<223> Particle size and distribution depending upon temperature elevation rate <224> After mixing 1.8g of iron oleate, 0.57g of oleic acid and 1.6g of oleyl alcohol with lOg of di phenyl ether , a temperature was raised to 250 ^° C with a temperature elevation rate of 5 ^° C/min and the mixture reacted at 250 ^° C for 30 minutes. As a result, non-uniform size iianoparticles including particles having almost 6nm ^~ size nanoparticles were synthesized. The other conditions are substantially the same as described in Example 1.

<225> COMPARATIVE EXAMPLE 3

<226> Synthesis of 12nni-size nanoparticles

<227> 12nm-size nanoparticles were synthesized by the same method as disclosed in J. Park et al . , Nat. Mater. 2004, 4, 891. More particularly, after mixing 1.8g of iron oleate and 0.28g of oleic acid with lOg of 1- octadecene, followed by raising a temperature to 318 ^° C with a temperature elevation rate of 3.3 ^° C/min and reacting and growing nanoparticles at 318 ^° C for 30 minutes. FIG. 20 shows a TEM image of the synthesized nanoparticles by observat ion.

<228> COMPARATIVE EXAMPLE 4

<229> Synthesis of iron oxide nanoparticles using behenic acid

<230> In order to inhibit excessive growth of nanoparticles by steric hindering and to try to produce small size particles, a bulky surfactant was used instead of a small fatty acid. Behenic acid having higher steric hindrance than oleic acid was added during synthesis, thus trying to inhibit excessive growth of the nanoparticles.

<23i> With regard to the synthesis, 1.8g of iron oleate and 0.34g of behenic acid were mixed with lOg of 1-octadecene, followed by raising a temperature to 318 ^° C with a temperature elevation rate of 3.3 ^° C/min and reacting the mixture at 318 ^° C for 30 minutes. As a result, iron oxide nanoparticles having a size of 12nm were observed and this size is substantially equal to that of nanoparticles obtained using oleic acid with relatively small steric hindrance. From the result, it was confirmed that the size of iron oxide nanoparticles is not controlled by using steric hindrance in the case where thermal decomposition of iron oleate is applied. FIG. 21 is a TEM image of the synthesized nanoparticles by observation. <232> COMPARATIVE EXAMPLE 5

<233> Synthesis of 7nm-size nanopart icles

^<234 > 7nm-size nanopar icles were synthesized by mixing 1.8g of iron oleate and 0.57g of oleic acid with lOg of 1-octadecene, followed by raising a temperature to 318 ^° C with a temperature elevation rate of 5°C/min and reacting the mixture at 318 ^° C for 30 minutes. FIG. 22 shows a TEM image of the synthesized nanopart icles by observation.

<235> COMPARATIVE EXAMPLE 6

<236> Preparation of hydrophi lie-modified capsules including aggregation of

4nm-size iron oxide nanopart icles

<237> After dispersing 40mg of 4nm-size iron oxide nanopart icles and 40mg of poly(lactic-co-glycolic acid)(PLGA) in an ethyl acetate solution, the dispersion was mixed with 4ml of Pluronic® F127 solution (BASF Corporation, Di functional Block Copolymer) and agitated, resulting in capsules. As a result of TEM observation (FIG. 23), it was found that several aggregates composed of nanopart icles are present in an encapsulated state. According to measurement through dynamic light scattering (DLS) (Maker: Malven), the nanopart icles have a hydrodynamic diameter (z-average) of 117nm.

<238> COMPARATIVE EXAMPLE 7

<239> Synthesis using 1,2-hexadecanediol

<240> 1.8g of iron oleate, 0.57g of oleic acid and 1.55g of 1,2- hexadecanediol were mixed with lOg of di henyl ether , followed by raising a temperature to 250 ^° C with a temperature elevation rate of 10 ^° C/min and reacting the mixture at 250 ^° C for 30 minutes to synthesize the nanopart icles. The other conditions are substantially the same as described in Example 1. As a result of TEM observation, it was found that slightly non-uniform size nanopart icles having a size of 6nm were synthesized. In the case where the synthesis was performed by replacing oleyl alcohol with 1,2-hexadecanediol having two hydroxyl groups, nanopart icles having a small size of about 4nm were not obtained, although thermal decomposition could be executed at a low temperature (FIG. 24). <242> While the present invention has been described with respect to embodiments, specific examples and accompanying drawings, these are only given for overall understanding and the present invention is not particularly limited thereto. Therefore, it will be apparent to those skilled in the art that modifications and variations may be possible from the foregoing.

<243> Accordingly, the spirit of the present invention is not restricted to the foregoing embodiments, various changes and modifications may be included in the scope of the present invention as defined in the appended claims and equivalents thereof.