Background Art A targeted drug delivery system is one of drug delivery systems, with which a drug is selectively, quantitatively accumulated in a target organ or tissue regardless of its administration routes and areas. This drug delivery system can increase local concentration of a drug at a target site, leading to maximum therapeutic efficacy of the drug, as well as reducing drug accumulation at a non-target tissue or organ, leading to minimum undesirable side effects of the drug. In addition, the targeted drug delivery system has the economic benefit of reducing the amount required to achieve the therapeutic efficacy of a drug and thus greatly reducing cost consumed upon therapy.

Typically, the targeted drug delivery system used in research and clinical applications includes one of the following basic schemes: (a) direct application of a drug to an affected site (organ or tissue) ; (b) passive accumulation of a drug via the leaky vasculature, for example, at the site of tumor, infarction or inflammation ; (c) physical targeting at a target site such as a tumor or inflammation site based on abnormal pH and/or temperature (e. g. , using a drug carrier sensitive to pH and temperature); (d) targeting of a drug attached to a magnetic carrier by the action of an external

magnetic field ; and (e) employment of a vector molecule with a highly specific affinity to an affected site.

Among the schemes, the targeted drug delivery system employing a magnetic field is particularly beneficial because it allows the concentration of drugs at a defined target site away from the reticular endothelial system with the aid of the magnetic field. U. S. Patent No. 4,345, 588 discloses a method of delivering a therapeutic agent to a target capillary bed of the body, which is characterized by preparing intravascularly-administrable, magnetically-localizable biodegradable microspheres containing a therapeutic agent and administering for permanently localization in the target capillary bed for release of the therapeutic agent therein. However, the particles disclosed in the above U. S. patent are limited in their application due to their micron size and thus incapable of penetrating the intact intracellular space. In addition, since the microparticles are encapsulated with natural substances such as lipids, proteins or carbohydrates, their isolation and washing are not easily accomplished.

International Patent Publication No. WO/01/56546 discloses a composition for treating a target site of a biological tissue comprising magnetoliposomes, a method of treating a biological tissue using the composition, and a method of preparing the composition. However, there is a problem with this publication, too. It requires a step to apply a magnetic field to a target site at which the magnetoliposomes have been concentrated to activate an inactive prodrug to an effective drug.

During the step of the application of the magnetic field, heat is generated, which may damage neighboring normal tissues or cells. Also, when the prodrug is released to the target site in a non- cleaved inactive form, it may be harmful to the body.

On the other hand, it has been known that encapsulation of therapeutic agents with magnetic nanoparticles is superior at reducing toxicity of the therapeutic agents. For example, Korean Patent Laid-open Publication No. 2001-0086811 discloses a method of preparing biodegradable microparticles by an emulsification-diffusion technique. In this Laid-open publication, the biodegradable microparticles may control the speed of drug release, and have a particle size ranging from several ten to several hundred nanometers, wherein this size allows the

particles to have strong affinity to biological tissues and deeply penetrate the tissues. However, the nanoparticles encapsulating only drugs by the method of the above-mentioned publication, as a targeted drug delivery system, are problematic in terms of not substantially concentrating the drugs only at a target site.

Disclosure of the Invention It is therefore an object of the present invention to provide a composition including magnetic nanoparticles which are capable of substantially concentrating a drug at a target site and deeply penetrating the target site to improve bioavailability of the drug and minimize the undesirable side effects of the drug.

In one aspect, the present invention provides a composition including magnetic nanoparticles each of which encapsulates a magnetic material and a drug with a biodegradable synthetic polymer.

In another aspect, the present invention provides a method of preparing magnetic nanoparticles, comprising the steps of : (a) dissolving a drug, a biodegradable synthetic polymer and a magnetic material in a partially water-soluble solvent (organic phase); (b) saturating the resulting organic solution to an aqueous solution (aqueous phase) of a stabilizing agent to reach equilibrium; (c) emulsifying the resulting saturated solution using a homogenizer; (d) adding water to the resulting emulsified solution to diffuse the partially water-soluble solvent into the aqueous phase; and (e) isolating magnetic nanoparticles from the resulting solution.

In a further aspect, the present invention provides a method of preparing magnetic nanoparticles, comprising the steps of : (a) dissolving a drug, a magnetic material and a first emulsifier in distilled water to provide a first aqueous phase; (b) dissolving a biodegradable synthetic polymer in a partially water-soluble solvent to provide an organic phase; (c) adding the first aqueous phase to the organic phase and mixing the resulting mixture with agitation to provide a primary W/O type emulsion; (d) adding to the primary W/O type emulsion distilled water (second aqueous phase) in

which a second emulsifier has been dissolved and mixing the resulting mixture with agitation to provide a W/O/W type double emulsion; and (e) isolating magnetic nanoparticles from the W/O/W type double emulsion.

In a still further aspect, the present invention provides a targeted drug delivery system, comprising the steps of : (a) administering a composition including magnetic nanoparticles each of which encapsulates a magnetic material and a drug with a biodegradable synthetic polymer to a subject ; and (b) applying an external or internal magnetic field to a target site for a predetermined time to locally concentrate the magnetic nanoparticles at the target site, allowing the biodegradable synthetic polymer to be degraded and thereby releasing the drug to the target site.

Brief Description of the Drawings The objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: FIG. 1 shows SEM images of magnetic nanoparticles prepared by a method of preparing magnetic nanoparticles based on an emulsmcation-difrusion method with an organic phase: aqueous phase ratio of 1: 1.5 (a), 1: 2 (b), 1: 3 (c) and 1: 4 (d); FIG. 2 shows SEM images of magnetic nanoparticles prepared by the method of preparing magnetic nanoparticles based on the emulsification-diffusion method with a magnetite : PCL ratio of 1 : 5 (a) and 2 : 5 (b); FIG. 3 is a graph showing average diameters of magnetic nanoparticles according to the ratio of magnetite to PCL, which are prepared by the method of preparing magnetic nanoparticles based on the emulsification-diffusion method; FIG. 4 shows TEM images of magnetic nanoparticles prepared by the method of preparing magnetic nanoparticles based on the emulsification-diffusion method with a magnetite : PCL ratio of 1 : 0 (a), 1 : 5 (b), 2: 5 (c) and 3 : 5 (d);

FIG. 5 shows SEM images of magnetic nanoparticles prepared by a method of preparing magnetic nanoparticles based on a multiple-emulsification method using polyvinylalcohol for a second aqueous phase in a concentration of 0.25% (a), 0.5% (b), 1 % (c) and 2% (d); FIG. 6 shows SEM images of magnetic nanoparticles prepared by the method of preparing magnetic nanoparticles based on the multiple-emulsification method with a magnetite: PCL ratio of 1 : 5 (a) and 2 : 5 (b); FIG. 7 is a graph showing average diameters of magnetic nanoparticles according to the ratio of magnetite to PCL, which are prepared by the method of preparing magnetic nanoparticles based on the multiple-emulsification method; FIG. 8 shows results of FTIR analysis of magnetite (a), pure PCL particles (b), magnetic nanoparticles (c) prepared based on the emulsification-diffusion method, and magnetic nanoparticles (d) prepared based on the multiple-emulsification method; FIG. 9 shows results of VSM analysis of magnetite (a), magnetic nanoparticles (b) prepared based on the emulsification-diffusion method, and magnetic nanoparticles (c) prepared based on the multiple-emulsification method; FIG. 10 shows release patterns of a drug (gemcitabin) encapsulated in magnetic nanoparticles (a) prepared based on the emulsification-diffusion method, and magnetic nanoparticles (b) prepared based on the multiple-emulsification method ; FIG. 11 shows release patterns of a drug (cisplastin) encapsulated in magnetic nanoparticles (a) prepared based on the emulsification-diffusion method, and magnetic nanoparticles (b) prepared based on the multiple-emulsification method; FIG. 12 is a photograph showing mice as a control group ( (a) and (b) ) administered with a composition according to the present invention and mice as a test group ( (c) and (d)) administered with the composition according to the present invention and then applied with a magnetic field at a left tumor, FIG. 13 shows results of blue iron staining of tumor tissues not applied with a magnetic field, which has been obtained from mice of the test group;

FIG. 14 shows results of blue iron staining of tumor tissues applied with a magnetic field, which has been obtained from mice of the test group; FIG. 15 shows results of blue iron staining of the spleen (a), the pancreas (b) and the liver (c), which all are not tumor tissues, which all have been obtained from mice of the test group; and FIG. 16 is a graph showing in vivo anti-tumor effect of magnetic nanoparticles containing gemcitabin.

Best Mode for Carrying Out the Invention To accomplish the aforementioned objective, the present invention provides a composition including magnetic nanoparticles each of which encapsulates a magnetic material and a drug with a biodegradable synthetic polymer.

Examples of the biodegradable synthetic polymer useful in the preparation of the magnetic nanoparticles according to the present invention include polyphospagen, polylactide, polyl (lactide- co-glycolide), polycaprolactone, polyanhydroride, polymaleic acid and derivatives thereof, polyalkylcyanoacrylate, polyhydroxybutylate, polycarbonate, and polyorthoester. Preferably, the biodegradable synthetic polymer is selected from polyesters including polylactide, polyl (lactide-co- glycolide), polycaprolactone and polyanhydroride, which are easily biodegraded by serum esterase.

Among these biodegradable polymers, polycaprolactone (hereinafter, referred to simply as "PCL") is a linear aliphatic polyester molecule that has five non-polar methylene groups and one comparatively polar ester group. Due to this molecular structure, PCL has several unique characteristics. PCL has the similar physical property to polyolepin due to its high olefin content, while the presence of the hydrolysis-unstable aliphatic ester linkage induces the polymer to be degraded in vivo. Also, the molecular structure allows PCL to be compatible with many other polymers. Thus, it is possible to prepare various PCL polymeric conjugates having unique properties. Furthermore, PCL is slowly degraded and does not form an acidic environment such as polylactide or poly (lactide-co-glycolide). It is degraded to a non-toxic, low molecular weight

byproduct accompanied by simultaneous drug release by biodegradation thereof. Therefore, most preferred is PCL as the biodegradable synthetic polymer in the preparation of the magnetic nanoparticles according to the present invention.

Examples of the magnetic material useful in the preparation of the magnetic nanoparticles according to the present invention include ferromagnetic compounds. Most preferred is magnetite (Fe304). In the present invention, the magnetic material is in the form of being finely divided into a hyperfine size of less than 100 nm, preferably less than 30 nm, and most preferably less than 10 nm.

Such very small sized magnetite may be prepared by a technique known in the art, for example, fine milling, vapor deposition, chemical precipitation, etc. Fine milling in ball mill may be used for preparation of colloidal suspension of magnetite. For example, magnetite fine powder or suspension is commercially available, for example, from the Ferrofluidics Corporation (Burlington, Massachusetts, U. S. A), which ranges from 10 to 20 nm in particle size.

The drug capable of being loaded into the magnetic nanoparticle of the present invention does not have any particular limitation in its kind and chemical properties, and may vary depending on diseases to be treated. The drug may be loaded into the magnetic nanoparticle in an aqueous form if being water-soluble or in the form of being dissolved in an organic solvent if being lipid- soluble. Therefore, the magnetic nanoparticles of the present invention may be used for delivery of various water-soluble drugs and hydrophobic drugs to target sites. In particular, since the composition including the magnetic nanoparticles according to the present invention is capable of concentrating drugs to desired sites, it is useful for delivery of drugs with restricted clinical applications due to their side effects, such as and-tumor drugs, immunosuppressors or anti- inflammatory drugs.

Examples of the and-tumor agents useful in the present invention include cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, busulfan, nitrosourea, diactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, etoposide, tamoxifen, taxol, taxotere, transplatinum, vincristin, vinblastin and irinotecan.

Gemcitabine and cisplatin used as the drug in an aspect of the present invention, each of

which is an organic platinum coordination complex, are used for treating small-cell lung cancer, metastatic ovarian tumor, pancreatic cancer, advanced bladder cancer, and other cancers. However, these drugs are known to have severe side effects to the kidney and the gastrointestinal system, and thus limited in their clinical applications.

Examples of the immunosuppressive drugs useful in the present invention include cyclophosphamide, azathiopurine, 6-mercaptopurin (6-MP), cytarabine, bromodeoxyuridine (BudR), fluorodeoxyuridine (FudR), methotrexate, mytomycin C, actinomycin D, cortisone, predonisolone, and dexamethasone.

Examples of the anti-inflammatory drugs useful in the present invention include naproxen, diclofenac, indomethacine, sulindac, piroxicam, ibuprofen, azapropazon, nabumeton, tiaprofenic acid, indoprofen, fenoprofen, flurbiprofen, pirazolac, zaltoprofen, nabumetone, bromfenac, ampiroxicam, and lomoxicam.

The magnetic nanoparticles according to the present invention may be prepared by a technique well known in the art. In a preferred aspect, the magnetic nanoparticles are prepared using an emulsification-diffusion technique or a multiple-emulsification technique. On the other hand, the diameter of the magnetic nanoparticle is typically smaller than 1,000 nm, but, in the present invention, its diameter is preferably smaller than 500 nm. Therefore the object of the present invention is to prepare magnetic nanoparticles each of which has a diameter of smaller than 500 nm and a uniform size distribution. The present methods meeting the requirements for the magnetic nanoparticles will be described in detail, as follows.

In an aspect, the present invention provides a method of preparing magnetic nanoparticles based on an emulsificationZiffusion technique, comprising the steps of : (a) dissolving a drug, a biodegradable synthetic polymer and a magnetic material in a partially water-soluble solvent (organic phase); (b) saturating the resulting organic solution to an aqueous solution (aqueous phase) with a stabilizing agent to reach equilibrium ; (c) emulsifying the resulting saturated solution using a homogenizer; (d) adding water to the resulting emulsified solution to diffuse the partially water- soluble solvent into the aqueous phase; and (e) isolating magnetic nanoparticles from the resulting

solution.

At step (e) of the above method, the magnetic nanoparticles, but are not limited to, may be isolated by a process including filtration of the solution obtained at step (d), serum replacement, centrifugation and vacuum drying.

In the above method, the term"partially water-soluble solvent"is intended to mean a solvent having both a polar group and a non-polar group such as propylenecarbonate, ethylacetate, benzylalcohol and methylethylketone. Among them, ethylacetate is particularly preferable because of having mild toxicity, proper solubility and low boiling point.

At step (b) of the above method, sodium lauryl sulfate, polyvinylalcohol, didodecyldimethyl ammonium bromide, and others, which all have low toxicity to the body are useful for the stabilizing agent. The stabilizing agent is preferably dissolved in the aqueous phase in a concentration ranging from about 1% to about 10% (w/v).

The magnetic nanoparticles prepared by the above method may vary in morphology and size according to the ratio of the organic phase to the aqueous phase. To prepare the magnetic nanoparticles according to the present invention, the aqueous phase is preferably prepared in a volume two to four times as much as the organic phase. If the aqueous phase is prepared in a two- fold smaller volume, the obtained particles are not changed in size but present at an aggregated state.

In contrast, if the volume of the aqueous phase exceeds four times the volume of the organic phase, the prepared particles have defects on their surface and are not uniform in size and morphology. In particular, when the ratio of the organic phase to the aqueous phase is about 1 : 2, the particles prepared have a relatively small uniform size distribution. Therefore, in the above method of preparing magnetic nanoparticles according to the present invention, the ratio of the organic phase to the aqueous phase is preferably about 1: 2.

The particles prepared by the above method may vary in morphology and size according to the ratio of the magnetic material to the biodegradable synthetic polymer. To prepare the magnetic nanoparticles according to the present invention, the magnetic material is preferably used in an amount 0.2 to 0.8 times as much as the biodegradable synthetic polymer. When the magnetic

material is used increasingly within the range, the magnetic nanoparticles prepared are slightly increased in size but greatly improved in encapsulation efficiency into the biodegradable synthetic polymer. However, in case that the magnetic material is used in an amount identical to or higher than the biodegradable synthetic material, magnetic nanoparticles are formed in a low yield and has a larger particle size. Therefore, in the above method of preparing magnetic nanoparticles according to the present invention, the magnetic material is most preferably used in an amount 0.8 times as much as the biodegradable synthetic polymer.

On the other hand, the present inventors found that, in case of using a hydrophobic drug, this drug is encapsulated with high efficiency by employing the emulsification-diffusion technique.

Thus, when magnetic nanoparticles containing a hydrophobic drug are to be prepared, the emulsification-diffusion technique is preferably employed. In addition, the present inventors found that the magnetic nanoparticles prepared by the emulsification-diusion technique have a relatively rapid drug release rate. Thus, when diseases requiring rapid release of drugs are to be treated, it is preferable to use the magnetic nanoparticles prepared based on the emulsification-diffusion technique.

In another aspect, the present invention provides a method of preparing magnetic nanoparticles based on a multiple-emulsification technique, comprising the steps of : (a) dissolving a drug, a magnetic material and a first emulsifier in distilled water to provide a first aqueous phase; (b) dissolving a biodegradable synthetic polymer in a partially water-soluble solvent to provide an organic phase; (c) adding the first aqueous phase to the organic phase and mixing the resulting mixture with agitation to provide a primary W/O type emulsion ; (d) adding to the primary W/O type emulsion distilled water (second aqueous phase) in which a second emulsifier has been dissolved and mixing the resulting mixture with agitation to provide a W/O/W type double emulsion; and (e) isolating magnetic nanoparticles from the W/O/W type double emulsion.

At step (e) of the above method, the magnetic nanoparticles, but are not limited to, may be isolated by a process including filtration of the solution obtained at step (d), serum replacement, centrifugation and vacuum drying.

In the above method, the term"partially water-soluble solvent"is intended to mean a solvent having both a polar group and a non-polar group, such as propylenecarbonate, ethylacetate, benzylalcohol and methylethylketone. Among them, ethylacetate is particularly preferable because of having mild toxicity, proper solubility and low boiling point.

In the above method, the first and second emulsifiers, respectively dissolved in the first aqueous phase and the second aqueous phase, are individually selected from among sodium lauryl sulfate, polyvinylalcohol, didodecyldimethyl ammonium bromide, and others, which all have low toxicity to the body.

The magnetic nanoparticles prepared by the above method may vary in morphology and size according to the concentration of the second emulsifier of the second aqueous phase. To prepare the magnetic nanoparticles according to the present invention, the emulsifier is preferably used in a concentration ranging from 0.25% to 2% (w/v). If the concentration of the emulsifier is less than 0.25%, the solution being under emulsification becomes unstable, and aggregates of particles are generated. On the other hand, as the concentration of the emulsifier increases within the range, the obtained particles have a smaller particle size and a uniform size distribution. When the emulsifier is used in a concentration higher than 2%, the generated magnetic nanoparticles have the similar morphology and size to those obtained by using the emulsifier in a concentration of 2%.

Therefore, in the above method of preparing magnetic nanoparticles according to the present invention, the emulsifier is most preferably used in a concentration of about 2%.

The particles prepared by the above method may vary in morphology and size according to the ratio of the magnetic material to the biodegradable synthetic polymer. To prepare the magnetic nanoparticles according to the present invention, the magnetic material is preferably used in an amount 0.2 to 0.8 times as much as the biodegradable synthetic polymer. When the magnetic material is used increasingly within the range, the prepared magnetic nanoparticles are slightly increased in size but greatly improved in encapsulation efficiency into the biodegradable synthetic polymer. However, in case that the magnetic material is used in an amount identical to or higher than the biodegradable synthetic material, magnetic nanoparticles are formed in a low yield and has a

larger particle size. Therefore, in the above method of preparing magnetic nanoparticles according to the present invention, the magnetic material is most preferably used in an amount 0.8 times as much as the biodegradable synthetic polymer.

On the other hand, the present inventors found that, in case of using hydrophilic drugs, they are encapsulated with high efficiency by employing the multiple-emulsification technique. Thus, when magnetic nanoparticles containing a hydrophilic drug are to be prepared, the multiple- emulsification technique is preferably employed. In addition, the present inventors found that the magnetic nanoparticles prepared by the multiple-emulsification technique have a relatively slow drug release rate. Thus, when diseases requiring sustained release of drugs are to be treated, it is preferable to use the magnetic nanoparticles prepared based on the multiple-emulsification technique.

The composition including the magnetic nanopardcles according to the present invention may be administered through various routes known in the art, and may be formulated into various pharmaceutical forms depending on their administration routes. The administration of the present composition may be carried out, for example, intravascularly, intralymphatically, parenterally, subcutaneously, intramuscularly, intranasally, intraperitoneally, interstitially, orally, intratumorly, and others. One preferred route of administration is intravascularly. For intravascular administration, the present composition is typically injected intravenously, but may be injected intraarterially as well. The present composition may be also injected interstitially or into any body cavity.

The present invention provides a targeted drug delivery system capable of specifically concentrating magnetic nanoparticles encapsulating a magnetic material and a drug with a biodegradable synthetic polymer to a target site using a magnetic field. The magnetic nanoparticles are locally concentrated at a target site (that is, a disease site) by application of an external or internal magnetic field to the target site for a predetermined time after administration of the present composition to a subject. Herein, the application time of the magnetic field indicates the time consumed for concentrating into a target site 70% of the dosage, preferably 80% of the dosage, and

most preferably 90% or higher of the dosage, wherein the time may vary depending on several factors, such as the distance between the administration region, the target site and the administration route. In addition, the strength of the magnetic field used for concentrating the magnetic nanoparticles of the present invention to a target site preferably ranges from about 0.2 to 0.3 Tesla.

The magnetic nanoparticles concentrated at a target site in this way are able to provide therapeutic efficacy by releasing a drug loaded'therein simultaneously with degradation by the action of an enzyme in serum.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLES I. Preparation of materials and drugs Magnetit (Fe304) and poly E-caprolactone (M. W. = 42,500 Da) was obtained from Idaho University in U. S. A. and purchased from Aldrich Chemical Co. (U. S. A. ), respectively. Ethylacetate and the non-ionic surfactant polyvinylalcohol (PVA) were purchased from Aldrich Chemical Co. (USA). The PVA had an average molecular weight of 15,000-20, 000 Da and a saponification degree of 88%. Ammonium thiocyanate, hydroperoxide, potassium phosphate (KH2PO4) and dibasic potassium phosphate (K2HPO4) were purchased from Duksan Pure Chemical Co. (Korea).

All of the chemicals were used without additional purification because of being provided as HPLC grade. Distilled and de-ionized (D. D. I) water prepared using Elgastat UHQ system (UK) was used in the present invention.

Gemcitabine hydrochloride (Gemzar, Lilly Co. , Indianapolis, U. S. A) and diamine- dichloroplatinum (cisplastin, Aldrich Chemical Co. , U. S. A) were used respectively as hydrophilic and hydrophobic drugs.

II. Comparison of the size and morphology of magnetic nanoparticles prepared with various ratios of an organic phase to an aqueous phase according to the method of preparing magnetic nanoparticles based on the emulsification-diffusion technique 50 mg of gemcitabine, 0.5 g (5% w/w) of PCL and 0.4 g of magnetite were dissolved in 10 ml of ethylacetate to provide an organic phase. Additionally, four aqueous phases with a final volume of 15,20, 30 or 40 ml were prepared using D. D. I water, each of which polyvinylalcohol (PVA) was dissolved to a concentration of 5%. Each of the aqueous phases was saturated with 10 ml of the organic phase to reach equilibrium. The saturated solution was emulsified for 10 min using a 30% power probe sonicator (ULH700S, Ulssohitech, Korea). Then, 200 ml water was added to the emulsified solution under gentle agitation (150 rpm) to diffuse the ethylacetate into the aqueous phase. Magnetic nanoparticles thus formed were isolated, as follows. The final solution was filtered with a 0. 45-pm membrane. After serum replacement, centrifugation was carried out at 10,000 rpm for 30 min three times. The resulting product was dried in a vacuum oven at 35°C.

On the other hand, the size and morphology of the isolated magnetic nanoparticles were evaluated by scanning electron microscopy (SEM). The results are given in FIG. 1.

As shown in FIG. 1, there was a tendency that the size of the magnetic nanoparticles increased along with the volume of the aqueous phase. When the organic phase and the aqueous phase were used at the ratio of 1 : 2 (b), the prepared particles had a relatively small particle size. When the ratio of the organic phase to the aqueous phase was 1: 4 (d), the prepared particles were found to have defects on their surface and be not uniform in size and morphology. m. Comparison of the size and morphology of magnetic nanoparticles prepared with various ratios of magnetite to PCL according to the method of preparing magnetic nanoparticles based on the emulsificationdiffusion technique Magnetic nanoparticles were prepared with various amounts of magnetite, ranging from 0.1 to 0.5 g, according to the same method as in Example II except that the ratio of the organic phase to the aqueous phase was 1 : 2. The morphology, average size and size distribution of the prepared

magnetic nanoparticles were evaluated by using SEM, TEM and DLS. The results are given in FIGS. 2,3 and 4, respectively.

As apparent from photographs of FIG. 2, the prepared magnetic nanoparticles based on the emulsification-diffusion technique were sleek, well individualized and had a uniform size distribution. In addition, as shown in FIG. 3, the magnetic nanoparticles prepared based on the emulsification-diffusion technique were found to have an average diameter which increased along with the amount of magnetite ranging from 0.1 to 0.5 g, that is, ranging from 150 to 170 nm, IV. Comparison of the size and morphology of magnetic nanoparticles prepared with various concentrations of an emulsifier for a secondary aqueous phase according to the method of preparing magnetic nanoparticles based on the multiple-emulsification technique 50 mg of gemcitabine, 0.4 g of magnetite and PVA (0.1% w/w) were dissolved in 2 ml D. D. I water to provide a first aqueous phase. PCL (5% w/w) was dissolved in 10 ml ethylacetate to provide an organic phase. The first aqueous phase was added to the organic phase, and the resulting solution was emulsified for 4 min using a 30% power probe sonicator, thus giving a W/O emulsion. Then, the W/O emulsion was added to 20 ml of a second aqueous phase containing PVA in an amount ranging from 0.05 to 0.4 mg, and the resulting solution was emulsified for 1 min using a 15% power probe sonicator, thus giving a W/O/W double emulsion. The W/O/W emulsion was diluted with 100 ml D. D. I water and maintained under gentle agitation. Magnetic nanoparticles thus formed were isolated, as follows. The resulting solution was filtered with a 0. 45-pu membrane. After serum replacement, centrifugation was carried out at 10,000 rpm for 30 min three times. The resulting product was dried in a vacuum oven at 35°C.

On the other hand, the size and morphology of the isolated magnetic nanoparticles were evaluated by scanning electron microscopy (SEM). The results are given in FIG. 5.

As shown in FIG. 5, the magnetic nanoparticles was found to decrease in size and be uniformly individualized as the concentration of the emulsifier in the second aqueous phase increased. When the concentration of the emulsifier in the second aqueous phase was less than

0.25% (a), aggregates of particles were generated. The use of about 2% emulsifier concentration in the second aqueous phase (b) resulted in production of proper magnetic nanoparticles.

V. Comparison of the size and morphology of magnetic nanoparticles prepared with various ratios of magnetite to PCL according to the method of preparing magnetic nanoparticles based on the multiple-emulsification technique Magnetic nanoparticles were prepared with various amounts of magnetite, ranging from 0.1 to 0.5 g, according to the same method as in Example IV except that the concentration of PVA in the second aqueous phase was 2%. The morphology, average size and size distribution of the prepared magnetic nanoparticles were evaluated by using SEM and DLS. The results are given in FIGS. 6 and 7, respectively.

As apparent from photographs of FIG. 6, the magnetic nanoparticles prepared based on the multiple-emulsification technique had wider size distribution range and were more aggregated than those prepared based on the emulsification diffusion technique. In addition, as shown in FIG. 7, the magnetic nanoparticles prepared based on the multiple-emulsification technique were found to have an average diameter which increased along with the amount of magnetite ranging from 0.1 to 0.5 g, that is ranging from 350 to 370 nm.

VI. Encapsulation efficiency of magnetite into magnetic nanoparticles The concentration of magnetite loaded into the magnetic nanoparticles prepared in Examples II (organic phase: aqueous phase = 1: 2) and IV (2% PVA in the second aqueous phase) were investigated by FTIR analysis. The results are given in FIG. 8.

As shown in (b) of FIG. 8, a spectrum of pure PCL particles showed a band at 1726 cm-1 corresponding to the carbonyl group and a band at 2943 cm''corresponding to the aliphatic carboxyl- hydro group. An IR spectrum (a) of magnetite was formed at a low frequency region (1000-300 cm'') due to its iron oxide structure. On the other hand, as shown in (c) and (d) of FIG. 8, through comparison of the spectra of the magnetic nanoparticles with the spectrum of magnetite for the most

specific band (570 cm~') and with the spectra of PCL, the magnetic particles prepared in Examples II and IV displayed all bands of the pure PCL particles and the most specific band of magnetite. These results demonstrate that magnetite is successfully loaded into the magnetic nanoparticles.

The practical concentration of magnetite loaded into the magnetic nanoparticles was determined by measuring absorbance at 480 nm. An HCl/H2O2 (2 : 3, v/v) solution was added to the magnetic nanoparticles. After oxidization of Fe2+ to Fe was allowed to take place, 1 % ammonium thiocyanate was added to the resulting solution. Then, assay for formed thiocyanate complexes was performed by measuring absorbance at 480 nm.

Table 1 (emulsification-diffusion technique) and Table 2 (multiple-emulsification technique) shows the encapsulation efficiency of magnetite into the magnetic nanoparticles which was calculated using the measured absorbance values, and the size of the magnetic nanoparticles below.

Both magnetic nanoparticle preparations were found to have an increased particle size along with the amount of magnetite, but this increase in size occurred in small scales. By contrast, with regard to the encapsulation efficiency for magnetite, the magnetic nanoparticles were found to vary according to the preparation methods and the amount of magnetite. The maximum encapsulation efficiency for magnetite was found to be respectively about 7.84% for the magnetic nanoparticles prepared based on the emulsification-diffusion technique and about 15.8% for the magnetic nanoparticles prepared based on the multiple-emulsification technique.

TABLE 1 Encapsulation efficiency of magnetite into the magnetic nanoparticles prepared based on the emulsification-diffusion technique Magnetite/PCL ratio (w/w) Average size (nm Magnetit loading (mg) Encapsulation efficiency (%) 1/5 150i0. 5 0.6 3.43 2/5 158~0 5 1. 3 4. 4 3/5 1640. 5 2.7 5.4 4/5 1670. 5 3.5 7.84 TABLE 2 Encapsulation efficiency of magnetite into the magnetic nanoparticles prepared based on the multiple-emulsification technique Average size Encapsulation Magnetite/PCL ratio (w/w) Magnetite loading (mg) (nm) efficiency (%) 1/5 322. 40. 5 1.86 1.18 2/5 3240. 5 3. 81 13. 32 3/5 329i 0. 5 5.56 14.83 4/5 339. 50. 5 6.7 15.80

Note: in Tables I and 2, magnetite loading : the amount of magnetite encapsulated into 1 mg of the magnetic nanoparticles ; and encapsulation efficiency : ratio of a magnetite loading to a theoretical loading VII. Magnetic properties of the magnetic nanoparticles according to the present invention The magnetic properties of magnetite and the magnetic nanoparticles prepared in Examples II (organic phase: aqueous phase = 1: 2) and IV (2% PVA in the second aqueous phase) were investigated by means of VSM (vibrating sample magnetomere). Magnetic hysteresis loops are given in FIG. 9.

As shown in (a) of FIG. 9, a magnetization curve for magnetite at room temperature did not display any magnetic hysteresis. This result has a correlation with the typical superparamagnetic property of 100 nm magnetite nanoparticles. A magnetic field of 10 kOe or higher basically saturated magnetic powder at room temperature, and a saturation magnetization of 48 emu/g was identified at a magnetic field of 6 kOe. As shown in (b) and (c) of FIG. 9, the magnetic nanoparticles were measured to have maximum magnetization of about 10.2 emu/g at a magnetic field of 6 kOe. Under this magnetic field, the curve did not exhibit any hysteresis, remanence and coercitivity. The magnetic nanoparticles were found to have the smaller saturation magnetization

than the magnetite with the huge saturation magnetization, but have the paramagnetic property.

VIII. Evaluation of drug release of the magnetic nanoparticles according to the present invention Magnetic nanoparticles were prepared based on the ratio of a drug to PCL, listed in Table 3, below, according to the same method as in Examples lI (organic phase: aqueous phase = 1: 2) and IV (2% PVA in the second aqueous phase).

Drug release from the magnetic nanopaticles were evaluated in a phosphate-buffered aqueous release medium at 370. 5°C. The dried magnetic nanoparticles were placed into a flask containing 30 ml of the aqueous release medium, and incubated in a shaking incubator (SI-900, J. O.

Tech. , Korea) at 150 rpm. 3 ml from the aqueous release medium was collected at intervals of 24 hrs, while the medium was supplemented with 3 ml D. D. I. water. The amount of the released drug was monitored by measuring absorbance at 267.8 nm for gemcitabin and at 270.6 nm for cisplatin using an UV spectrometer (UV16A, Shimadzu, Japan) with a standard calibration curve. The results are given in FIGS. 10 and 11.

As shown in FIGS. 10 and 11, there was a large difference in drug release profiles of the magnetic nanoparticles according to their preparation methods. When the magnetic nanoparticles were prepared based on the emulsification-diffusion technique, the magnetic nanoparticles displayed a very rapid drug release rate. It results from the location of the drug which is placed near the surface of the nanoparticles. The preparation of the magnetic nanoparticles based on the multiple- emulsification technique resulted in a very slow drug release. This slow drug release is believed to result from that the drug is located in the polymeric matrix and released by degradation of the polymeric matrix.

IX. Encapsulation efficiencies of drugs into the magnetic nanoparticles of the present invention Encapsulation efficiencies of drugs into the magnetic nanoparticles according to the present invention were investigated by the drug release test of the Example VIII. In this test, total amounts of gemcitabin and cisplatin released from the magnetic nanoparticles for 30 days were measured.

The results are given in Table 3, below.

As shown in Table 3, the prepared magnetic nanoparticles based on the emulsification- diffusion technique had maximum encapsulation efficiencies of about 18.6% for gemcitabin and about 52.2% for cisplatin. Also, when prepared based on the multiple-emulsification technique, the magnetic nanoparticles had maximum encapsulation efficiencies of about 71.4% for gemcitabin and about 30.4% for cisplatin. Cisplastin (hydrophobic) and gemcitabin (hydrophilic) were found to be encapsulated into the magnetic nanoparticles with high efficiencies by the emulsification-diffusion technique and the multiple-emulsification technique, respectively. These results indicate that the present method based on the multiple-emulsification technique is suitable for encapsulation of water- soluble drugs, and the present method based on the emulsification-diffusion technique is suitable for encapsulation of water-immiscible drugs.

TABLE 3 Encapsulation efficiencies of drugs into the magnetic nanoparticles of the present invention according to the preparation methods Method Drug Drug/PCL ratio Drug loading Encapsulation (mg) efficiency (%) 0.01 0. 11 10.8 Gemcitabine Emulsification-0. 1 1. 93 18.6 diffusion 0. 01 0. 39 39. 2 Cisplatin 0.1 4.74 52. 2 0.01 0.49 49.4 Gemcitabine Multiple-0. 1 6.58 71.4 emulsification 0. 01 0. 24 24. 6 Cisplatin 0. 1 2. 8 30.4 Note: drug loading : the amount of a drug encapsulated into 1 mg of the magnetic nanoparticles ; and encapsulation efficiency: ratio of a drug loading to a theoretical loading

X. Antitumor effect of the magnetic nanoparticles according to the present invention (1) Pancreatic cancer cell culture HPAC (human pancreatic cancer cell line, ATCC No. CRL-2119) is a moderately differentiated human adenocarcinoma of pancreatic ductal origin. HPAC cells were grown in DMEM: F-12, which is a mixture of DMEM and Ham's F-12 nutrient medium (1 : 1) containing 1.2 g/liter NaHCO3 and 15 mM HEPES. The culture medium was supplemented with 5% fetal bovine serum and 1% antibiotics. Cells were cultured at 37°C in a humidified atmosphere of 5% C02- enriched air, and the medium was replaced every three days.

(2) Xenogaft 1x10 HPAC cells were subcutaneously injected into both flaks of BLAC/c nude mice.

Tumor mass was formed along with the costal ridge of each mouse, while covering most of both flaks of the mice. All mice were maintained under identical conditions.

(3) Drug administration and magnetic field application Fourteen days after the tumor cell injection, mice developed the graft of HPAC tumor cells.

The mice were then randomly assigned into one of two treatment groups: (a) control (No. = 7) and (b) test group (No. = 8). The day on which the mice were randomly grouped into different treatment protocols was designated as"zero day". On days 0,7, 14 and 20, the mice all were intravenously administered with a composition including the magnetic nanoparticles prepared in Example n via their tail vessels using a 26-gauge needle (dosage of the magnetic nanoparticles : 120 mg/kg).

Immediately after administration, each mouse of the test group was fixed to minimize their movement, attached with a magnet of 0.25 Tesla on the skin on the left tumor mass using a convenient tape, and maintained under the magnetic field for two hours. The control group was also

administered with the same dosage of the composition by the same procedure as in the test group, but did not receive the magnetic field treatment (FIG. 12).

The applied magnetic field strength was determined based on the in vitro study resulted in that a magnetic field of 0.25 Tesla is most effective in targeting the magnetic nanoparticles to the tumor mass.

(4) Tumor excision and blue iron staining On day 21, the survived mice were sacrificed by cervical dislocation. The developed tumor was excised and collected from each mouse, and fixed with para-formaldehyde and cryo- sectioned for detection of iron deposition by Prussian blue staining.

The frozen section of the excised tumor tissue was fixed with acetone, treated with H202, and stained with Prussian blue according to the Gomori's iron reaction. The blue iron staining results are given in FIGS. 13,14 and 15.

As shown in FIGS. 13,14 and 15, the magnetic nanoparticles according to the present invention were found to be locally concentrated at the magnetic field-applied region.

(5) Data and statistical analysis Tumor size was two-dimensionally measured in length and longest width by using calipers.

Tumor volume was calculated according to Equation 1, below, and the results are given as a graph in FIG. 16. Mean values were calculated for each of the test and control groups and compared with each other by an unpaired t test with Welch's correction.

[Equation 1] V = (ax2b) /2 [a= length (cm); b= width (cm) ] In the present invention, the present inventors performed two comparisons for tumor size.

First, the left tumor (applied with the magnetic field) and the right tumor (not applied with the

magnetic field) were compared with each other in each mouse of the test group. The second comparison was carried out between the tumor of the control group and the left tumor of the test group.

In the first comparison, the present inventors found that the magnetic field-applied tumor was more decreased in size than the tumor not applied with the magnetic field. In the second comparison, there was a larger decrease in size of the magnetic field-applied tumor than that of the tumor of the control group. On the other hand, the difference observed in the first comparison was found to be greater than that in the second comparison. These results are believed to result from that, in the test group, relatively many magnetic nanoparticles are concentrated at the left tumor by the application of the magnetic field to the left tumor, whereas, in the control group, the magnetic nanoparticles are distributed in similar concentrations at the left tumor and the right tumor. Taken together, the concentration of the magnetic nanoparticles was found to increase in a sequence of the right tumor of the test group, the right and left tumors of the control group and the left tumor of the test group. The anti-tumor effect of the magnetic nanoparticles was found to increase with their increased concentration at the tumor site. In particular, the test group, the left tumor was growth- inhibited and degenerated.

Industrial Applicability As described hereinbefore, the composition including the magnetic nanoparticles according to the present invention is capable of substantially concentrating drugs only at disease sites and deeply penetrating into tissues of the disease sites the drugs, thereby minimizing side effects of the drugs and enhancing therapeutic efficacy of the drugs at the disease sites.