WO2011138975A1

WO2011138975A1 - High density poly-alkylene glycol-coated iron oxide gold core-shell nanoparticle

Info

Publication number: WO2011138975A1
Application number: PCT/JP2011/061123
Authority: WO
Inventors: Kazunori Kataoka; Tridib Kumar Sarma; Michiaki Kumagai; Horacio Cabral; Masaki Sekino; Takehiko Ishii
Original assignee: The University Of Tokyo
Priority date: 2010-05-07
Filing date: 2011-05-09
Publication date: 2011-11-10
Also published as: JP2013525477A

Abstract

The present invention provides a composite particle comprising as a core a magnetic iron-oxide particle, a gold shell on said magnetic iron-oxide particle, and a poly(alkylene glycol) coating on said gold shell, wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.05-1.0 nm²/poly(alkylene glycol) molecule.

Description

HIGH DENSITY POLY-ALKYLENE GLYCOL-COATED IRON OXIDE GOLD CORE-SHELL NANOPARTICLE

CROSS REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. provisional Applications Serial Nos. 61/332346 and 61/332888, each filed on 7 May 2010 and 10 May 2010, which are hereby incorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention relates to a high-density poly(alkylene glycol)-coated iron-oxide-gold core-shell nanoparticles and the use thereof as a magnetic resonance image (MRI) contrasting agent.

BACKGROUND ART

Pancreatic cancer is highly aggressive cancer characterized by high invasiveness, acute resistance to chemo and radiotherapy and consequently represents one of the most difficult malignancies to detect and treat [^{1 2}]. Patient prognosis is often dismal due to late diagnosis and a lack of effective therapies. This outlook could be improved by realization of diagnostic tools useful at earlier stages of the disease.

Magnetic resonance imaging (MRI) is a powerful and noninvasive technique for medical imaging of soft tissues. MRI offers clinical feasibility for molecular imaging because it provides superb anatomic resolution and contrast for visualizing tissue morphology and anatomical details of organs in vivo [^3~6]. Development of contrast agents has been central to advances in magnetic resonance imaging techniques for early diagnosis of cancer and detection of biologically active processes at the cellular and molecular level. Particularly, iron oxide nanoparticles are becoming increasingly attractive for applications of in vivo MRI, due to their low toxicity and excellent magnetic susceptibility that results in strong T₂ and T₂* contrast for enhanced MRI. In order to enhance the in vivo utility and include use for tumors, the iron oxide nanoparticles should be highly biocompatible of appropriate size, and should have sufficiently long blood circulation time allowing for passive tumor accumulation through the Enhanced Permeability and Retention (EPR) effect [^7~9]. The surface modification of iron oxide nanoparticles with biocompatible polymers can avoid or effectively reduce the recognition by reticuloendothelial systems (RES), improving their circulating property.

Poly(ethylene glycol) (PEG) has found widespread clinical use as a biocompatible, non-specific protein resistant material that prolongs the circulation time of protein therapeutics [^{10 11}]. PEG-based block copolymers and PEGylated liposomes have been used to improve the stability and pharmacokinetics of iron oxide nanoparticles in the physiological environment [^12~13]. Many groups showed negative enhancement at the hypervascular tumor site in the tumor-bearing mice using PEG- coated iron oxide nanoparticles in T₂-weighted MR images.

Recently, gold-coated superparamagnetic core-shell nanoparticles have attracted considerable attention for biomedical application [^16"19]. Au and iron oxide nanoparticles are known to be biocompatible and have been used extensively for optical and magnetic application, respectively. Furthermore, gold coating on the magnetic nanoparticles can be stabilized in biological conditions and readily functionalized through the Au-S bonding.

Nakagawa et al reported an MRI contrasting agent having a magnetized iron- oxide core surrounded with gold layer and PEG coating on the gold layer and that the agent successfully provided a negative enhancement of MR image of subcutaneously transplanted fibrosarcoma in mice [ 28 ].

However, there are no papers to enhance the MR image at the pancreatic tumor site using PEG-coated iron oxide nanoparticles without targetable biomolecules, because PEG-coated iron oxide nanoparticles developed so far has limited circulating property in blood compartment and a size not small enough to penetrate into pancreatic tumor models. In fact, without intrapenitoneal administration of TGF-β inhibitor, PEG- coated iron oxide nanoparticles, which had about 100 nm size, failed to accumulate in the subcutaneous model of BxPC3 pancreatic tumor, which is characterized by hypo vascularity and thick fibrosis [¹⁵].

DISCLOSURE OF THE INVENTION

PROBLEM TO BE SOLVED BY THE INVENTION

Under these circumstances, the development of an MRI contrasting agent having a prolonged circulating property in blood and capable of providing an enhancement on pancreatic cancer tissue is awaited.

The present inventors previously reported PEG-coated iron oxide nanoparticles with a hydrodynamic diameter of ~ 100 nm as a negative contrast agent and successful MR imaging of subcutaneous colon tumor models [¹⁴].

Also, the present inventors recently found that the size upper limit of the nanoparticle system for passive targeting to pancreatic tumor models was found to be ~ 50 nm (unpublished data). Hence, the present inventors hypothesized that with proper control of the size and surface properties of iron oxide based nanoparticle systems, development of effective T₂- weighed MRI contrast agents for in vivo detection of pancreatic tumors could be possible.

The present inventors extensively studied to realize the above mentioned hypothesis and finally developed PEG-coated iron oxide-gold core-shell nanoparticles (PEG-AuION) system for use as a T₂-weighed MRI contrast agent for imaging of pancreatic tumor models.

In the method developed by the present inventors, the overall size of the nanoparticle was controlled by coating iron oxide nanoparticles with Au that allowed for reaction with methoxy-PEG-thiol (MeO-PEG-SH) and subsequent formation of a high density PEG coating on the surface through strong Au-S bonding, without the formation of any higher order assemblies. The results obtained using the nanoparticle demonstrated that PEG-AuION showed prolonged blood circulation and MR imaging of orthotopic pancreatic tumor and subcutaneous colon tumor model. Present findings suggest that the above mentioned method, that allows precise control of hydrodynamic size and effective PEG density on the iron oxide-gold core-shell nanoparticles (AuION), could be a promising method for development of MRI contrast agents for various tumor types including pancreatic cancer.

MEANS TO SOLVE THE PROBLEM

Accordingly, the present invention provides the followings:

[I] A composite particle comprising as a core a magnetic iron-oxide particle, a gold shell on said magnetic iron-oxide particle, and a poly(alkylene glycol) coating on said gold shell, wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.05-1.0 nm²/poly(alkylene glycol) molecule.

[2] The composite particle recited in [1], wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.125-0.7 nm²/poly(alkylene glycol) molecule.

[3] The composite particle recited in [1], wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.2-0.4 nm²/poly(alkylene glycol) molecule.

[4] The composite particle recited in [1], having an average diameter of no more than 60 nm.

[5] The composite particle recited in [1], having an average diameter of 10-50 nm.

[6] The composite particle recited in [1], having an average diameter of 15-40 nm.

[7] The composite particle recited in [1], wherein said magnetic iron-oxide is one selected from a group consisting of Fe₃0₄, y-Fe₂0₃, MnFe₂0₃ and ferrite.

[8] The composite particle recited in [1], wherein said poly(alkylene glycol) is one selected from a group consisting of poly(methylene glycol), poly(ethylene glycol) and poly(propylene glycol).

[9] The composite particle recited in [1], wherein said poly(alkylene glycol) molecule is conjugated to said gold molecule by thiol-gold coupling.

[10] The composite particle recited in [1], wherein said poly(alkylene glycol) has an average molecular size in a range of 500-5000 dalton.

[I I] The composite particle o recited in [1], which produces a negative enhancement of more than one type of cancer selected from the group consisting of brain tumor, pharyngeal cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, pancreatic cancer, biliary cancer, duodenal cancer, colon cancer, liver cancer, uterus cancer, ovarian cancer, prostate cancer, renal cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma and cutaneous cancer.

[12] The composite particle recited in [1], for use in a method for diagnosing more than one type of cancer selected from the group consisting of pancreatic cancer, colon cancer, renal cancer, liver cancer, lung cancer, prostate cancer, breast cancer, stomach cancer, brain tumor and ovarian cancer.

[13] A method of producing the composite particle recited in [1], comprising the steps of:

applying a gold shell onto a magnetic iron-oxide particle; and

applying a poly(alkylene glycol) coating onto said gold shell at a density of 0.05-1.0 nm²/poly(alkylene glycol) molecule.

[14] The method recited in [13], wherein the application of said gold shell onto said magnetic iron-oxide particle is performed by a reduction of a gold-complex in a solution containing said magnetic iron-oxide particle, said gold complex and a reducing agent selected from the group consisting of oleylamine, oleic acid, dodecanoic acid, stearic acid, octylamine, NaBH₄, dodecylamine and dodecanethiol.

[15] The method recited in [13], wherein the application of said poly(alkylene glycol) coating onto said gold shell is repeated at least once.

[16] The method recited in [13], wherein said poly(alkylene glycol) molecule is conjugated to said gold molecule by thiol-gold coupling.

[17] An MRI contrasting agent comprising the composite particle recited in any one of [l] to [11].

[18] The MRI contrasting agent recited in [17], wherein said composite particle maintains an average diameter of no more than 60 nm in a solution having an NaCl concentration of 0-1.5M.

[19] A method of diagnosing cancer comprising the steps of:

administering the MRI contrasting agent recited in [17] to a subject;

obtaining a magnetic resonance image of said subject using an MRI system; and

detecting a tumor tissue from said magnetic resonance image.

[20] The method recited in [19], wherein said magnetic resonance image is T₂- weighted MRI.

EFFECT OF THE INVENTION

The composite particle of the present invention having a controlled sub-50 nm size and coated with a dense PEG brush can be used as an MRI contrast agent for variety of tumors, including pancreatic tumor, without attachment of any tumor targeting biomolecules. The relatively small hydrodynamic diameter along with a high poly(alkylene glycol) density allows the composite particle of the invention to show prolonged circulation. The composite particle of the invention accumulates into the pancreatic models through the EPR effect and thus provides MR image contrast enhancement of the tumors in the pancreas.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 : Schematic representation of the synthetic procedure, Au shell formation on the y-Fe₂0₃ nanoparticles and subsequent coating with MeO-PEG-SH to form biocompatible nanoparticles.

Figure 2: X-ray diffraction pattern of y-Fe₂0₃ (A) and AuION (B). The Braggs's reflections for each nanoparticle are shown.

Figure 3: UV- visible spectra of (I) y-Fe₂0₃ nanoparticles in chloroform (II) AuION in chloroform (III) PEG- AuION in aqueous medium.

Figure 4: Transmission electron microscopy of (A) y-Fe₂0₃ nanoparticles (B) AuION in chloroform (C) PEG-AuION in aqueous medium shows the MeO-PEG-SH coating on the AuION surface. The nanoparticles were stained with 1 % phosphotungstic acid solution. Scale bar is 50 nm in all the TEM images.

Figure 5: Particle size distribution of AuION in chloroform (solid line) and PEG-AuION in water (dotted line). Z-average size distribution of PEG-AuION were measured by dynamic light scattering.

Figure 6: Change in the particle size with increased NaCl concentration.

Figure 7: Time dependency of the relative hydrodynamic diameter of PEG- AuION. Fe concentration 1 mrnol/L, in Tris-HCl buffer at 37 °C containing 10 % fetal bovine serum.

Figure 8: Relaxivity r₂ of the PEG-AuION using Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence at 25 °C, 0.59 T.

Figure 9: Biodistribution of the PEG-AuION. The levels of nanoparticles in blood, tumor, liver, spleen, kidney and muscles are shown as percentage of dose at each time after intravenous injection.

Figure 10: In vivo MR imaging of cancer T₂- weighted images of subcutaneously implanted C26 murine carcinoma (tumor sites are circled by red dotted line) at 4 hours after intravenous injection of the PEG-AuION (a) and Feridex^® (b). T₂- weighted images of orthotopic MiaPaCa-2 human pancreatic cancer model (tumor sites are circled by red dotted line) at 4 hours after intravenous injection of the PEG-AuION (c) and Feridex^® (d). All images were obtained in a magnetic field strength of 4.7 T.

Figure 11 : Time dependencies of relative signal intensities at the tumor site in T₂-weighted images after injection of the PEG-AuION and Feridex^® in C26 subcutaneous model (a) and in MiaPaCa-2 orthotopic model (b). Figure 12: T₂-weighted images of mice implanted MiaPaCa-2 human pancreatic tumor cells metastasized to liver (tumor sites are circled by red line) at 2 hours after intravenous injection of the PEG-AuION (A) and Feridex^® (B). All images were obtained in a magnetic field strength of 4.7 T.

Figure 13: Histological sections of metastatic focus on Haematoxylin & Eosin staining (panels A and B) and silver staining (panel C, brown: Au). The distribution of PEG-AuION was examined 24 h after the administration.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will be described in more detail below. The following embodiments are illustrated to describe the present invention, and it is not intended to limit the present invention thereto. The present invention can be implemented in various modes, without departing from the spirit of the present invention.

Composite Particle

The composite particle of the present invention comprises at least three types of materials: a magnetic iron oxide particle as a core, gold as a shell surrounding said core, and poly(alkylene glycol) as an outer coating-layer surrounding said gold shell (see Figure 1).

The composite particle of the present invention has a highly dense poly(alkylene glycol) coating so that said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.05-1.0 nm² of gold shell surface/poly(alkylene glycol) molecule. Preferably, the poly(alkylene glycol) is conjugated to said gold shell at a density of 0.075-0.9 nm², 0.1-0.8 nm², 0.125-0.7 nm², 0.15-0.6 nm², 0.175-0.5 nm², 0.2- 0.4 nm², 0.225-0.3 nm² or 0.23-0.27 nm² of gold shell surface/poly(alkylene glycol) molecule.

With this dense poly(alkylene glycol) coating, the particle of the present invention has a high biocompatibility and a prolonged circulating ability in the circulating system of the subject.

The composite particle of the present invention has an average diameter of no more than 60 nm, preferably in a range of 5-55 nm, preferably 10-50 nm, more preferably 15-40 nm, still more preferably 15-35 nm, 15-30 nm, 20-30 nm, 20-27.5 nm or 20-25 nm.

As such the composite particle of the present invention has surprisingly smaller size compared to those of composite particles of MRI contrasting agents according to the prior art such as one described in Nakagawa et al having an average diameter of 234.0 nm so that the composite particle of the present invention is able to penetrate into intractable tumors such as pancreatic cancer. The composite particle of the present invention has a relaxivity r² ranging from 100 x 10^"3 to 200 x 10^"3 M^"1 -s^"1, preferably 125 x 10^"3 to 175 x 10^"3 M^"' -s^"1, more preferably 140 x 10^"3 to 160 x lO^ M^"1 -s^"1 and still more preferably 145 x 10^"3 to 155 x 10^"3 M^"1 -s^"1, thus providing a strong contrast on MR images.

Further, the composite particle of the present invention shows a tumor specific biodistribution when administered to a subject. The biodistribution of the composite particle can be confirmed by measuring Au content in blood and tissues using inductively coupled plasma mass-spectrometry (ICP-MS).

The composite particle of the present invention is stable in salt-containing solutions. As demonstrated in the Example of the present invention, the composite particle of the present invention maintains its particle size, i.e. diameter, in aqueous NaCl solutions containing 0-1.5 M NaCl. Further, the composite particle of the present invention is highly stable under physiological conditions so that it provides a long lasting MR image enhancement. The composite particle of the present invention is also stable in a wide range of pHs such as pH3-10.

The composite particle of the present invention can be produced basically by two steps: applying a gold shell onto the magnetic iron-oxide particle; and applying a poly(alkylene glycol) coating onto the gold shell. The magnetic iron-oxide particle may be one commercially available such as Feridex^® or prepared from appropriate iron complexes. The details of the preparation of the magnetic iron-oxide particle, the application of the gold shell onto the magnetic iron-oxide particle and the application of poly(alkylene glycol) coating onto the gold shell are discussed below.

Magnetic Iron Oxide

The magnetic iron oxide used for the composite particle of the present invention may be any materials as long as it is magnetized and contains iron-oxide. Accordingly, the magnetic iron-oxide particle may be one commercially available such as Feridex^®. Examples of magnetic iron oxide include Fe₃0₄, y-Fe₂0₃, MnFe₂0₃ and ferrite. Preferably the magnetic iron oxide is y-Fe₂0₃.

The magnetic iron oxide particle used for the composite particle of the present invention has an average diameter of 7.5-15 nm, preferably 8.0-14 nm, more preferably 8.5-13.5 nm and still more preferably 9.0-11 nm.

The magnetic iron oxide particle can be prepared by a thermal decomposition of iron complex such as Fe(CO)₅, FeCl₃ and FeS0₄ in the presence of one or more of capping agents.

Examples of the capping agents include oleylamine, oleic acid, dodecanoic acid, stearic acid, octylamine, 1,2-hexadecanediol, 3-mercapto-l-propane Sulphonic acid, dodecylamine and dodecanethiol. Preferably examples of the capping agents are oleylamine and oleic acid, which are advantageously used in the ratio of 5: 1 (=oleylamine: oleic acid). The presence of oleic acid may help in obtaining uniformly shaped magnetic iron oxide particles, in particular, particles of y-Fe₂0₃.

Preferably the thermal decomposition of the iron complex is performed under aerobic conditions.

The formation of magnetic iron oxide particles can easily be confirmed by electron microscopic observation.

Gold Shell

The gold shell on the magnetic iron oxide particle consists of Au molecules and has an average thickness of 0.5-1.8 nm, preferably 0.7-1.5 nm, more preferably 1.0-1.4 nm, and still more preferably 1.2- 1.4 nm.

The application of the gold shell onto the magnetic iron-oxide particle may be performed by a reduction of a gold-complex in a solution containing the magnetic iron- oxide particle, the gold complex and a reducing agent.

Examples of the gold-complex include HAuCLt and Au(0₂CCH₃)₃. A preferable example of the gold-complex is HAuCl₄. However, the gold-complex is not limited to the above as long as it can form a complex with Au molecule by providing ligands and produce Au⁺ ion in appropriate solutions.

Alternatively, Au molecule may be provided in the form of salts such as nitrate, chloride, acetate and citrate of gold.

Examples of the reducing agent are selected from the group consisting of oleylamine, oleic acid, dodecanoic acid, stearic acid, octylamine, NaBH₄, dodecylamine and dodecanethiol, but are not limited thereto.

The solvent used for the solution include water, water-containing alcohol or alcohols such as methanol, ethanol and n-propanol; acids such as hydrochloric acid, sulfuric acid and nitric acid; and organic solvents such as benzene, phenol, toluene, chloroform and dioctylether.

The compound listed above may be substituted with one or more of halogen groups. A preferable example of the solvent is 1,2-dichloro benzene.

Stimulation such as gamma ray, electron beam or ultrasonic wave may optionally be applied to the solution in order to facilitate the formation of the gold shell on the magnetic iron-oxide particle.

The thus prepared gold- shelled magnetic iron-oxide particle may be washed by an appropriate means such as dispersion centrifugation in order to remove excess ligands, reducing agents or solvent.

The formation of the gold shell can be confirmed via X-ray diffraction measurement by detecting Bragg's diffraction peaks from the face-centered cubic (fee) lattice structure of Au molecule. Alternatively, the formation of the gold shell can be confirmed by electron- microscopic observation by measuring the increase in the thickness of the magnetic iron-oxide particle, or UV-vis spectroscopy

Poly(Alkylene Glycol)

The poly(alkylene glycol) used for the composite particle of the present invention may be any materials as long as endows hydrophilicity and biocompatibility to the composite particle. Examples of the poly(alkylene glycol) include, but not limited to polymers of lower (Ci-C₆) alkylene glycols such as poly(methylene glycol), poly(ethylene glycol) and poly(propylene glycol). In one embodiment, the poly( alkylene glycol) is poly(ethylene glycol) (PEG).

Preferably the poly(alkylene glycol) has an average molecular size in a range of 500-5000 daltons, 750-4000 daltons or 1000-3000 daltons.

The poly( alkylene glycol) may contain one or more of SH-groups so as to be conjugated to the surface of the gold shell by thiol-gold coupling reaction. Preferably the poly(alkylene glycol) contains one SH-group at its one end. The poly(alkylene glycol) may optionally contain an alkoxy-group at the other end. Preferable examples of the alkoxy group include Ci-C₄ alkoxy groups: methoxy, ethoxy, propoxy and butoxy groups. In one embodiment, the alkoxy group is methoxy.

The thus obtained composite particles coated with the poly(alkylene glycol) is already water soluble, however, the coating step may be repeated at least once in order to increase the density of the poly(alkylene glycol) on the surface of the gold shell so that the stability of the composite particle in aqueous conditions will be further enhanced.

By using lower alcohol such as methanol, ethanol or propanol as the solvent for the repetitive cycles of poly(alkylene glycol) coating step, the size of the composite particle of the present invention can be well controlled.

On the other hand, any solvent may be used for the 1 st round of poly(alkylene glycol) coating such as organic solvents including phenol or chloroform or aqueous solvents such as water. A preferable example of the solvent for the 1st round of poly( alkylene glycol) coating is chloroform.

As a result the composite particle of the invention has 500-5000 poly( alkylene glycol) molecules/particle on average, preferably, 1000-4000, 1500-3000, 2000-2750 or 2250-2600 poly(alkylene glycol) molecules/particle on average. The number of poly(alkylene glycol) molecules/particle can be calculated by thermogravimetric analysis (TGA) of the composite particle under N₂ atmosphere.

The average thickness of the poly(alkylene glycol) coating would be 3-10 nm, preferably 4-8 nm, and more preferably 5-7 nm. The formation of the poly(alkylene glycol) coating on the gold shell can be confirmed by either electron microscopic observation with phosphotungstic acid staining.

Application of the Composite Particle

As discussed above, the composite particle of the present invention is characterized with a dense poly(alkylene glycol) coating on its gold shell. Owing to the dense poly(alkylene glycol) coating, the composite particle of the present invention has an excellent biocompatibility and prolonged blood circulation ability so that it provide a long lasting MR image enhancement.

Further, the composite particle of the present invention shows a tumor specific bio distribution when administered to a subject.

In addition, since the composite particle of the present invention has a small particle size (much less than 50 nm), it can penetrate into tumor tissues, which are intractable for the MRJ contrast agent used in a conventional method, and provide a clear image enhancement of said tumor tissues.

With these properties, the composite particle of the present invention can beneficially be used as an MR image contrasting agent, especially for diagnosing tumor of subjects.

Since the composite particle of the present invention comprises as its core a magnetic iron oxide particle which promotes proton relaxation to reduce MRI signal with longer echo time, it can advantageously be used as a T₂-weighted MRI contrast agent.

The subject to be diagnosed using the composite particle include mammals such as human, monkeys, primates other than human, domestic animals such as horse, cow, sheep, goat and pig, pet animals such as dog and cat, and experimental model animals such as mouse, rat, guinea pig and rabbit, however, are not limited thereto.

Examples of tumors visualized by the composite particle of the present invention may be either benign or malignant. Examples of malignant tumors include brain tumor, pharyngeal cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, pancreatic cancer, biliary cancer, duodenal cancer, colon cancer, liver cancer, uterus cancer, ovarian cancer, prostate cancer, renal cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma and cutaneous cancer, however, are not limited thereto. These cancers may be either primary or metastatic. In one embodiment, the cancer to be diagnosed is pancreatic cancer. In another embodiment, the cancer to be diagnosed is colon cancer.

The composite particle may be administered to the subject either by oral or parenteral route. For oral administration, the composite particle of the present invention may be formulated as a tablet, capsule, granule, powder or syrup. For parenteral administration, the composite of the present invention may be formulated as an injection, suppository, eye-drops, preparations for pulmonary administration such as one to be administered using nebulizer, for nasal administration and for transdermal administration such as a cream and ointment.

In the form of an injection, the composite particle of the present invention may be administered either systemically or tropically via intravenous injection including infusion, intramuscular injection, intraperitoneal injection or subcutaneous injection. The composite particle of the present invention may be formulated together with one or more of pharmaceutically acceptable additives such as an excipient, lubricant, decomposer, stabilizer, binding agent, flavoring agent and diluents.

Examples of the excipient include starches such as potato starch and maize starch, lactose, crystalline cellulose and calcium hydrogen phosphate.

Examples of the lubricant include ethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, shellac, tare, carnauba wax and paraffin.

Examples of the binder include polyvinylpyrrolidone, macrogol and the compounds listed as the examples of the excipients.

Examples of the decomposer include the compounds listed as the examples of the excipients and chemically-modified starch or cellulose such as croscarmellose sodium, carboxymethyl starch sodium and polyvinylpyrrolidone.

Examples of the stabilizer include esters of ara-hydroxybenzoic acid such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; phenols such as phenol and cresol; thimerosal; dehydro acetic acid; and sorbic acid.

Examples of the flavoring agent include routinely used sweetenings, acidulants and flavors.

As for preparation of a liquid formulation of the composite particle of the present invention, solvents such as ethanol, phenol, chloroform, chlorocresol, purified water and distilled water can be used. Alternatively the composite particle of the present invention may be dissolved in phosphate saline butter or Tris-HCl butter. The liquid formulation may also contain surfactants and/or emulsions such as polysorbate 80, polyoxyl 40 stearate and lauromacrogol.

The above mentioned additives may be used alone or in combination therebetween depending on the formulation of the composite particle of the present invention.

The dose of the composite of the present invention to be administered to the subject varies depending on the age, gender and condition of the subject, the route and frequency of administration, and the type of formulation of the composite particle of the invention. The dose schedule may be adequately determined by those skilled in the art such as physicians, surgeons, veterinary surgeons, biomedical researchers and especially radiologists. The effective dose of the composite particle of the invention ranges from 1 to 500 mg/kg of the subject body weight, preferably from 1 to 20 mg/kg of the subject body weight, however, is not limited thereto.

Upon administration of the composite particle of the invention to a subject, tumor tissues are visualized with negative enhancement, i.e. as dark images.

Together with the composite particle of the present invention, additional agents such as TGF-β inhibitors may optionally be co-administered to subject. Coadministration of the additional agents may be done before or after the administration of the composite particle of the present invention. The additional agents may be coadministered while the composite particle of the present invention is administered to a subject.

The present invention will be further described in more detail by way of the following illustrative examples, which are not intended to limit the scope of the invention.

EXAMPLES

1. Materials and Methods

Materials

Iron pentacarbonyl (Fe(CO) ) and hydrogen tetrachloroaurate (III) tetrahydrate (HAuC ) were purchased from Kanto Chemical Co., Inc., (Tokyo, Japan). 1,2- dichlorobenzene (ODCB) was purchased from Sigma-Aldrich Corporation (St. Louis, Mo, USA). Oleylamine, oleic acid, ethanol, methanol, and chloroform were purchased from WAKO Pure Chemical Industries (Osaka, Japan). Methoxy-PEG- thiol (MeO- PEG-SH; Mw = 2,000) was purchased from NOE Corporation (Tokyo, Japan).

Synthesis of iron oxide-gold core shell nanoparticles (AuION)

In a typical synthesis, 4 mmol of Fe(CO)₅ (0.53 ml) was dissolved in 3 ml of (not deoxygenated) ODCB and then rapidly injected into a hot solution containing 15 ml of ODCB, 8 mmol of oleylamine (2.63 ml) and 1.6 mmol of oleic acid (0.5 ml). The resulting mixture was slowly heated to 180 °C under aerobic conditions. The initial orange color of the solution gradually turned to black during the process. After 9 hours, the resulting black solution was cooled to room temperature and ethanol was added to precipitate the nanoparticles. The resulting black flocculate was isolated by centrifugation at 8,000 rpm for 10 minutes and the y-Fe₂0₃ pellet was redispersed into ODCB.

To prepare an Au shell on the y-Fe₂0₃ nanoparticle surface, first prepared was a solution of HAuC (2.5 mmol) in 5 ml ODCB in presence of 1 mmol oleylamine and then vortexed this solution for several minutes to allow complete dissolution of HAuCl₄ in ODCB. Next, this solution was added slowly to a solution containing 50 mg of γ- Fe₂0₃ nanoparticles and 2 mmol of oleylamine in 10 ml of ODCB. The resulting mixture was stirred at room temperature for 1 hour and then 140 °C for 2 hours. The color of the solution turned from black to faint pink. The mixture was cooled to room temperature, centrifuged at 8,000 rpm for 5 minutes and the resulting small amount of precipitate was discarded. Ethanol was added to the remaining solution followed by centrifugation at 14,000 rpm for 15 minutes. The nanoparticles were washed by several cycles of dispersion-centrifugation to remove excess oleylamine from the surface of nanoparticles. Finally, the nanoparticles were dispersed in 10 ml of chloroform.

PEG coating onto AuION

PEG was attached to the surface of nanoparticles using well known thiol-gold chemistry. To an AuION solution in chloroform, 100 mg of MeO-PEG-SH was added, and the mixture was agitated by shaking for 24 hours at room temperature followed by removal of chloroform under vacuum. The PEG coated AuION could be solubilized in aqueous medium. However, partial salt-induced agglomeration of the nanoparticles was observed 12 hours after addition of 150 mM NaCl to the nanoparticle solution. Nanoparticles were subjected to a second PEGylation process in order to increase the PEG density on the surface. Vacuum-dried PEG coated AuION (after chloroform evaporation) were dispersed in methanol, and 50 mg of MeO-PEG-SH was added. The solution was mixed by shaking for 24 hours. Methanol was exchanged with water by dialysis (molecular weight cut off 10,000) overnight. Nanoparticles were readily dispersed in aqueous medium and no precipitate was observed even after several months. Unbound MeO-PEG-SH was removed by ultrafiltration (MWCO 200 000; poly sulfone membrane, Toyo Roshi Co. Ltd.,Tokyo, Japan).

Physicochemical characterization of PEGylated y-Fe₂0₃/Au core-shell nanoparticles (PEG-AuION).

UV-visible spectra were recorded on a UV-visible-NIR spectrometer (V-570, JASCO Corporation, Hachioji, Tokyo, Japan) using a quartz cuvette. The morphology and size distribution of the nanoparticles were recorded by Transmission electron microscopy (H-7000, Hitachi, Ltd., Tokyo, Japan) at an accelerating voltage of 100 kV. TEM samples were prepared by mounting a drop of the nanoparticles (10 μΐ) in different solvents on carbon-coated 400 mesh Cu grids and allowing them to dry in air. X-ray diffraction patterns were recorded using a MAC Science M18XHF diffractometer with Cu-Κα radiation (λ = 1.54056 A) at 293 K. The Au and Fe content in the AuION were determined by ion coupled plasma-mass spectroscopy (ICP-MS, 4500, Hewlett Packard, Pao Alto, CA, USA). Dynamic light scattering and ζ-potential measurements were performed at 25 °C using a Zetasizer NanoZs instrument equipped with a DTS5001 cell on a Malvern 4700 system. The amount of polymer adsorbed onto AuION was measured by thermogravimetric analysis (TGA) (EXSTAR6200 TG/DTA, Seiko Instuments Inc., Chiba, Japan) under nitrogen atomosphere with a heating rate of 10 °C/min in the temperature range of 25-600 °C. The proton relaxivity r₂ of nanoparticle solutions in water was evaluated by the Carr-Purcell-Meiboom-Gill (CPMG) sequence at 25 °C using 25 MHz pulse NMR (JNM MU25A, JEOL Ltd., Akishima, Japan).

Cancer cell lines and animals

All animal experimental protocols were performed in accordance with the policies of the Animal Ethics Committee of the University of Tokyo. C26 cell line, derived from murine colon adenocarcinoma, was supplied by Dr. Y. Matsumura, the National Cancer Center Research Institute East, Japan (Kashiwa, Japan). MiaPaCa-2 human pancreatic adenocarcinoma cell line was obtained from the American Type Culture Collection (Manassas, VA). C26 and MiaPaCa-2 cells were cultured in bottle- necked flasks in DMEM with 10% FBS at 37 °C in 5 % C0₂. BALB/c nude mice, 5-6 weeks of age, were obtained from Charles River Laboratories, (Tokyo, Japan). The BALB/c nude mice (female) were inoculated subcutaneously with C26 cells (1 x 10⁶ cells, 50 Ε volume). The xenografts were used 12 days after inoculation. Orthotopic pancreatic tumors were prepared by orthotopic inoculation of MiaPaCa-2 cells (1 x 10⁷ cells, 50 μL volume) into the pancreas of nude mice, and allowed to grow for 2-3 weeks to reach proliferative phase. Liver metastasis tumor model was developed by injecting MiaPaCa-2 cancer cells (1 x 10⁷ cells, 50 volume) into the mesenteric vein. Tumors were allowed to grow for 3 weeks and proper metastasis formation was detected macro scopically. Mice having metastatic nodes of more than 20 mm³ were selected for magnetic resonance imaging.

Biodistribution studies

Tumor bearing mice were prepared by transplanting C26 cells into the abdominal region of BALB/c nude mice (female, 6 weeks old, n = 5, Charles River, Japan). When the tumor volume reached 100 mm³, the PEG-AuION were intravenously injected at a dose of 0.8 mgAu/kg. After nanoparticle injection, the tumor and major organs (kidney, liver, muscle, spleen) were collected at 0.25, 0.5, 1, 2, 4, 8 and 24 hours. Blood was collected from the inferior vena cava, heparinized and centrifuged to obtain the plasma. The plasma and all organs were decomposed in aquaresia, evaporated and redissolved in 1 M hydrochloric acid solution to prepare samples for ICP-MS measurement. The area under the curve (AUC) for plasma and tissue Au concentration was calculated based on the trapezoidal rule up to 24 hours.

In vivo MRI

MR imaging of tumors was conducted with a 4.7 T scanner (INOVA200, Varian, Inc., Palo Alto, CA, USA). For T₂-weighted MR images of live mice the following parameters were adopted: point resolution = 234 x 234 / m, section thickness = 2.0 mm, TE - 60 ms, TR = 3000 ms, number of acquisitions = 5. After magnetic nanoparticles (PEG-AuION and Feridex^®, 450 g of Fe) were administered intravenously to the tumor-bearing mice, T₂ weighted MR images of the tumor region were taken at different temporal points (e.g. preinjection, ~ 5 min post-injection and every 15 minutes up to 4 hours of injection).

Histology

The excised samples were fixed overnight in 4 % paraformaldehyde and then paraffin-embedded to prepare them for Haematoxylin & Eosin (H&E) staining. Silver staining of the sections was carried out on de-waxed mounted sections using the Silver Enhancer Kit for Microscopy Applications (Kirkegaard & Perry Laboratories, Inc. Gaithersburg, Maryland, USA). Samples were observed by using an Olympus (Tokyo, Japan) AX80 microscope.

2. Results and Discussion

Synthesis of iron oxide-gold core shell nanoparticles (AuION)

Figure 1 shows the schematic representation for preparation of the PEG- AuION in this study. Synthesis of the nanoparticles performed in two sequential steps in organic solvent at high temperature, which resulted in the formation of highly monodisperse nanoparticles with controlled size, crystallinity and magnetic properties. First, nearly monodisperse y-Fe₂0₃ nanoparticles with average diameter of 10.5 ± 1.6 nm were synthesized by thermal decomposition of Fe(CO)₅ in the presence of capping agents oleylamine and oleic acid (5: 1) under aerobic conditions with a slight modification of a previously reported method [²⁰]. To avoid the formation of y-Fe₂0₃ nanoparticles with different shapes, a small amount of oleic acid was added into the reaction mixture. The crystal structure of y-Fe₂0₃ nanoparticles were confirmed by X- ray diffraction measurement as shown in Figure 2. In the next step, the Au was coated onto the y-Fe₂0₃ nanoparticle surface by reduction of HAuCL_t with oleylamine at 140 °C in an organic solvent where oleylamine functions as both the reducing agent and the stabilizer [¹⁸]. The oleylamine coated AuION thus synthesized was dispersible into organic solvents for further modification.

The formation of Au shell on the y-Fe₂0₃ surface was confirmed by UV-Vis spectroscopy of the nanoparticles in different media. As shown in Figure 3, AuION did not show any characteristic plasmon resonance in organic medium as the AuION is protected by the long chain alkyl ligands. However, when the AuION was transferred to the aqueous medium, a plasmon resonance band appears with absorption at 515 nm, probably due to a change of the dielectric constant in water. The formation of an Au shell on the y-Fe₂0₃ nanoparticles was also confirmed by X-ray diffraction measurement as shown in Figure 2. Evolution of Bragg's diffraction peaks from the fee lattice structure of Au are clearly visible from the X-ray diffraction spectra of AuION. Using the Debye-Scherer equation, the thickness of Au on the y-Fe₂0₃ surface was calculated to be 1.3 nm. TEM studies (Figure 4 (A, B)) showed that the average particle size increased by 1.5 ± 0.6 nm following Au shell deposited onto the y-Fe₂0₃ nanoparticles, which is comparable to the thickness of Au calculated from the X-ray diffraction peaks.

After the single cycle of PEG modification in chloroform followed by the evaporation under vacuum, the PEG-AuION was readily soluble in aqueous medium. However, when the nanoparticles were incubated for 12 hours in 150 mM NaCl solution, agglomeration occurred, suggesting incomplete coating of MeO-PEG-SH on the nanoparticle surface (data not shown). Therefore, nanoparticles were subjected to two PEG coating procedures to achieve a high density PEG coating. No salt-induced agglomeration of the PEG-AuION was observed following transfer to aqueous media through dialysis, suggesting the formation of the appreciably high density PEG layer around the AuION surface. The PEG-AuION stored in 10 mM Tris-HCl buffer containing 0.03 % bovine serum albumin were stable for several months without any notable aggregation. The TEM image of PEG-AuION transferred to water after surface modification with MeO-PEG-SH (Figure 4 (C)) showed no apparent agglomeration. Furthermore, the PEG coating was observed surrounding the AuION core after negative staining with 1 % phosphotungstic acid. Eventually, the thickness of the PEG layer was determined to be 6.5 ± 1.2 nm by TEM.

Synthesis and Characterization of PEG-coated iron oxide-gold core-shell nanoparticles (PEG-AuION)

The PEG-AuION showed a Z-average hydrodynamic diameter of 42 nm as measured by dynamic light scattering (DLS) measurement in 10 mM Tris-HCl buffered saline, as shown in dotted line of Figure 5. The results showed no aggregation of the PEG-AuION under physiological environment, while there was observed an increase of 29 nm in the hydrodynamic diameter compared to AuION dispersed in chloroform (solid line in Figure 5, Z-average size 13 nm, PDI 0.071). This increase could be a contribution of PEG shell surrounding the iron oxide core. The thickness of the PEG layer determined from DLS studies are higher when compared to the thickness observed from TEM studies (Figure 4). These results showed that some AuION might assemble in water. The neutral ζ-potential of 0.49 ± 0.12 mV of the PEG-AuION in water showed the complete passivation of nanoparticle surface with PEG layers. The number of PEG molecules (molecular weight = 2 kDa) on the surface of 12.9 nm AuION was calculated to be ~2500 by TGA analysis of the sample under N₂ atmosphere. This surface coverage corresponds to a footprint area of 0.25 nm² per PEG molecule, significantly lower than previously reports for MeO-PEG-SH conjugated on Au nanoparticle surfaces (2.42 nm²) [²¹-²²]. The dispersion stability of the PEG-AuION against increased NaCl concentration in the solution was evaluated using DLS. The hydrodynamic diameter of PEG-AuION did not change significantly up to 1.5 M NaCl (Figure 6). To examine the stability of the PEGylated nanoparticles under physiological conditions, the PEGylated nanoparticles were incubated them in 10 mM Tris-HCl buffer containing 10 % fetal bovine serum at pH 7.4, 37 °C for 12 hours and then measured the hydrodynamic diameter (Figure 7). The hydrodynamic volume did not change over the 12 hr period, indicating excellent particle stability. The nanoparticles were also stable within the pH range of 3-10. To examine the possibility of using PEG-AuION as a T₂ MRI contrast agent, the relaxivity r₂ of the PEG-AuION was evaluated and the value of which was determined to be 149.32 mM^'V¹ (Figure 8), comparable to the commercially available T₂ contrast agent Feridex^® (dextran-coated iron oxide nanoparticles.).

Coating of MeO-PEG-SH on the AuION was performed in two steps, to ensure the formation of a dense PEG layer on the nanoparticle surface. It has been reported that the process involving repetitive PEG adsorption significantly contribute to increase the PEG surface density on gold surface [²³]. This repetitive process of PEG coating onto the Au surface of the AuION was employed for this study. Repetitive addition of PEG to the AuION in different solvents, first in chloroform and then in methanol led to the formation of highly dense PEG layer on nanoparticle surface. Use of methanol as the solvent for the second cycle of PEG coating also proved to be crucial for controlling particle size and dispersity on the PEGylated nanoparticles. Applying the second PEG coating of the AuION in water resulted in low quality particles with large hydrodynamic diameter [66.4 ±12.2 nm] and high polydispersity index. On the other hand, application of the second PEG coating in methanol followed by transferring the nanoparticles to aqueous medium through dialysis allowed proper control of the hydrodynamic size of the nanoparticle system (~ 40 nm) with a narrow size distribution.

Biodistribution of PEGylated Fe₂0₃-Au core-shell nanoparticles

Figure 9 shows the concentration of gold in the plasma over time after i.v. administration of PEG-AuION. The gold concentration measurement in plasma indicates that PEG-AuION stably circulates in the plasma compartment after i.v. administration with 8 % of the injected dose observed even after 24 hours. Prolonged blood circulation of the PEG-AuION was reasonably associated with high stability of the PEG-AuION at physiological conditions at 37 °C (Figure 7). Accumulation of the PEG-AuION within solid tumor and normal tissues (liver, kidney, spleen and muscle) is also shown in Figure 9. Notably, PEG-AuION showed continuous accumulation with time into solid tumors, while its accumulation to normal tissues was somewhat limited.

To assess the selectivity towards the solid tumors, the area under the Au concentration-time curve (AUC) and AUC ratios of the tumor to normal tissues at 24 hours after injection were determined in Table 1. The PEG-AuION exhibited ratios UQumor/AUCorgan > 1.0 for spleen, kidney, and kidney, indicating selectivity to the tumor (AUCtumor AUCorgan measurement of 2.84, 1.21, and 67.27 with respect to spleen, kidney, and muscle). These AUCtumor AUC₀rgan ratios are comparable to those observed for stealth drug carrier [²⁴]. However, AUC_tumor AUCii_ver ratio is 0.95, indicating no selectivity to the tumor. The bio distribution of PEG-AuION in the liver included a part of circulating PEG-AuION in the blood. Liver has about one-fifth of blood volume in the body. These AUCtumor AUC₀rgan ratios are comparable to those observed for stealth drug carrier [²⁴], which is still rare in the literature as inorganic nanoparticles modified with biocompatible polymers. These results suggest that the PEG-AuION showed an appreciably low uptake to RES located at the organs such as liver and spleen.

Table 1. Accumulation area under the curve (AUC) and AUC ratios between tumor and normal tissues at 24 h after administration of the PEG-AuION.

Tumor selectivity

AUC^a (AUC_ft_/mo;-/ AUC_organ)

Blood 263.54

Tumor 289.79

Liver 304.02 0.953

Spleen 102.16 2.836

Kidney 237.94 1.218

Muscle 4.27 67.27

a AUC denotes the area under a concentration curve that is obtained from the pharmacokinetic study with time points at 0.25, 0.5, 1, 2, 4, 8, and 24 h. Values were calculated on the basis of the trapezoidal rule up to 24 h after intravenous injection. The unit for AUC is defined as % dose/mL plasma*h or % dose/g organ*h for the blood or other tissues (tumor, liver, spleen, kidney, and muscle), respectively.

* Tumor selectivity of the PEG-AuION was determined by calculating the relative accumulated concentrations between the tumor tissues and each organ

In vivo tumor imaging

In order to study the efficacy of PEG-AuION for dynamic in vivo MRI, imaging of tumor tissue was conducted and compared with the commercially available MRI contrast agent Feridex^® (dextran-coated iron oxide nanoparticles). First, in vivo MRI was performed on nude mice bearing subcutaneously inoculated murine colon adenocarcinoma (C26) cells (Figure 10 (a, b)). Negative enhancement in T₂ weighted tumor site gradually increased after 5 min of injection of PEG-AuION with a maximum negative enhancement of 66 % observed 4 hours postinjection (Figure 10 (a) and Figure 11 (a), Table 2). In contrast, Feridex failed to show any negative enhancement in the tumor even after 4 hours (Figure 10 (b) and Figure 11 (a), Table 2), presumably due to high non-specific accumulation in the RES.

Table 2. Percentile rate of negative enhancement in MR signals in the tumor and intestine at 4h post-injection of PEG-AuION and Feridex into mice bearing a subcutaneous model of murine colon adenocarcinoma (C26) cells

Negative enhancement

in MRI signals

Tumor Intestine

PEG-AuION 60% 25%

Next the efficacy of the PEG-AuION system in an orthotopic model of pancreatic cancer was tested using MiaPaCa-2 cell line derived from human pancreatic cancer. T₂-weighed MR images of the tumor region over time are shown in Figure 10 (c, d). Noticeable negative enhancement ( ~ 25 %) was observed only 5 min after nanoparticle administration and continued eyen 4 hours after administration (Figure 10 (c) and Figure 11 (b), Table 3), demonstrating efficient nanoparticle accumulation into tumors. For a control experiment, the effect of Feridex^® on the time-dependent MR imaging in the same tumor model was studied. As shown in Figure 10 (d) and Table 3, Feridex^® failed to show any negative tumor enhancement even 4 hours after administration, indicating poorer particle accumulation into the tumor.

Table 3. Percentile rate of negative enhancement in MR signals in the tumor and intestine at 4h post-injection of PEG-AuION and Feridex into mice bearing an orthotopic model of human pancreatic adenocarcinoma (MiaPaCa-2) cells

Negative enhancement in MRI signals

Tumor (whole) Tumor (arrow head⁰') Intestine

PEG-AuION 25% 47% 18% Feridex 3%

a⁾The rate of negative enhancement of MRI signals in the regions indicated by red arrowhead in Figure 10 (c) was calculated.

In vivo MRI using liver metastasis model

The efficacy of the PEG-AuION system comparing to Feridex^® was tested in a model representing metastatic foci in liver using MiaPaCa-2 cell line. T₂-weighed MR images of the tumor region over time are shown in Figure 12 (A, B). Noticeable negative enhancement ( ~ 25 %) was observed only 5 min after nanoparticle administration and continued even 4 hours after administration (Figure 12 A), demonstrating efficient nanoparticle accumulation into tumors. On the other hand, Feridex^® failed to show any negative tumor enhancement even 4 hours after administration, indicating poorer particle accumulation into the tumor.

To further verify the accumulation of PEG-AuION in the metastatic foci in liver, H&E staining and silver staining of the metastatic foci in liver were performed so as to detect gold, which stains brown. Figure 13 (panels A and B) showed that H&E staining of the metastatic foci in liver. As shown in Figure 13 (panel C), positive staining of the metastatic foci in liver for gold, which was same section as H&E staining shown in Figure 13 (panel B), was obvious in case of administration of the PEG-AuION. The presence of gold was consistent with the MRI results. Gold was observed around the blood vessels.

3. Conclusion

In this study, it is demonstrated that AuION of controlled sub-50 nm size and coated with a dense PEG brush can be used as an MRI contrast agent for variety of tumors, including pancreatic tumor, without attachment of any tumor targeting biomolecules. The relatively small hydrodynamic diameter along with a high PEG density allowed the PEG-AuION to show prolonged circulation. MR images showed that the PEG-AuION accumulates into the pancreatic models through the EPR effect. These results imply that, with proper control of nanoparticle structural and surface properties, it is feasible to develop effective MRI contrast agents for the diagnosis of intractable tumors, such as pancreatic cancer, even without attachment of cell-targeting ligands. The presence of an Au shell on the magnetic nanoparticle not only offers the possibility for surface modification with various biomolecules for biomarker-targeted imaging, but also offers another detection modality through various techniques such as X-ray tomography [^25~26] (CT) and surface enhanced Raman scattering (SERS) [²⁷]. The ultimate goal of nanoparticle medical research is to develop high performance nanoparticle systems for both detection and treatment of biological events such as cancer metastasis and real time visualization of biological events at the cellular and molecular level, leading to better prognosis in patients bearing tumor.

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[28] Japanese Unexamined Patent Publication No. 2008-266214

Claims

1. A composite particle comprising as a core a magnetic iron-oxide particle, a gold shell on said magnetic iron-oxide particle, and a poly(alkylene glycol) coating on said gold shell, wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.05-1.0 nm²/poly(alkylene glycol) molecule.

2. The composite particle of claim 1, wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.125-0.7 nm /poly(alkylene glycol) molecule.

3. The composite particle of claim 1, wherein said poly(alkylene glycol) is conjugated to said gold shell at a density of 0.2-0.4 nm /poly(alkylene glycol) molecule.

4. The composite particle of claim 1, having an average diameter of no more than 60 nm.

5. The composite particle of claim 1, having an average diameter of 10-50 nm.

6. The composite particle of claim 1, having an average diameter of 15-40 nm.

7. The composite particle of claim 1, wherein said magnetic iron-oxide is one selected from a group consisting of Fe₃0₄, y-Fe₂0₃, MnFe₂0₃ and ferrite.

8. The composite particle of claim 1, wherein said poly(alkylene glycol) is one selected from a group consisting of poly(methylene glycol), poly(ethylene glycol) and poly(propylene glycol).

9. The composite particle of claim 1, wherein said poly(alkylene glycol) molecule is conjugated to said gold molecule by thiol-gold coupling.

10. The composite particle of claim 1, wherein said poly(alkylene glycol) has an average molecular size in a range of 500-5000 dalton.

11. The composite particle of claim 1, which produces a negative enhancement of more than one type of cancer selected from the group consisting of brain tumor, pharyngeal cancer, lung cancer, breast cancer, esophageal cancer, stomach cancer, pancreatic cancer, biliary cancer, duodenal cancer, colon cancer, liver cancer, uterus cancer, ovarian cancer, prostate cancer, renal cancer, bladder cancer, rhabdomyosarcoma, fibrosarcoma, osteosarcoma, chondrosarcoma and cutaneous cancer.

12. The composite particle of claim 1, for use in a method for diagnosing more than one type of cancer selected from the group consisting of pancreatic cancer, colon cancer, renal cancer, liver cancer, lung cancer, prostate cancer, breast cancer, stomach cancer, brain tumor and ovarian cancer.

13. A method of producing the composite particle of claim 1, comprising the steps of:

applying a gold shell onto a magnetic iron-oxide particle; and

applying a poly(alkylene glycol) coating onto said gold shell at a density of 0.05-1.0 nm /poly(alkylene glycol) molecule.

14. The method according to claim 13, wherein the application of said gold shell onto said magnetic iron-oxide particle is performed by a reduction of a gold-complex in a solution containing said magnetic iron-oxide particle, said gold complex and a reducing agent selected from the group consisting of oleylamine, oleic acid, dodecanoic acid, stearic acid, octylamine, NaBH₄, dodecylamine and dodecanethiol.

15. The method according to claim 13, wherein the application of said poly(alkylene glycol) coating onto said gold shell is repeated at least once.

16. The method according to claim 13, wherein said poly(alkylene glycol) molecule is conjugated to said gold molecule by thiol-gold coupling.

17. An MRI contrasting agent comprising the composite particle according to any one of claims 1 to 11.

18. The MRI contrasting agent of claim 17, wherein said composite particle maintains an average diameter of no more than 60 nm in a solution having an NaCl concentration of 0- 1.5M.

19. A method of diagnosing cancer comprising the steps of:

administering the MRI contrasting agent of claim 17 to a subject;

obtaining a magnetic resonance image of said subject using an MRI system; and

detecting a tumor tissue from said magnetic resonance image.

20. The method according to claim 19, wherein said magnetic resonance image is T₂-weighted MRI.