EP1934609A4

EP1934609A4 - Magnetic nanoparticle having fluorescent and preparation method thereof and use thereof

Info

Publication number: EP1934609A4
Application number: EP06798700A
Authority: EP
Inventors: Jin-Kyu Lee; Myung-Haing Cho; Seung-Bum Park; Tae-Jong Yoon; Jun-Sung Kim; Byung-Geol Kim; Kyeong-Nam Yu
Original assignee: BITERIALS CO Ltd
Current assignee: BITERIALS Co Ltd
Priority date: 2005-09-08
Filing date: 2006-09-08
Publication date: 2009-06-24
Also published as: EP1934609A1; CA2621352A1; AU2006288048A1; WO2007029980A1

Abstract

Disclosed herein are magnetic nanoparticles (MNPs) having fluorescence, and preparation and use thereof. The magnetic nanoparticles according to the present invention have both optical and magnetic properties and are therefore applicable to a variety of bio-fields. Via the surface treatment of silica shells of the magnetic nanoparticles with a water-soluble material, a variety of chemical functional groups can be introduced into nano-scale materials. Further, via the use of the thus-chemically modified nano-scale materials, it is possible to increase or decrease the pene¬ trability of the magnetic nanoparticles into cells, and it is also possible to impart a selectivity to act only on desired specific cells.

Description

MAGNETIC NANOPARTICLE HAVING FLUORESCENT AND PREPARATION METHOD THEREOF AND USE THEREOF

Technical Field

[1] The present invention relates to magnetic nanoparticles (MNPs) having fluorescence, and preparation and use thereof. Background Art

[2] Magnetic materials are important in conventional biological applications including medical diagnoses and bio-sensors. Therefore, a great deal of research and study has been recently focused on cell staining using nanoparticles (bio-imaging), cell separation, in vivo drug delivery and in vivo gene delivery. In particular, approach toward bio-fields utilizing nanoparticles has received a great deal of attention, starting from the study of external determination of the fluorescence of quantum dots via intracellular uptake of the quantum dots using luminescent quantum dot nanoparticles (see US Patent No. 6194213, issued to Barbera-Guillem Emilio, entitled "Lipophilic, Functionalized Nanocrystals and Their Use for Fluorescence Labeling of Membranes"; and US Patent No. 6306610, issued to Bawendi Moungi G., Mikulec Frederic V., and Sundar Vikram C, entitled "Biological applications of quantum dots").

[3] However, since most of nanoparticles including quantum dots are composed of heavy metals such as cadmium (Cd), zinc (Zn), cobalt (Co) and the like, surfaces of the synthesized nanoparticles should be made biocompatible, in order to enhance the applicability thereof to bio-fields. Therefore, various attempts for the surface treatment of the nanoparticles have been actively undertaken, such as for example, an increased hy- drophilicity of the nanoparticles as well as an increased in vivo circulation time, by the introduction of an inorganic or organic compound such as silica (SiO ) or polyethylene glycol (PEG), which is known to be non-toxic to living organisms, into the surface of the synthesized nanoparticles (Shuming Nie et al., In vivo Cancer Targeting And Imaging With Semiconductor Quantum Dots Nat. Biotechnol., 2004 (22), 969).

[4] However, such a conventional synthesis technique of quantum dots involves very complicated and sophisticated synthesis conditions and suffers from a problem of a very low overall yield through a surface treatment process.

[5] In recent years, an attempt has been made which is directed to a study of recognizing cancer cells by the introduction of an antibody into the surface of the quantum dots, such that a certain cancer cell can be bound thereto. The detection and localization method of light emitting from the quantum dots, which is one of the most difficult points in this study, is of significance for in vitro studies, but suffers from limitations for in vivo studies. This is because there is a difficulty associated with the detection of light emitted from the quantum dots, due to barriers of deep and thick biological tissues (Mark Stroh et al., "Zooming In and Out With Quantum Dots", Nat. Biotechnol., 2004 (22), 959).

[6] As another approach to overcome the problems as discussed above, a study has been made on magnetic nanoparticles. This is because the in vivo introduction of the magnetic nanoparticles facilitates the detection of magnetic properties of a magnetic material by application of a strong external magnetic field, such as by magnetic resonance imaging (MRI) (see US Patent No. 5565215, issued to Gref Ruxandra et al., entitled "Biodegradable Injectable Particles for Imaging").

[7] Hence, domestic and foreign research and study groups and institutes have recently made many efforts to overcome the problems associated with the approach toward bio- fields utilizing quantum dots, for example by synthesizing nanoparticles having magnetic properties and introducing a silica shell thereto to be suited for bio-fields. Unfortunately, such a method utilizing the strong magnetic field from the outside, as opposed to the surface treatment of nanoparticles, is not easily applicable to the in vitro studies such as cell research.

[8] Another conventional field using magnetic properties has employed a material (in the form of pudding) having the incorporation of several magnetic nanoparticles into a polymer agglomerate having a size ranging from 300 nm to several D. Introduction of a certain compound such as vancomycin into the surface of the above-treated material has presented the possibility to recognize and separate certain bacteria by application of the external magnetic filed. However, organic polymers have in vivo toxicity and are not suitable for circulation via blood vessels due to an excessively large size of the resulting material, thereby posing problems associated with the in vivo studies.

[9] In addition, such a material having an organic polymer shell must undergo a very complicated synthesis process in order to achieve a desired level of surface treatment and therefore suffers from various limitations in application thereof. That is, the size and surface treatability of the synthesized material are very important factors that should be considered to ensure application potential such as in vivo drug delivery and gene delivery.

Disclosure of Invention Technical Problem

[10] Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide magnetic nanoparticles having fluorescence.

[11] It is another object of the present invention to provide magnetic nanoparticles wherein magnetic nanoparticles having fluorescence are bound to negatively charged genes or nucleic acid molecules, and a gene delivery system comprising the same.

[12] It is a further object of the present invention to provide magnetic nanoparticles wherein magnetic nanoparticles having fluorescence are bound to negatively charged nucleic acid molecules, and a gene delivery system comprising the same .

[13] It is a still further object of the present invention to provide magnetic nanoparticles wherein magnetic nanoparticles having fluorescence are bound to antibodies, and a cell staining agent comprising the same.

[14] It is yet another object of the present invention to provide a method for preparing the above-mentioned magnetic nanoparticles. Technical Solution

[15] That is, as a result of a variety of extensive and intensive studies and experiments to develop magnetic nanoparticles which are capable of solving the problems as described above and can be used in both in vivo and in vitro applications, the inventors of the present invention have succeeded in synthesis of magnetic nanoparticles having a silica shell modified with polyethylene glycol (PEG) to impart biocompatibility and published (Tae-Jong Yoon et al., "Multifunctional Nanoparticles Possessing a Magnetic Motor Effect for Drug or Gene Delivery", Angew. Chem. In. Ed. 2005 (44), 1068-1071). However, PEG has no electric charge and thus has suffered from a difficulty in the binding with electrically charged biomolecules such as DNA molecules.

[16] To this end, the inventors of the present invention have investigated a method of modifying a surface of magnetic nanoparticles, which contain organic fluorescent materials and are coated with silica shells, with an electrically charged material. As a result, we have synthesized magnetic nanoparticles coated with silica containing the organic fluorescent materials and having a surface modified with the electrically charged material, and have confirmed that upon introduction of such magnetic nanoparticles into cells, it is possible to locate and control the introduced nanoparticles by application of an external magnetic field, and simultaneously it is also possible to efficiently apply such particles to both in vivo and in vitro studies via easy and convenient detection of the fluorescence. The present invention has been completed based on these findings.

[17] In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of magnetic nanoparticles having a core containing a magnetic material and a surface-modified silica shell containing an organic fluorescent material and coated on the core, wherein the nanoparticles have a size of less than 100 nm and are water-soluble. [18] In accordance with another aspect of the present invention, there are provided magnetic nanoparticles wherein the magnetic nanoparticles are bound to negatively charged genes or nucleic acid molecules, and a gene delivery system comprising the same.

[19] In accordance with a further aspect of the present invention, there are provided magnetic nanoparticles wherein magnetic nanoparticles having fluorescence are bound to negatively charged nucleic acid molecules, and a gene delivery system comprising the same.

[20] In accordance with a still further aspect of the present invention, there are provided magnetic nanoparticles wherein magnetic nanoparticles having fluorescence are bound to antibodies, and a cell staining agent comprising the same.

[21] In accordance with yet another aspect of the present invention, there is provided a method for preparing the above-mentioned magnetic nanoparticles.

Advantageous Effects

[22] Magnetic nanoparticles according to the present invention have both optical and magnetic properties and are applicable to bio-fields. By the high hydrophilicity of the nanoparticles and a simple chemical surface treatment technique, chemical functional groups can be introduced into nano-scale materials, using a variety of compounds. Use of the thus-chemically modified nano-scale materials can lead to an increased or decreased penetrability of magnetic nanoparticles into cells. Further, the magnetic nanoparticles can be usefully used as a gene delivery system by transfer of a desired plasmid DNA into a target cell using nano-scale materials having a positive charge, and can also be usefully used in cell staining, based on a technique which is capable of performing selective binding of nanoparticles to certain cells and recognition of the nanoparticle-bound cells, using an appropriate surface treatment technique. In addition, the selectively recognized cells can be separated and purified by application of a strong external magnetic field. Brief Description of the Drawings

[23] The above and other 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:

[24] FIG. 1 is a view showing a process for preparing magnetic nanoparticles

(MNP @ SiO (RITC or FITC)) containing organic fluorescent materials and coated with silica shells;

[25] FIGS. 2A to 2C are transmission electron micrographs (TEMs) of magnetic nanoparticles (MNP @ SiO (RITC or FITC)) containing organic fluorescent materials and coated with silica shells; [26] FIG. 3 is a view showing a chemical treatment process of a surface of magnetic nanoparticles (MNP @ SiO (RITC)) according to the present invention with various silicon compounds;

[27] FIG. 4 is a graph showing zeta-potential for the measurement of changes in a surface charge of all the magnetic nanoparticles, due to various surface treatments of magnetic nanoparticles (MNP@SiO (RITC)) according to the present invention (Black: Non-surface treated MNP@SiO (RITC), Red: (CH O) Si-PEG-surface treated MNP @ SiO (RITC)-PEG, Light green: (CH O) Si-PMP-surface treated MNP @ SiO (RITC)-PMP, and Blue: (CH O) Si-PTMA-surface treated MNP@SiO (RITC)-PTMA);

[28] FIGS. 5A to 5D are confocal laser scanning micrographs showing a penetration rate of MNP @ SiO (RITC)-PEG, MNP @ SiO (RITC)-PTMA, MNP @ SiO (RITC) and MNP @ SiO (RITC)-PMP into breast cancer cells;

[29] FIGS. 6 A to 6H are confocal laser scanning micrographs showing intracellular location of nanoparticles, upon injection of MNP@Si0 (RITC)-PEG and MNP@Si0 (RITC)-PMP into breast cancer cells at the same amounts under the same conditions (6A to 6D: Micrographs for injection of MNP@Si0 (RITC)-PEG, and 6E to 6H: Micrographs for injection of MNP @ SiO (RITC)-PMP; 6A and 6E: Red fluorescence micrographs, 6B and 6F: Optical micrographs, 6C and 6G: Fluorescence micrographs confirming DAPI nuclear staining, and 6D and 6F: Overlapping micrographs of 6A to 6C and 6E to 6G, respectively);

[30] FIG. 7 is a bar graph showing results of cytotoxic test (MTT assay) after treatment of MNP@SiO (RITC), MNP@SiO (RITC)-PEG, MNP@SiO (RITC)-PMP, and MNP @ SiO (RITC)-PTMA on a breast cancer cell line (MCF-7), a lung cancer cell line (A549) and a normal (non-malignant) lung epithelial cell line (NL20), respectively;

[31] FIG. 8 is a view showing a process using MNP @ SiO (RITC)-PTMA as a gene delivery system, by binding of it to a plasmid DNA;

[32] FIGS. 9 A to 9D are confocal laser scanning micrographs of transfected cells after gene delivery using plasmid DNA-bound MNP @ SiO (RITC)-PTMA (9A: Blue fluorescence micrograph, 9B: Optical micrograph, 9C: Red fluorescence micrograph, and 9D: Overlapping micrograph of 9 A, 9B and 9C);

[33] FIG. 10 is a view showing a process of co-treating a surface of MNP@Si0 (FITC) with (CH O) Si-PEG and 3-aminopropyltriethoxysilane (APS), introducing a maleimide group into an amine group of the MNP @ SiO (FITC) surface, and introducing an antibody for recognition of a certain cell thereto;

[34] FIGS. 1 IA to 1 ID are confocal laser scanning micrographs showing utilization of antibody-bound MNP@Si0 (FITC)-PEG/APS-MaI in cell staining (HA: Blue fluorescence micrograph, HB: Optical micrograph, HC: Red fluorescence micrograph, and 1 ID: Overlapping micrograph of 1 IA, 1 IB and HC); wherein a material penetrated into cells is MNP @ SiO (RITC) emitting red fluorescence, and a cell membrane-bound material is MNP @ SiO (FITC)-PEG/APS-MaI-Her2 emitting blue

Ab fluorescence (hereinafter, referred to as MNP @ SiO (FITC)-Her2 ));

2 Ab

[35] FIGS. 12A to 12F are micrographs showing a selectivity of MNP@SiO

(FITC)-CDlO , having a CD-IO antibody, being capable of selectively binding to a

Ab leukemia cell (SP2/O) membrane, introduced into a blue nanoparticle, wherein MNP @ SiO (FITC)-CD 10 selectively binds to a cell wall of a leukemia cell (SP2/O)

2 Ab

(12A to 12C), but does not bind to a lung cancer cell (A549) (12D to 12F); [36] FIGS. 13A and 13B are optical micrographs showing that MNP@SiO

(FITC)-CDlO is selectively recognized by a cell wall of a leukemia cell and is then

Ab attracted by an external magnetic field (A: non- application of external magnetic field, and B: application of external magnetic field to a red dotted line region, thus causing the movement of cells to a specific location); and

[37] FIG. 14 shows results of MRI analysis at predetermined time intervals after intraperitoneal injection of MNP @ SiO (RITC) into mice, wherein a control is a micrograph of a mouse with no injection of magnetic nanoparticles, and the remainder are micrographs taken 15 min, 30 min, 1 hour, 1 day and 3 days after synthesized magnetic nanoparticles were injected into mice. Best Mode for Carrying Out the Invention

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

[39] Magnetic nanoparticles of the present invention contain a magnetic material inside the particle and the outside of the core thereof is coated with a non-magnetic silica shell containing an organic fluorescent material and having a surface modified with an electrically charged material. Therefore, the magnetic nanoparticles of the present invention have both optical and magnetic properties and can be applied to a variety of bio-fields.

[40] The magnetic nanoparticles of the present invention can be prepared by a method comprising the steps of:

[41] 1) treating a surface of water-soluble magnetic nanoparticles with a polyvinylpyrrolidone (PVP) polymer, converting the nanoparticles into ethanol- dispersible particles, followed by centrifugation;

[42] 2) dispersing the polymer- stabilized magnetic nanoparticles separated in Step 1 in ethanol for silica coating;

[43] 3) adding to the solution prepared in Step 2 a solution of an organic fluorescent material treated with 3-aminopropyltriethoxysilane (APS) and a solution of tetraethoxysilane (TEOS), and adding NH OH to a mixed solution to thereby induce

4 formation of silica on the surface of the magnetic nanoparticles containing the organic fluorescent material; and

[44] 4) treating the surface of the silica shells of the magnetic nanoparticles obtained in

Step 3 with a silicon compound.

[45] Hereinafter, the method for preparing the magnetic nanoparticles of the present invention will be described in more detail according to the corresponding steps.

[46] In Step 1, the water-soluble magnetic nanoparticles may be prepared according to any conventional method known in the art, such as wet, dry or vacuum method. Examples of such a method may include, but are not limited to, grinding of large size materials, precipitation from a solution, co-precipitation, microemulsification, polyol process, high-temperature degradation of organic precursors, solution techniques, aerosol/bubble methods, spray pyrolysis, plasma atomization and laser pyrolysis. Preferably, the water-soluble magnetic nanoparticles of the present invention may be prepared by co-precipitation.

[47] The water-soluble magnetic nanoparticles are composed of cobalt (Co) and iron

(Fe) oxides, and may include an oxide of a transition metal such as manganese (Mn), zinc (Zn), nickel (Ni), copper (Cu) or the like.

[48] In Step 3, the organic fluorescent material is preferably Rhodamine B isoth- iocyanate (RITC) or fluoresceine isothiocyanate (FITC), but is not limited thereto and may include chemical modifications of the existing organic fluorescent materials. For example, mention may be made of Alexa Fluor, Rhodamine Red-X, Texas Red, Tetramethylrhodamine, Cascade Blue, DAPI (4',6-diamidino-2-phenylindole), coumarine, Lucifer Yellow and Dansylaminde.

[49] An increase in an amount of TEOS, a raw material of silica in the present invention, leads to an increase in the thickness of the silica shells of the magnetic nanoparticles. Therefore, it is possible to control the size of the magnetic nanoparticles by controlling the amount of TEOS.

[50] In Step 4, the silicon compound used in the surface modification of the silica shell is preferably an electrically charged material, i.e. an organic silicon compound having ionic functional group(s). For example, such an organic silicon compound may include specific functional compounds such as ionic compounds, water soluble compounds and drugs, to which a (CH O) Si- functional group is introduced. Specifically, the silicon compound that can be used in the present invention may include, but is not limited to, one compound selected from the group consisting of (CH O) Si-PEG [(CH O) SiCH CH 2 CH 20(CH 2 CH 2 O) 6-9 CH 3 ], (CH 3 O) 3 Si-PMP [(CH 3 O) 3 SiCH 2 CH 2 CH 2 PO 2 (OCH 3 )Na],

(CH 3 O) 3 Si-PTMA [(CH 3 O) 3 SiCH 2 CH 2 CH 2 N⁺(CH 3 ) 3 Cl^"] and

3 - aminopropyltriethoxy silane (APS ) . [51] The magnetic nanoparticles according to the present invention exhibit no toxicity for all kinds of cells including a breast cancer cell line (MCF-7), a lung cancer cell line (A549) and a normal (non-malignant) lung epithelial cell line (NL20).

[52] Upon exposure of the cells to the external magnetic field following the uptake of the magnetic nanoparticles of the present invention into the cells, the number of the magnetic nanoparticles is not sufficient to cause cytotoxicity, but is enough to provide cells exhibiting magnetically induced movement. The above-mentioned cell may include eukaryotic cells, human cells, animal cells and plant cells. The magnetic particles may have an average particle size of less than about 100 nm, particularly about 30 to 80 nm.

[53] The cells to which the magnetic nanoparticles were penetrated are moved at a rate of 0.5 to 1 mm/sec by application of the external magnetic field (ca. 0.3 tesla, T). The external magnetic field strength and the moving rate are not limited to the above- specified range.

[54] On the other hand, the magnetic nanoparticles according to the present invention, coated with silica shells containing an organic fluorescent material and having a surface modified with the electrically charged material, can be used for various applications, by binding the surface of the surface-modified silica shell to various materials such as negatively charged genes or nucleic acid molecules and antibodies.

[55] For example, on the surface of the surface-modified silica shells of the magnetic nanoparticles according to the present invention, positively charged MNP @ SiO (RITC)-PTMA can be bound to the negatively charged genes.

[56] The magnetic nanoparticles bound to the negatively charged genes can be prepared by a method comprising the steps of:

[57] 1) introducing and incubating negatively charged genes and positively charged

MNP @ SiO (RITC)-PTMA in an HEPES [N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid)] buffer solution;

[58] 2) adding CaCl to the solution incubated in Step 1 and further incubating the solution for 2 hours; and

[59] 3) adding Dulbecco's Modified Eagle Medium (DMEM) to the incubated solution of Step 2, adjusting a Ca ⁺ ion concentration of the solution to 4.5 mM, further incubating the solution for 4 hours and washing the solution with a phosphate-buffered saline (PBS) solution.

[60] When the magnetic nanoparticles bound to the negatively charged genes {plasmid

DNA-[MNP@SiO (RITC)-PTMA] } enter the target cells, they pass through the cell membrane and deliver the negatively charged genes into the cells and are then separated from the genes and remain as the magnetic nanoparticles in the cytoplasm (red fluorescence). In addition, it can be confirmed that a blue protein was synthesized in the cytoplasm by the delivered DNA (see FIG. 8). [61] Preferably, the negatively charged gene includes, but is not limited to, a plasmid

DNA, specifically pcDNA3.1/CT-GFP. Therefore, the magnetic nanoparticles according to the present invention may be bound to various genes.

[62] The magnetic nanoparticles according to the present invention may also be bound to negatively charged nucleic acid molecules, in addition to negatively charged genes.

[63] Hence, the magnetic nanoparticles according to the present invention may be usefully used as a gene delivery system, by attachment of the particles to the negatively charged genes or nucleic acid molecules.

[64] Further, via the introduction of an antibody into the surface of the surface-modified silica shells of the magnetic nanoparticles according to the present invention, the magnetic nanoparticles may be selectively bound to certain cells. The magnetic nanoparticles-introduced cells may be separated via the induction of a movement thereof by application of an external magnetic field.

[65] The antibody-bound magnetic nanoparticles can be prepared by a method comprising the steps of:

[66] 1) co-treating a surface of magnetic nanoparticles containing organic fluorescent materials with Si-PEG/3-aminopropyltriethoxysilane (APS);

[67] 2) reacting the magnetic nanoparticles obtained in Step 1 with maleimidobutyric acid, thereby introducing a maleimide (MaI) group into an amine group present on the surface of the silica shells of the magnetic nanoparticles;

[68] 3) reacting an antibody with 2-mercaptoethylamine to form an antibody having a thiol group; and

[69] 4) binding the antibody obtained in Step 3 to the maleimide (MaI) group present on the surface of the silica shells of the magnetic nanoparticles obtained in Step 2.

[70] Examples of the antibody that can be used in Step 4 may include CD-10 antibody against leukemia cells and Her2 antibody against breast cancer cells. However, the antibodies that can be used in the present invention are not limited thereto, and therefore may include antibodies of various cells including stem cells.

[71] Therefore, the magnetic nanoparticles according to the present invention may be usefully used as a cell staining agent, by binding of the nanoparticles to the antibodies of interest.

[72] In addition, the magnetic nanoparticles according to the present invention are observed as a black magnetic signal in the liver of mice, following intraperitoneal administration of the nanoparticles into the animals.

[73] Hence, the magnetic nanoparticles according to the present invention may be usefully used in cell staining (bio-imaging), cell separation, in vivo drug delivery and in vivo gene transfer.

[74] Further, the magnetic nanoparticles according to the present invention may be used as an assay reagent which is capable of simultaneously performing fluorescence analysis and MRI analysis. Mode for the Invention

[75] EXAMPLES

[76] Now, the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

[77]

[78] Example 1: Preparation of magnetic nanoparticles according to the present invention

[79] 1. Preparation of magnetic nanoparticles containing organic fluorescent materials and coated with silica shells

[80] 34.7 mL of an aqueous cobalt ferrite magnetic nanoparticle solution (20 mg/mL) was added to 0.65 mL of an aqueous polyvinylpyrrolidone (PVP) solution (concentration of 25.6 g/L) and stirred at room temperature for 1 day. A solution of water and acetone in a ratio of 1 : 10 was added to the solution of magnetic nanoparticles stabilized with polyvinylpyrrolidone, and the resulting mixed solution was centrifuged at 4000 rpm for 10 min. A supernatant was decanted, and precipitated nanoparticles were re-dispersed in 10 mL of ethanol. To the resulting dispersion were added a solution of an organic fluorescent material, e.g. RITC (Rhodamine B isoth- iocyanate) or FITC(fluoresceine isothiocyanate) treated with 3-aminopropyltriethoxysilane (APS), and a solution of tetraethoxysilane (TEOS) in ethanol (mole ratio of 0.04:0.3). 0.86 mL of NH OH containing 30% by weight of NH was added to the mixed solution, thereby inducing the formation of silica on the surface of the magnetic nanoparticles. The magnetic nanoparticles coated with the organic fluorescent material-containing silica shells were centrifuged at 18,000 rpm for 30 min, using a high-speed centrifuge, and the precipitates were purified by water and ethanol washing. The resulting material was readily dispersible in water or an alcohol.

[81] FIG. 1 shows a process for preparing the magnetic nanoparticles coated with the organic fluorescent material-containing silica shells (MNP @ SiO (RITC or FITC)), and FIG. 2 shows TEMs of the magnetic nanoparticles coated with the organic fluorescent material-containing silica shells (MNP @ SiO (RITC or FITC)).

[82] As shown in FIG. 2, an increase in an amount of tetraethoxysilane (TEOS) used as a raw material of silica has led to an increase in a size of the magnetic nanoparticles. Therefore, it can be seen that the shell thickness of the magnetic nanoparticles can be adjusted to the desired range by controlling the amount of TEOS. [83]

[84] 2. Preparation of magnetic nanoparticles coated with silica shells containing organic fluorescent materials and having a surface modified with electrically charged material

[85] 45 mg of magnetic nanoparticles (MNP@SiO (RITC)) prepared in Section 1 was dispersed in 10 rnL of ethanol, each 0.02 mmol silicon compound [125 mg of (CH O) Si-PEG, (CH O) SiCH CH CH 0(CH CH O) CH ; 238 mg of (CH O) Si-PMP, (CH

3 3 2 2 2 2 2 6-9 3 3 3 3

O) SiCH CH CH PO (OCH )Na; and 257 mg of (CH O) Si-PTMA, (CH O) SiCH CH

3 2 2 2 2 3 ^& 3 3 3 3 2 2

CH N⁺(CH ) Cl ] was added to the dispersion, and the acidity of the solution was then adjusted to a pH of 12 using NH OH. The solution was vigorously stirred at 6O⁰C for 3

4 hours. Then, using a high speed centrifuge, the solution was rotated at 18,000 rpm for 30 min to thereby precipitate the surface-treated nanoparticles. Excessive amounts of silicon compounds remained in the filtrate. The precipitated nanoparticles were washed three times with water and ethanol, purified and separated. The thus-prepared nanoparticles exhibited a fairly high stability in water.

[86] FIG. 3 shows a chemical treatment process of a surface of magnetic nanoparticles according to the present invention with various silicon compounds, and FIG. 4 shows a graph of zeta-potential for the measurement of changes in a surface charge of all the magnetic nanoparticles, due to various surface treatments of magnetic nanoparticles according to the present invention.

[87] As shown in FIGS. 3 and 4, MNP@SiO (RITC) (Black line), representing a non- surface treatment, exhibited a charge value of -16.8 mV, (CH O) Si-PEG-surface treated MNP @ SiO (RITC)-PEG (Red line) exhibited a charge value of 2.4 mV, (CH O) Si-PMP-surface treated MNP@SiO (RITC)-PMP (Light green) exhibited a charge value of -50 mV, and (CH O) Si-PTMA-surface treated MNP @ SiO (RITC)-PTMA (Blue line) exhibited a charge value of +35.7 mV.

[88]

[89] Experimental Example 1: Cell penetrability of magnetic nanoparticles according to the present invention

[90] In order to examine the cell penetrability of magnetic nanoparticles according to the present invention, the following experiments were carried out.

[91] A breast cancer cell line (MCF-7) was purchased from ATCC (American Type

Culture Collection). The breast cancer cell line was cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 40 D of 10% fetal bovine serum (FBS), and 2 mg/mL of non- surface treated magnetic nanoparticles [MNP@SiO (RITC)] and 2 mg/ mL of silicon-surface treated magnetic nanoparticles [MNP @ SiO (RITC)-PEG, MNP @ SiO (RITC)-PMP, or MNP @ SiO (RITC)-PTMA], prepared in Example 1. AU the cells were cultured in a Lab-Tek glass chamber slide and observed under a confocal laser scanning microscope (CLSM).

[92] Upon injection of the non-surface treated magnetic nanoparticles [MNP @ SiO

(RITC)] and the silicon-surface treated magnetic nanoparticles [MNP @ SiO (RITC)-PEG, MNP@SiO (RITC)-PMP or MNP@SiO (RITC)-PTMA] into the breast cancer cells at the same amounts under the same conditions, the penetrability of the nanoparticles into the cells is shown in FIG. 5. In addition, upon injection of MNP @ SiO (RITC)-PEG and MNP @ SiO (RITC)-PMP into the breast cancer cells at the same amounts under the same conditions, the penetrability of the nanoparticles into the cells is shown in FIG. 6.

[93] As shown in FIG. 5, upon injection of the silicon-surface treated magnetic nanoparticles into the cells at the same amounts under the same conditions, the magnitude in the cell penetrability of the nanoparticles was measured in the order of MNP@SiO (RITC)-PEG > MNP@SiO (RITC)-PTMA = MNP@SiO (RITC) > MNP @ SiO (RITC)-PMP.

[94] In addition, as shown in FIG. 6, 6A to 6D are micrographs for injection of

MNP @ SiO (RITC)-PEG, and 6E to 6H are micrographs for injection of MNP @ SiO (RITC)-PMP. 6A and 6E are red fluorescence micrographs, 6B and 6F are optical micrographs, 6C and 6G are fluorescence micrographs confirming DAPI nuclear staining, and 6D and 6F are overlapping micrographs of 6A to 6C and 6E to 6G, respectively. The (CH O) Si-PEG-surface treated magnetic nanoparticles have neutral electrical properties and are therefore irregularly distributed in the cytoplasm upon penetration thereof into cells, whereas the (CH O) Si-PMP-surface treated magnetic nanoparticles have anionic properties and are therefore exist around the nuclear membrane.

[95] That is, since the magnetic nanoparticles of the present invention coated with silica shells containing the organic fluorescent materials and having a surface modified with the electrically charged material have different locations in the cells, depending upon kinds of modifying components, it is possible to induce changes in the intracellular location of the nanoparticles by using the surface charge of the magnetic nanoparticles according to the present invention.

[96]

[97] Experimental Example 2: Cytotoxic test (MTT assay)

[98] In order to examine cytotoxicity of magnetic nanoparticles according to the present invention, the following experiments were carried out.

[99] A breast cancer cell line (MCF-7), a lung cancer cell line (A549) and a normal

(non-malignant) lung epithelial cell line (NL20) were purchased from ATCC (American Type Culture Collection). The MCF-7 cell line was cultured in DMEM (Dulbecco's Modified Eagle's Medium) containing 40 D of 10% fetal bovine serum (FBS) and 2 mg/mL of nanoparticles according to the present invention. The A549 cell line and the NL20 cell line were cultured in RPMI containing 10% FBS, 2 rnM L- glutamine, 1 mM sodium pyruvate, 1 non-essential amino acids and 5 mM 2-mercaptoethanol under the same culture conditions. All the cell lines were cultured in a Lab-Tek glass chamber slide to facilitate observation under a confocal laser scanning microscope (CLSM).

[100] Each cell line was cultured in a 96- well plate, and at the end of incubation, 50 D of

MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] was added to each well and phosphate-buffered saline (PBS, 0.2 mg/mL, pH 7.2) was then added to make a final MTT concentration of 0.4 mg/mL. Cells were further incubated in a 5 % CO environment at 37⁰C for 4 hours. The culture medium was carefully removed by pipetting, and formazan crystals, formed by the action of the mitochondrial dehydrogenase which is responsible for cellular respiration of viable cells, were dissolved in 150 D of DMSO. The resulting solution was stirred for about 10 min using a stirrer, and an optical density (OD) was measured at 490 nm and 620 nm, respectively.

[101] The results thus obtained are given in FIG. 7.

[102] As shown in FIG. 7, the magnetic nanoparticles according to the present invention exhibited no cytotoxicity for all kinds of cells [a breast cancer cell line (MCF-7), a lung cancer cell line (A549) and a normal (non-malignant) lung epithelial cell line (NL20)].

[103]

[104] Example 2: Preparation of plasmid DNA-bound MNP @ SiO (RITCVPTMA

[105] As the plasmid DNA gene, pcDNA3.1/CT-GFP was used.

[106] The plasmid DNA and MNP@SiO (RITC)-PTMA were placed in 30 D of an

HEPES [N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid)] buffer solution (pH of ca. 7.4), and the resulting hybridization product was incubated at 4⁰C for 2 hours, followed by addition of 30 D of 100 mM CaCl The resulting solution was incubated for another 2 hours, and transferred to a 24- well plate. Thereafter, 0.6 mL of DMEM was added thereto, and a concentration of Ca ⁺ ions was adjusted to 4.5 mM. After further incubation at 37⁰C for 4 hours, the DNA-bound nanoparticles were washed with a PBS solution. The DNA-bound nanoparticles were added to cells and gene delivery signals were observed.

[107] FIG. 8 shows a process using, as a gene delivery system, MNP@SiO

(RITC)-PTMA, obtained by surface treatment of the magnetic nanoparticles according to the present invention with (CH O) Si-PTMA, via binding of it to plasmid DNA. FIG. 9 shows confocal laser scanning micrographs of the transfected cells after the gene delivery using the plasmid DNA-bound MNP @ SiO (RITC)-PTMA.

[108] As shown in FIG. 8, once entered into the cells, the positively charged nanoparticles (MNP@SiO (RITC)-PTMA), bound to the plasmid DNA gene, pcDNA3.1/CT-GFP, pass through the cell membrane and are then separated from the plasmid DNA, thereby leaving the magnetic nanoparticles in the cytoplasm (red fluorescence) and consequently transferring the plasmid DNA to the cells. A blue protein was synthesized in the cytoplasm by the thus -transferred DNA.

[109] As shown in FIG. 9, 9A is a blue fluorescence micrograph, 9B is an optical micrograph, 9C is a red fluorescence micrograph, and 9D is an overlapping micrograph of 9 A, 9B and 9C. Red dots correspond to MNP @ SiO (RITC)-PTMA, and a blue color shows that GFP fluorescence appears in the cytoplasm by DNA transfection.

[110] Therefore, the magnetic nanoparticles according to the present invention can be usefully used as a gene delivery system by binding with the plasmid DNA gene.

[I l l]

[112] Example 3 : Preparation of MNP @ SiO (TITCV PEG/APS-Mal material

[113] MNP @ SiO (FITQ-PEG/APS was prep^~ared in the same manner as in Example 1 , except that 3-aminopropyltriethoxysilane (APS) was co-treated upon treatment of magnetic nanoparticles (MNP@SiO (RITC)) with a (CH O) Si-PEG compound in Section 1 of Example 1.

[114] A solution of MNP@SiO₂(FITC)-PEG/APS in anhydrous DMF [36.5 mL; Si-

PEG/ APS = 5/1 (mole ratio), 22.9 mg/mL, amine concentration of 6.5 mmol/g] was added to a solution of maleimidobutyric acid (0.96 g, 1.4 mmol), PyBOP (benzotriazol-l-yl-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate) (0.43 g, 0.826 mmol) and HOBt (N-hydroxybenzotriazole) (0.19 g, 1.4 mmol) in anhydrous DMF. Next, purified diisopropylethylamine (0.2 mL) was added to the mixture which was then stirred at room temperature for 20 hours. The reaction materials were transferred to an Eppendorf tube and washed several times with DMF. The nanoparticles were re-dispersed in 0.8 mL of DMF, and stored at room temperature under shielding of light.

[115] FIG. 10 shows a process involving co-treatment of (CH O) Si-PEG and APS on the surface of the magnetic nanoparticles, introduction of a maleimide group into an amine group of the surface of the magnetic nanoparticles and introduction of an antibody for recognition of a certain cell thereto.

[116]

[117] Example 4: Introduction of antibody biomolecules into magnetic nanoparticles according to the present invention and Cell staining

[118] An antibody (CD-IO or Her2 Ab ) solution (200 D/mL) in PBS was pre-treated with 10

D of 0.5 M EDTA. A solution of 2-mercaptoethylamine (5 D, 0.779 mmol) in 500 D of PBS was added to the antibody solution which was then incubated at 37⁰C for 90 min. Through this process, the antibody was divided into two halves which were purified using Sephadex G-25, placed in MNP@SiO₂(FITC)-PEG/APS-MaI (0.8 mL, 22.9 mg/ niL PBS) prepared in Example 3 and incubated at 37⁰C for 20 hours. Antibody-bound nanoparticles were centrifuged to be precipitated at 13,000 rpm for 20 min, followed by filtration. 1 rnL of PBS was added to the filtrate in order to re-disperse antibody- bound nanoparticles which were then stored at 4⁰C.

[119] FIG. 11 shows confocal laser scanning micrographs showing utilization of the antibody-bound magnetic nanoparticles in cell staining.

[120] FIG. 12 shows confocal laser scanning micrographs showing utilization of the antibody-bound magnetic nanoparticles in cell staining for a leukemia cells and lung cancer cells.

[121] As shown in FIG. 11, a material penetrated into cells is MNP @ SiO (RITC) emitting red fluorescence, and a cell membrane-bound material is MNP @ SiO (FITC)-Her2 emitting blue fluorescence. 1 IA: Blue fluorescence micrograph, 1 IB:

Ab

Optical micrograph, HC: Red fluorescence micrograph, and HD: Overlapping micrograph of 1 IA, 1 IB and 11C.

[122] As shown in FIG. 12, the CD-10 antibody selectively bound to a membrane of a leukemia cell (SP2/O) (12A to 12C), but did not bind to a lung cancer cell (12D to 12F).

[123]

[124] Example 5: Intraperitoneal administration of magnetic nanoparticles according to the present invention into mice ( in vivo experiment)

[125] In order to examine the in vivo action of magnetic nanoparticles according to the present invention, the following experiments were carried out.

[126] As laboratory animal strains for this experiment, specific-pathogens-free, 4- week old ICR male mice (n = 12) were raised in a laboratory breeding room maintained at a temperature of 22+3⁰C, humidity of 55+10%, and a light-dark cycle of 12 : 12 hours. Mice were allowed to acclimate to new environment of the breeding room for one week prior to the experiment. Animals were fed a laboratory animal feed (for mouse, available from Cheiljedang Corporation, Seoul, Korea) and water ad libitum after sterilization thereof.

[127] Mice were given MNP@SiO (RITC) according to the present invention via intraperitoneal injection and observed at intervals of 15 min via MRI. A control group was not given nanoparticles of the present invention.

[128] The results thus obtained are shown in FIG. 14.

[129] As shown in FIG. 14, the thus-administered magnetic nanoparticles were observed as a black magnetic signal in the liver of mice. Industrial Applicability

[130] The magnetic nanoparticles according to the present invention have both optical and magnetic properties and are applicable to various bio-fields. By the high hy- drophilicity of the nanoparticles and a simple chemical surface treatment technique, chemical functional groups can be introduced into nano-scale materials, using a variety of compounds. Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

[I] Magnetic nanoparticles having a core containing a magnetic material and a surface-modified silica shell containing an organic fluorescent material and coated on the core, wherein the nanoparticles have a size of less than 100 nm and are water-soluble.

[2] The nanoparticles according to claim 1, wherein the magnetic nanoparticles include one metal oxide selected from the group consisting of cobalt (Co), iron (Fe), manganese (Mn), zinc (Zn), nickel (Ni) and copper (Cu) oxides.

[3] The nanoparticles according to claim 1, wherein the organic fluorescent material is Rhodamine B isothiocyanate (RITC) or fluoresceine isothiocyanate (FITC).

[4] The nanoparticles according to claim 1, wherein the silica shell is surface- modified with an electrically charged material.

[5] The nanoparticles according to claim 4, wherein the electrically charged material is an organic silicon compound having ionic functional group(s).

[6] The nanoparticles according to claim 5, wherein the organic silicon compound having ionic functional group(s) is one compound selected from the group consisting of (CH O) Si-PEG [(CH O) SiCH CH CH 0(CH CH O) CH ], (CH ^to 3 3 3 3 2 2 2 2 2 6-9 3 3

O) Si-PMP [(CH O) SiCH CH CH PO (OCH )Na], (CH O) Si-PTMA [(CH O)

3 3 3 2 2 2 2 3 3 3 3 3

SiCH CH CH N⁺(CH ) Cl ] and 3-aminopropyltriethoxysilane (APS). [7] The nanoparticles according to claim 1, wherein the magnetic nanoparticles penetrate into cells and are moved at a rate of 0.5 to 1 mm/sec by application of an external magnetic field (0.3 tesla, T). [8] The nanoparticles according to claim 7, wherein the cells are eukaryotic cells, human cells, animal cells or plant cells. [9] Gene-bound magnetic nanoparticles wherein a negatively charged gene is bound to the surface of the surface-modified silica shells of the magnetic nanoparticles of any one of claims 1 to 8. [10] The nanoparticles according to claim 9, wherein the negatively charged gene is a plasmid DNA.

[I I] The nanoparticles according to claim 10, wherein the plasmid DNA is pcDNA3.1/CT-GFP.

[12] A gene delivery system comprising the gene-bound magnetic nanoparticles of claim 9. [13] Nucleic acid-bound magnetic nanoparticles wherein a negatively charged nucleic acid molecule is bound to the surface of the surface-modified silica shells of the magnetic nanoparticles of any one of claims 1 to 8. [14] A gene delivery system comprising the nucleic acid-bound magnetic nanoparticles of claim 13. [15] Antibody-bound magnetic nanoparticles wherein an antibody is bound to the surface of the surface-modified silica shells of the magnetic nanoparticles of any one of claims 1 to 8. [16] The nanoparticles according to claim 15, wherein the surface-modified silica shell is surface-modified by 3-aminopropyltriethoxysilane (APS). [17] The nanoparticles according to claim 16, wherein an amine group of

3-aminopropyltriethoxysilane (APS) is treated with maleimidobutyric acid, thereby introducing a maleimide group into the amine group. [18] The nanoparticles according to claim 17, wherein the antibody has a thiol group via reaction with 2-mercaptoethylamine. [19] The nanoparticles according to claim 18, wherein the antibody is a CD-10 antibody against a leukemia cell or an Her2 antibody against a breast cancer cell. [20] A cell staining agent comprising the antibody-bound magnetic nanoparticles of claim 15. [21] A method for preparing magnetic nanoparticles comprising:

1) treating a surface of water-soluble magnetic nanoparticles with a polyvinylpyrrolidone (PVP) polymer, converting the nanoparticles into ethanol- dispersible particles, followed by centrifugation;

2) dispersing the polymer- stabilized magnetic nanoparticles separated in Step 1 in ethanol for silica coating;

3) adding to the solution prepared in Step 2 a solution of an organic fluorescent material treated with 3-aminopropyltriethoxysilane (APS) and a solution of tetraethoxysilane (TEOS), and adding NH OH to a mixed solution to thereby induce formation of silica on the surface of the magnetic nanoparticles containing the organic fluorescent material; and

4) treating the surface of the silica shells of the magnetic nanoparticles obtained in Step 3 with a silicon compound.

[22] A method for preparing negatively charged gene-bound magnetic nanoparticles comprising:

1) introducing and incubating negatively charged genes and positively charged MNP @ SiO (RITC)-PTMA in an HEPES [N-(2-hydroxyethyl)-piperazine-N'-(2-ethanesulfonic acid)] buffer solution;

2) adding CaCl to the solution incubated in Step 1 and further incubating the solution for 2 hours; and

3) adding Dulbecco's Modified Eagle Medium (DMEM) to the incubated solution of Step 2, adjusting a Ca ⁺ ion concentration of the solution to 4.5 mM, further incubating the solution for 4 hours and washing the solution with a phosphate-buffered saline (PBS) solution. [23] A method for preparing antibody-bound magnetic nanoparticles comprising:

1) co-treating a surface of magnetic nanoparticles containing organic fluorescent materials with Si-PEG/3-aminopropyltriethoxysilane (APS);

2) reacting the magnetic nanoparticles obtained in Step 1 with maleimidobutyric acid, thereby introducing a maleimide (MaI) group into an amine group present on the surface of the silica shells of the magnetic nanoparticles;

3) reacting an antibody with 2-mercaptoethylamine to form an antibody having a thiol group; and

4) binding the antibody obtained in Step 3 to the maleimide (MaI) group present on the surface of the silica shells of the magnetic nanoparticles obtained in Step 2.

[24] An assay reagent which is capable of simultaneously performing fluorescence analysis and MRI analysis using the magnetic nanoparticles of any one of claims l to 8.