WO2009088250A2

WO2009088250A2 - Porous hollow silica n anop articles, preparation method of the silica nanoparticles, and drug carriers and pharmaceutical composition comprising the silica nanoparticles

Info

Publication number: WO2009088250A2
Application number: PCT/KR2009/000123
Authority: WO
Inventors: Seung Joo Haam; Jin Suck Suh; Yong-Min Huh; Ho-Geun Yoon; Jae Moon Yang; Jae Won Lee; Jin Young Kang
Original assignee: Industry-Academic Cooperation Foundation, Yonsei University
Priority date: 2008-01-10
Filing date: 2009-01-09
Publication date: 2009-07-16
Also published as: CN101687632A; WO2009088250A3; KR100950548B1; CN101687632B; KR20090077159A; JP2010509404A

Abstract

Porous hollow silica nanoparticles are provided. Each of the silica nanoparticles comprises a core having a large inner cavity and a porous silica shell surrounding the core. Due to the presence of the cavities in the cores, the porous hollow silica nanoparticles exhibit much higher drug loading efficiency than conventional particles for drug carriers. In addition, the pore size range of the porous silica shells is optimized by surface functional groups bonded to the silica shells and/or a biocompatible polymer bonded to the silica shells via the surface functional groups to ensure a steady and stable release of a drug from the silica nanoparticles. Therefore, the porous hollow silica nanoparticles can be effectively used for the preparation of drug carriers. Further provided are a method for the preparation of the silica nanoparticles and drug carriers comprising the silica nanoparticles.

Description

[DESCRIPTION] [Invention Title]

POROUS HOLLOW SILICA N ANOP ARTICLES, PREPARATION METHOD OF THE SILICA NANOPARTICLES, AND DRUG CARRIERS AND PHARMACEUTICAL COMPOSITION COMPRISING THE SILICA

NANOPARTICLES

[Technical Field]

The present invention relates to porous hollow silica nanoparticles, each comprising a core having a large inner cavity and a silica shell surrounding the core wherein the shell has pores whose size is controlled to allow a large amount of a drug to be released in a steady and stable manner. The present invention also relates to a method for the preparation of the silica nanoparticles, and drug carriers and a pharmaceutical composition comprising the silica nanoparticles.

[Background Art]

Since 1970, remarkable progress has been made in the development of drug delivery systems based on controlled release technology. A great many products relating to drug delivery systems are commercially available and being developed at present.

Extensive research on drug carriers is actively underway in various fields, including non-parenteral drug delivery systems, such as oral, pulmonary, nasal and ophthalmic delivery of drugs, as well as parenteral drug delivery systems. However, drug carriers known hitherto are very large in size, which makes it difficult to deliver drugs to target tissues through mucosal membranes or systemic circulation. Under these circumstances, techniques associated with the preparation of nanometer-sized particles as drug carriers have drawn considerable attention for their potential applications in the controlled release delivery systems of drugs.

In recent years, there has been research on various biodegradable polymer- based drug delivery systems, some of which are already present on the market. According to a newly developed sol-gel-based technology, a biologically active agent may be introduced into a silica xerogel at room temperature, proposing a new possibility of controlling the release profile of the agent from the gel matrix (S. B. Nicoll., et al, In vitro release kinetics of biologically active transforming growth factor-βl from a novel porous glass carrier, Biomaterial 18 (1997) 853-859, etc.). The sol-gel technology is advantageous in terms of cost, simplicity and versatility, and the silica xerogel is attractive because of its non-toxicity and biocompatibility

(P. Kortesuo., et al, Silica Xerogel as implantable carrier for controlled drug deliver evaluation of drug distribution and tissue effects after implantation, Biomaterial 21 (2000) 193-198). Based on these advantages, research has been concentrated on silica xerogel systems as materials for controlled release delivery of various therapeutic agents such as heparin (M. S. Ahola., et al, In vitro release of heparin from silica xerogels, Biomaterials 22 (2001) 2163-2170).

Most of the presently known systems using silica xerogels are carriers containing therapeutic agents entrapped or absorbed therein or carriers prepared from polymers chemically bonded to therapeutic agents on the surfaces thereof. Therefore, the preparation of the systems necessitates the use of crosslinking agents and involves the control of processing parameters {e.g., temperature and pH). This process control increases the risk of adverse effects of the loaded drugs, thus limiting the clinical utility of the systems.

As an approach to solve these problems, a method may be considered in which porous hollow silica particles prepared by various methods (e.g., templating) are used as drug carriers (F. Caruso., et al, Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating, Science 282 (1998) 1111-1114). A drawback encountered with the use of templates in the current technology is that it is difficult to prepare nanosized hollow particles in a stable form. Another drawback is that only a very limited amount of drugs is loaded within the nanosized hollow particles because of very small inner cavities of the particles. Further, there is a difficulty in controlling the pore size of shells surrounding the cavities, thus making it difficult to control the release profiles of the drugs. Therefore, the nanosized hollow particles are not suitable for use as drug carriers.

[Disclosure] [Technical Problem]

The present invention has been made in view of the problems of the prior art, and it is an object of the present invention to provide hollow silica nanoparticles, each being composed of a core having a large inner cavity and a porous silica shell in which the core can be stably formed, the size of the cavity is freely controllable and the pore size of the silica shell is controlled, thus achieving an effective release profile of a drug.

A further object of the present invention is to provide a method for the preparation of the silica nanoparticles.

Another object of the present invention is to provide drug carriers comprising the silica nanoparticles.

Still another object of the present invention is to provide a pharmaceutical composition comprising the silica nanoparticles.

[Technical Solution] In accordance with one aspect of the present invention for accomplishing the above objects, there are provided porous hollow silica nanoparticles, each comprising a core having a cavity with a diameter of 1 to 100 nm and a porous silica shell having functional groups on the surfaces thereof (hereinafter, referred to simply as 'surface functional groups').

In accordance with another aspect of the present invention, there are provided porous hollow silica nanoparticles, each comprising a core having a cavity with a diameter of 1 to 100 nm and a porous silica shell whose surface is modified with a biocompatible polymer.

In a preferred embodiment, the biocompatible polymer is selected from the group consisting of polyalkylene glycol (PAG), polyetherimide (PEI), polyvinylpyrrolidone (PVP), hydrophilic vinyl polymers, copolymers thereof, and mixtures thereof.

In a preferred embodiment, the hollow silica nanoparticles have a diameter of 20 to 250 nm.

In accordance with another aspect of the present invention, there is provided a method for preparing hollow silica nanoparticles which comprises mixing magnetic nanoclusters with a silica precursor (first step), forming silica shells using the silica precursor on the respective magnetic nanoclusters (second step), removing the magnetic nanoclusters present inside the silica shells (third step), and introducing functional groups on the surfaces of the silica shells (fourth step).

In an embodiment, the hollow silica nanoparticles may be surface-modified with a biocompatible polymer (fifth step).

In an embodiment, the magnetic nanoclusters can be prepared by (1) dissolving magnetic nanoparticles in an organic solvent to prepare an oily phase, (2) dissolving an amphophilic compound in an aqueous solvent to prepare an aqueous phase, (3) mixing the oily phase with the aqueous phase to prepare an emulsion, and (4) separating the oily phase from the emulsion.

In accordance with another aspect of the present invention, there are provided drug carriers comprising the hollow silica nanoparticles and a pharmaceutically active ingredient loaded in the cavities of the cores or the pores of the silica shells.

In a preferred embodiment, the pharmaceutically active ingredient has a loading fraction of 1 to 100%, as calculated by Equation 1 : c , .. . . ,„.. Weight of the pharmaceutically active ingredient , _nn /, \ o Loading fractoin (%) = — ^≥ x 100 (.1 ;

Weight of the silica nanoparticles

In accordance with yet another aspect of the present invention, there is provided a pharmaceutical composition comprising the hollow silica nanoparticles and at least one pharmaceutically acceptable carrier.

In an embodiment, the pharmaceutical composition may further comprise a0 pharmaceutically active ingredient loaded in the cavities of the cores or the pores of the silica shells.

[Advantageous Effects]

Due to the presence of large cavities in the cores, the porous hollow silica5 nanoparticles of the present invention exhibit much higher drug loading efficiency than conventional particles for drug carriers. In addition, the pore size range of the porous silica shells is optimized by surface functional groups bonded to the silica shells and/or a biocompatible polymer bonded to the silica shells via the surface functional groups to ensure a steady and stable release of a drug from the silica0 nanoparticles. Therefore, the porous hollow silica nanoparticles of the present invention can be effectively used for the preparation of drug carriers or a pharmaceutical composition. Furthermore, the cavity size of the cores can be freely controlled by the use of magnetic nanoclusters as templates for the preparation of the silica nanoparticles. 5

[Description of Drawings] In the figures, FIG. 1 is a schematic diagram depicting the procedure for the preparation of a hollow silica nanoparticle whose surface is modified with polyethylene glycol in Example 1 ;

FIG. 2 shows diagrams depicting different kinds of nanoparticles prepared in Example 1 ;

FIG. 3 shows transmission electron micrographs (TEM) of (a) magnetic nanoparticles, (b) magnetic nanoclusters, (c) magnetic silica particles and (d) hollow silica nanoparticles, all of which were prepared in Example 1 ;

FIG. 4 shows (a) a graph illustrating the size distributions and zeta potentials of products prepared in respective steps in Example 1 , and (b) photographs of silica nanoparticles before and after the removal of magnetic materials present inside the silica nanoparticles in Example 1 ;

FIG. 5 is a graph showing the results of X-ray diffraction analysis for (a) magnetic silica nanoparticles and (b) hollow silica nanoparticles prepared in Example 1 ;

FIG. 6 is a graph showing the results of Fourier transform infrared spectroscopy (FT-IR) for products ((a) magnetic nanoclusters, (b) magnetic silica nanoparticles and (c) hollow silica nanoparticles) prepared in respective steps in Example 1 ; FIG. 7 is a graph showing the results of thermo gravimetric analysis (TGA) for (a) magnetic silica nanoparticles and (b) hollow silica nanoparticles prepared in Example 1 ;

FIG. 8 shows graphs illustrating the results of X-ray photoelectron spectroscopy for (a) magnetic nanoclusters (MKs), (b) magnetic silica particles (MSNPs) and (c) hollow silica nanoparticles (HSNPs), all of which were prepared in

Example 1 , and a graph (d) showing the composition ratios of Si and Fe in the products (a), (b) and (c); FIG. 9 is a graph showing the amounts of nitrogen adsorbed/desorbed in particles prepared in Example 1;

FIG. 10 shows (a) a graph and (b) a semi-logarithmic graph illustrating the amounts of a drug released from drug carriers prepared in Test Example 3 as a function of time; and

FIG. 11 shows (a) a fluorescence micrograph of anticancer agent-loaded nanoparticles prepared in Test Example 3, (b) a photograph of the anticancer agent- loaded nanoparticles precipitated by centrifugation, and (c) a photograph of the anticancer agent-loaded nanoparticles dispersed in water. The abbreviations are as follows:

MKs = magnetic nanoclusters

MSNPs = magnetic silica nanoparticles

HSNPs = hollow silica nanoparticles

HSNPs-PEG = hollow silica nanoparticles surface-modified with polyethylene glycol

[Best Mode]

The present invention is directed to porous hollow silica nanoparticles (hereinafter, also referred to as "PHSNs" or "HSNPs"), each comprising a core having a cavity with a diameter of 1 to 100 nm and a porous silica shell having functional groups on the surface thereof. Due to the presence of large cavities in the cores, the silica nanoparticles of the present invention exhibit much higher drug loading efficiency than conventional particles for drug carriers. In addition, the pore size range of the porous silica shells is optimized by the surface functional groups and/or a biocompatible polymer bonded to the silica shells via the surface functional groups to ensure a steady and stable release of a drug from the porous hollow silica nanoparticles. Therefore, the porous hollow silica nanoparticles of the present invention can be effectively used for the preparation of drug carriers.

The porous hollow silica nanoparticles of the present invention will now be described in greater detail.

The cores are surrounded by the porous silica shells and have cavities with a diameter of from 1 to 100 nm, preferably from 40 to 100 nm. The term "cavities" refers to inner spaces surrounded by porous silica constituting shells (i. e. the porous silica shells) of the porous hollow silica nanoparticles. The silica particles of the present invention can be used to prepare drug carriers with markedly improved drug loading efficiency because of the large-volume inner cavities, compared to that of conventional drug carriers. If the diameter of the cavities is less than 1 nm, the drug loading efficiency is excessively low, which may render meaningless the formation of the cavities. Meanwhile, if the diameter of the cavities is more than 100 nm, the drug release behavior may be difficult to control.

Functional groups are introduced on the surfaces of the porous silica shells surrounding the hollow cores, and a biocompatible polymer may be introduced via the surface functional groups. The introduction of the surface functional groups or the biocompatible polymer enables control of the pore size of the silica shells, and as a result, the release rate of a drug, which is to be introduced into the hollow cores and/or the pores, can be controlled. There is no particular restriction on the kind of the surface functional groups. Any surface functional group capable of being chemically bonded to the silica shells and the biocompatible polymer, which will be described below, can be used without any particular limitation. Examples of suitable surface functional groups include -COOH, -CHO, -NH₂, -SH, -CONH₂, - PO₃H, -PO₄H, -SO₃H, -SO₄H, -OH, -NR₄ ⁺X^", sulfonate, nitrate, phosphonate, succinimidyl, maleimide and alkyl groups. These functional groups may be introduced alone or in combination thereof. The silica nanoparticles having the surface functional groups preferably have a diameter of 10 to 200 nm. If the diameter is smaller than 10 nm, there is the risk that the pore size may be difficult to control. Meanwhile, if the diameter is larger than 200 nm, the in vivo applicability may be limited.

The present invention is also directed to porous hollow silica nanoparticles, each comprising a core having a cavity with a diameter of 1 to 100 nm and a porous silica shell surface-modified with a biocompatible polymer. The surfaces of the porous silica shells may be modified with the biocompatible polymer via surface functional groups of the silica shells. This surface modification enables additional control of the pore size of the shells. It is preferred that the biocompatible polymer has a weight average molecular weight of 100 to 100,000. The biocompatible polymer having a weight average molecular weight lower than 100 may show toxicity in vivo. Meanwhile, the biocompatible polymer having a weight average molecular weight higher than 100,000 undesirably suffers from a difficulty in its applicability. The kind of the biocompatible polymer is not particularly limited, so long as the weight average molecular weight of the biocompatible polymer is in the range defined above. For example, the biocompatible polymer is selected from the group consisting of polyalkylene glycol (PAG), polyetherimide (PEI), polyvinylpyrrolidone (PVP), hydrophilic vinyl polymers, copolymers thereof, and mixtures thereof. The use of polyalkylene glycol is more preferred. Examples of preferred polyalkylene glycols include polyethylene glycol (PEG) and monomethoxy polyethylene glycol (mPEG).

The biocompatible polymer is preferably present in an amount of 5 to 50% by weight, based on the weight of the silica particles. The amount of the biocompatible polymer smaller than 5% by weight may be insufficient in controlling the pore size of the silica shells, while the amount of the biocompatible polymer larger than 50% by weight may cause an excessive reduction in the size of the inner cavities and pores. In addition, the hollow cores of the nanoparticles surface- modified with the biocompatible polymer have cavities with a diameter of 1 to 100 nm and preferably 40 to 100 nm. If the diameter of the cavities is smaller than 1 nm, it may be meaningless to form the cavities. Meanwhile, if the diameter of the cavities is larger than 100 nm, the drug release behavior may be difficult to control. Further, the silica shells surface-modified with the biocompatible polymer preferably have a pore size of 1 to 100 A. When the pore size of the silica shells is smaller than 1 A, there is the risk that the release efficiency of a substance {e.g., a drug) may drop. Meanwhile, when the pore size of the silica shells is larger than 100 A, there is the risk that the release efficiency of a substance {e.g., a drug) may be difficult to control. It is preferred that the porous silica shells have a thickness in the range of 1 to 50 nm. If the porous silica shells are thinner than 1 nm, the drug loading stability may drop. Meanwhile, if the porous silica shells are thicker than 50 nm, the drug release rate may be excessively slow. The silica nanoparticles surface-modified with the biocompatible polymer preferably have a diameter of 20 to 250 nm and more preferably 80 to 250 nm. If the diameter of the silica nanoparticles is less than 80 nm, the pore size may be difficult to control. Meanwhile, the diameter of the silica nanoparticles is more than 250 nm, the in vivo applicability may be limited.

The silica nanoparticles of the present invention may further comprise a tissue-specific binding substance introduced on the surfaces thereof. The term

"tissue-specific binding substance" as used herein means a substance that is capable of specific binding to a target living tissue. The introduction of the tissue-specific binding substance allows drug carriers comprising the silica nanoparticles to more easily reach a desired site. Examples of suitable tissue-specific binding substances for use in the present invention include, but are not limited to, antigens, antibodies,

RNAs, DNAs, haptens, avidin, streptavidin, neutravidin, protein A, protein G, lectin, selectin, radioisotope-labeling components, and substances capable of specific binding to tumor markers. The term "tumor markers" used herein refers to particular substances that are expressed and/or secreted from tumor cells and are less or not at all produced in normal cells. Numerous tumor markers and substances capable of specific binding to the tumor markers are already known in the art. Such tumor markers can be classified into ligands, antibodies, receptors and encoding nucleic acids thereof, depending on the mechanism of action of the tumor markers, as summarized in Table 1. Table 1

Kind Tumor marker Tissue-specific binding substance

Ligands C2 domains of synaptotagmin I Phosphatidyl serine

Annexin V

Integrin Integrin receptor

VEGF VEGFR

Angiopoietin 1, 2 Tie 2 receptor

Somatostatin Somatostatin receptor Vasointestinal peptide Vasointestinal peptide receptor Antibodies Carcinoembryonic antigen Herceptin (Genentech, USA)

HER2/neu antibody Prostate-specific membrane Rituxan (Genentech, USA) antigen Receptor Folic acid receptor Folic acid

In the case where the tumor marker is a ligand, a substance capable of specific binding to the ligand can be introduced into the silica nanoparticles, and an example thereof may be a receptor or an antibody capable of specific binding to the ligand. Examples of such ligands and receptors capable of specific binding to the ligands that can be employed in the present invention include, but are not limited to, C2 domains of synaptotagmin and phosphatidyl serine, annexin V and phosphatidyl serine, integrin and its receptors, vascular endothelial growth factors (VEGFs) and their receptors, angiopoietin and Tie2 receptors, somatostatin and its receptors, and vasointestinal peptide and its receptors.

In the case where the tumor marker is an antigen, a substance capable of specific binding to the antigen can be introduced into the silica nanoparticles, and an example thereof may be an antibody. Examples of such antigens and antibodies capable of specific binding to the antigens that can be employed in the present invention include, but are not limited to, carcinoembryonic antigen (colorectal cancer marker antigen) and Herceptin (Genentech, USA), HER2/neu antigen (breast cancer marker antigen) and Herceptin, and prostate-specific membrane antigen (prostate cancer marker antigen) and Rituxan (IDCE/Genentech, USA). A representative example of a receptor as the tumor marker is a folic acid receptor expressed in ovarian cancer. A substance capable of specific binding to the receptor (a folic acid receptor for folic acid) may be introduced into the silica nanoparticles, and examples thereof include ligands and antibodies capable of specific binding to the receptor. As described above, an antibody is particularly preferred as the tissue- specific binding substance in the present invention. An antibody has the ability to selectively and stably be bound to a particular subject only. When it is intended to introduce an antibody into the silica nanoparticles, -NH₂ of lysine, -SH of cysteine and -COOH of asparaginic acid and glutamic acid in the Fc domain of the antibody are useful sites.

Such antibodies are commercially available or can be produced by suitable methods known in the art. For example, an antibody is produced by the following procedure. First, a mammal (e.g., mouse, rat, goat, rabbit, horse or sheep) is immunized once or more times with an appropriate amount of an antigen. After a certain time, serum is collected from the mammal when the titer reaches an optimum level. If desired, the serum may be purified by any known process and stored in a frozen buffer solution until use. The details of this method are well known in the art.

The term "nucleic acids" is intended to include RNAs and DNAs coding for the above-mentioned ligands, antigens, receptors and at least a portion thereof. As known in the art, a nucleic acid has the ability to form base pairs between complementary sequences. Based on this ability, a nucleic acid having particular base sequences can be detected using a nucleic acid having base sequences complementary to the particular base sequences. In the present invention, a nucleic acid having base sequences complementary to a nucleic acid encoding one of the enzymes, ligands, antigens and receptors can be used as the tissue-specific binding substance of the silica nanoparticles. In addition, functional groups (such as -NH₂,

-SH and -COOH) located at the 5'- and 3'- ends of a nucleic acid are useful for the introduction of the nucleic acid into the nanoparticles. Such nucleic acids can be synthesized by standard methods known in the art, for example, using automatic DNA synthesizers available from Biosearch, Applied Biosystems, etc. Specifically, a phosphorothioate oligonucleotide can be synthesized by the method described in

Stein et al. Nucl. Acids Res. 1988, vol.16, p.3209, and a methylphosphonate oligonucleotide can be synthesized using controlled glass as a polymer support (Sarin et al. Proc. Natl. Acad. Sci. U.S.A. 1988, vol.85, p.7448).

As explained earlier, the tissue-specific binding substance can be introduced via the surface functional groups of the shells of the silica nanoparticles according to the present invention. Alternatively, when the shells of the silica nanoparticles are surface-modified with the biocompatible polymer, specific binding domains are introduced into the biocompatible polymer and then the tissue-specific binding substance can be introduced via the specific binding domains. The binding domains are determined depending on the kind of the tissue-specific binding substance introduced, and non-limiting examples thereof are counter functional groups of the surface functional groups.

The present invention is also directed to a method for preparing hollow silica nanoparticles which comprises mixing magnetic nanoclusters with a silica precursor (first step), forming silica shells using the silica precursor on the respective magnetic nanoclusters (second step), removing the magnetic nanoclusters present inside the silica shells (third step), and introducing functional groups on the surfaces of the silica shells (fourth step).

The method of the present invention may further comprise modifying the surfaces of the hollow silica nanoparticles with a biocompatible polymer (fifth step).

Hereinafter, the individual steps of the method according to the present invention will be explained in more detail.

In the first step, magnetic nanoclusters are mixed with a silica precursor. This mixing induces binding of the silica precursor to the magnetic nanoclusters and hydrolysis of the silica precursor. The magnetic nanoclusters serve as templates to form hollow cores of the final nanoparticles. The use of the magnetic nanoclusters as templates enables the formation of larger cavities within the nanoparticles than those formed by conventional methods. In addition, this templating step offers an advantage in that the size of the cavities can be freely controlled.

No particular limitation is imposed on the preparation process of the magnetic nanoclusters. For example, the magnetic nanoclusters can be prepared by the following procedure.

First, magnetic nanoparticles are dissolved in an organic solvent to prepare an oily phase, and an amphiphilic compound is dissolved in an aqueous solvent to prepare an aqueous phase. Then, the oily phase is mixed with the aqueous phase to prepare an emulsion. The oily phase is ^"separated from the emulsion to leave the desired magnetic nanoclusters.

There is also no particular restriction on the preparation process of the magnetic nanoparticles. For example, the magnetic nanoparticles can be prepared by (a) reacting a nanoparticle precursor with an organic surface stabilizer in a solvent and (b) thermally decomposing the reaction product. In step (a), the reaction allows the organic surface stabilizer to be coordinated to the surfaces of the nanoparticles.

The kind of the nanoparticle precursor is not specifically limited. As the nanoparticle precursor, there may be exemplified a metal compound bound with at least one ligand selected from -CO, -NO₃ -C₅H₅, alkoxides and other known ligands. Specific examples of such organometallic compounds include metal carbonyl compounds, such as iron pentacarbonyl (Fe(CO)₅), ferrocene and manganese carbonyl (Mn₂(CO)₁₀), and metal acetylacetonate compounds, such as ferric acetylacetonate (Fe(acac)₃). Another example of the nanoparticle precursor is a salt composed of a metal ion and a known counter anion (e.g., Cl^" or NO₃ ^"). Such metal salts include ferric chloride (FeCIs), ferrous chloride (FeCl₂) and ferric nitrate (Fe(NO₃)₃). If needed, a mixture of two or more kinds of the aforementioned metal precursors may be used to synthesize alloy nanoparticles or composite nanoparticles.

The organic surface stabilizer can be selected from the group consisting of alkyl trimethylammonium halides, saturated and unsaturated fatty acids, trialkylphosphine oxides, alkyl amines, alkyl thiols, sodium alkyl sulfates, and sodium alkyl phosphates. These surface stabilizers may be used alone or as a mixture thereof. Preferably, the solvent has a high boiling point close to the thermal decomposition temperature of the complex compound, in which the organic surface stabilizer is coordinated to the surface of the nanoparticle precursor. Examples of solvents suitable for use in the present invention include: ether compounds, such as octyl ether, butyl ether, hexyl ether and decyl ether; heterocyclic compounds, such as pyridine and tetrahydrofuran (THF); aromatic compounds, such as toluene, xylene, mesitylene and benzene; sulfoxide compounds, such as dimethylsulfoxide (DMSO); amide compounds, such as dimethylformamide (DMF); alcohols, such as octyl alcohol, and decanol; hydrocarbons, such as pentane, hexane, heptane, octane, decane, dodecane, tetradecane and hexadecane; and water. The reaction conditions in step (a) are not specifically limited, and may be appropriately varied depending on the kinds of the nanoparticle precursor and the surface stabilizer. For example, the reaction may proceed at or below room temperature. The reaction is typically conducted while maintaining the temperature at about 30 to 200⁰C.

In step b), the complex compound, in which the organic surface stabilizer is coordinated to the surface of the nanoparticle precursor, is thermally decomposed to grow nanoparticles. The reaction conditions may be appropriately varied such that the nanoparticles are uniform in size and shape. The thermal decomposition temperature may be suitably varied depending on the kinds of the nanoparticle precursor and the surface stabilizer. Preferably, the thermal decomposition is conducted at about 50 to 500⁰C. The nanoparticles thus prepared may be separated and purified by known means.

The kind of the magnetic nanoclusters is determined by the kind of the nanoparticle precursor used. Examples of suitable materials for the magnetic nanoclusters include, but are not particularly limited to, metal materials, magnetic materials, and magnetic alloys. Suitable metal materials are Pt, Pd, Ag, Cu and Au, suitable magnetic materials are Co, Mn, Fe, Ni, Gd, Mo, MM'₂O₄ and M_xOy (M and

M' are each independently represents Co, Fe, Ni, Mn, Zn, Gd, or Cr, 0 < x < 3, and 0 < y < 5), and suitable magnetic alloys are CoCu, CoPt, FePt, CoSm, NiFe and NiFeCo. These materials may be used alone or in combination of two or more thereof. Specific conditions for the preparation of the magnetic nanoclusters using the magnetic nanoparticles are not particularly limited in the method of the present invention. That is, the magnetic nanoclusters can be prepared using an organic solvent (e.g., chloroform), an aqueous solvent (e.g., ultrapure water) or an amphiphilic compound (e.g., polyvinyl alcohol) by an emulsion process known in the art. Further, the magnetic nanoclusters may be prepared in the presence of a suitable surfactant selected from: soaps, such as potassium oleate and sodium oleate; anionic detergents, such as aerosol^® OT, sodium cholate and sodium caprylate; cationic detergents, such as cetylpyridinium chloride, alkyltrimethylammonium bromides, benzalkonium chloride and cetyldimethylethylammonium bromide; zwitterionic detergents, such as N-alkyl-N,N-dimethylammonio-l-propanesulfonates and CHAPS; and non-ionic detergents, such as polyoxyethylene esters, polyoxyethylenesorbitan esters, sorbitan esters, and various tritons (e.g., TX-100,

TX-114); and mixtures thereof. These surfactants function to reduce the interfacial tension between the aqueous and oily phases, rendering the oily or aqueous phase dispersed in the emulsion thermodynamically stable.

In the first step, the magnetic nanoclusters are mixed with a silica precursor, which constitutes silica shells in the subsequent step, in a suitable solvent to induce binding of the silica precursor to the magnetic nanoclusters and hydrolysis of the silica precursor.

As the solvent, any aqueous solvent and organic solvent known in the art may be used without any particular limitation. A mixed solvent of water and alcohol is preferably used. The water of the mixed solvent serves to hydrolyze the silica precursor. In this step, hydroxyl groups are introduced into the silicon atoms of the silica precursor to participate in condensation and gelling reactions. It is common that the silica precursor is previously mixed with a suitable organic solvent (e.g., alcohol) due to its water insolubility. The alcohol dissolves both the water and the silica precursor to homogeneously mix the water and the silica precursor, so that the silica precursor can be sufficiently hydrolyzed. The water and the alcohol may be mixed in any ratio, and an optimum mixing ratio thereof can be readily determined by those skilled in the art.

Any material may be used without particular limitation as the silica precursor so long as it can form silica shells on the respective magnetic nanoclusters. An alkoxysilane such as tetramethoxysilane, tetraethoxysilane or a mixture thereof is preferred as the silica precursor. Tetraethoxysilane is more preferred. In the first step, the amount of the alkoxysilane used can be properly varied by those skilled in the art to control the thickness of shells to be formed. Conditions for the hydrolysis of the silica precursor are not particularly limited. Generally, the silica precursor is hydrolyzed under reflux with stirring. The hydrolysis of the silica precursor can be accelerated by the addition of a suitable catalyst, for example, an acid catalyst (e.g., HCl or CH₃COOH) or a base catalyst (e.g., KOH or NH₄OH).

In the second step, the hydrolysis product of the silica precursor is condensed and gelled to form silica shells on the respective magnetic nanoclusters. As a result of the condensation and gelling, siloxane bonds (-Si-O-Si-) are formed on the surfaces of the clusters.

The condensation reactions can be divided into dehydration condensation and alcohol condensation. In the dehydration condensation, hydrogen bonding occurs between the hydroxyl groups (OH) introduced into the precursor during hydrolysis in the first step to form siloxane bonds and water is eliminated from the precursor. In the alcohol condensation, the hydroxyl groups are bonded to alkoxy groups (OR) to form siloxane bonds and alcohol is eliminated from the precursor. Conditions for the condensation and gelling reactions are not especially limited. For example, the mixture is stirred at an optimum temperature for condensation and gelling.

In the third step, the magnetic nanoclusters present inside the particles on which the silica shells are formed are removed to leave hollow silica nanoparticles.

Any method may be used to remove the magnetic nanoclusters. For example, the magnetic nanoclusters are removed by the treatment with a material (e.g., hydrochloric acid or sulfuric acid) capable of dissolving the magnetic materials. In the third step, the hollow silica nanoparticles may be fired at a high temperature to remove remaining organic residues, if needed.

In the fourth step, functional groups are introduced on the surfaces of the silica shells of the hollow silica nanoparticles. Hydroxyl groups remain on the surfaces of the silica particles prepared by hydrolysis of the silica precursor (e.g., an alkoxysilane). In the fourth step, the surfaces of the particles are treated with a precursor material capable of introducing functional groups thereon. There is no particular restriction on the kind of the precursor material, and general precursor materials corresponding to surface functional groups to be introduced may be used without limitation. For example, an aminoalkylalkoxysilane can be used as the precursor material when it is intended to introduce amino groups on the surfaces of the silica shells. The amount of the precursor material used is not especially limited. Preferably, the precursor material is used in an amount such that the number of intended surface functional groups is 5% or more of the number of the hydroxyl groups introduced on the surfaces of the particles after the third step.

The thus prepared hollow silica nanoparticles can be separated and/or purified by standard methods known in the art. Preferably, the method of the present invention further comprises modifying the surfaces of the hollow silica nanoparticles with a biocompatible polymer (fifth step).

The surface modification with a biocompatible polymer may be performed by any suitable method. For example, a variety of functional groups capable of bonding to the surface functional groups present on the silica shells of the porous hollow silica nanoparticles are introduced into the biocompatible polymer, and then the biocompatible polymer is introduced on the surfaces of the hollow silica nanoparticles via the introduced functional groups. The kind of the functional groups introduced into the biocompatible polymer is not particularly limited. Depending on the kind of the surface functional groups of the nanoparticles, the kind of the counter functional groups can be readily selected by those skilled in the art. Representative kinds of the surface functional groups present on the surface of the nanoparticles and the counter functional groups and their bonding interactions are summarized in Table 2.

I Q Table 2

Surface functional group Counter functional group Bonding interaction

(R': Biocompatible polymer)

R-NH₂ R'-COOH R-NHCO-R'

R-SH R'-SH R-SS-R

R-OH R'-(epoxy) R-OCH₂C(OH)CH₂-R'

RH-NH₂ R'-(epoxy) R-NHCH₂C(OH)CH₂-R'

R-SH R'-(epoxy) R-SCH₂C(OH)CH₂-R'

R-NH₂ R'-COH R-N=CH-R'

R-NH₂ R'-NCO R-NHCONH-R'

R-NH₂ R'-NCS R-NHCSNH-R'

R-SH R'-C0CH₂ R'-COCH₂S-R

R-SH R'-O(C=O)X R-OCH₂(C=O)O-R'

R-(aziridine) R'-SH R-CH₂CH(NH₂)CH₂S-R'

R-CH=CH₂ R'-SH R-CH₂CHS-R'

R-OH R'-NCO R'-NHCOO-R

R-SH R'-COCH₂X R-SCH₂CO-R'

R-NH₂ R'-C0N₃ R-NHCO-R'

R-COOH R'-COOH R-(C=O)O(C=O)-R' + H₂O

R-SH R'-X R-S-R'

R-NH₂ R¹CH₂C(NH²⁺)OCH₃ R-NHC(NH²⁺)CH₂-R'

R-OP(O^2")OH R' -NH₂ R-0P(0^2")-NH-R'

R-CONHNH₂ R'-COH R-CONHN=CH-R'

R-NH₂ R'-SH R-NHCO(CH₂)₂SS-R'

Any suitable method may be utilized to introduce the counter functional groups into the biocompatible polymer. For example, a suitable crosslinking agent corresponding to functional groups to be introduced may be used to freely introduce the functional groups into the biocompatible polymer. Examples of such crosslinking agents include 1 ,4-diisothiocyanatobenzene, 1 ,4-phenylene diisocyanate, 1 ,6-diisocyanatohexane, 4-(4-maleimidophenyl)butyric acid N- hydroxysuccinimide ester, phosgene solution, 4-(maleinimido)phenyl isocyanate, 1,6-hexanediamine, p-nitrophenyl chloroformate, N-hydroxysuccinimide, 1,3- dicyclohexylcarbodiimide, lj '-carbonyldiimidazole, 3-maleimidobenzoic acid N- hydroxysuccinimide ester, ethylenediamine, bis(4-nitrophenyl)carbonate, succinyl chloride, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride, N₅N'- disuccinimidyl carbonate, N-succinimidyl 3-(2-pyridyldithio)propionate₅ and succinic anhydride.

The present invention is also directed to drug carriers comprising the silica nanoparticles and a pharmaceutically active ingredient loaded in the cavities of the cores or the pores of the silica shells. The drug carriers can load a much larger amount of the pharmaceutical active ingredient in the silica nanoparticles than already known drug carriers. In addition, the pores of the silica nanoparticles can be controlled such that the drug is steadily and stably released therethrough. Therefore, the silica nanoparticles can be efficiently used to prepare the drug carriers.

As described previously, the nanoparticles have the large cavities whose size is freely controllable, if needed. Therefore, the amount of the drug loaded in the nanoparticles can be freely determined according to the intended application of the drug carriers. For example, the drug has a loading fraction of 1 to 100%, as calculated by Equation 1 :

. . ,„ .. Weight of the pharmaceutically active ingredient , „ . /u

Loading fractoin (%) — x lOO U J

Weight of the silica nanoparticles

There is no particular restriction on the kind of the pharmaceutically active ingredient introduced into the drug carriers. Any ingredient known to be pharmaceutically active in the art can be used in the present invention. For example, the pharmaceutically active ingredient can be selected from the group consisting of anticancer agents, antibiotics, hormones, hormone antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphotoxins, urokinase, streptokinase, tissue plasminogen activators, protease inhibitors, alkylphosphocholines, radioisotope-labeling components, surfactants, cardiovascular system drugs, gastrointestinal system drugs, nervous system drugs, and mixtures thereof. Specific examples of the anticancer agents include, but are not limited to, epirubicin, docetaxel, gemcitabine, paclitaxel, cisplatin, carboplatin, taxol, procarbazine, cyclophosphamide, dactinomycin, daunorubicin, etoposide, tamoxifen, doxorubicin, mitomycin, bleomycin, plicomycin, transplatinum, vinblastin, and methotrexate. The pharmaceutically active ingredient may be introduced into the silica nanoparticles by any suitable method. For example, a mixture of the nanoparticles and the pharmaceutically active ingredient in a proper solvent may be introduced into the silica nanoparticles.

The drug carriers of the present invention can be used for the treatment of diseases. Non-limiting examples of diseases to which the drug carriers of the present invention are applied include, but are not particularly limited to, gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer and cervical cancer. The kind of the pharmaceutically active ingredient contained in the drug carriers of the present invention may be changed according to the intended applications. That is, the drug carriers of the present invention can be used in a variety of medical applications.

The present invention is also directed to a pharmaceutical composition comprising the drug carriers and at least one pharmaceutically acceptable carrier.

The kind of the pharmaceutically active ingredient loaded in the pharmaceutical composition of the present invention and the kind of diseases to which the pharmaceutical composition of the present invention is applied are not specifically limited, and for example, are the same as those in the drug carriers.

No particular limitation is imposed to the kind of carriers that can be used in the pharmaceutical composition. General carriers and vehicles available in medical applications can be used in the present invention, and specific examples thereof include, but are not limited to, ion-exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g. , human serum albumin), buffer materials (e.g. , phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids), water, salts and electrolytes (e.g. , protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose- based substrates, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, and lanoline. The pharmaceutical composition of the present invention may further comprise at least one additive selected from lubricants, wetting agents, emulsifiers, suspending agents and preservatives.

In an embodiment, the drug carriers or the pharmaceutical composition of the present invention may be prepared into a water-soluble solution for parenteral administration. In a preferred embodiment, the water-soluble solution may be

Hanks' solution, Ringer's solution or a buffer solution such as physically buffered saline. In an alternative embodiment, the drug carriers or the pharmaceutical composition of the present invention may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. The aqueous injectable suspension may further contain a thickener, such as sodium carboxymethylcellulose, sorbitol or dextran. The suspension may be formulated using a suitable dispersant or wetting agent (e.g. , Tween 80) and a suspending agent in accordance with a known technique in the art. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butandiol. Vehicles and solvents available in the present invention are mannitol, water, Ringer's solution and an isotonic sodium chloride solution. In addition, sterile nonvolatile oil is commonly used as a solvent or suspending medium. For this purpose, less irritable, nonvolatile oil products including synthetic mono- or diglyceride may be used.

[Mode for Invention]

Hereinafter, the present invention will be explained in more detail with reference to the following examples. However, these examples are not to be construed as limiting the scope of the invention.

EXAMPLES Example 1 Hollow silica nanoparticles whose surfaces were modified with polyethylene glycol were prepared in accordance with the following procedure. The preparation procedure is diagrammatically depicted in FIG. 1. FIG. 2 shows schematic diagrams of products prepared in the respective steps: (A) a hollow silica nanoparticle having hydroxyl groups on the surface thereof, (B) a hollow silica nanoparticle having amine groups on the surface thereof, and (C) a hollow silica nanoparticle surface-modified with polyethylene glycol.

(1) Preparation of magnetic nanoparticles Ferric triacetylacetonate (0.2 mol),t 1 ,2-hexadecanediol (1 mol), dodecanoic acid (0.6 mol) and dodecylamine (0.6 mol) were subjected to thermal decomposition at 15O⁰C for 30 minutes and at 29O⁰C for 30 minutes to synthesize magnetite (Fe₃O₄) having a size of 12 nm. The magnetic nanoparticles were purified with pure ethanol. A transmission electron micrograph (TEM) of the magnetite (3a) is shown in FIG. 3 (a).

(2) Preparation of magnetic nanoclusters

Magnetic nanoclusters (MKs) were prepared using the magnetic nanoparticles by an oil/water (O/W) emulsion method. Specifically, 200 mg of polyvinyl alcohol as an amphiphilic polymer was dissolved in 100 ml of ultrapure water as an aqueous solvent, and 5 mg of the magnetic nanoparticles were dissolved in chloroform as an organic solvent. The two solutions were mixed to prepare an emulsion. The emulsion was stirred for about 6 hr to remove the oily phase. Impurities were removed by repeated centrifugation to prepare highly sensitive magnetic nanoclusters. Transmission electron microscopy reveals that the MKs were surrounded by the polyvinyl alcohol and had a round shape (FIG. 3(b)). The MKs were found to have a size distribution of 45.3 ± 5.9 nm, as measured using a particle size analyzer, and a zeta potential close to zero, which is because the MKs were surrounded by the non-ionic polyvinyl alcohol.

(3) Coating of MKs with silica The MKs were dispersed in a mixture of ultrapure water (1 ml), ethanol (4 ml) and aqueous ammonia (0.1 ml), and then 0.1 ml of tetraethoxysilane (TEOS) was slowly injected into the dispersion to prepare magnetic silica nanoparticles (MSNPs) in which the MKs were coated with silica. Transmission electron microscopy (TEM) of the MSNPs reveals that the MKs were completely surrounded by the silica (FIG. 3(c)). The MSNPs were found to have a size distribution of 80.9 ± 9.3 nm and a zeta potential of -35.6 ± 7.8 mV. The reason why the zeta potential of the MSNPs had a larger negative value than that of the MKs is due to hydroxyl groups formed on the surface of the MSNPs during the sol-gel process. The crystallinity of the MSNPs was determined by X-ray diffraction (XRD) analysis (see 'a' in FIG. 5).

(4) Preparation of hollow silica nanoparticles

50 mg of the MSNPs were dispersed in a mixture of ultrapure water (5 ml) and hydrochloric acid (4 ml). After a while, the MKs present inside the MSNPs began to dissolve. At this time, the solution turned from dark brown (T in FIG.

4(b)) to light yellow ('ii' in FIG. 4(b)) in color. After the MKs were completely dissolved, centrifugation was repeated several times. The precipitate was collected, purified with ultrapure water, lyophilized, and fired at 300⁰C to remove organic impurities. As a result, hollow silica nanoparticles (HSNPs-OH) having surface hydroxyl groups were prepared. FIG. 3(d) is a transmission electron micrograph of the HSNPs-OH. The image confirms that the inner portions of the HSNPs-OH were completely empty to leave spherical cavities (hollow cores). The HSNPs-OH were found to have a size distribution of 83.1 ± 8.9 nm and a zeta potential of -43.0 ± 3.2 mV (FIG. 4(a)).

(5) Introduction of surface functional groups 10 mg of the HSNPs-OH and 0.05 ml of 3-aminopropyltrimethoxysilane were stirred in ultrapure water at 7O⁰C for 12 hours and purified by repeated centrifugation to substitute the hydroxyl groups of the silica surfaces with amine groups. The nanoparticles (HSNPs-NH₂) thus prepared were found to have a size distribution 85.9 ± 7.1 nm and a zeta potential of -4.2 ± 2.2 mV.

(6) Surface modification with biocompatible polymer

Polyethylene glycol (PEG-diCOOH, 0.05 mol) having carboxyl groups was added to 10 ml of dioxane, and then succinic anhydride (0.2 mol), 4- dimethylaminopyridine (0.1 mol) and triethyl amine (0.1 mol) were added thereto to activate the PEG-diCOOH. The mixture was allowed to react at room temperature' for 24 hr. The reaction mixture was passed through a filter, purified with carbon tetrachloride, precipitated with ethyl ether, and dried under vacuum to prepare a standard buffer. 20 mg of the HSNPs-NH₂ were dispersed in 0.5 ml of the carboxylate-polyethylene glycol standard buffer (0.1 mol), and N-3- dimethylaminopropyl-N-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide

(EDC/NHS, 0.2 mol) was added thereto. The mixture was incubated for 4 hr. As the reaction proceeded, the amine groups were reacted with the carboxyl groups of the polyethylene glycol on the surfaces of the silica shells to form amide bonds (O=C-N-H), which were confirmed by IR spectroscopy. The unreacted materials were removed by filtration and centrifugation to give, hollow silica nanoparticles

(HSNPs-PEG) surface-modified with the polyethylene glycol. The HSNPs-PEG were found to have a size distribution of 91.3 ± 8.1 nm and a zeta potential of 1.3 ± 3.2 mV.

Test Example 1

X-ray diffraction analysis, IR spectroscopy, thermo gravimetric analysis, and X-ray photoelectron spectroscopy were used to confirm whether the desired hollow silica nanoparticles were prepared in the respective steps.

(1) X-ray diffraction analysis

X-ray diffraction analysis was performed to identify the complete removal of the MKs present inside the MSNPs by acid treatment and firing in (4) of Example 1. FIG. 5 shows the results of the MSNPs containing the MKs (a) and the particles having undergone acid treatment and firing (b). From the graph of FIG. 5, it can be seen that silicate and spinel magnetic nanoparticles were present before removal of the MKs, but the magnetic nanoclusters and organic substances were not present after acid treatment and firing, demonstrating complete removal of the magnetic materials present inside the MSNPs.

(2) IR spectroscopy

FIG. 6 is a graph showing the results of Fourier transform infrared spectroscopy (FT-IR) for (a) MKs, (b) MSNPs and (c) HSNPs. The graph shows that no peaks at about 580 cm^"1 corresponding to Fe-O bonds of the magnetite were observed in the silica particles free of the MKs, indicating that the magnetic materials present inside the MSNPs were completely removed.

(3) Thermogravimetric analysis Thermogravimetric analysis was performed on the MSNPs and the HSNPs, and the results are shown in FIG. 7. The graph shows that the curve (a) of the MSNPs steeply falls at 260⁰C due to the decomposition of the organic substances such as dodecanoic acid and polyvinyl alcohol but the curve (b) of the HSNPs having undergone at 300⁰C slowly falls, indicating a relatively low content of the organic substances. These results suggest that the magnetic materials present inside the MSNPs were removed.

(4) X-ray photoelectron spectroscopy

The iron contents of the particles prepared in the respective steps were measured by energy-dispersive X-ray spectroscopy (EDX). FIG. 8 shows the EDX analytical results and the composition ratios Si/Fe of (a) MKs, (b) MSNPs and (c) HSNPs. As shown in FIG. 8, MKs, MSNPs and HSNPs had iron contents of 96.92

± 1.13%, 38.70 ± 0.77% and 2.86 ± 0.46%, respectively.

Test Example 2

The amounts of nitrogen adsorbed/desorbed in the HSNPs were measured by a BET method to determine the volumes of the cavities of the cores and the silica pores of the shells. The results are shown in FIG. 9. As is apparent from FIG. 9, the amounts of nitrogen adsorbed in the HSNPs-PEG and the MSNPs were > 198.7 cm /g and 92.7 cm Ig, respectively. The amounts of nitrogen adsorbed in the silica particles (HSNPs) before surface modification were smaller than those of nitrogen adsorbed in the particles (HSNPs-PEG) after surface modification. This is because the pore size was reduced by the polyethylene glycol molecules present on the

HSNPs-PEG. The nitrogen adsorption/desorption experimental results also show that the HSNPs-PEG and the MSNPs had average pore sizes of about 1.64 nm and about 2.3 nm, respectively. These results are believed to be because the pore size was reduced after the surface modification with the polyethylene glycol. That is, the size of the inner cavities of the cores and the pore size of the silica shells were reduced by the polyethylene glycol molecules. Test Example 3

10 mg of each of the three kinds of particles (i. e. HSNPs-OH, HSNPs-NH₂ and HSNPs-PEG) were reacted with 5 mg of doxorubicin as an anticancer agent in 4

5 ml of the standard buffer solution for 24 hr to introduce the anticancer agent into the nanoparticles, followed by centrifugation. Thereafter, the loading fraction and efficiency of the anticancer agent in the nanoparticles were calculated by Equations

2 and 3, respectively. r ,. - . ,„ „ Weight of the drug in the nanoparticles . . _ ,_nΛ

Loading fi-actoin (%) = x 100 (2)

Weight of the nanoparticles

_{1 n} ,. „ . .. .. Weight of the drug loaded in the nanoparticles _{- ΛΛ} /Q \

J- U Loading efficiency (%) = x 100 \->)

Weight of the drug used

The results are shown in Table 3.

Table 3 Loading fraction (%) Loading efficiency (%)

HSNPs-OH 23.5 47.7 HSNPs-NH₂ 21.6 42.3 HSNPs-PEG 19^5 38/7

As can be seen from Table 1, the cavity size of the hollow cores and the pore 15 size of the shells were controlled by the modification of the hollow silica nanoparticles with the surface functional groups (amine groups) and the biocompatible polymer (polyethylene glycol), and as a result, the loading fraction and efficiency of the drug (doxorubicin) were changed.

Subsequently, the different kinds of anticancer agent-load particles were 20 tested for drug release. Specifically, 2 ml of the anticancer agent-loaded particles were dispersed in a dialysis tube and 10 ml of the standard buffer was added thereto.

The amount of the anticancer agent released was measured using a UV-Vis spectrometer at a wavelength of 480 nm while maintaining the temperature at 37⁰C.

At this time, sonication was performed at room temperature to induce the release of 25 the anticancer agent from the anticancer agent-loaded particles, and the released anticancer agent was collected by centrifugation. FIG. 10 shows release profiles of the anticancer agent from the different kinds of nanoparticles with the loading fractions and efficiencies indicated in Table 3. Specifically, FIG. 10 shows (a) a graph and (b) a semi-logarithmic graph illustrating the amounts of the drug released from the respective drug carriers as a function of time.

The drug release profiles of the different kinds of nanoparticles were calculated by Equation 4:

in which X_t and X;_nf represent particular time points, t represents the drug release time, inf means infinite (i. e. the time when the physical release of the drug was completed), and k is the rate constant.

The results are shown in Table 4. Table 4

Rate K^a (Ln%/day) R² Rate K^a (Ln%/day) R²

Period (days) 0- 1 2-15

HSNPs-OH 2.2956 0.9781 0.1040 0.8607

HSNPs-NH₂ 0.7108 0.9734 0.1051 0.9958

HSNPs-PEQ 0.2270 0.9401 0.0636 0.9765

From the results in FIG. 10 and Table 4, it can be known that the drug was released from the HSNPs-OH in one day, whereas the HSNPs-NH₂ showed a slower release rate and the HSNPs-PEG showed a much slower release rate than the HSNPs- OH. Particularly, the HSNPs-PEG showed no sudden release profile at the initial stage. On the first day of the test, the release rate of the anticancer agent from the HSNPs-OH was 2.2956 Ln%/day, which is about three times higher than that

(0.7108 Ln%/day) for HSNPs-NH₂ and is about ten times higher than that (0.2270 Ln%/day) for the HSNPs-PEG. From the second day after the test, the HSNPs-PEG showed a zero-order reaction profile and a release rate of 0.0636 Ln%/day, which is lower than that (0.1040 Ln%/day) of the HSNPs-OH. These results demonstrate that the drug was steadily released from the hollow silica nanoparticles, suggesting

3D that the hollow silica nanoparticles are effective as drug carriers. Furthermore, taking advantage of the ability of the anticancer agent (doxorubicin) to emit fluorescence, the dispersibility of the anticancer agent-loaded silica particles was observed under a fluorescence microscope (FIG. 1 1). It can be confirmed from FIG. 11 that the doxorubicin-loaded particles were stably dispersed in water.

Claims

[ CLAIMS] [Claim 1 ]

Porous hollow silica nanoparticles, each comprising a core having a cavity with a diameter of 1 to 100 nm and a porous silica shell having functional groups on the surfaces thereof.

[ Claim 2]

The silica nanoparticles according to claim 1, wherein the surface functional groups are selected from the group consisting of -COOH, -CHO, -NH₂, -SH, - CONH₂, -PO₃H, -PO₄H, -SO₃H, -SO₄H, -OH, -NR₄ ⁺X^", sulfonate, nitrate, phosphonate, succinimidyl, maleimide and alkyl groups.

[ Claim 3 ]

Porous hollow silica nanoparticles, each comprising a core having a cavity with a diameter of 1 to 100 nm and a porous silica shell whose surface is modified with a biocompatible polymer.

[ Claim 4]

The silica nanoparticles according to claim 3, wherein the biocompatible polymer has a weight average molecular weight of 100 to 100,000.

[Claim 5]

The silica nanoparticles according to claim 3, wherein the biocompatible polymer is selected from the group consisting of polyalkylene glycol, polyetherimide, polyvinylpyrrolidone, hydrophilic vinyl polymers, copolymers thereof, and mixtures thereof. [ Claim 6]

The silica nanoparticles according to claim 3, wherein the biocompatible polymer is polyalkylene glycol.

[Claim 7]

The silica nanoparticles according to claim 3, wherein the biocompatible polymer is present in an amount of 5 to 50% by weight, based on the weight of the silica particles.

[ Claim S]

The silica nanoparticles according to claim 3, wherein the silica nanoparticles have a diameter of 20 to 250 nm.

[ Claim 9] The silica nanoparticles according to claim 1 or 3, further comprising a tissue-specific binding substance introduced into the silica shells.

[ Claim 10]

A method for preparing hollow silica nanoparticles, the method comprising mixing magnetic nanoclusters with a silica precursor, forming silica shells using the silica precursor on the respective magnetic nanoclusters, removing the magnetic nanoclusters present inside the silica shells, and introducing functional groups on the surfaces of the silica shells.

[ Claim 11 ]

The method according to claim 10, further comprising modifying the surfaces of the hollow silica nanoparticles with a biocompatible polymer.

[Claim 12]

The method according to claim 10, wherein the magnetic nanoclusters are prepared by dissolving magnetic nanoparticles in an organic solvent to prepare an oily phase, dissolving an amphiphilic compound in an aqueous solvent to prepare an aqueous phase, mixing the oily phase with the aqueous phase to prepare an emulsion, and separating the oily phase from the emulsion.

[Claim 13]

The method according to claim 12, wherein the magnetic nanoclusters are composed of a metal material, a magnetic material or a magnetic alloy.

[ Claim 14] The method according to claim 13, wherein the metal material is selected from the group consisting of Pt, Pd, Ag, Cu, Au and mixtures thereof, the magnetic material is selected from the group consisting of Co, Mn, Fe, Ni, Gd, Mo, MM'₂O₄, M_xO_y (M and M' are each independently represents Co, Fe, Ni, Mn, Zn, Gd, or Cr, 0 < x < 3, and 0 < y < 5) and mixtures thereof, and the magnetic alloy is selected from the group consisting of CoCu, CoPt, FePt, CoSm, NiFe, NiFeCo and mixtures thereof.

[ Claim 15]

The method according to claim 10, wherein the silica precursor is an alkoxysilane.

[Claim 16] Drug carriers comprising the silica nanoparticles according to any one of claims 1 to 8 and a pharmaceutically active ingredient loaded in the cavities of the cores or the pores of the silica shells.

[Claim 17]

The drug carriers according to claim 16, wherein the pharmaceutically active ingredient has a loading fraction of 1 to 100%, as calculated by Equation 1 :

,. _r . ,„,. Weight of the pharmaceutically active ingredient _{< ΛΛ /} I N

Loading fractoin (%) = — - x 100 ( 1 )

Weight of the silica nanoparticles

[Claim 18]

The drug carriers according to claim 16, wherein the pharmaceutically active ingredient is selected from the group consisting of anticancer agents, antibiotics, hormones, hormone antagonists, interleukins, interferons, growth factors, tumor necrosis factors, endotoxins, lymphotoxins, urokinase, streptokinase, tissue plasminogen activators, protease inhibitors, alkylphosphocholines, radioisotope- labeling components, surfactants, cardiovascular system drugs, gastrointestinal system drugs, nervous system drugs, and mixtures thereof.

[Claim 19] The drug carriers according to claim 18, wherein the anticancer agent is selected from the group consisting of epirubicin, docetaxel, gemcitabine, paclitaxel, cisplatin, carboplatin, taxol, procarbazine, cyclophosphamide, dactinomycin, daunorubicin, etoposide, tamoxifen, doxorubicin, mitomycin, bleomycin, plicomycin, transplatinum, vinblastin, methotrexate, and mixtures thereof.

[Claim 20] A method for loading a pharmaceutically active ingredient, the method comprising mixing the hollow silica nanoparticles according to any one of claims 1 to 8 with the pharmaceutically active ingredient in a solvent.

[ Claim 21 ]

A pharmaceutical composition comprising the drug carriers according to claim 16 and at least one pharmaceutically acceptable carrier.