KR20160063706A - Target-specific ligands conjugated stimuli-responsive hydrogel nanoparticles containing magnetic nanoparticles - Google Patents

Abstract

The present invention relates to a target-specific ligand-conjugated stimuli-responsive hydrogel nanoparticle which contains a magnetic nanoparticle, and more specifically, to a stimuli-responsive hydrogel nanoparticle comprising: a temperature-responsive hydrogel nanoparticle core (11); a magnetic nanoparticle (12) distributed inside the temperature-responsive hydrogel nanoparticle core; a solvent (13) swelling inside the temperature-responsive hydrogel nanoparticle; a shell (14), which is a modified surface of the temperature-responsive hydrogel nanoparticle core; and a target-specific ligand (15) attached to the surface of the shell. The stimuli-responsive hydrogel nanoparticle has a core which has a network structure and is made of tempearture-responsive hydrogel, such that a large amount of any kind of drug dispersed in an aqueous solution can be contained, and by containing magnetic nanoparticles inside temperature-responsive hydrogel, the initiation/termination of stimuli can be instantaneous such that localized control is possible, and the target-specific ligand attached to the surface of the shell of the hydrogel nanoparticle can deliver a drug to specific cancer cells, thereby reducing the risk of side effects and increasing the efficacy of the drug. Therefore, the present invention can be beneficially used as a contrast agent and also a drug carrier.

Description

The present invention relates to a target-specific ligands conjugated stimuli-responsive hydrogel nanoparticles containing magnetic nanoparticles containing magnetic nanoparticles and a target-

The present invention relates to a method of preparing a drug delivery vehicle having a target material and a functional material capable of changing its physical or chemical properties in response to an external stimulus, and more particularly, To a hydrogel nanoparticle having a selectivity for a specific cancer cell by binding a target-directed ligand.

The drug delivery system is intended to target and deliver a drug to a specific site in the body, and it is focused on the development of a drug delivery system using nanotechnology because it suppresses unnecessary distribution of drugs to maximize the efficacy while reducing side effects. In particular, studies on stimuli-sensitive drug delivery systems are becoming more popular because they can release drugs at a desired rate at a desired location by external stimuli.

As prior art related to drug delivery agents that are sensitive to external stimuli, non-patent document Du, Pengcheng, et al. Journal of Materials Chemistry B, 2013, 39, 5298-5308 discloses a dual-responsive drug delivery system comprised of a temperature sensitive polymeric layer surrounding the pH-sensitive microgel center. In the above non-patent reference, a pH-responsive microgel center was coated with silica and the surface was surrounded with a thermosensitive polymer, N-isopropylacrylamide, to prepare a target-oriented ligand. However, the drug delivery system which is sensitive to temperature and pH has a disadvantage in that the start / termination of stimulation is not immediate and local control is impossible because the characteristics of the whole solution in which the particles are dispersed are changed when the drug is released by stimulation.

Recently, in the field of nano-bio, magnetic nanoparticles have been used in various fields such as biomaterial separation, magnetic resonance imaging probes, biosensors including giant magnetoresistive sensors, microfluidic sensors, drug / gene transfer and magnetic high temperature treatment .

Specifically, the magnetic nanoparticles can be used as diagnostic probes (contrast agents) for magnetic resonance imaging. Magnetic nanoparticles have the effect of amplifying a magnetic resonance image signal by shortening the spin-spin relaxation time of hydrogen atoms contained in the surrounding water molecules.

Magnetic nanoparticles can also act as probe materials for giant magnetic resistance (GMR) sensors. When magnetic nanoparticles sense and combine patterned biomolecules on the surface of a giant magnetoresistive biosensor, the current signal of the giant magnetoresistive sensor is changed by the magnetic particles, and biomolecules can be selectively detected using the same (US Pat. No. 6,452,763 B1, US 6,940,277 B2, US 6,944,939 B2, US 2003/0133232 A1).

Magnetic nanoparticles can also be applied to the separation of biomaterials. For example, when a cell that expresses a specific biomarker and a variety of other cells are mixed, the magnetic nanoparticles are selectively bound to a specific biomarker, and when a magnetic field is externally applied, only the desired cell is separated in the magnetic field direction (US 4,554,088, US 5,665,582, US 5,508,164, US 2005/0215687 A1). Furthermore, it can be applied not only to separation of cells but also to separation of various biological materials such as proteins, antigens, peptides, DNA, RNA and viruses. Magnetic nanoparticles can also be applied to magnetic microfluidic sensors to isolate and detect biomolecules. By creating very small channels on the chip and flowing magnetic nanoparticles into it, it is possible to detect and isolate from the micro-fluid system.

On the other hand, magnetic nanoparticles can also be used for biotherapy through delivery of drugs or genes. The drug or gene is loaded onto the magnetic nanoparticle through chemical bonding or adsorption and is moved to a desired position using an external magnetic field to release a drug and a gene to a desired site, thereby providing a selective therapeutic effect (US Pat. 6,855,749).

Another example of application of magnetic nanoparticles to biotherapy is high temperature treatment using magnetic spin energy (US 6,530,944 B2, US 5,411,730). Magnetic nanoparticles emit heat through a spin flipping process when an alternating current of radio frequency is applied from the outside. At this time, when the temperature around the nanoparticles reaches 40 ° C or higher, the cells die due to high heat, thereby selectively killing diseased cells.

As such, magnetic nanoparticles are widely used in nano-biotechnology. In order for magnetic nanoparticles to be efficiently used in the above-mentioned applications, they must have excellent magnetic properties and must be stably transported and dispersed in a living body, that is, in an aqueous environment , It should be able to bind easily with bioactive substance.

In this connection, in Korean Patent No. 10-1351331 entitled " Magnetic Nanoparticles for Target-Oriented Drug Delivery and Method for Producing Drug Delivery Materials Using the Same ", the surface of the magnetic nanoparticles is combined with the thermosensitive polymer to form a target-oriented drug delivery system For example. The patent discloses that spherical magnetic nanoparticles are prepared by coating the surface of a liposome impregnated with Fe ³⁺ with silica and reduced, and the surface of the prepared magnetic nanoparticles is bonded with a thermosensitive polymer, N-isopropylacrylamide, Delivery system. However, since liposomes contain only a small amount of drug to be loaded and are only bonded to a thermosensitive polymer, the characteristics of the entire swollen liquid change upon release of the drug due to temperature changes, and it is disadvantageous in that immediate stimulation is initiated / terminated and local control is difficult .

Also, Korean Patent No. 10-1079235 entitled " Magnetic Nanocomposite, Contrast Agent Composition Containing the Same, and Pharmaceutical Composition Containing the Magnetic Nanocomposite " And a hydrogel present on the surface of the particulate polymer, a contrast agent composition and a pharmaceutical composition containing the same. Specifically, in the above patent, a hydrophilic polymer (PEG) is introduced on the surface of a particulate polymer (lactide-glycolide copolymer (PLGA)) in which magnetic nanoparticles and a pharmaceutically active ingredient are encapsulated and then a hydrogel component Discloses a magnetic nanocomposite in which hyaluronic acid (HA) is introduced. However, such a magnetic nanocomposite is also disadvantageous in that the hydrogel component is only selective for a specific disease marker in the living body, and since the drug-bearing PLGA is not sensitive to external stimuli, it is difficult to start / have.

Accordingly, the present inventors have made efforts to produce a drug delivery system capable of increasing the loading amount of a drug, targeting only to specific cancer cells, responding to various external stimuli, initiating / terminating stimulation promptly and locally, Nanoparticles incorporating target-oriented ligands on the surface of temperature-responsive hydrogel nanoparticles loaded with nanoparticles control the swelling and contraction of the hydrogel by various external stimuli, thereby providing an on-demand It is possible to induce the release of the drug, to initiate / terminate the stimulation promptly and locally, and to transfer the drug only to specific cancer cells due to the target-oriented ligand bound to the surface of the hydrogel, It was completed.

In order to solve the above problems, it is an object of the present invention to provide a stimulus-sensitive hydrogel gel nanoparticle having magnetic nanoparticles therein and a target-oriented ligand bound to the surface thereof.

The present invention also provides a method for producing the above-mentioned stimulus-sensitive hydrogel nanoparticles.

The present invention also provides a contrast agent composition comprising the stimulus-sensitive hydrogel nanoparticles.

It is another object of the present invention to provide a pharmaceutical composition comprising the above-mentioned stimulus-sensitive hydrogel nanoparticles.

In order to solve the above problems, the present invention provides, as one aspect,

A thermosensitive hydrogel nanoparticle core 11; Magnetic nanoparticles (12) distributed in the thermosensitive hydrogel nanoparticle core; A solvent (13) swollen in the thermosensitive hydrogel nanoparticles; A shell 14 modified on the surface of the thermosensitive hydrogel nanoparticle core; And a target-oriented ligand (15) bonded to the shell surface.

More preferably, the temperature-sensitive hydrogel nanoparticle core 11 is N - isopropyl acrylamide, N -n- propyl acrylamide, N, N '- dimethyl acrylamide, N, N' - diethyl acrylamide, At least one monomer selected from the group consisting of methacrylic acid, 2-hydroxymethacrylic acid, N -2-diethylaminoethyl acrylate and 1-vinyl-2-pyrrolidinone is copolymerized.

More preferably, the temperature-sensitive hydrogel nanoparticle core 11 has a diameter of 20 nm to 1 μm.

More preferably, the temperature-sensitive hydrogel nanoparticle core 11 has a diameter of 50 nm to 500 nm.

More preferably, the magnetic nanoparticles 12 include a metal material, a magnetic material, or a magnetic alloy that has a photo-thermal conversion effect and is sensitive to an external stimulus.

More preferably, the magnetic nanoparticles 12 are made of at least one magnetic material selected from iron oxide (II) nanoparticles and iron (III) oxide nanoparticles.

More preferably, the magnetic nanoparticles 12 are contained in an amount of 0.1 to 20 parts by weight based on 100 parts by weight of the thermosensitive hydrogel.

More preferably, the solvent (13) is a solution in which a drug, gene or biomaterial is dispersed or dissolved; Distilled water; Buffer solution; And a precursor solution of the transfer material.

More preferably, the drug is selected from the group consisting of anticancer agents, antibiotics, hormones, hormone antagonists, interleukins, interferon, growth factors, tumor necrosis factor, endotoxin, lymphotoxin, urokinase, Choline, a radioactive isotope labeling component, a surfactant, a cardiovascular drug, a gastrointestinal drug, and a nervous system drug.

More preferably, the anticancer agent is selected from the group consisting of epirubicin, docetaxel, gemcitabine, paclitaxel, cisplatin, carboplatin, taxol, procarbazine, cyclophosphamide, diazinomycin, diunorubicin, etoposide, At least one selected from the group consisting of diphenylmethane, diphenol, doxorubicin, mitomycin, bleomycin, plicomycin, transplatinum, vinblastine and methotrexate.

More preferably, the shell 14 is characterized in that a monomer having at least one functional group selected from the group consisting of a hydroxyl group (-OH), an amino group (-NH ₂ ) and a carboxyl group (-COOH) is copolymerized.

More preferably, the target-directed ligand 15 is at least one selected from the group consisting of a carbohydrate residue, transperine, folic acid, hyaluronic acid, and aptamer.

According to another aspect of the present invention,

A first step of preparing a stimulus-sensitive hydrogel nanoparticle core having magnetic nanoparticles mounted on thermosensitive hydrogel nanoparticles; A second step of preparing a shell modified with the surface of the stimulable nano particle core; And a third step of binding the target-oriented ligand to the surface of the shell. The present invention also provides a method for preparing a stimulable sensitized hydrogel nanoparticle.

According to another aspect of the present invention,

The above-mentioned stimulus-sensitive hydrogel nanoparticles; And a pharmaceutically acceptable carrier.

According to another aspect of the present invention,

The above-mentioned stimulus-sensitive hydrogel nanoparticles; And a pharmaceutically acceptable carrier.

More preferably, the pharmaceutically acceptable carrier is selected from the group consisting of ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins, buffer substances, water, salts, electrolytes, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone , A cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax or wool.

The stimulus-sensitive hydrogel nanoparticles according to the present invention are formed of a thermosensitive hydrogel having a core in the form of a network, so that the amount of the drug to be loaded is large. When the drug is dispersed in an aqueous solution, The magnetic nanoparticles are placed inside the thermosensitive hydrogel to convert the external magnetic field or external stimuli such as light into heat, thereby controlling the swelling and contraction of the hydrogel, It is possible to induce on-demand emission so as to narrow the target-oriented range, to start / stop the stimulation promptly, thereby enabling local control, Targeting ligands can deliver drugs only to specific cancer cells, reducing side effects and increasing drug efficacy. Therefore, it can be used not only as a contrast agent but also as a drug delivery system.

1 is a schematic diagram of a stimulus-sensitive hydrogel nanoparticle comprising a magnetic nanoparticle according to the present invention and a target-oriented ligand bound thereto.
2 is a graph showing the size distribution of the stimulus-sensitive hydrogel nanoparticles prepared according to an embodiment of the present invention.
FIG. 3 is a transmission electron microscope (TEM) image of the stimulus-sensitive hydrogel nanoparticles prepared according to one embodiment of the present invention.
4 is a fluorescence image showing the specific binding of HeLa cells to the stimulus-sensitive hydrogel nanoparticles prepared according to one embodiment of the present invention
FIG. 5 is a graph showing drug release behavior due to initiation / termination of light of the stimulus-sensitive hydrogel nanoparticles prepared according to an embodiment of the present invention.

As used herein, the term " thermosensitive hydrogel "refers to a hydrogel capable of responding to changes in temperature and capable of controlling the release of the solvent swelled in the hydrogel, and the term" Refers to a hydrogel capable of being placed in the thermosensitive hydrogel to control the temperature as well as other external stimuli to control the release of the swollen solvent in the hydrogel.

Hereinafter, the present invention will be described in detail.

FIG. 1 shows a schematic diagram of a stimulus-sensitive hydrogel according to the present invention.

As shown in FIG. 1, the stimulus-sensitive hydrogel nanoparticle according to the present invention comprises a temperature-sensitive hydrogel nanoparticle core 11; Magnetic nanoparticles (12) distributed in the thermosensitive hydrogel nanoparticle core; A solvent (13) swollen in the thermosensitive hydrogel nanoparticles; A shell 14 modified on the surface of the thermosensitive hydrogel nanoparticle core; And a target-oriented ligand (15) bonded to the shell surface. The hydrogel nanoparticles of the core-shell structure may incorporate various functional groups to bind the target-directed ligand to the shell surface without changing the properties of the hydrogel core itself.

The hydrogel nanoparticles have a characteristic of absorbing and swelling an aqueous solution because hydrophilic polymers have a three-dimensional network structure through chemical or physical crosslinking. Hydrogel nanoparticles are similar to living tissues in a state of absorbing a large amount of water, and thus are excellent in biocompatibility. The water retention ability of the hydrogel nanoparticles is suitable as a drug delivery vehicle because it can induce proper diffusion of the drug. In addition, since it has a network structure, it has a large amount of drugs to be loaded and swells by absorbing moisture, so that it can be mounted on any kind of drug in a form dispersed in an aqueous solution.

The thermosensitive hydrogel nanoparticles according to the present invention are characterized in that the thermosensitive hydrogel nanoparticles are polymers having a volumetric phase transition property according to temperature and include N -isopropylacrylamide, N -n-propylacrylamide, N , N ' Acrylamide, N , N' -diethylacrylamide, methacrylic acid, 2-hydroxymethacrylic acid, N -2-diethylaminoethylacrylate and 1-vinyl-2-pyrrolidinone The selected one or more monomers may be copolymerized. These monomers are monomers capable of producing thermosensitive hydrogels, and some of the monomers which do not exhibit thermosensibility may be copolymerized. Therefore, other monomers other than the above-mentioned monomers may also be copolymerized under the condition of maintaining the temperature sensitivity of the hydrogel.

Further, in linking the monomers, a crosslinking agent may be optionally used. The crosslinking agent is preferably selected from the group consisting of N , N , -methylenebisacrylamide, diethylene glycol diacrylate and dimethacrylate, ethylene glycol diacrylate and dimethacrylate, tetra (ethylene glycol) diacrylate, Hexanediol diacrylate, divinylbenzene, trimethylolpropane triacrylate, and poly (ethylene glycol) diacrylate.

The thermosensitive hydrogel nanoparticles exhibit a rapid change in temperature with temperature due to the phase transition near the low critical temperature (LCST). When the temperature of the hydrogel nanoparticles rises above the lower critical temperature, the hydrophobic bond of the polymer chain is strong The volume is reduced and the drug is released along with the swelling liquid.

In the prior art, a drug delivery system which binds a thermosensitive polymer and emits a drug responsive to an external temperature was prepared. However, in the case of the drug delivery system which is sensitive only to the external temperature, there is a disadvantage in that the start / end of stimulation is not immediate and local control is difficult.

In order to solve this problem, the present invention prepared hydrogel nanoparticles capable of responding to various external stimuli by mounting magnetic nanoparticles on thermosensitive hydrogel nanoparticles.

In the stimulus-sensitive hydrogel nanoparticle according to the present invention, since the temperature-sensitive hydrogel nanoparticles 11 on which the magnetic nanoparticles 12 are mounted can be arranged in the target site according to the distribution of the external magnetic field, The magnetic nanoparticles generate heat due to Neel relaxation or brownian relaxation, and the heat causes the thermosensitive hydrogel nanoparticles to shrink, and when the crossed magnetic field is applied, Due to the shrinkage of these thermosensitive hydrogel nanoparticles, the swollen solvent and the loaded drug are released. This allows for the placement of hydrogel nanoparticles by applying an external magnetic field to a desired site and controlling onset and termination of stimulation to induce on-demand drug release which releases as many drugs as desired .

In addition, in the stimulus-sensitive hydrogel nanoparticle according to the present invention, the magnetic nanoparticles 12 may be used as a photo-thermal conversion material having a photo-thermal conversion effect that absorbs light and emits heat. When the magnetic nanoparticles mounted on the thermosensitive hydrogel nanoparticles absorb heat to generate heat, the thermosensitive hydrogel nanoparticles shrink due to the heat. Just like a magnetic field, it is possible to start / stop an immediate stimulus, and local control is possible by adjusting the size and position of the light. Since the thermosensitive hydrogel nanoparticles loaded with the magnetic nanoparticles having the photo-thermal conversion effect exhibit a sensitivity in proportion to the intensity of light in the visible light and the near-infrared light, the volume change of the hydrogel nanoparticles It is possible to release the drug with precise control, and the degree of influence on the activity of the biomaterial is lower than temperature or pH, which is advantageous for application in the biotechnology field. The above-mentioned magnetic nanoparticles may be a metal material, a magnetic material or a magnetic alloy having a photo-thermal conversion effect, preferably iron oxide (II) nanoparticles, iron oxide (III) ) Nanoparticles may be used.

The magnetic nanoparticles may be prepared by a general method known in the art. For example, magnetic nanoparticles may be prepared by coprecipitation without limitation.

The magnetic nanoparticles 12 are preferably contained in an amount of 0.1 to 20 parts by weight based on 100 parts by weight of the thermosensitive hydrogel. If the content is less than 0.1 part by weight, the overall magnetic properties of the hydrogel nanoparticle core may deteriorate. If the content exceeds 20 parts by weight, the overall stability of the core may be decreased due to excessive magnetic materials, There is a possibility that the sealing efficiency of the sealing member becomes difficult.

If the particle size of the thermosensitive hydrogel nanoparticle core 11 is too small, it is discharged to the outside of the cell before the drug is released. If it is too large, it can not pass through the loose blood vessel gap around the cancer cell and is not suitable for application in the body. Lt; RTI ID = 0.0 > nm, < / RTI >

In the stimulus-sensitive hydrogel nanoparticles according to the present invention, the solvent (13) swollen in the thermosensitive hydrogel nanoparticles is based on a solution in which a substance such as drug, gene, or biomaterial is dispersed or dissolved, , A buffer solution, a precursor solution of a substance to be delivered, and the like, may be used without limitation.

Herein, the type of the drug that can be used in the present invention is not particularly limited, and examples thereof include anticancer drugs, antibiotics, hormones, hormone antagonists, interleukins, interferon, growth factors, tumor necrosis factor, endotoxin, lymphotoxin, urokinase, , A tissue plasminogen activator, a protease inhibitor, an alkylphosphocholine, a radioactive isotope labeled component, a surfactant, a cardiovascular drug, a gastrointestinal drug, and a nervous system drug.

Specific examples of the medicinal cancer drug include epirubicin, docetaxel, gemcitabine, paclitaxel), cisplatin, carboplatin, taxol, But are not limited to, procarbazine, cyclophosphamide, dactinomycin, daunorubicin, etoposide, tamoxifen, doxorubicin, mitomycin, mitomycin, bleomycin, plicomycin, transplatinum, vinblastin, and methotrexate.

It is possible to produce a drug carrier having selectivity for a specific cancer cell by imparting target orientation to the hydrogel nanoparticles sensitive to external stimuli. Targeted drug delivery systems are an optimized way to deliver drugs only to target sites while protecting other sites by suppressing unnecessary distribution of drugs. They are used as antibodies, peptides or ligands that can bind specifically to proteins expressed in cancer cells And the method of binding to a specific cancer cell receptor is referred to as active target orientation.

Active target orientation is achieved by the interaction of drug carriers and protein receptors present in target cells, where successful targeting is determined by high affinity and specificity. Representative examples of ligand-receptor interactions include carbohydrate residue-lectin, transferrin-transferrin receptor, folate-folate receptor, hyaluronan-CD44 Proteins. Thus, in the stimulable sensitized hydrogel nanoparticles according to the present invention, the target-directed ligand 15 can be selected from the group consisting of carbohydrate residues, transperin, folic acid, hyaluronic acid, and high affinity and specificity to various types of target ligands, Aptamer, which is a small (20 to 60 nucleotide) single stranded nucleic acid (DNA or RNA) fragment having binding characteristics can be used.

In the present invention, the shell 14 is a surface-modified portion for introducing a functional group for target-oriented ligand binding, and has a hydroxyl group (-OH), an amino group (-) on the surface of the hydrogel nanoparticle core, NH ₂ ), a carboxyl group (-COOH), or the like, as a functional group, can be copolymerized to prepare a surface-modified shell.

The method of binding the target-oriented ligand to the shell is not particularly limited. For example, a known crosslinking agent corresponding to each binding region may be used.

In addition, the stimulus-sensitive hydrogel nanoparticle according to the present invention comprises a first step of preparing a stimulus-sensitive hydrogel nanoparticle core having magnetic nanoparticles mounted on thermosensitive hydrogel nanoparticles; A second step of preparing a shell modified with the surface of the stimulable nano particle core; And a third step of binding the target-oriented ligand to the surface of the shell.

More specifically, in the present invention, the first step is a step of preparing a stimulus-sensitive hydrogel nanoparticle core, wherein the stimulus-sensitive hydrogel nanoparticle core is prepared by dissolving a thermosensitive hydrogenco monomer in a solvent (swelling solution) Particles and optionally a cross-linking agent, and thereafter adding a polymerization promoter and an initiator in an inert atmosphere, polymerizing the mixture, and gelling the mixture. The solvent, thermosensitive hydrogel monomer, magnetic nanoparticle, cross-linking agent and the like used herein are as described above, and the polymerization promoter and initiator can be those conventionally used in the art.

In the present invention, the second step is a step of preparing a shell modified with the surface of the hydrogel nanoparticle core. Specifically, the hydrogel nanoparticle core is coated with a hydroxyl group (-OH), an amino group (-NH ₂ ) -COOH) or the like having a functional group as a functional group can be copolymerized to prepare a surface-modified shell. In this case, the monomer used in the shell preparation is not particularly limited, and those conventionally used in the art can be used.

In the present invention, the third step is a step of binding the target-orienting ligand to the shell, wherein the method of binding the target-orienting ligand to the shell is not particularly limited, and a known crosslinking agent A method commonly used in the art can be used.

The thus prepared stimulus-sensitive hydrogel nanoparticles are composed of a thermosensitive hydrogel having a core in the form of a network, so that the amount of drug that can be loaded is large. When the drug is dispersed in an aqueous solution, By mounting magnetic nanoparticles inside a thermosensitive hydrogel, magnetic nanoparticles convert external stimuli such as external magnetic field or light into heat, thereby controlling the swelling and contraction of the hydrogel, On-demand emissions can be induced in the target area, and the target-oriented range can be narrowed, and the start / end of the stimulation can be immediately performed, so that local control is possible (see FIG. 5) Directed ligand bound to the surface of the tumor (see FIG. 4) Because of the efficacy can be increased, as well as the contrast agent can be useful as a drug delivery system.

The present invention also provides a contrast agent composition comprising the above-described stimulus-sensitive hydrogel nanoparticle according to the present invention and a pharmaceutically acceptable carrier; Or a pharmaceutical composition.

The type of carrier that can be used in the embodiment of the composition of the present invention is also not particularly limited, and carriers and vehicles commonly used in the medical field can be used. Examples of such carriers include ion exchange, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids ), Water, salts or electrolytes (such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substrate, polyethylene glycol, sodium carboxymethyl Cellulose, polyarylate, wax or wool, and the like. The compositions of the present invention may further comprise additives such as lubricants, wetting agents, emulsifying agents, suspending agents or preservatives in addition to the above-mentioned components.

In one embodiment, the compositions of the present invention may be prepared as an aqueous solution for parenteral administration. Preferably, a buffer solution such as Hank's solution, Ringer's solution or physically buffered saline can be used. Water-soluble injection suspensions may suitably be added with a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Another preferred embodiment of the composition of the present invention may be in the form of a sterile injectable preparation of an aqueous or oily suspension. Such suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Vehicles and solvents that may be used include mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally used as a solvent or suspending medium. For this purpose, any non-volatile oil including synthetic mono or diglycerides and less irritant may be used.

The method of using the respective compositions of the present invention is not particularly limited, and conventional methods known in this field can be used.

For example, in the case of the contrast agent composition according to the present invention, (A) the composition is administered to a living body or a sample; And (B) detecting a signal emitted from the magnetic nanocomposite of the living body or the composition contained in the sample to obtain an image.

In addition, in the step of injecting the composition into a living body or a sample in the above-described use mode, it can be administered through a route commonly used in the medical field, and parenteral administration is preferable. For example, intravenous, intraperitoneal, , Subcutaneously, or via a local route.

In addition, the signal emitted from the magnetic nanocomposite in the above-described embodiment can be detected by various apparatuses using a magnetic field, and a magnetic resonance imaging (MRI) apparatus is particularly preferable.

MRI is a method of inserting a living body into a strong magnetic field and irradiating a radio wave of a specific frequency so that the atomic nucleus such as hydrogen in the living tissue absorbs the energy to make the state high in energy and then the radio wave is stopped, And converts the energy into a signal, which is then processed by a computer and imaged. Since the magnetic or electromagnetic waves are not disturbed by the bone, a sharp three-dimensional monolayer image can be obtained at the end, transverse and arbitrary angle with respect to the hard bone around or the brain and the bone marrow. In particular, the MRI apparatus is preferably a T2 spin-spin relaxation MRI apparatus.

In the present invention, by using a substance capable of selectively reacting with a specific disease agent as the hydrogel, the pharmaceutical composition can be selectively used for simultaneous diagnosis and treatment of a specific disease. For example, when a drug such as magnetic nanoparticles and an anticancer agent is sealed in a hydrogel capable of selectively reacting with tumor cells and the like, the magnetic nanocomposite is selectively moved only to a site where tumor cells are present , It acts as a contrast agent through magnetic signals from the magnetic substance contained therein to instantly diagnose the onset area and further reliably and selectively releases the anticancer drug contained in the particulate polymer to selectively and efficiently treat the tumor cell And has an advantage that the therapeutic effect can be directly confirmed from the outside.

Hereinafter, the present invention will be described in detail with reference to examples, but these examples should not be construed as limiting the scope of the present invention.

<Production example> Production of magnetic nanoparticles

Iron (II) chloride and iron (III) chloride were dissolved in sodium hydroxide solution and reacted with 30 mL of distilled water at a 1: 2 molar ratio. To remove residual oxygen, a nitrogen atmosphere was made for 15-20 minutes and then 1 M sodium hydroxide was adjusted to pH 10. After stirring for 30 minutes, 2 wt% sodium oleate, a surfactant, was dissolved in 20 mL and dropped at a spray rate of 1 mL / min. The mixture was stirred at 80 ° C for 1 hour and then at room temperature. 1 M hydrochloric acid was added dropwise to adjust the pH to 5-5.5, followed by washing with distilled water, and 2 wt% dodecylbenzenesulfonate was added to the 10 mL aqueous solution to form a physical adsorption layer. And stirred for 30 minutes to obtain stable magnetic nanoparticles in the water phase.

<Examples> Preparation of stimulus-sensitive hydrogel nanoparticles containing magnetic nanoparticles and a target-oriented ligand bound thereto

Step 1: Manufacture of hydrogel nanoparticle core with magnetic nanoparticles

At room temperature, 150 mg of N -isopropylacrylamide as a monomer and 6 mg of methylene bisacrylamide as a crosslinking agent were dissolved in 20 ml of distilled water. The magnetic nanoparticles prepared in Preparation Example were mixed at a concentration of 0.84% by weight. The prepared solution was deoxygenated using a pressure reducer and then stirred at 70 DEG C at 250 rpm. 4.8 mg of potassium persulfate as an initiator was added and stirred for 3 hours and then lyophilized to obtain a hydrogel nanoparticle core loaded with magnetic nanoparticles.

Step 2: Preparation of a shell modified with the surface of the hydrogel nanoparticle core

In order to modify the surface of the hydrogel nanoparticle core with an amine group, 10 ml of the solution in which the hydrogel nanoparticle core was dispersed was deoxygenated by using a pressure reducer and then stirred at 70 ° C at 250 rpm. Next, 75 mg of N-isopropylacrylamide as a monomer, 17 μl of allylamine as a comonomer and 3 mg of methylene bisacrylamide as a crosslinking agent were added to 10 ml of tertiary distilled water and completely dissolved to prepare a mixed solution. Thereafter, the oxygen-removed mixed solution was added to the solution in which the hydrogel nanoparticle core was dispersed, followed by the addition of 4.8 mg of potassium persulfate as an initiator, followed by stirring for 6 hours. After stirring, the mixture was cooled at room temperature, sealed with a pre-washed dialysis membrane (15 kDa molecular weight cut-off), and dialyzed with primary distilled water for 24 hours to remove residual monomers and initiator to obtain a core- Particles were obtained.

Step 3: Preparation of target-oriented ligand-conjugated hydrogel nanoparticles

6mg folate, N - (3- dimethylaminopropyl) - N '- ethyl carbodiimide hydrochloride (EDC) 2.2mg, N- hydroxy-dimethyl the succinimide (NHS) 1.3mg sulfoxide (DMSO) completely 2ml After dissolving, the mixture was stirred in a dark place for 2 hours. Next, 5 ml of the solution prepared by dispersing the surface-modified hydrogel nanoparticles prepared above and 20 ml of phosphate buffered saline (PBS, pH 4.7) were mixed and then oxygen was removed using a pressure reducer, followed by stirring. The folate mixed solution, which was activated for 2 hours, was added to the solution in which the surface-modified hydrogel nanoparticles were dispersed and stirred for 16 hours in the dark. After stirring, sodium hydroxide was added to prepare a pH 9 solution. The mixture was sealed with a pre-washed dialyzed membrane (15 kDa molecular weight cut-off) and dialyzed for 7 days in primary distilled water to remove residual substances. .

<Analysis>

1. Identification of size distribution of stimulus-sensitive hydrogel nanoparticles

The hydrodynamic diameters of the stimulus-sensitive hydrogel nano-particles prepared according to the present invention were measured at room temperature using dynamic light scattering (DLS). The results are shown in FIG.

As shown in FIG. 2, the average particle size of the prepared hydrogel nanoparticles was 230 nm, and it was confirmed that the particle size distribution was uniform.

2. Identification of magnetic nanoparticle-containing stimuli-sensitive hydrogel nanoparticle core

The stimulus-sensitive hydrogel nanoparticle core obtained in Example 1 was photographed by a transmission electron microscope (TEM) and is shown in FIG.

As shown in FIG. 3, it was confirmed that the stimulus-sensitive hydrogel nanoparticles prepared according to the present invention are spherical bodies, and the magnetic nanoparticles are contained in the hydrogel nanoparticles.

<Experimental Example 1> Target-directed experiment of stimulus-sensitive hydrogel nanoparticles

In order to confirm the cell specific binding of the stimulus-sensitive hydrogel nanoparticles according to the present invention, the following experiment was conducted.

Specifically, the hydrogel nanoparticles according to one embodiment of the present invention in which folic acid is coupled as a target-directed ligand and the core-shell hydrogel nanoparticles having no folic acid conjugated thereto are cultured in HeLa cells for 24 hours to compare fluorescence images The results are shown in Fig.

As shown in FIG. 4, more fluorescence could be observed in HeLa cells in which folic acid-bound hydrogel nanoparticles were cultured. From this, it can be seen that the hydrogel nanoparticles to which the target-oriented ligand is bound according to the present invention can specifically bind to HeLa cells and thus can be actively targeted.

Experimental Example 4 Drug Release Behavior of Stimulation-Sensitive Hydrogel Nanoparticles

In order to investigate drug release behavior of the stimulus-sensitive hydrogel nanoparticles according to the present invention, the following experiment was conducted.

The hydrogel nanoparticles according to the present invention loaded with indomethacin as a drug were dispersed in a phosphate buffered saline solution. Then, the hydrogel nanoparticles were irradiated with visible light for 30 minutes, and the period for not irradiating visible light for 2 hours was 4 The absorbance was measured by UV / VIS spectroscopy, and the results are shown in FIG. 5.

As shown in FIG. 5, indomethacin was released at 20.1% at the first light start, 18.3% at the second light start, 13.5% at the third start, and 7.6% at the fourth light start. As the cycle repeats, the amount of indomethacin released decreases because the amount of indomethacin contained in the hydrogel nanoparticles decreases. When light was initiated / terminated repeatedly, indomethacin was released 14 hours after release, while indomethacin was released after 20 hours without stimulation. This means that when the visible light is irradiated, the volume of the hydrogel nanoparticles shrinks rapidly to increase the amount of the released drug and release the drug in a short time than in the case of not irradiating the visible light. It can be seen that it is possible to control the amount of released drug by adjusting the start / end time of light.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

11: core of hydrogel nanoparticles
12: Magnetic nanoparticles
13: swelling liquid
14: Shell
15: Targeted ligand

Claims (15)

A thermosensitive hydrogel nanoparticle core 11; Magnetic nanoparticles (12) distributed in the thermosensitive hydrogel nanoparticle core; A solvent (13) swollen in the thermosensitive hydrogel nanoparticles; A shell 14 modified on the surface of the thermosensitive hydrogel nanoparticle core; And a target-directed ligand (15) bonded to the shell surface. The method according to claim 1,
The thermosensitive hydrogel nanoparticle core 11 may include at least one selected from the group consisting of N -isopropylacrylamide, N -n-propylacrylamide, N , N' -dimethyl acrylamide, N , N' -diethylacrylamide, Wherein at least one monomer selected from the group consisting of hydroxymethacrylic acid, N -2-diethylaminoethyl acrylate and 1-vinyl-2-pyrrolidinone is copolymerized. The method according to claim 1,
Wherein the thermosensitive hydrogel nanoparticle core (11) has a diameter of 20 nm to 1 μm. The method according to claim 1,
The magnetic nanoparticle (12) comprises a metal material, a magnetic material or a magnetic alloy having a photothermal conversion effect and being sensitive to external stimuli. 5. The method of claim 4,
Wherein the magnetic nanoparticles (12) are made of at least one magnetic material selected from iron oxide (II) nanoparticles and iron oxide (III) nanoparticles. The method according to claim 1,
Wherein the magnetic nanoparticles (12) are contained in an amount of 0.1 to 20 parts by weight based on 100 parts by weight of the thermosensitive hydrogel. The method according to claim 1,
The solvent 13 may be a solution in which a drug, gene or biomaterial is dispersed or dissolved; Distilled water; Buffer solution; And a precursor solution of a carrier substance. 8. The method of claim 7,
The drug may be selected from the group consisting of anticancer agents, antibiotics, hormones, hormone antagonists, interleukins, interferons, growth factors, tumor necrosis factor, endotoxin, lymphotoxin, urokinase, streptokinase, tissue plasminogen activator, protease inhibitor, Wherein the stimulable compound is at least one selected from the group consisting of a labeling agent, a surfactant, a cardiovascular drug, a gastrointestinal drug, and a nervous system drug. 9. The method of claim 8,
Wherein the anticancer agent is selected from the group consisting of epirubicin, docetaxel, gemcitabine, paclitaxel, cisplatin, carboplatin, taxol, procavazine, cyclophosphamide, diazinomycin, diunorubicin, etoposide, Wherein the stimulable compound is at least one selected from the group consisting of mitomycin, bleomycin, plicomycin, transplatinum, vinblastine and methotrexate. The method according to claim 1,
Wherein the shell (14) is copolymerized with a monomer having at least one functional group selected from the group consisting of a hydroxyl group (-OH), an amino group (-NH ₂ ) and a carboxyl group (-COOH). The method according to claim 1,
Wherein the target-directed ligand (15) is at least one selected from the group consisting of a carbohydrate residue, transperine, folic acid, hyaluronic acid, and aptamer. A first step of preparing a stimulus-sensitive hydrogel nanoparticle core having magnetic nanoparticles mounted on thermosensitive hydrogel nanoparticles; A second step of preparing a shell modified with the surface of the stimulable nano particle core; And a third step of binding the target-oriented ligand to the surface of the shell. 11. The stimulable compound according to any one of claims 1 to 11, And a pharmaceutically acceptable carrier. 11. The stimulable compound according to any one of claims 1 to 11, And a pharmaceutically acceptable carrier. 15. The method of claim 14,
The pharmaceutically acceptable carrier may be selected from the group consisting of ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins, buffer substances, water, salts, electrolytes, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, Polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax or wool.

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