KR20160142976A

KR20160142976A - Method to prepare nontoxic fluorescent nano-particles coated hydrophilic silicon containing polymers complexed with Lanthanide

Info

Publication number: KR20160142976A
Application number: KR1020150078917A
Authority: KR
Inventors: 김도경; 김민철
Original assignee: 건양대학교산학협력단
Priority date: 2015-06-04
Filing date: 2015-06-04
Publication date: 2016-12-14
Also published as: KR101749805B1

Abstract

The present invention relates to fluorescent nanoparticles and a method for manufacturing fluorescent nanoparticles comprising the steps of: (a) synthesizing a copolymer including a silicon-based compound and a water soluble polymer; (b) preparing a core material of nanoparticles; (c) manufacturing a lanthanide metal complex; (d) mixing respective materials obtained in the step (a) and (c) at a weight ratio of 1 : 0.2 to 1 : 0.5, mixing the mixture with the core material obtained in the step (b) at a weight ratio (mixture : core material) of 1 : 0.5 to 1 : 1, and performing coating; and (e) heating the particles obtained in the step (d) and crosslinking the same.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for preparing fluorescent nanoparticles coated with a water-soluble polymer containing a lanthanide metal complex and silicon,

The present invention relates to a fluorescent nanoparticle and a method for producing the same, and more specifically, to a method for producing a fluorescent nanoparticle and a method for producing the same by preparing a magnetic nanoparticle, a water-soluble polymer containing silicon in a core material, and a lanthanide metal complex, And a method for producing the fluorescent nanoparticles.

Recently, fluorescent nanoparticles are similar in size to functional biomaterials, so biocompatible nanoparticles can predict breakthroughs in biomedical and medical applications. For example, semiconducting quantum dots are far superior to organic light-emitting materials used in conventional bio-optical imaging, and their emission color changes according to their size. Therefore, biological characteristics such as image technology, sensor technology, and microarray As a fluorescent probe and a photon source for interest, there is a growing interest.

In order to apply fluorescent nanoparticles to various fields, it is required to be arranged in a high-dimensional complex structure or to be able to bind with biological macromolecules, and single-molecule nanoparticles capable of controlling excitation and luminescence should be studied.

In general, the fluorescence of quantum dots is generated by the electrons falling from the conduction band to the valence band and shows many properties different from general fluorescent dyes. Although the quantum dots are composed of the same material center, the fluorescence wavelength changes according to the size of the particles. As the particle size becomes smaller, the fluorescence of a shorter wavelength is emitted. By controlling the particle size of the quantum dots, Can be implemented. Also, unlike a general organic fluorescent compound, fluorescence can be obtained even if an emission wavelength is arbitrarily selected. Therefore, when various quantum dots are coexisted, fluorescence of various colors can be observed at the same time when light is emitted with a single wavelength.

However, in spite of the excellent characteristics of the quantum dot, cadmium, which is a regulated substance, has limited use except for research purposes or large-scale photovoltaic cells, and it is necessary to develop more secure fluorescent nanoparticles that can solve this problem.

Generally, the quantum dots generated in the narrow wavelength range of fluorescence that is much stronger than the fluorescent material are particles in which the nanoscale II-IV semiconductor particles (CdSe, CdTe, CdS, etc.) form the core and the lanthanide- It has excellent physico-chemical properties such as luminescence time, strong luminescence, long Stokes shift and narrow half-width (FWHM), and has been applied to a variety of preparations such as fluorescent labels and OLED.

Fluorescence-based imaging technology has been widely used in the field of medicine, and it has been studied in a variety of applications such as diagnostic kits using micro- or nanoparticles, and fluorescent dyes for tracking molecular-based materials. Recently, there has been a demand for a special labeling technique capable of diagnosing a specific activity by maximizing a fluorescence intensity value at a desired emission wavelength band by removing background noise. In addition to fluorescent materials, the development of new forms of nanoparticle-based fluorescent labeling technology can improve the sensitivity of instrumental analysis.

Recently, studies have been conducted to bond lanthanide complexes with submicron sized particles and to combine silica particles labeled with terbinafine (III) and europium (III) with prostate cancer specific antigens.

The electronic structure of the lanthanide ion (Ln ³⁺ ) has a structure of 4f ⁿ (n = 1 to 14) in which the 4f orbital functions are sequentially filled from the [Xe] structure. Such lanthanide-based fluorescent materials are attracting attention because of their narrow half-width (FWHM) and high fluorescence intensity, and thus many industrial applications are possible with these intrinsic spectroscopic characteristics. In particular, ligands that form complexes with lanthanide-based materials have been reported, such as Cryptands, calixarenes, β-diketones, macrocyclic ligands, carboxylic acid derivatives and heterobiary ligands. Among them, β-diketones and lanthanide Many studies on complexes have been made.

In addition, superpowder nanoparticles are materials that can be used in various fields such as nanomedical fields, such as magnetic resonance image contrast agent, target-oriented drug delivery system, and high frequency thermal cancer therapy. Particularly, super-magnetic nanoparticles are recognized as a breakthrough technology capable of transferring a therapeutic drug into a cell in an active or passive target-oriented form. Particularly, receptor-mediated intracellular penetration and penetration by diffusion is a type of passive cell membrane permeation, which is caused by the accumulation of substances outside the cell such as nanoparticles and biomolecules. Unlike direct receptive cell uptake, drug delivery by receptor - mediated intracellular delivery is a more broadly applicable technique because it is not limited by particle size or molecular weight.

As a conventional technique for fluorescent nanoparticles, a hydrophobic fluorescent polymer and a silica precursor are mixed in a solvent in which an organic solvent, a surfactant and water are mixed to form a fluorescent polymer core and a fluorescent polymer core, To provide core-shell nanoparticles in the form of silica shells.

However, since nanoparticles of various wavelengths can be produced, there is a problem of a high fluorescence intensity and a long fluorescence lifetime. In the present invention, fluorescent nanoparticles containing lanthanide metal complexes and super magnetic iron oxide nanoparticles are produced, .

Published Patent No. 10-2012-0072671 (July 4, 2012)

The present invention provides a non-toxic fluorescent nanoparticle comprising a lanthanide metal complex having improved fluorescence stability and a method of producing the same.

The present invention also provides a method for producing a fluorescent material which emits red light under UV excitation by crosslinking defects of a copolymer and a lanthanide metal complex on the surface of nanoparticles through thermal curing.

The present invention relates to a process for producing a nanoparticle, comprising the steps of: (a) synthesizing a copolymer comprising a silicone compound and a water-soluble polymer, (b) producing a core material of nanoparticles, (c) preparing a lanthanide metal complex, (1) and (2) are mixed at a ratio of 1: 0.2 to 1: 0.5 by mass of the respective materials obtained in the steps (a) and (c) 1: 1, and (e) crosslinking the particles obtained in the step (d) by applying heat to the fluorescent nanoparticles.

In one embodiment, the copolymer comprising the silicone compound and the water-soluble polymer in the step (a) may be represented by the following formula (1).

[Chemical Formula 1]

In one embodiment, in the step (b), the core material may be any one selected from super magnetic iron oxide nanoparticles or polystyrene, polymethylmethacrylate, silica, porous silica, zirconia, and titania.

In one embodiment, the lanthanide metal complex in the step (C) may be represented by the following formula (2).

(2)

The present invention relates to a process for producing a nanoparticle, comprising the steps of: (a) synthesizing a copolymer comprising a silicone compound and a water-soluble polymer, (b) producing a core material of nanoparticles, (c) preparing a lanthanide metal complex, (1) and (2) are mixed at a ratio of 1: 0.2 to 1: 0.5 by mass of the respective materials obtained in the steps (a) and (c) 1 to 1; and (e) cross-linking the particles obtained in the step (d) by applying heat to the fluorescent nanoparticles.

In the present invention, by combining a water-soluble polymer copolymer containing a lanthanide metal complex and a silicone compound on the surface of the super-magnetic iron oxide nanoparticles, the effect of high fluorescence intensity and long fluorescence lifetime is exhibited.

In addition, the fluorescent nanoparticles of the present invention can be applied to a wide variety of fields such as LEDs, OLEDs, and photovoltaic power generation devices as well as nano-medicine-related non-toxic fluorescent markers.

FIG. 1 is a schematic diagram of a method for manufacturing fluorescent nanoparticles according to an embodiment of the present invention.
FIG. 2 is a graph showing the relationship among the super-magnetic nanoparticles and the fluorescent nanoparticles
It is a transmission electron microscope photograph.
3 is a DSC thermal analysis graph of the copolymer and fluorescent nanoparticles prepared according to one embodiment of the present invention.
4 is a light emission and excitation spectrum according to the concentration of the mixture and the fluorescent nanoparticles prepared according to an embodiment of the present invention.
5 is a photograph of a mixture and fluorescent nanoparticles prepared according to an embodiment of the present invention under fluorescent lamp and UV condition.
6 shows the results of toxicity test of fluorescent nanoparticles prepared according to one embodiment of the present invention.
FIG. 7 is a photograph of the morphology of cells obtained by culturing the fluorescent nanoparticles prepared according to an embodiment of the present invention with U373MG cells and observing the shape of cells using a coprecipitation microscope.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, which illustrate preferred embodiments in which the present invention can be readily practiced by those skilled in the art. In the drawings of the present invention, the sizes and dimensions of the structures are enlarged or reduced from the actual size in order to clarify the present invention, and the known structures are omitted so as to reveal the characteristic features, and the present invention is not limited to the drawings . DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. In the following description, well-known functions or constructions are not described in detail to avoid obscuring the subject matter of the present invention.

As can be seen from FIG. 1, the present invention is a fluorescent nanoparticle having a structure in which a synthetic polymer composed of a polymer copolymer and a lanthanide metal complex is coated on the surface of a core material of nanoparticles.

The present invention relates to a process for producing a nanoparticle, comprising the steps of: (a) synthesizing a copolymer comprising a silicone compound and a water-soluble polymer, (b) producing a core material of nanoparticles, (c) preparing a lanthanide metal complex, (synthetic polymer: core material) 1: 0.2 to 1: 0.5 by mass of the respective materials obtained in steps (a) and (c) and the core material obtained in step (b) 0.5 to 1: 1; and (e) crosslinking the particles obtained in the step (d) by applying heat to the nanoparticles.

In the step (a) of synthesizing the copolymer comprising the silicone compound and the water-soluble polymer, the copolymer may be represented by the following formula (1).

[Chemical Formula 1]

In Formula 1,

R ₁ to R ₃ are the same or different from each other and independently selected from the group consisting of hydrogen and an alkyl group having 1 to 5 carbon atoms,

X is any one selected from oxygen, sulfur and nitrogen,

Y is any one selected from the group consisting of polyethylene glycol, dextran, polyvinyl pyrrolidone, polypropylene glycol, and polyethylene glycol,

l, m and n are integers of 1 to 10,000,

p and q may be an integer of 1 to 20.

Here, Y may preferably be polyethylene glycol, and it may be water-soluble and have an anti-biofouling action to prevent binding of proteins and cells in vivo to nanoparticles.

In the present invention, the silicone compound may be an acryl-containing silane compound, and examples thereof include 3-methoxysilylpropyl methacrylate, 3-ethoxysilylpropyl methacrylate, 3-methoxysilylpropyl acrylate, 3- Methoxysilylbutyl methacrylate, 3-methoxysilylbutyl methacrylate, 3-methoxysilylbutyl acrylate, 3-ethoxysilylbutyl acrylate, 3-methoxysilylpentyl methacrylate, 3-methoxysilylhexyl methacrylate, 3-methoxysilylhexyl methacrylate, 3-ethoxysilylhexyl methacrylate, 3-methoxysilylhexyl methacrylate, , 3-methoxysilylhexyl acrylate, 3-methoxysilylhexyl acrylate, 3-methoxysilyl heptyl methacrylate, 3-ethoxysilyl heptyl methacrylate, 3- Et Methoxysilyloctyl acrylate, 3-methoxysilyloctyl acrylate, 3-methoxysilyl octyl methacrylate, 3-methoxysilyl octyl methacrylate, 3-methoxysilyl octyl acrylate, 3- Methoxysilylnonyl acrylate, 3-ethoxysilylnonyl methacrylate, 3-methoxysilylnonyl acrylate, or 3-ethoxysilylnonyl acrylate, preferably 3-trimethoxysilylpropyl Methacrylate, but are not limited thereto.

The copolymer is a water-soluble polymer having a silicon-based compound covalently bonded thereto, and may be the main skeleton in the formula (1). Here, the water-soluble polymer may be any one selected from the group consisting of polyacrylic acid, polyacrylic acid derivatives, polymethacrylic acid, polymethacrylic acid derivative polyacrylamide, polyacrylamide derivatives, polyundecanoic acid, polyundecenoic acid derivatives, And may be derivatives or copolymers thereof. Preferably, the water-soluble polymer may be a polyacrylic acid or a derivative thereof, or a copolymer of the polymer, preferably, polymethacrylic acid.

In the present invention, the copolymer may have a structure of a block copolymer in which a repeating unit containing silicon and a repeating unit including a polymer of polyethylene glycol are sequentially arranged, and may be a random copolymer.

In the step (a), the copolymer may contain 25 to 35% by weight of silicon, 50 to 70% by weight of polyethylene glycol, and 5 to 15% by weight of a water-soluble polymer. In addition, the radical reaction initiator azobisisobutyronitrile azobisisobutyronitrile (AIBN) may be added to the reaction mixture, and the polymerization reaction may be carried out, preferably in the form of poly (TMSMA-r-PEGMA-r-MAA). In addition, the process for preparing the copolymer in the step (a) may be as shown in Reaction Scheme 1.

[Reaction Scheme 1]

In the above Reaction Scheme 1, l, m and n are the same as 1, m and n in the above-described formula (1).

In the present invention, in the step (b) of producing the core material of the nanoparticles (b), the core material may be either a super-magnetic iron oxide nanoparticle or any one selected from polystyrene, polymethylmethacrylate, silica, porous silica, zirconia, And may be preferably a super magnetic iron oxide nanoparticle.

The superpowder iron oxide nanoparticles may be any one selected from iron oxide (II), iron oxide (III), cobalt ferrite, zinc ferrite, nickel ferrite, manganese ferrite, iron, cobalt, nickel, manganese, FeAu, FePt, And may preferably be iron oxide (II), iron oxide (III).

In the present invention, in the step of preparing the (C) lanthanide metal complex, the lanthanide metal complex may be represented by the following formula (2).

(2)

In Formula 2,

R ₁₁ to R ₁₃ are the same or different from each other, and are each independently selected from among hydrogen, an alkyl group having 1 to 20 carbon atoms, and a phenyl group,

X is any one selected from an aromatic hydrocarbon ring having 6 to 50 carbon atoms and an aromatic heterocycle having 3 to 40 carbon atoms,

Y is a fluorohydrocarbon having 1 to 10 carbon atoms,

Ln is any one selected from lanthanide group rare earth elements,

r and s are integers of 1-6.

Here, Ln is preferably any one selected from europium, terbium, gadolinium, samarium, yttrium, neodymium, lanthanum and cerium, and more preferably europium.

In the present invention, the ligand forming the complex with the lanthanide-based material may be any one selected from Cryptands, calixarenes,?-Diketones, macrocyclic ligands, carboxylic acid derivatives, and heterobiary ligands, preferably?-Diketones complex , More preferably trioctylphosphine oxide.

In general, β-diketones have been studied extensively for lanthanide-based materials, and excitation wavelengths and energy transfer efficiencies can be affected by substituents added to the skeleton of β-diketones. That is, the sensitized luminescence intensity of the β-diketone complex can be greatly affected by various substituents such as methyl, trifluoromethyl, phenyl, thiophenyl, polyaromatic units, etc. In the present invention, 4,4-trifluoro-1- (2-naphthyl) -1,3-butadione.

In particular, in the case of complexes composed of β-diketones ligands and lanthanide-based materials, lipid-soluble substances can not be used in an aqueous solution. Therefore, β-diketones ligands and lanthanide complexes The dispersibility of the aqueous solution can be improved.

In order to prepare the lanthanide metal locating agent, europium, 4,4,4-trifluoro-1- (2-naphthyl) -1,3-butadione and trioctylphosphine oxide are preferably used in a molar ratio of 1: 2: To 1: 3: 3. After the mixing, the mixture can be heated at 50 to 70 ° C for 30 to 60 minutes through a hot water bath method.

In the present invention, (d) the mass of the composite material obtained by mixing the respective materials obtained in the steps (a) and (c) at a mass ratio of 1: 0.2 to 1: 0.5 and the core material obtained at the step (b) Polymer: core material) 1: 0.5 to 1: 1; And (e) cross-linking the particles obtained in (d) by applying heat to the particles.

The synthetic polymer obtained by mixing the respective materials obtained in steps (a) and (c) in the step (d) may be represented by the following formula (3).

(3)

In Formula 3, 1, m, and n are the same as 1, m, and n in Formula 1 described above, and x is an integer of 1 to 10.

Also, by mixing the synthetic polymer and the core material obtained in the step (b), the synthetic polymer can be coated on the surface of the core material, and heat can be applied to complete the coating.

Here, the water-soluble polymer containing silicon is thermally cured on the surface of the core material through cross-linking between the silicon elements by heat, that is, physical adhesion is established between the silicon element and the core material, The stability of the core material itself can be greatly increased. At this time, the thermosetting may be performed at a temperature of 70 to 90 ° C.

The present invention relates to a process for producing a nanoparticle, comprising the steps of: (a) synthesizing a copolymer comprising a silicone compound and a water-soluble polymer, (b) producing a core material of nanoparticles, (c) preparing a lanthanide metal complex, (1) to (1): 0.5 to 1: 0.5 by mass of the respective materials obtained in the steps (a) and (c) : 1; and (e) a step of cross-linking the particles obtained in the step (d) by applying heat to the fluorescent nanoparticles.

Hereinafter, the present invention will be described in detail by way of examples.

(Example)

Example 1

Example 1-1. Synthesis of Polyethylene Glycol-Silicon-Carboxylic Acid (PEG-Silicone-COOH) Copolymer

0.25 g (1 mmol) of 3-trimethoxysilylpropyl methacrylate (TMSMA), 0.475 g (1 mmol) of polyethylene glycol methyl ether methacrylate (PEGMA) and 0.086 g (1 mmol) of methacrylic acid After dissolving in 10 ml of tetrahydrofuran (THF), flow nitrogen gas for 20 minutes. Thereafter, 10 mg (0.1 mmol) of azobisisobutyronitrile (AIBN) was added and the reaction was allowed to proceed at 70 ° C for 24 hours to obtain a water-soluble polyethylene glycol-silicone (PEG-Silicone) .

Example 1-2: Synthesis of super magnetic iron oxide nanoparticles

After removing oxygen by adding nitrogen gas to 20 mL of distilled water for 30 minutes, 0.05 g (0.185 mmol) of FeCl ₃ ㅇ 6H ₂ O and 0.0184 g (0.0925 mmol) of FeCl ₂ ㅇ 4H ₂ O were added and dissolved. To prepare an iron stock solution.

After that, 2 mL of ammonia water cooled in the iron stock solution is added, and the temperature is raised while stirring, and the reaction is carried out at 85 ° C for 1 hour. Thereafter, after washing with distilled water three times or more, the obtained precipitate is redispersed in distilled water using ultrasonic waves, and then stored at 4 ° C.

Example 1-3: Eu (NTA) ₃ (TOPO) ₃ synthesis

0.3569 g (1 mmol) of Europium oxide (Eu ₂ O ₃ , Fw = 356.92 g / mol) was dissolved in 1 mL of nitric acid and heated at 50 ° C in a hot water bath. Then, 5 mL of H ₂ O was added, Lt; / RTI > The solution is then made to a final concentration of 0.02 mmol.

Further, 0.2662 g (1 mmol) of 4,4,4-trifluoro-1- (2-naphthyl) -1,3-buthadione (NTA) was dissolved in ethanol and the solution was adjusted to a final concentration of 0.02 mmol I make it.

Further, 0.3866 g (1 mmol) of trioctylphosphine oxide (TOPO) was dissolved in ethanol, and the solution was made to have a final concentration of 0.02 mmol.

Mix 1 mL of the prepared Eu (0.02 mmol), 3 mL of NTA (0.02 mmol) and 3 mL of TOPO (0.02 mmol), and heat the mixture at 60 ° C for 1 hour.

Example 1-4: Synthesis of poly (TMSMA-r-PEGMA-r-MAA) Eu (NTA) ₃ (TOPO) ₃ Coated Fluorescent Nanoparticle Synthesis

30 mg of the poly (TMSMA-r-PEGMA-r-MAA) prepared in Example 1-1 is dissolved in a mixture of 10 mL of distilled water and 10 mL of ethanol. Then, 10 mg of Eu (NTA) ₃ (TOPO) ₃ prepared in Example 1-3 was mixed and stirred.

Then, 30 mg of the super-magnetic iron oxide nanoparticles prepared in Example 1-2 and 30 mg of the synthetic polymer were mixed in distilled water and heated at 80 ° C for 1 hour. Thereafter, the osmotic bag having a molecular weight of 12,500 cut off is used to wash until there is no further ion detection.

(Experimental Example)

Evaluation of fluorescent nanoparticles

The size and shape of the nanoparticles were analyzed by transmission electron microscopy. The excitation, emission wavelength and intensity of the coated iron oxide nanoparticles were analyzed by fluorescence spectroscopy. The uptake characteristics and toxicity of the iron oxide nanoparticles coated with poly (TMSMA-r-PEGMA-r-MAA) Eu (NTA) ₃ (TOPO) ₃ were also investigated. uptake was observed.

Experimental Example 1: Measurement of particle size and shape using a transmission electron microscope

In the present invention, particle size and shape according to whether or not the fluorescent nanoparticles were coated were analyzed with a transmission electron microscope (JEOL 2100F, 200 kV), as shown in FIG.

2 (a), the supercritical oxide nanoparticles prepared in Example 1-2, and (b) the transmission electron microscope photographs of the iron oxide nanoparticles prepared in Example 1-4. As a result, ) And (b), spherical particles of 13 nm in average were identified, and in FIG. 2 (b), it was confirmed that particles were aggregated after coating but there was no change in particle size. It is believed that the size of the particles is less than 15 nm at maximum, which facilitates injection into the mold.

Experimental Example 2: Measurement of Polymer Coating Using DSC Thermal Analysis

In the present invention, the physicochemical properties of the fluorescent nanoparticles and the coating of the surface were confirmed by DSC thermal analysis, as shown in FIG.

FIG. 3 shows the results of DSC analysis of the iron oxide nanoparticles prepared in the copolymer prepared in Example 1-1 and 1-4, wherein two endothermic peaks in the case of the copolymer and five endothermic peaks in the case of the iron oxide nanoparticles can be identified . The PEG-related peaks at 50 ° C were not observed in the analysis of the iron oxide nanoparticles. It can be seen that the PEG endothermic peak disappeared after being coated on the surface. Through the endothermic peak near 80.5 ° C, . &Lt; / RTI > The peaks at 106.7 ° C and 111.8 ° C are NTA and TOPO, which are used as europium complexing agents, and the strong peaks at around 127 ° C, are peaks due to the physical adsorption water on the surface of the iron oxide nanoparticles. As a result, .

Experimental Example 3: Fluorescence spectral analysis

In the present invention, the excitation, emission wavelength and intensity of the iron oxide nanoparticles coated with the fluorescent material according to the concentration change were analyzed using a fluorescence spectrometer (Shimadz, RF-5301PC), which is shown in FIG.

FIG. 4 (a) is the emission wavelength spectrum of the fluorescent nanoparticle coated with the synthetic polymer according to the concentration change in Example 1-4. FIG. 4 (a), all the luminescence spectra exhibit typical f-transition characteristics of europium ³⁺ and emit red light. The most intense luminescence peak is in the range of 621 nm where ⁵ D ₀ → ⁷ F ₂ transition occurs, ⁵ D ₀ → ⁷ F ₁ transition at 592 and 597 nm, ⁵ D ₀ → ⁷ F ₀ transition at 580 and 587 nm And ⁵ D ₁ → ⁷ F ₆ transition occurs at 700 nm.

FIG. 4 (b) shows the excitation wavelength spectrum of the fluorescent nanoparticles coated with the synthetic polymer in Examples 1-4, the excitation wavelength is wide ranging from 300 to 400 nm, and the maximum excitation wavelength is about 350 nm Can be confirmed. Here, when the excitation wavelength is wide, it is considered to be a characteristic of a typical europium complex.

Experimental Example 4: Measurement of cytotoxicity by MTT

The cytotoxicity of the fluorescent nanoparticles prepared in Example 1-4 was measured and shown in FIG. Cytotoxicity measurements for U373MG cells in DMEM (10% fetal bovine serum ( FBS), 2 mM L-glutamine, 1% penicillin / streptomycin) 5% CO 2, 37 ℃ atmosphere with the moisture in 100 mm cell flask using Cells were cultured. Cell viability was measured using MTT assay. U373MG cells are cultured in 96-well flasks at 1 × 10 ⁵ cells for 24 hours at 37 ° C in 5% CO ₂ . After the nanoparticles are added to the cells, the nanoparticles are incubated for 24 hours under the same conditions. Then, 10 μL of MTT solution (5 mg / mL) is added to the flask and incubated for 4 hours under the same conditions. 100 uL of Formazan dissolved in DMSO was added and measured by ELISA at 540 nm.

5 of the present invention, the sample (1) is a fluorescent substance prepared by coating the europium complex and the polymer, which are synthesized in Example 1-4, and the europium complex and the polymer-binding substance, (B) photographs under UV conditions. In the case of the sample, (a) it is visible under fluorescent lamps, (b) it has a red fluorescence under UV, and (b) in the case of (2) .

Generally, in the case of a fluorescent material, fluorescence intensity is significantly lowered or disappears when coexisting with an inorganic material. However, the non-toxic fluorescent nanoparticles developed in the present invention show no significant change in light emission intensity.

Experimental Example 5: Measurement of intracellular magnetic nanoparticle uptake and cell viability by coprecipitation microscope

FIG. 6 is a graph showing cell survival rate of the fluorescent nanoparticles prepared in Example 1-4 by the toxicity test according to the contents of the super-magnetic iron oxide nanoparticles.

For toxicity experiments, 10 ⁵ cells per cover slip were cultured in a 24 - well plastic dish and the nanoparticles were incubated with cells at a fixed concentration. After incubation at 37 ° C for 24 hours, the cells were washed twice with PBS. The cells were fixed in 4% paraformaldehyde solution at 4 ° C for 15 minutes and the upper layer was mounted with cover glass to prevent further discoloration.

As can be seen from FIG. 6, the cell viability was 80% at a concentration ranging from 10 to 300 ug / mL, and the cell viability was decreased at a concentration of 500 ug / mL.

7 is a graph showing the results of observing the shape of cells using a coprecipitation microscope after incubating the fluorescent nanoparticles prepared in Example 1-4 with U373MG cells for 24 hours. As a result, (TMSMA-r-PEGMA-r-Eu (NTA) ₃ (MAA) (TOPO) ₃ ) @ SPIONs are well penetrated into the cytoplasmic region inside the cell. The results show that the particles do not become toxic and increase cell labeling efficiency.

Claims

(a) synthesizing a copolymer comprising a silicone compound and a water-soluble polymer;
(b) preparing a core material of nanoparticles;
(c) preparing a lanthanide metal complex;
(d) mixing the respective materials obtained in the steps (a) and (c) at a ratio of 1: 0.2 to 1: 0.5 by mass to prepare a core material and a core material (mixture: core material) 1: 0.5 to 1: 1; And
(e) crosslinking the particles obtained in (d) by applying heat thereto; Wherein the fluorescent nanoparticle is a fluorescent nanoparticle.

The method according to claim 1,
The fluorescent nanoparticle according to claim 1, wherein the copolymer comprising the silicone compound and the water-soluble polymer in the step (a) is represented by the following formula (1).
[Chemical Formula 1]

In Formula 1,
R ₁ to R ₃ are the same or different from each other and independently selected from hydrogen and an alkyl group having 1 to 5 carbon atoms;
X is any one selected from oxygen, sulfur and nitrogen;
Y is any one selected from polyethylene glycol, dextran, polyvinylpyrrolidone, polypropylene glycol, and polyethylene glycol;
l, m and n are integers of 1 to 10,000,
p and q are an integer of 1-20.

The method according to claim 1,
Wherein the core material in the step (b) is any one selected from the group consisting of super magnetic iron oxide nanoparticles or polystyrene, polymethylmethacrylate, silica, porous silica, zirconia, and titania.

The method according to claim 1,
Wherein the lanthanide metal complex in the step (C) is represented by the following formula (2).
(2)

In Formula 2,
R ₁₁ to R ₁₃ are the same or different from each other and independently selected from the group consisting of hydrogen, an alkyl group having 1 to 20 carbon atoms, and a phenyl group,
X is any one selected from an aromatic hydrocarbon ring having 6 to 50 carbon atoms and an aromatic heterocycle having 3 to 40 carbon atoms,
Y is a fluorohydrocarbon having 1 to 10 carbon atoms,
Ln is any one selected from lanthanide group rare earth elements,
r and s are integers of 1 to 6;

The method of claim 3,
The superparamagnetic iron oxide nanoparticles are any one selected from iron oxide (II), iron oxide (III), cobalt ferrite, zinc ferrite, nickel ferrite, manganese ferrite, iron, cobalt, nickel, manganese, FeAu, FePt, As fluorescent nanoparticles.

3. The method of claim 2,
In the formula 1, the main skeleton is selected from among polyacrylic acid, polyacrylic acid derivatives, polymethacrylic acid, polymethacrylic acid derivative polyacrylamide, polyacrylamide derivatives, polyundecanoic acid, polyundecenoic acid derivatives and copolymers thereof Wherein the fluorescent nanoparticle is one or more fluorescent nanoparticles.

(a) synthesizing a copolymer comprising a silicone compound and a water-soluble polymer;
(b) preparing a core material of nanoparticles;
(c) preparing a lanthanide metal complex;
(d) mixing the respective materials obtained in the steps (a) and (c) at a ratio of 1: 0.2 to 1: 0.5 by mass to prepare a core material and a core material (mixture: core material) 1: 0.5 to 1: 1; And
(e) crosslinking the particles obtained in (d) by applying heat thereto; Wherein the fluorescent nanoparticles have a molecular weight of about 5,000 or less.