KR20220015844A - Fluorescent magnetic nanoparticles using porous silica and preparation method thereof - Google Patents

Abstract

The present invention relates to fluorescent magnetic nanoparticles using porous silica and a manufacturing method thereof and, more specifically, to: fluorescent magnetic nanoparticles using porous silica, having low cytotoxicity, excellent biocompatibility, red-light emission and magnetic properties to enable tracking with a confocal microscope in-vitro and non-invasive tracking of particles using MRI in-vivo; and a manufacturing method of the fluorescent magnetic nanoparticles. To this end, the method comprises: (a) a step of using fatty acid and a metal precursor to prepare fatty acid-coated magnetic nanoparticles; (b) a step of coating the surface of the fatty acid-coated magnetic nanoparticles with phosphatidylcholine; (c) a step of adding the phosphatidylcholine-coated magnetic nanoparticles and a silica precursor to a positive ionic surfactant template to form a magnetic nanoparticle core/porous silica shell; (d) a step of introducing a methacrylate functional group into the porous silica shell using a silane coupling agent and then adding acrylic acid to activate a carboxyl group; (e) a step of synthesizing a europium complex; and (f) a step of making the materials obtained in (d) and (e), respectively, react to prepare fluorescent magnetic nanoparticles.

Description

Fluorescent magnetic nanoparticles using porous silica and manufacturing method thereof

The present invention relates to fluorescent magnetic nanoparticles using porous silica and a method for manufacturing the same, and more particularly, to form a mesoporous silica shell layer on the surface of a magnetic core and at the same time to introduce a functional group capable of chemical bonding on the silica surface to introduce lanthana Fluorescent magnetic nanoparticles using porous silica that can be detected with a confocal microscope when applied to the in-vitro field by coating an id metal complex and provide excellent spatial resolution as an MRI contrast agent in in-vivo, and a method for manufacturing the same will be.

In recent years, nanomaterials have been attracting attention for their use in various applications such as biosensors, bioimaging, drug delivery, and nanodevices. In particular, in the field of bio-imaging, development of imaging devices and suitable contrast agents has been continued in order to overcome the detection limit at the molecular and cellular level in the early stages of disease.

Bio-imaging diagnostic equipment, including X-ray, magnetic resonance imaging (MRI), positron emission tomography (PET), and computer tomography (CT), is widely used in the medical field.

In particular, MRI equipment is known as one of the excellent equipment that can apply non-invasive diagnostics and provides high spatial resolution and tomography function, but has a disadvantage in that it has a relatively low detection limit compared to a fluorescent bioimaging probe.

This drawback of MRI can be addressed by using magnetic contrast agents such as gadolinium (T1 contrast agent) and superparamagnetic iron oxide nanoparticles (SPION) (T2 contrast agent) to enhance the signal response induced by external magnetic fields.

It has been reported that gadolinium-based contrast agents have limitations in detecting high-resolution MRI images because of their short residence time in blood vessels and in vivo. In order to replace gadolinium, a study on an MRI contrast agent based on SPION with relatively low toxicity and unique magnetic properties was conducted.

The synthesis of SPION can be prepared by various methods such as microemulsion, co-precipitation, and thermal decomposition. It is known to be synthesized by thermal decomposition of a metal oleate precursor, which is known as a general method for synthesizing monodisperse SPION. This method is known to generate hydrophobic SPIONs with polar tail groups attached to the surface of magnetic nanoparticles. The advantage of SPION coated with oleic acid is its easy to control physical and chemical properties.

However, the properties of SPION coated with a hydrophobic material limit its application in vivo and its use in aqueous solutions.

Therefore, many studies are being conducted to modify the surface of the oleic acid-coated SPION with a hydrophilic material such as dextran, chitosan, polyethylene glycol and silica.

In particular, porous silica is widely used as a SPION surface coating material due to its chemical stability, easy surface functionalization, biocompatibility, biodegradability and drug loading ability.

Nanoparticles with a magnetic core/porous silica shell structure coated with lanthanide (III) (Eu ^{3 +} , Sm ^{3 +} , Tb ^{3 +} and Dy ^{3 +} ) complexes are being actively developed as multifunctional bioimaging probes, such Fluorescent magnetic nanoparticles are applicable in in-vivo and in-vitro fields,

For this reason, there is a need for intensive development of research on multi-purpose nanoparticles in order to improve the disadvantages of the existing MRI contrast agents.

In addition, the Eu ³⁺ complex in lanthanide (III ⁾ emits a very strong red emission under UV irradiation with anti-photobleaching due to the effective intermolecular energy transfer from the coordination ligand. The photoluminescence ^{(PL) properties of Eu 3+ complexes} are highly dependent on the composition of organic ligands.

In addition, the carboxylic acid group of acrylic acid chelates strongly with Eu ^{3+ than the Eu 3+ complex} component molecule, ^and protects the dissociation of ^the Eu ³⁺ complex from various chemical fragmentation factors ^. Based on the above theory, a study was reported to prepare carboxylic acid-activated polystyrene-based copolymer nanoparticles ^{and then to synthesize fluorescent nanoparticles through coordination bonding of carboxylic acid and chelated Eu 3+ complex} .

As a result, this approach has proven that the final composition has excellent anti-photobleaching properties and very high luminescence properties, but studies on fluorescent probes with various core (SPION)/shell (mSiO ₂ ) structures have been conducted so far, but mSiO ₂ @ A study on Eu(TTA) ₃ (P(Oct) ₃ ) ₃ chelated to SPION has not been reported.

Republic of Korea Patent Registration No. 10-1749805 (June 15, 2017)

An object of the present invention is to provide a fluorescent magnetic nanoparticle using porous silica, which can be detected with a confocal microscope when applied to the in-vitro field, and provides excellent spatial resolution as an MRI contrast agent in-vivo, and a method for manufacturing the same.

According to the features for achieving the above object, the present invention, (a) using a fatty acid and a metal precursor to prepare a fatty acid-coated magnetic nanoparticles: (b) the fatty acid-coated magnetic nanoparticles coating the surface of phosphatidylcholine; (c) adding phosphatidylcholine-coated magnetic nanoparticles and a silica precursor to a cationic surfactant template to form a magnetic nanoparticle core/porous silica shell; (d) introducing a methacrylate functional group into the porous silica shell using a silane coupling agent and then adding acrylic acid to activate the carboxyl group; (e) A solution of europium chloride in distilled water, a solution of 2-(4,4,4-trifluoroacetoacetyl)naphthalene in ethanol, and a solution of trioctylphosphine in ethanol were mixed, and then acrylic acid was added. and synthesizing a europium complex by adding ammonia to adjust the pH and heating in a bath; and (f) reacting each of the materials obtained in steps (d) and (e), and covalently bonding the europium complex to the inside and outside of the pores of the porous silica shell to prepare magnetic fluorescent nanoparticles.

And the silica precursor is characterized in that TEOS (Tetraethyl orthosilicate).

The magnetic nanoparticles are preferably at least one selected from iron (II) oxide, iron (III) oxide, cobalt ferrite, zinc ferrite, nickel ferrite, manganese ferrite, iron, cobalt, nickel, manganese, FeAu, FePt, and CoNi.

In addition, the magnetic nanoparticles may be prepared by mixing iron (II) and iron (III) in a molar ratio of 1:2.

In addition, in step (b), the phosphaticholine is preferably added in an amount of 100 to 1000 parts by weight based on 100 parts by weight of the fatty acid.

According to the fluorescent magnetic nanoparticles using porous silica and a method for manufacturing the same according to the present invention, there is an effect of showing excellent performance in tracing light emitted from a molecule after excitation of a specific wavelength through a fluorescent probe having high sensitivity and spatial resolution.

In addition, according to the present invention, it is possible to track in-vitro with a confocal microscope and non-invasively track particles using MRI in-vivo. Not only can the lesion be identified by using it, but it also has the advantage of accurately and completely resecting the lesion by tracking the exact lesion under UV during surgery.

In addition, according to the present invention, multifunctional nanoparticles with low cytotoxicity, excellent biocompatibility, red light emission and magnetic properties can be used as a dual-function contrast agent and have the advantage of being used in a diagnostic kit for amplifying low signals. .

1 is a view showing a process of synthesizing fluorescent magnetic nanoparticles using porous silica according to the present invention;
2 is a transmission electron microscope and scanning electron microscope images and EDX results of fluorescent magnetic nanoparticles using porous silica according to the present invention;
3 is a Fourier-transform infrared spectroscopy (FT-IR) graph showing the unique chemical characteristics of nanoparticles prepared according to an embodiment of the present invention;
Figure 4 is a core / shell structure according to the present invention mSiO ₂ @SPION and Eu(TTA) ₃ (p(Oct) ₃ ) ₃ Thermal analysis (DSC), (TGA) graph of doped mSiO ₂ @SPION,
5 is a graph measuring the specific surface area and pore size (BET) of mesoporous silica nanoparticles having a magnetic core according to an embodiment of the present invention;
6 is a graph of excitation and emission wavelengths of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION and an image of fluorescent magnetic nanoparticles emitting light in red under ultraviolet excitation according to an embodiment of the present invention;
7 is a graph of the cytotoxicity results of europium complex coordinated mSiO ₂ @SPION in H460 cells according to an embodiment of the present invention;
8 is an image detected by confocal microscopy of intracellular uptake and distribution according to the concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION in H460 cells according to an embodiment of the present invention .

Hereinafter, the present invention will be described in detail based on Examples. The terms, examples, etc. used in the present invention are merely exemplified to explain the present invention in more detail and help those of ordinary skill in the art to understand, and the scope of the present invention should not be construed as being limited thereto.

Technical terms and scientific terms used in the present invention represent meanings commonly understood by those of ordinary skill in the art to which this invention belongs, unless otherwise defined.

The following embodiments are provided so that the disclosed content may be thorough and complete and the spirit of the present invention may be sufficiently conveyed to those skilled in the art, and also include complementary embodiments thereof.

In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, the terms 'comprise' and/or 'comprising' do not exclude the presence or addition of one or more other components.

In the description of the specific embodiments below, several specific contents have been written to more specifically describe and help the understanding of the invention, but those skilled in the art may be used without these various specific contents. can recognize that In some cases, it is mentioned in advance that parts which are commonly known and not largely related to the invention in describing the invention are not described in order to avoid confusion in describing the invention.

1 is a view showing a synthesis process of magnetic fluorescent nanoparticles using porous silica according to the present invention.

As shown in FIG. 1, the present invention relates to a fluorescent magnetic nanoparticle using porous silica and a method for preparing the same.

To this end, first (a) using a fatty acid and a metal precursor, a step of preparing the fatty acid-coated magnetic nanoparticles is performed.

More specifically, the manufacturing of the magnetic nanoparticles includes: (a-1) synthesizing a metal precursor; (a-2) adding a fatty acid and a metal precursor to an organic solvent to produce magnetic nanoparticles by thermal decomposition It can consist of steps.

(a-1) In the step of forming the metal precursor, it is preferable to synthesize iron chloride oleate by ion exchange of iron chloride hexahydrate (FeCl ₃ .6H ₂ O) and sodium oleate (NaOl).

And (a-2) in the step of preparing magnetic nanoparticles by thermal decomposition by adding the fatty acid and the metal precursor to the organic solvent, the metal precursor and the fatty acid are added to and dissolved in the organic solvent to achieve a uniform and monodisperse supernatant by thermal decomposition. Magnetic iron oxide nanoparticles can be synthesized.

In addition, the magnetic nanoparticles are characterized in that at least one selected from iron (II) oxide, iron (III) oxide, cobalt ferrite, zinc ferrite, nickel ferrite, manganese ferrite, iron, cobalt, nickel, manganese, FeAu, FePt, CoNi and nanoparticles prepared in an aqueous phase by the coprecipitation method.

In addition, the magnetic nanoparticles are magnetic iron oxide nanoparticles having superparamagnetism, and are preferably prepared by mixing iron (II) and iron (III) in a molar ratio of 1:2.

In this case, the solvent used for synthesizing the magnetic nanoparticles may be one or a mixture of one or more selected from distilled water, methanol, and ethanol.

More specifically, the fatty acid is preferably oleic acid, the metal precursor is preferably iron chloride oleate (FeOl), and the organic solvent is preferably octadecene having a high boiling point.

That is, in step (a-2), oleic acid-coated superparamagnetic iron oxide nanoparticles can be synthesized by pyrolysis by adding iron chloride oleate and oleic acid, which are metal precursors, in octadecene.

Next, (b) proceeds with the step of coating the phosphatidylcholine on the surface of the fatty acid-coated magnetic nanoparticles.

In step (b), the fatty acid-coated magnetic nanoparticles are phase-changed from hydrophobicity to hydrophilicity by adding phosphatidylcholine.

More specifically, primary micelles are formed by van der Waals action between phosphatidylcholine and an alkyl chain present on the surface of the magnetic nanoparticles.

Here, the phosphaticholine is a phospholipid, and the fatty acid composed of choline as the head group and glycerophosphoric acid as the tail group, and various types of fatty acids is preferably composed of saturated fatty acids or unsaturated fatty acids, and saturated fatty acids and unsaturated fatty acids It can also be prepared as a mixture of

The phosphatidylcholine is added in an amount of 100 to 1000 parts by weight based on 100 parts by weight of the fatty acid, and more specifically, the fatty acid: phosphatidylcholine is preferably prepared in a weight ratio of 1:1.

When the phosphatidylcholine is added in less than 100 parts by weight based on 100 parts by weight of the fatty acid, the coating of phosphatidylcholine is not made on the surface of the fatty acid-coated magnetic nanoparticles, and when it exceeds 1000 parts by weight, the core/shell is not formed.

More specifically, it is possible to form primary micelles by introducing phosphatidylcholine to the surface of the superparamagnetic iron oxide nanoparticles coated with oleic acid.

In addition, the phosphatidylcholine may be added after pyrolysis of fatty acids and metal precursors, after washing impurities, and then heated and sequentially coated. It can also be coated.

Next, (c) phosphatidylcholine-coated magnetic nanoparticles and silica precursors are added to the cationic surfactant-based template to form a magnetic nanoparticle core/porous silica shell. In this case, it is preferable that the silica precursor is tetraethyl orthosilicate (TEOS), and the cationic surfactant is CTAB.

And (d) a methacrylate functional group is introduced into the porous silica shell present on the surface of the magnetic core using a silane coupling agent, and then acrylic acid is added to activate the carboxyl group.

In step (d), a methacrylate functional group may be introduced into the pores and the surface of the porous silica shell existing on the surface of the magnetic nanoparticle core by using a silane coupling agent.

More specifically, in step (d), the inside and the surface of the pores of the porous silica shell of the magnetic nanoparticles obtained in step (c) are coated with tetramethoxysilylpropyl meta-rate using acetic acid and subjected to free radical polymerization. Acrylic acid can be bound by

In addition, it is preferable to remove the CTAB from the silica shell layer using NH ₄ NO ₃ after modifying the surface of mSiO ₂ @SPION using a silane coupling agent.

Next, (e) synthesizing the europium complex is a step of preparing a complex using europium and β-diketone, and acrylic acid or a derivative thereof, which is a compound containing a carboxyl group and an acrylic group, that is, polyethylene-co-acrylic Acid, polyacrylamide-co-acrylic acid, poly-n-isopropylacrylamide-co-acrylic acid, polyacrylic acid, methacrylic acid n-hydroxysuccinimide ester, polyethylene-co-methacrylic acid, 3 -(2-Puri)Acrylic acid can be used in any one or a combination, and the use thereof is a structure that is difficult to separate from each other in any case, and finally the physical/chemical stability of the structure is greatly increased.

In step (e), a solution of europium chloride in distilled water, a solution of 2-(4,4,4-trifluoroacetoacetyl)naphthalene in ethanol, and a solution of trioctylphosphine in ethanol are mixed. Then, acrylic acid is added, pH is adjusted by adding ammonia, and then the europium complex is synthesized by heating in a bath.

More specifically, a solution obtained by dissolving 20 mmol of europium chloride in distilled water, 20 mmol of 2-(4,4,4-trifluoroacetoacetyl) naphthalene in ethanol, and 20 mmol of trioctylphosphine in ethanol After mixing in a certain ratio, it is preferable to add acrylic acid and add ammonia.

Next, (f) mix and react each material obtained in steps (d) and (e) to share the europium complex obtained in step (e) inside and outside the pores of the porous silica shell obtained in step (d) Proceed to the step of preparing fluorescent magnetic nanoparticles by binding.

In the present invention, fluorescent magnetic nanoparticles using porous silica were prepared according to the manufacturing method described above. In particular, oleic acid-coated magnetic nanoparticles, which are immiscible layers for TEOS, were changed to a miscible layer using phosphatidylcholine, and then The core-shell structure of mSiO ₂ @SPION was successfully synthesized by forming micelles on the surface of superparamagnetic nanoparticles.

Fluorescent magnetic nanoparticles using porous silica prepared in this way form a silicate shell layer on the surface of the magnetic core and at the same time introduce a functional group capable of chemical bonding to the silica surface and coat the lanthanide metal complex with a lanthanide metal complex in-vitro. When applied to the field, it can be detected with a confocal microscope and can be applied as a multifunctional fluorescent probe that provides excellent spatial resolution as an MRI contrast medium in in-vivo.

The Eu ³⁺ complex supported inside the silica pores can extend the fluorescence properties for several months and reduce cytotoxicity due to indirect exposure to UV and laser, which are used to visualize biological samples ^. For this reason, the structure proposed in the present invention, Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION, is a multifunctional nanoparticle with low cytotoxicity, excellent biocompatibility, red light emission and magnetic properties. It is a potential candidate for a functional contrast agent, and shows that it can be used as a fluorescent nanomaterial to prepare a diagnostic kit for amplifying low signals.

Hereinafter, a method of manufacturing the fluorescent magnetic nanoparticles will be described in more detail through examples.

[Example 1]

(a) using a fatty acid and a metal precursor to prepare a fatty acid-coated magnetic nanoparticle

(a-1) synthesizing a metal precursor

An iron chloride oleate (FeOl) complex was synthesized as follows.

FeOl was produced by ion exchange of iron (III) chloride hexahydrate (FeCl ₃ 6H ₂ O) with sodium oleate (NaOl). Iron chloride hexahydrate (10.8 g, 40 mmol) and NaOl (36.5 g, 120 mmol) were dissolved in 80 mL EtOH, 60 mL distilled water and 140 mL n-hexane.

The 500 mL beaker containing the mixture was sealed and stirred at 70° C. for 3 hours. Upon completion of the reaction, the resulting solution was stored in a freezer for 5 hours.

The liquid in the lower layer was removed from the frozen organic layer, and 80 mL of distilled water was added to wash the organic layer. The solution was frozen in a freezer for 2 hours and again the upper layer containing FeOl was separated from the lower layer.

The above process was repeated 3 times to collect the upper layer containing FeOl. The collected FeOl solution was dried overnight at room temperature to evaporate residual n-hexane, and FeOl in the form of a solid wax was stored in a freezer.

(a-2) adding a fatty acid and a metal precursor to an organic solvent to prepare magnetic nanoparticles by thermal decomposition

Monodisperse superparamagnetic iron oxide nanoparticles were synthesized by thermal decomposition as follows.

Oleic acid (OA) ligand-capped SPIONs were synthesized by thermal decomposition of metal precursors in organic solvents. In a 100 mL flask (two-necked round bottom), 3.6 g of the synthesized FeOl precursor and 0.57 g of oleic acid (OA) were dissolved in 20 g of octadisin.

After connecting the proportional integral derivative (PID) control module to the heating mantle and magnetic stirrer, a 100 mL flask containing the mixture was placed in the heating mantle and stirred at 110° C. for 1 hour to physically evaporate the water in the mixture.

The mixture containing the FeOl precursor was then heated to reflux at 310°C and the temperature held at 310°C for 30 minutes, and then the solution was cooled to room temperature.

The synthesized SPION was washed several times using n-hexane, ethanol and acetone (1:2:1, V/V/V), and the solution was centrifuged at 21,500 g for 20 minutes.

(b) coating phosphatidylcholine on the surface of the fatty acid-coated magnetic nanoparticles

SPION coated with a hydrophobic oleic acid ligand was changed from hydrophobic to hydrophilic by adding liposome-based phosphatidylcholine.

Briefly, 0.5 mL of SPION dispersed in chloroform was placed in each 2 mL EP tube. Then 1 mL ether and 0.5 mL ethanol were added to the EP tube.

A 2 mL EP tube containing SPION was sonicated for 3 min. The samples were then precipitated by centrifugation at 21,500 g for 5 minutes. After precipitation, the supernatant was removed and 0.5 mL of chloroform and 80 mg of phosphatidylcholine were added into the EP tube containing the precipitated SPION.

The mixture was sonicated at 50 °C for 1 h, then 0.5 mL ether and 1 mL ethanol were added to the EP tube and centrifuged at 21,500 g for 10 min. The SPION-coated phosphatidylcholine was redispersed in 2 mL of chloroform.

(c) magnetic nanoparticles core-porous silica by adding phosphatidylcholine-coated magnetic nanoparticles and a silica precursor to a cationic surfactant shell step to form

240 mL of 45.73 mM CTAB (cationic surfactant, cetyl trimethy lammonium bromide) aqueous solution was placed in a 500 mL two-necked round-bottom flask, and then 1.76 mL of 2M NaOH was added. The mixture solution was sonicated at 70° C. for 1 hour.

After sonication, SPION coated with phosphatidylcholine dispersed in 2 mL chloroform was added very slowly. The mixture solution was sonicated at 70° C. for 1 hour to evaporate residual chloroform.

A flask containing the mixture was placed in a water bath placed on a magnetic stirrer and stirred under reflux at 40°C for 1 hour at a stirring rate of 750 r.p.m. To form a porous silica layer on the surface of SPION, 400 μL TEOS was injected very slowly and maintained for 30 minutes.

After that, 800 μL TEOS was further added at a rate of 1 drop per 5 seconds and reacted for 2 hours. After 2 hours, the temperature was increased to 80° C. and reacted for 2 hours. The reaction flask was cooled to room temperature with stirring.

Samples were washed with ethanol (200 mL×3) and centrifuged. Then, the precipitated mSiO ₂ @SPION was redispersed in 20 mL of ethanol and stored in a 50 mL tube.

(d) silane coupling agent cast using porous silica in the shell methacrylate After introducing a functional group, adding acrylic acid to activate the carboxyl group

TMSPMA was used for coating of silanol groups on mSiO ₂ . mSiO ₂ @SPION dispersed in 20 mL ethanol at a concentration of 0.6 g/mL was placed in a 50 mL tube, 100 μL of TMSPMA was added, 0.6 mL diluted acetic acid (1:10 acetic acid: DI water) was added, and ultrasonicated for 5 minutes. dealt with

The samples were transferred to a 2mL EP tube and centrifuged at 21,500g for 5 minutes. After precipitation, the supernatant was removed, redispersed in ethanol and centrifuged again. The washing procedure was repeated 3 times.

The obtained silane-modified mSiO ₂ was dispersed in a 250 mL flask by adding 100 mL of ethanol (6 g/L, ammonium nitrate/ethanol), and then the flask was sonicated at 80° C. for 3 hours to remove CTAB. After 3 h, the mixture was washed with ethanol (200 mL×3) and precipitated by centrifugation.

Samples were redispersed in 30 mL distilled water and stored in 50 mL tubes. Activation of carboxyl groups on the surface of mSiO ₂ @SPION surface-modified with TMSPMA was formed by free radical polymerization with AAc. Transfer TMSPMA-coated mSiO ₂ @SPION dispersed in 30 mL distilled water to a 100 mL flask (two-necked round bottom), followed by 27 mL distilled water, 3 mL potassium persulfate (PPS, 1.75 g PPS dissolved in 50 mL of distilled water) ) 0.6 mL of acrylic acid was added.

The reaction flask was sonicated at 75° C. for 1 hour. Impurities and unreacted chemicals were dialyzed against 5 L of distilled water using a dialysis bag (Mw. Cutoff = 12,400). The dialyzed distilled water was exchanged with fresh 5L distilled water 5 times every 2 hours.

(e) a solution of europium chloride in distilled water, and 2-(4,4,4- trifluoroacetoacetyl ) a solution of naphthalene in ethanol, trioctylphosphine A step of synthesizing a europium complex by mixing a solution dissolved in ethanol, adding acrylic acid, adjusting the pH by adding ammonia, and heating in a bath

1 mmol (0.366 g ^{) Eu 3+ was} dissolved in 50 mL of ethanol. A stock solution of 4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione (TTA) was prepared by dissolving 1 mmol (0.222 g) of TTA in 50 mL of ethanol. 1 mmol (0.37 g) Trioctylphosphine P(Oct) ₃ was dissolved in 50 mL ethanol to prepare a P(Oct) ₃ stock solution. The concentrations of the prepared stock solutions were as follows; Eu ^{3 +} :20 mM, TTA: 20 mM, P(Oct) ₃ :20 mM. 10 mL of distilled water and 100 μL of (20 mM) Eu ³⁺ stock solution and 300 μL of (20 mM) TTA and 300 μL of ⁽ 20 mM) P(Oct) ₃ were each added to a 20 mL glass vial.

Then 20 μL of concentrated ammonia solution was added. The glass vial containing mixture was stirred with a magnetic stirrer at 600 rpm at 60 °C in a water bath. After stirring for 2 hours, the resulting β-diketone Eu ^{3 +} complex was stored at room temperature.

(f) reacting each material obtained in steps (d) and (e) above, porous silica of the shell The europium inside and outside the pores lubrication Covalent bonding to prepare fluorescent magnetic nanoparticles

The chemical structure of the β-diketone europium complex was chelated with a carboxylic acid ligand on mSiO ₂ @SPION. Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ For the synthesis of @SPION, 50 mL of mSiO ₂ @SPION solution dispersed in distilled water was added to a 100 mL flask, and then 1 mL of Eu(TTA) ₃ (TOP) ₃ , 0.5 μL of ammonia solution was added.

And the flask containing the mixture was stirred in a water bath at 70 ° C. at 700 rpm for 1 hour, and the prepared Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION was a dialysis bag (Mw. Cutoff = 12,400) was washed with 5 L of distilled water. At this time, distilled water was replaced 5 times every hour, and then maintained for 24 hours.

[ Experimental example 1] Transmission electron microscopy and scanning electron microscopy images and EDX Analysis

Transmission electron microscope and scanning electron microscope images and EDX analysis were performed according to the embodiment, which is shown in FIG. 2 .

2A is a TEM image of an OA-capped SPION formed by thermal decomposition of an FeOl precursor, and it can be confirmed that magnetic nanospheres having an average size of about 12 nm were successfully synthesized.

2 (b) is a TEM image of mSiO ₂ @SPION, it was confirmed that the average size was 120 nm, the measured average pore size was 30 Å, and the standard deviation was 0.47.

2(c) is a TEM image of mSiO ₂ @SPION chelated with a europium complex, and according to this, no distinct morphological difference is seen after chelation with Eu(TTA) ₃ (P(Oct) ₃ ) ₃ .

In addition, (d) of FIG. 2 is an SEM image of mSiO ₂ @SPION, and through this, it can be confirmed that mSiO ₂ @SPION has a spherical structure with a magnetic core.

2(e) is an EDX analysis result of mSiO ₂ @SPION, and it can be confirmed that the peaks of zinc and copper are coming out of the sample holder.

[ Experimental example 2] FT-IR spectrum measurement

An FT-IR spectrum according to an embodiment of the present invention was measured, which is shown in FIG. 3 .

On the graph of FIG. 3, (a) is oleic acid-coated magnetic nanoparticles, (b) is magnetic nanoparticles coated with pasphatidylcholine on the surface, (c) is mSiO ₂ @SPION , (d) is Eu(TTA) ) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION.

The peak at 578 cm ^-1 in Fig. 3 (a) is due to the presence of Fe-O bonds from Fe ₃ O ₄ in the FT-IR spectrum. And the peak at 1438 cm ^-1 corresponds to the symmetrical vibration of iron carboxylate in SPION, the peak at 1540 cm ^-1 indicates COO-, and the peak at 1700 cm ^-1 indicates the presence of C=O stretch of COO-. . In addition, the peak at 2850 cm ^-1 and the peak at 2921 cm ^-1 represent CH scratching peaks from OA.

The broad peak at 3240cm ^-1 in (b) of FIG. 3 corresponds to the OH group on mSiO ₂ @SPION. And the peak at 1736 cm ^-1 corresponds to the C=O elongation of the ester group indicating the presence of phosphatidylcholine, and the peak at 1262 cm ^-1 corresponds to the POR bond. In addition, the peak at 1170 cm ^-1 corresponds to CO, and the peak at 970 cm ^-1 indicates the presence of choline containing phospholipids. In addition, the peak at 518 cm ^-1 represents a phosphoric acid group (OPO).

The peaks shown at 1087 cm ^-1 , 789 cm ^-1 , and 470 cm ^-1 in (c) of FIG. 3 indicate a Si—O—Si bond. And the peaks at 3442 cm ^-1 and 958 cm ^-1 are due to stretching vibrations of Si-OH and free silanol groups due to hydrogen bonding to the respective nanoparticles.

In Fig. 3(d), the peak at 1595 cm ^-1 and 1410 cm ^-1 represents Eu ³⁺ chelated with carboxylic acid of AAc formed by free radical polymerization of TMSPMA present on mSiO ₂ SPION. And the peak at 1700cm ^-1 corresponds to the C=O stretch.

These results mean that the chelation of the Eu ³⁺ complex to Eu ⁽ TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION was successfully performed.

[ Experimental example 3] mSiO ₂ @SPION and Eu (TTA) ₃ (p(Oct) ₃ ) ₃ @ mSiO ₂ @SPION's thermal analysis ( DSC -TGA)

4 (a) is a DSC-TGA graph of mSiO ₂ @SPION, and FIG. 4 (b) is a DSC-TGA graph of Eu(TTA) ₃ (p(Oct) ₃ ) ₃ @mSiO ₂ @SPION.

The TGA curves of FIGS. 4(a) and 4(b) show rapid weight loss at room temperature due to dehydration of water absorbed by the particles in the 130°C region. And secondary weight loss appears in the range of 300°C to 500°C corresponding to oxidation of -CH3 and evaporation of residual organic solvent.

The weight loss of mSiO ₂ @SPION is much lower than that of Eu(TTA) ₃ (p(Oct) ₃ ) ₃ chelated mSiO ₂ @SPION, which is due to the presence of chelated Eu ³⁺ complex on mSiO ₂ @SPION ^. means

The weight loss is small at around 600°C, indicating that organic molecules such as TEOS remain in the synthetic nanoparticles.

As observed in the TGA curve, the endothermic peak of DSC observed at 130 °C corresponds to the dehydration of absorbed water. In the temperature range of 250~500℃, the exothermic peak is related to the decomposition of organic molecules, ^{and it can be estimated that the heat of reaction increased due to the presence of the Eu 3+ complex} chelated on mSiO ₂ @SPION.

[ Experimental example 4] having a magnetic core mesoporous of silica nanoparticles specific surface area class fore Size (BET) measurement

BET and mean pore diameter (dp) were determined using adsorption isotherms in the relative pressure range of 0.05 to 0.3.

In Fig. 5(a), the nitrogen physical absorption isotherm of mSiO ₂ @SPION shows an isotherm with a strong absorption tendency near P/P ^o =0.9-1.0, which means the presence of a typical mesoporous structure.

And Fig. 5 (b) is a graph of the pore size and distribution curve of mSiO ₂ @SPION, the average pore size of mSiO ₂ @SPION is about 28Å, and the specific surface area is 464.96m ² /g.

[ Experimental example 5] Eu (TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION excitation and emission wavelength graph and image of fluorescent magnetic nanoparticles emitting red light under UV excitation

6 (a) is an excitation (309 nm) spectrum according to the concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION, FIG. 6 (b) is Eu(TTA) ₃ (P( Oct) ₃ ) ₃ @mSiO ₂ Shows the photoluminescence (PL) characteristics of the emission (621 nm) spectrum of @SPION.

In Fig. 6(a), the excitation spectrum of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION shows a typical optical trend due to the ff transition of the Eu ³⁺ complex with strong red emission characteristics ^.

The strong emission peak at 621 nm in FIG. 6(b) is due to the ⁵ D ₀ → ⁷ F ₂ transition, and the lower peak at 592 nm can be assigned to the D ₀ → ⁷ F ₁ transition.

6(b) shows that Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION has strong PL properties at the ⁵ D ₀ → ⁷ F ₂ transition from a stable Eu ^{3 +} complex.

As shown in FIG. 6(a), the excitation spectrum of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION is identified as a maximum peak at 309 nm, and shown in FIG. 6(b). As shown, it can be seen that the PL intensity at 621 nm increases as the concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION gradually increases.

And Figure 6 (c) is a photograph of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION diluted in DI Water in an indoor light, (d) of Figure 6 is Di water in UV light It is a photograph of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION diluted in .

[ Experimental example 6] Cytotoxicity assay

The cytotoxicity of europium complex coordination-bound mSiO ₂ @SPION in H460 cells was measured, which is shown in FIG. 7 .

At this time, the concentrations of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION were 10μg/mL, 50μg/mL, 100μg/mL, 200μg/mL, 300μg/mL, 500μg/mL, and 10 H460 cell viability was greater than 90% in the concentration range of ~500 μg/mL.

That is, Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION does not induce cytotoxicity and is biocompatible.

[ Experimental example 7] in H460 cells Eu (TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ Confocal microscopy images according to the concentration of @SPION

8 is an image detected by confocal microscopy of the intracellular uptake and distribution according to the concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION in H460 cells, Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ The concentration of @SPION is 10 μg/mL in (a) of FIG. 8, 100 μg/mL in (b), 300 μg/mL in (c), and 500 μg/mL in (d). was made with

As shown in (b) of FIG. 8, as the concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION increased to 100 μg/mL, a red emission part appeared due to endocytosis. started

And as shown in (a) to (d) of FIG. 8 , it was confirmed that the fluorescence intensity increased as the concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION increased, as a result. It was confirmed that the optimal concentration of Eu(TTA) ₃ (P(Oct) ₃ ) ₃ @mSiO ₂ @SPION for visualization was about 500 μg/mL.

In addition, the rights of the present invention are not limited to the above-described embodiments, but are defined by the claims, and those of ordinary skill in the art can make various modifications and adaptations within the scope of the claims. It is self-evident that it can

Claims (6)

(a) using a fatty acid and a metal precursor to prepare a fatty acid-coated magnetic nanoparticle:
(b) coating phosphatidylcholine on the surface of the fatty acid-coated magnetic nanoparticles;
(c) adding phosphatidylcholine-coated magnetic nanoparticles and a silica precursor to a cationic surfactant template to form a magnetic nanoparticle core/porous silica shell;
(d) introducing a methacrylate functional group into the porous silica shell using a silane coupling agent and then adding acrylic acid to activate the carboxyl group;
(e) A solution of europium chloride in distilled water, a solution of 2-(4,4,4-trifluoroacetoacetyl)naphthalene in ethanol, and a solution of trioctylphosphine in ethanol are mixed, and then acrylic acid is added. and synthesizing a europium complex by adding ammonia to adjust the pH and heating in a bath;
(f) reacting each of the materials obtained in steps (d) and (e) to covalently bond the europium complex inside and outside the pores of the porous silica shell to prepare magnetic fluorescent nanoparticles; A method for producing fluorescent magnetic nanoparticles using porous silica.
According to claim 1,
The method for producing fluorescent magnetic nanoparticles using porous silica, characterized in that the silica precursor is TEOS (Tetraethyl orthosilicate).
According to claim 1,
The magnetic nanoparticles are porous, characterized in that at least one selected from iron (II) oxide, iron (III) oxide, cobalt ferrite, zinc ferrite, nickel ferrite, manganese ferrite, iron, cobalt, nickel, manganese, FeAu, FePt, CoNi A method for manufacturing fluorescent magnetic nanoparticles using silica.
According to claim 1,
The magnetic nanoparticles,
A method for producing fluorescent magnetic nanoparticles using porous silica, characterized in that it is prepared by mixing iron (II) and iron (III) in a molar ratio of 1:2.
According to claim 1,
In step (b),
The phosphaticholine is a method for producing a magnetic fluorescent nanoparticles using porous silica, characterized in that the addition of 100 to 1000 parts by weight based on 100 parts by weight of the fatty acid.
[Claim 6] Fluorescent magnetic nanoparticles using porous silica, characterized in that it is prepared by the method according to any one of claims 1, 2, 3, 4, and 5.

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