KR101875474B1 - Functionalized magnetic nanoparticles and preparing method of thereof - Google Patents

Abstract

Provided are modified magnetic nanoparticles and a method for producing the same. The present invention comprises: magnetic nanoparticles of a core-shell structure; and a polymer layer adhered to the outer periphery of the magnetic nanoparticles. The present invention is characterized in which cytotoxicity and physicomechanical properties are changed by a magnetic field. Therefore, the present invention can greatly improve biocompatibility by modifying the surface of the magnetic nanoparticles of which use is limited due to bio-toxicity.

Description

[0001] The present invention relates to modified magnetic nanoparticles and methods for preparing the same,

The present invention relates to a process for producing magnetic nanoparticles and a modified magnetic nanoparticle produced thereby.

Magnetic nanoparticles are very useful for contrast agents and targeted drug delivery systems due to their appropriate size, surface charge, and magnetism.

Biosensing, which binds magnetic nanoparticles to target cells of magnetic nanoparticles, is an important characteristic of magnetic nanoparticles. The dynamic behavior of the magnetic nanoparticles attached to the surface of the cell can be controlled by controlling the direction and intensity of the magnetic field. The particles are not intertwined in the magnetic field and are readily sterilizable. This cell separation method is called MACS (Magnetic Activated Cells Sorting).

Methods for labeling and isolating cells in complex cell mixtures using fluorescent nanoparticles are also being studied. A method called FACS (Fluorescence Activated Cells Sorting) has the advantage of being able to visualize a fluorescent dye bonded to a cell.

Therefore, increasing the intracellular absorption of magnetic nanoparticles enables various physiological applications.

However, these various uses are limited, but their bioavailability is limited due to the toxicity of magnetic nanoparticles themselves.

In particular, there is a problem in that active oxygen (ROS) is formed which promotes cell death with high reactivity of hydrogen peroxide after magnetic absorption of magnetic nanoparticles (Non-Patent Document 1), so that biomedical application is very difficult.

There is also no report on the change in the biomechanical properties of cells in the presence or absence of a magnetic field before and after treatment with magnetic particles and polymer coated magnetic particles.

Therefore, it is very urgent to develop new magnetic nanoparticles capable of reducing cytotoxicity of magnetic nanoparticles and increasing absorption into cells.

Korean Patent Publication No. 2014-0110463 (published on September 17, 2014)

 Quinlan TR, Marsh JP, Janssen Y, Borm PA, Mossman BT. 1994. Oxygen radicals and asbestos-mediated disease. Environ Health Persp 102 (Suppl 10): 107.

Accordingly, the present invention provides a modified magnetic nanoparticle having improved biocompatibility by modifying the surface of magnetic nanoparticles, and a method for producing the same.

The problems to be solved by the present invention are not limited to the above-mentioned problem (s), and another problem (s) not mentioned can be understood by those skilled in the art from the following description.

In order to solve the above-mentioned problems, the present invention provides a magnetic nanoparticle having a core-shell structure; And a polymer layer adhered to the outer periphery of the magnetic nanoparticles, wherein the cytotoxicity and physicomechanical properties of the modified nanoparticle are changed by a magnetic field.

The magnetic nanoparticles may be formed of nanoparticles of core-shell structure by coprecipitation of Fe ^{2 +} and Fe ^{3 +} ions.

The molar ratio of Fe ^{2 +} and Fe ^{3 +} ions may be 1: 2.

The polymer layer may also be formed of poly (lactic-co-glycolic acid) or alginate.

The polymer layer may be adsorbed on the surface of the magnetic nanoparticles and negatively charged.

The negative charge may be -14 to -8 mV.

Also, the change in the physiochemical properties may be cell size, Young's modulus and cell roughness.

According to another aspect of the present invention, there is provided a magnetic nanoparticle comprising: (a) preparing magnetic nanoparticles; (b) adding a polymer to the magnetic nanoparticles to form a polymer layer on the surface of the magnetic nanoparticles; And (c) recovering and washing the modified magnetic nanoparticles and drying the modified magnetic nanoparticles.

Also, the step (a) may form magnetic nanoparticles in the form of a core-shell by coprecipitation of Fe ^{2 +} and Fe ^{3 +} ions.

The Fe ^{2 +} and Fe ^{3 +} ions may have a molar ratio of 1: 2.

Also, in the step (b), the polymer layer may be poly (lactic-co-glycolic acid) or alginate.

When the polymer layer is poly (lactic-co-glycolic acid), the polymer layer may be prepared by mixing chloroform with the poly (rat-co-glycolic acid) Poly (vinyl alcohol) and a duplicate emulsion evaporation through ultrasonic treatment.

The ultrasonic treatment may also be performed with a pulse of 5 seconds interval for 10 minutes.

When the polymer layer is alginate, it may be formed by mixing sodium alginate with ethylene glycol and electrospraying it.

The electric injection may be performed at a flow rate of 45 to 50 μL min ^-1 and an applied voltage of 15 to 19 kV.

According to the present invention, it is possible to improve the biocompatibility by modifying the surface of magnetic nanoparticles whose use is limited due to biotoxicity.

Surface modified magnetic nanoparticles can be used for intracellular delivery, target-oriented drug delivery systems, and imaging, and can provide a useful means of identifying cellular mechanisms.

Biocompatibility can also confirm the biophysical and biomechanical properties of cells due to the interaction of the surface-modified magnetic particles with the cells.

Magnetic fields can be used to perform forced absorption into cells to increase cell stiffness and allow for intracellular uptake of target cells and tissues.

FIG. 1 is a schematic view showing a method of manufacturing a surface-modified magnetic nanoparticle according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of field emission scanning electron microscopy, atomic force microscopy, high-resolution transmission electron microscopy, and magnetic field hysteresis loop analysis according to the types of modified magnetic nanoparticles in the method for producing modified magnetic nanoparticles according to an embodiment of the present invention Fig.
3 shows the cytotoxicity of the modified magnetic nanoparticles according to the embodiment of the present invention.
4 is a graph showing cell uptake of modified magnetic nanoparticles according to an embodiment of the present invention.
5 is a 3D photograph of an atomic force microscope showing cell behavior of modified magnetic nanoparticles according to an embodiment of the present invention.
6 is a graph showing biophysical mechanical analysis of modified magnetic nanoparticles according to an embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention and the manner of achieving it will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings.

The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail.

The present invention relates to a poly (lactic-co-glycolic acid) copolymer having magnetic nanoparticles (hereinafter referred to as 'MNP'). (Hereinafter abbreviated as 'aMNP') by ion-crosslinking through electrospinning using surface-modified magnetic nanoparticles (hereinafter referred to as 'pMNP') and alginate, do.

The modified magnetic nanoparticle according to the present invention includes a core-shell structure magnetic nanoparticle and a polymer layer attached to the outer periphery of the magnetic nanoparticle.

The magnetic nanoparticles may exhibit cytotoxicity, and the cytotoxicity depends on the concentration of the magnetic nanoparticles.

In one experimental example of the present invention, when cells were treated with MNP, extensive cell death occurred at 512 to 1024 μg / mL.

Therefore, when the MNP is intracellularly absorbed, a problem of cytotoxicity may occur, but the cytotoxicity can be greatly reduced by a modification method of forming a polymer layer on the surface of the magnetic nanoparticles.

The magnetic nanoparticles may be formed into a core-shell structure by coprecipitation of Fe ^{2 +} and Fe ^{3 +} ions.

The Fe ^{2 +} is oxidized Fe ³⁺ can be present, the Fe ^{+ 2} and Fe ^{+ 3} ions, the molar ratio of 1: 2 days.

When the magnetic nanoparticles are formed in the molar ratio range, the polymer may be adsorbed to form a polymer layer.

The polymer layer may be a poly (lactic-co-glycolic acid) or an alginate.

When the polymer layer is PLGA, it can have a low density and a large particle size, so that it can greatly increase the biocompatibility when it is absorbed into a cell, and can maintain the surface as a negative charge in a magnetic field.

When the polymer layer is selected as PLGA, a polymer layer can be formed on the surface of the magnetic nanoparticles by emulsification.

When the polymer layer is alginate, the metal ion can be very well received, and in particular, the cation metal can be accommodated and stored.

Particularly, when the polymer layer is selected as alginate, the modified magnesium nanoparticles can be produced by one-spot formation by electrospray.

The coating layer formed of a polymer other than the polymer layer is difficult to form a polymer layer at 2 to 6 nm through emulsification and electrospray on the surface of the magnetic nanoparticles. When the coating layer is out of the range, the magnetic nanoparticles It is difficult to change the cytotoxic and physiochemical properties.

In addition, the polymer layer is adsorbed on the surface of the magnetic nanoparticles and can be negatively charged.

The negative charge may be -14 to -8 mV.

The morphology of the morpho-modified magnetic nanoparticles can be changed by electrostatic repulsion between the magnetic nanoparticles and the polymer layer within the negative charge range. When the morphology of the nanoparticles is changed, the cytotoxicity and physicochemical properties Can be changed.

Since the polymer layer is formed and the intracellular absorption can be greatly increased in the magnetic field due to the surface charge, it is possible to use various materials for intracellular mass transfer.

On the other hand, the change in the physical properties may be cell size, Young's modulus and cell roughness.

When the polymer layer is formed, it can be absorbed into cells to change the cell size and Young's modulus in the magnetic field.

The cell size can be reduced in the magnetic field, and the Young's modulus of the cell membrane elasticity can be increased to greatly increase intracellular absorption.

Therefore, the modified magnetic nanoparticles formed with the polymer layer can affect various cell functions in the cell only by the magnetic field.

 According to another aspect of the present invention, there is provided a magnetic nanoparticle comprising: (a) preparing magnetic nanoparticles; (b) adding a polymer to the magnetic nanoparticles to form a polymer layer on the surface of the magnetic nanoparticles; And (c) recovering and washing the modified magnetic nanoparticles and drying the modified magnetic nanoparticles.

The step (a) may form magnetic nanoparticles in the form of a core-shell by coprecipitation of Fe ^{2 +} and Fe ^{3 +} ions.

The Fe ^{2 +} and Fe ^{3 +} ions may have a molar ratio of 1: 2, and the polymer may be adsorbed at the molar ratio to form a polymer layer.

In this case, when the modified magnetic nanoparticles are absorbed into a cell, their morphology may be changed due to the polymer layer and the electrostatic attraction in the magnetic field, and the physicomechanical properties may be changed.

In the step (b), the polymer layer may be poly (lactic-co-glycolic acid) or alginate.

When the polymer layer is a poly (lactic-co-glycolic acid), the polymer layer is formed by mixing chloroform with the poly (ricic-co-glycolic acid) (Vinyl alcohol) and a duplicate emulsion evaporation through ultrasonic treatment.

It is very difficult to form a polymer layer through emulsification when the polymer layer is not poly (ric-co-glycolic acid).

The ultrasonic treatment can be performed with a pulse of 5 seconds interval for 10 minutes, and the polymer layer can be formed by the ultrasonic treatment in the range, and the thickness of the polymer layer can not be controlled if the range is out of the range.

When the polymer layer is alginate, it may be formed by mixing sodium alginate with ethylene glycol and electrospray.

The electric injection may be performed at a flow rate of 45 to 50 μL min ^-1 and an applied voltage of 15 to 19 kV.

When the polymer layer is selected as alginate, a polymer layer may be formed by an electrospray method in order to improve the biocompatibility of the alginate. If the polymer layer is out of the range, the thickness of the polymer layer can not be controlled and it is difficult to control the charge on the surface.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited to the following examples.

< Example  1> Synthesis of Surface-Modified Magnetic Nanoparticles

First, magnetic nanoparticles were prepared by coprecipitation of Fe ^{2 +} and Fe ^{3 +} ions under hydrothermal synthesis conditions.

600 mg of iron (II) chloride tetra-hydrate (FeCl 2 .4H 2 O, MW: 198.27, Sigma Aldrich,> 99%) and iron (III) chloride hexa-hydrate (FeCl ₃ .6H ₂ O, MW: 270.30, Sigma Aldrich, >97%; Fe ²⁺ : Fe ^{3 +} molar ratio of 1: 2) was added to a 100 mL beaker. The solution was dissolved in 30 mL of water and finally the iron was formed at 0.3 M concentration. The prepared mixed solution was stirred at 700 rpm for 60 minutes at 30 ° C.

And 7.5 mL of an ammonium hydroxide solution (NH ₄ OH, MW: 35.05, Sigma Aldrich, content in aqueous NH ₃ solution, w / w 28-30%) was used to achieve chemical precipitation using a 25 G needle- Was added to the mixed solution under the same conditions as the magnetic stirring.

The pH was maintained at 10 during the course of the reaction. After adding the ammonium hydroxide solution, the mixed solution was heated at 80 ° C for 30 minutes.

The manufacturing solution containing magnetic nanoparticles was sonicated for 5 minutes using a Sonic Dismembrator Model 500 (Fisher Scientific).

After ultrasonic treatment, the mixture was centrifuged at 12,000 rpm for 10 minutes to obtain magnetic nanoparticles (hereinafter, referred to as 'MNP').

After removing the supernatant, it was completely dried at 80 ° C for more than 3 hours using a vacuum oven (JEIO TECH, OV-11) and stored in a vacuum desiccator at 4 ° C.

FIG. 1 is a schematic view showing a method of manufacturing a surface-modified magnetic nanoparticle according to an embodiment of the present invention.

1, the surface-modified magnetic nanoparticles in (B) were prepared by adding poly (lactic-co-glycolic acid) to chloroform (CHCl ₃ , MW: 119.38, DAEJUNG,> 99.5% glycolic acid], (PLGA 50:50; Boehringer Ingelheim, Germany) was prepared in a 6% w / v oily phase and mixed with 100 mg MNP in a 15 mL centrifuge tube in a 5% Alcohol) (PVA; 87% - 89% hydrolyzed, MW 13,000 - 23,000; Aldrich) was prepared and ultrasonicated through the overlapping emulsion evaporation.

The contents were ultrasonicated by applying a pulse at a 5-second interval for 10 minutes using a tip sonicator.

And centrifuged at 12,000 rpm for 15 minutes to obtain magnetic nanoparticles whose surface was modified with a polymer and then washed four times with deionized water.

The air was circulated in an oven at 30 ° C, dried and stored in a vacuum desiccator.

Referring to FIG. 1 (C), the magnetic nanoparticles modified with sodium alginate were prepared by using an electro-spraying system (NanoNC, eS-robot electrospinning spray system, Seoul, Korea).

3 mL of ethylene glycol (HOCH ₂ CH ₂ OH, MW: 62.07, Sigma Aldrich,> 99%) was mixed with 100 mg of MNP in a 10 mL syringe with 2% of liquid sodium alginate [(C ₆ H ₈ O ₆ ) n, 500-600 cp, Wako Chemicals].

The aMNP was manufactured with a scale gauge of 28G, a flow rate of 50 μL min ^-1 , an applied voltage of 19 kV, and a gap between the nozzle and the collector of 8 cm.

Electroprojected aMNP was immediately cross-linked in 1% w / v calcium chloride (Daehan Scientific) solution.

aMNP was recovered using a centrifuge, washed with deionized water and stored at 4 ° C.

< Experimental Example  1> Reformed  Physical properties of magnetic nanoparticles

(1) Analysis method

The morphology of the modified magnetic nanoparticles was investigated using an optical microscope (Nikon Eclipse Ti-U), a field emission scanning electron microscope (FE-SEM; Jeol 7500F, USA), a high-resolution transmission electron microscope (HR-TEM; Tecnai G2 F30 (Bio-AFM) (Nanowizard II, JPK Instruments, Berlin, Germany).

For atomic force microscopy, samples were loaded onto a mica plate, dried and then scanned in air contact mode using a silicon sensor (Budget sensor, long cantilever).

For the scanning electron microscopic analysis, the samples were dropped onto a cleaned silicon substrate, dried and then plated with platinum.

The average size and zeta potential of the nanoparticles were measured using a laser diffraction particle analyzer (Malvern Mastersizer 2000, Malvern, UK).

The concentration of the used particles was 0.1 mg / ml in deionized water and the prepared nanoparticles were analyzed by ultraviolet-visible spectroscopy (Jasco v-730) and X-ray diffraction analysis (XRD, Rigaku.co) FT-IR infrared spectroscopy (FT-IR, Jasco, FT / IR 4600).

To obtain the FT-IR spectrum (500-4000 cm ^-1 ) of each sample, an average of 32 scans were taken at 4 cm ^-1 spectral resolution through a Fourier transform infrared spectroscope. Where the samples were automatically measured through a reference background scan.

 The magnetic properties of the prepared nanoparticles were confirmed at room temperature using a magnetometer using a vibration sample (VSM, Lakeshore) and a hysteresis loop in a magnetic field with a period of -1.5T to +1.5T.

Prior to the measurement, the nanoparticles were prepared from pellets having a diameter of 4.4 mm and MNP, aMNP and pMNP were prepared by varying the thickness to 1.46 mm, 0.49 mm, and 0.39 mm, respectively.

An experimental apparatus for determining the microwave magnetic properties was provided with a transmission type S-band rectangular cavity resonator and a network analyzer.

The pellet was attached to the end of the glass membrane and vibrated in an electric field.

(2) Analysis results

MNP was synthesized by coprecipitation under the thermal conditions of the given scheme according to Scheme 1 below.

[Reaction Scheme 1]

Fe ^{2 +} + 2Fe ^{3 +} + 8OH ^- ? Fe ₃ O ₄ +4H ₂ O

The molar ratio of Fe ^{2 +} and Fe ^{3 +} was determined at 1: 2, and iron ions were oxidized with hydroxide as a base.

pMNP and aMNP were synthesized according to the scheme of FIG. 1 through complex emulsification and electrospinning, respectively.

FIG. 2 is a graph showing the results of field emission scanning electron microscopy, atomic force microscopy, high-resolution transmission electron microscopy, and magnetic field hysteresis loop analysis according to the types of modified magnetic nanoparticles in the method for producing modified magnetic nanoparticles according to an embodiment of the present invention Fig.

 (A), (B) and (C) are hysteresis loops (VSM) of FE-SEM, AFM, HR-TEM photograph and VSM, (F) is the RMS roughness, and (G) is the FT-IR image of MNP, pMNP, and aMNP. Data were determined by standard deviation, and the experiment was repeated 3 times, indicating significant differences between the samples * p <0.05 and *** p <0.001.

Referring to FIG. 2, as a result of the morphological analysis, MNP was identified as homogeneous and spherical.

Referring to FIGS. 2 (A) to 2 (C), the nanostructures of the modified pMNP and aMNP were very clearly visualized using HR-TEM.

(B), the PLGA shell was slightly darker than the more transparent alginate shell.

(A), the coagulation of MNP was confirmed by the magnetic field and electrostatic attraction on the negatively charged surface.

The FE-SEM, AFM, and HR-TEM data were in good agreement.

The AFM height scale was 10 nm, 90.7 nm, and 126.7 nm for MNP, pMNP, and aMNP, respectively.

VSM also showed magnetic field hysteresis of MNP, aMNP, and pMNP when a magnetic field of 1.5 T was formed.

All three samples were enriched with 0.8 T and were listed on the easy axis of planar magnetization.

All samples exhibited strong magnetism and MNP samples showed the highest magnetization of ~ 36 emu / cc, while the magnetization of pMNP and aMNP were found to be ~ 8 emu / cc and ~ 12 emu / cc, respectively.

MNP samples showed the strongest magnetism, while pMNP and aMNP were thought to decrease magnetism due to polymer surface coating, respectively.

The PLGA thickness and alginate shell thickness were 2-3 nm and 5-6 nm for pMNP and aMNP, respectively.

Referring to (D) in Figure 2, the nanoparticle size for MNP, pMNP, and aMNP was distributed between 15.25 nm, 55.75 nm, and 135.155 nm.

Referring to FIG. 2 (E), the surface charge of the nanoparticles was charged at -8.7 mV, -14 mV, and -8 mV.

Referring also to (F) in FIG. 2, the RMS roughnesses were 12 nm, 33 nm, and 87 nm, and the roughness was consistent with the nanoparticle size trend.

MNP ultraviolet-visible spectrum showed nanoscale with an absorption band of 300-400 nm and PLGA and alginate signals were masked by MNP peaks.

XRD results are respectively ~ 30 °, ~ 35 °, ~ 43 ° (corresponding to 220, 311, 400, 422, 511, , ~ 55 °, ~ 58 °, ~ 63 °, and ~ 75 ° to confirm the cubic inverse spinel structure of MNP.

The structure is a structure in which the bonding of iron oxide is filled in the cube, Fe 2+ is disposed in the octahedral space, and Fe ³⁺ is disposed in the octahedral tetrahedral space.

PLGA and alginate are amorphous structures and exhibit a broad peak in the range of 20-30 DEG derived from the glass cover slip used as the substrate.

Weak and relative intensities of all pMNP and aMNP diffraction signals were well matched with the MNP decision criterion data.

Referring to (G) in FIG. 2, the FT-IR results showed elongated bands corresponding to Fe-O between 500 and 600 cm ^-1 in all groups.

A strong asymmetric-symmetric vibration peak of PLGA (C- (C = O) -O) in pMNP was observed at 1150-1300 cm ^{- 1} and a peak at 1750 cm- ¹ showed elongation of C = O.

< Experimental Example  2> Cell survival rate assay

(1) Test method

Fetal Bovine Serum (FBS) and antibiotics (100 U / mL penicillin, 100 μg / mL) were prepared at a seeding density of 2 × 10 ⁴ cells / cm ² . mL streptomycin) in Dulbecco's modified eagle medium (DMEM).

Cell culture conditions were evaluated using the LIVE / DEAD Viability / Cytotoxicity kit (MP03224, Invitrogen) according to the manufacturer's instructions. Cell culture conditions were 37 ° C and 5% CO ₂ .

Cells were treated with nanoparticles at concentrations of 1024, 512, 256, and 128 μg / mL, respectively, and placed in an on-off magnetic field and incubated for 24 hours.

Each cell was directly cultivated in LIVE / DEAD [calcein AM and ethidium homodimer-1 (EthD-1)] for 30 min and then observed for live (green) and dead (red) by confocal laser scanning microscopy LSM 700, Carl Zeiss Micro-Imaging GmbH, Germany).

 In addition, NIH3T3 proliferation was assessed using a cell counting kit (CCK-8, Dojindo, CK04) after treatment with serially diluted nanoparticles from 1024 μg / mL to 16 μg / mL.

CCK-8 solution (10% medium volume per well) was added to each well according to the manufacturer's instructions and incubated at 37 ° C for 1 hour.

A sample of 200 μL was taken and absorbance at 450 nm was measured using a Multiskan microplate reader (Thermo Scientific, Rockford, Ill., USA).

(2) Cytotoxicity

3 shows the cytotoxicity of the modified magnetic nanoparticles according to the embodiment of the present invention.

(D), (E), and (F) show the results of a cell-free survival assay of MNP, pMNP And aMNP, respectively, and (G), (H), and (I) were formed through magnetic field treatment.

(J) is the confirmation of cell proliferation by MNP, pMNP and aMNP by CCK-8 assay.

All results were expressed as standard deviations. The experiment was repeated 3 times, and the significant difference between the control and the test samples was expressed as *** (p <0.001).

Rat fibroblast (NIH3T3) was used to test the cytotoxicity of nanoparticles. Cells cultured in tissue culture dishes were treated with serially diluted nanoparticles at 1024, 512, 256, and 128 μg / mL.

Referring to FIG. 3, a magnetic field of 100 mT was formed to confirm the effect of the magnetic field on the cytotoxicity of the nanoparticles. The magnetic plates were sterilized and placed inside the incubator 6 hours before the experiment.

Referring to FIG. 3 (C), after the cells were treated with nanoparticles, the culture dish was firmly fixed on the magnetic plate.

Referring to FIG. 3 (D), the cell death assay showed that the MNP concentration of 1024 μg / mL showed minute cytotoxicity due to dead cells (red mark).

Referring to FIG. 3 (G), it was confirmed that the cytotoxicity of MNP decreased with decreasing concentration, and MNP solution showed cytotoxicity depending on the concentration. At the same time, the cytotoxicity of MNP was observed at 1024 and 512 μg / mL MNP concentration in the magnetic field, but the diluted solution showed negligible cytotoxicity.

The results show strong evidence that the magnetic field affects cytotoxicity as a result of compelling cell uptake of MNP.

To further confirm that modifying with polymers increases biocompatibility, pMNP and aMNP were also tested in the same way.

Referring to FIGS. 3 (E) and 3 (F), the cytotoxicity was negligible even at a very high concentration of 1024 μg / mL. Referring to FIGS. 3 (H) and (I), the presence of the magnetic field formed microscopic cytotoxicity and showed an increase in the uptake of the forced nanoparticles.

Referring to FIG. 3 (J), the cell proliferation divided into different groups did not show any significant difference at low concentrations (<32 μg / mL), but when the concentration exceeded 64 μg / mL, Respectively.

Cell viability was reduced more rapidly within the magnetic field. The cell proliferation was 1.2-fold reduced by MNP treatment in the magnetic field. However, the cells treated with pMNP and aMNP showed a better cell viability than the cells treated with MNP even in the magnetic field, and biocompatibility was increased due to the polymer coating.

< Experimental Example  3> Assessment of intracellular absorption

(1) Intracellular absorption

Intracellular uptake was assessed by Prussian blue assay and intracellular iron content analysis.

The coverslips coated with gelatin were cultured at 2 × 10 ⁴ cells / cm ² , treated with 256 μg / ml nanoparticles, and cultured for 24 hours.

Cells were washed with PBS to remove unabsorbed nanoparticles and fixed with 4% cold paraformaldehyde for 30 min.

 Washed with PBS and incubated with freshly prepared stain (1: 1 V / V, 10% soluble prussian blue: 20% hydrochloric acid) for 30 minutes.

Finally, the cells were washed with PBS and the cells were observed with CLSM. For the measurement of iron content in the cells, the cells were washed with PBS, collected and counted.

After centrifugation at 7000 rpm for 5 minutes, the cell pellets collected were dispersed in 100 μL of 12% hydrochloric acid aqueous solution and cultured at 60 ° C for 4 hours.

After incubation, the suspension was centrifuged at 12000 rpm for 10 minutes and the supernatant was recovered to increase the iron concentration.

The sample solution was put into a 96-well plate at a volume of 50 μL, and ferrous ions (Fe ^{2 +} ) were oxidized by adding 1% ammonium sulfate (Sigma aldrich).

Finally, 100 μL of 0.1 M potassium thiocyanate (Sigma aldrich) was added to the sample solution and aligned for 5 minutes to form iron-cyanate red. Absorbance was measured at 490 nm using a Multiskan microplate reader.

(2) Absorption evaluation

4 is a graph showing cell uptake of modified magnetic nanoparticles according to an embodiment of the present invention.

(A), (C), (E) and (G) are cells treated with MNP, pMNP and aMNP in the absence of a magnetic field, And cells treated with MNP, pMNP, and aMNP, respectively, in the magnetic field.

Absorbed nanoparticles were tinted blue by the Prussian blue test.

(I) and (J) illustrate the intracellular absorption mechanism of nanoparticles and cell behavior in a magnetic field.

(K) is a quantitative analysis of the intracellular iron content. All results were expressed as standard deviation. The experiment was repeated 3 times, and the significant differences between the control and the test samples were shown as * p <0.05, ** p <0.01, *** p <0.001.

Intracellular absorption mechanisms are crucial for all nanoparticle-based biological applications. This determines the internalization of nanoparticles and drug delivery into cells.

 Intracellular uptake of MNP, pMNP, and aMNP (256 μg / mL) was measured daily using prussian blue staining.

The iron content in the cells was quantitatively analyzed by thiocyanate colorimetric assay. Cells treated with MNP showed the highest intracellular uptake regardless of magnetic field formation, and were confirmed by 3D-reconstruction confocal photographs of FIGS. 4 (C) and 4 (D).

The cross section more clearly shows the dispersion of MNP internalized in the cell cytoplasm.

The zeta potential of MNP was -8.7 mV, and it was judged that MNP was undergoing electrostatic repulsion against the cell membrane.

Referring to FIG. 4 (K), the results showed that the absorption of MNP into the cells was increased and the iron content in the cells was 10 μg / mL.

Referring to (E), (K) and (G) of FIG. 4, pMNP and aMNP exhibited somewhat low intracellular uptake in the absence of a magnetic field, and were higher than the control group.

Referring to Figure 4 (I), differences in the intracellular iron content were found in the cells treated with pMNP and aMNP. The lower iron content is due to the increased size of pMNP and aMNP and is due to the strong electrostatic repulsion between the cell membrane and pMNP / aMNP.

Finally, the content of iron due to intracellular uptake was found to be the control <pMNP <aMNP <MNP, and the cells treated with MNP showed a large decrease (~ 1 (18.7 μg / mL) of the iron content Times).

It was confirmed that the cells treated with pMNP and aMNP in FIGS. 4 (F), (H), (J), and (K)

< Experimental Example  4> Cell morphology

(1) Morphology analysis method

NIH3T3 cells were cultured on glass gelatin coated coverslips.

The cells were incubated in 256 μg / mL nanoparticles for 24 hours according to cell lysate discrimination test, the medium was removed, and the samples were washed three times with PBS.

Live cells were scanned using bio-AFM using the HYDRA2R-50NG probe. The photographs were obtained by isolating whole cells, nucleus, and cytoplasm.

The scan size was adjusted to 100 μm × 100 μm, 40 μm × 40 μm, and 10 μm × 10 μm according to the typical surface measurement sequence while maintaining high resolution.

For FE-SEM measurements, the cells were fixed, washed, and then dehydrated to the maximum with successive dilutions (60%, 70%, 80%, 90%, 99%) of ethanol. After dehydration, the samples were coated with platinum and pictures of the three compartments of the cells were taken.

(2) Microindentation test

AFM spectroscopy was performed, cells were cultured, and biophysical (cell size, spreading area and roughness) properties and biomechanical properties (cell membrane elasticity) were analyzed using Bio-AFM.

A CONT-S spherical probe (5 μm radius; Nanoworld services GmbH, Erlangen, Germany) with a force of 0.4 N / m was used to measure the Young's modulus. After scanning the cell compartment, Respectively.

The size and the dropping speed of the lamp were determined at 1 μm and 1 μm / s. In order to prevent defects on the cell surface and to prevent the limitation of the Hertz model, indentations in the range of 0.5 to 1.5 μm with a continuous force of 0.5 to 10 nN The depth was adjusted.

Tip - Sample separation curves and Young's moduli were determined using the Hertz contact model using JPK data processing software (JPK instruments) with Poisson's ratio adjusted to 0.5.

For cell size measurements, a cell morphology size scale of AFM was used.

The roughness of the cell surface was calculated using JPK photo processing software.

For cell spreading analysis, Image J software (NIH) was used.

(3) Biophysical biomechanical changes

5 is a 3D photograph of an atomic force microscope showing cell behavior of modified magnetic nanoparticles according to an embodiment of the present invention.

(B), (C) and (D) of the left panel are photographs treated with MNP, pMNP, and aMNP in the absence of a magnetic field, and (F) (G) and (H) are photographs treated with MNP, pMNP, and aMNP in the magnetic field.

5 (A) to 5 (H), cells were treated with atomic force microscope without forming or forming a magnetic field with 256 μg / mL of nanoparticles for one day.

Cell Bio-AFM images were scaled to 100 × 100 μm, 40 × 40 μm, and 10 × 10 μm, respectively. Changes in the morphology of the cells treated and treated with nanoparticles were clear.

Referring to FIGS. 5 (A) to 5 (D), the magnitude of the structural change of the cells was measured.

5 (A) to 5 (H), no change was observed in the filopodia, lamellipodia, and invadopodia of the cells along with the skeletal structure of the different cells in all the nanoparticle treatment groups except for the case where a magnetic field was formed .

The cytoskeleton is a dynamic structure that plays an important role in cell activity such as cell adsorption, morphology, mobility and cellular uptake process.

6 is a graph showing biophysical mechanical analysis of modified magnetic nanoparticles according to an embodiment of the present invention.

Referring to FIGS. 5A and 5E and FIGS. 6A and 6D, the cell skeleton in which the magnetic field is not formed is normal, but the cells are elongated in the magnetic field. These results indicate that the magnetic field affects cell functions such as cell size (from 2.5 μm to 2.2 μm) and cell membrane elasticity (from 3 kPa to 4 kPa).

 Atomic force microindentation was measured using a 5-μm diameter spherical tip around the nucleus in the cell compartment.

Referring to Figure 6 (A), the mean cell size was reduced to 1.7 μm after MNP treatment and further reduced to 0.6 μm in the magnetic field.

Referring to (H) of FIG. 6, MNP slightly decreased the spreading region, and the cell morphology was slightly changed into a circular shape in which the cytoskeleton collapsed.

In addition, the cell roughness was 100 nm according to the formation of the magnetic field, and the formation of the magnetic field did not affect the roughness of the cell.

Referring to FIG. 6 (C), the cell roughness was increased to 170 nm when treated with MNP, and it was further adhered to the cell surface by magnetic field induction in the magnetic field and further increased to 220 nm.

Referring to FIGS. 5 (B), (F) and 6 (D), MNP treatment significantly reduces the elasticity of the cell membrane to 2 kPa, which is due to collapse of the cytoskeleton by MNP adsorption and cell membrane separation Can be explained by cytotoxicity.

Referring to FIGS. 5 (C) to (H) and 6 (A), the Young's modulus of the MNP-treated cells was reduced to 1.3 kPa in the magnetic field and increased cell uptake of MNP and subsequent cytotoxic effects .

Referring to FIGS. 5 (C) to 5 (H) and 6 (A), when pMNP and aMNP were treated, the average cell sizes were 0.75 μm and 1.23 μm, respectively, and further decreased to 0.5 μm or 0.87 μm in the magnetic field .

Referring to FIG. 6 (H), in the case of cells treated with pMNP and aMNP, the spreading region increased regardless of the influence of the magnetic field.

Referring to Figure 4 (J), this phenomenon was due to the polymer chains forming layers at the surface of the cells. The spreading region of the cells was further increased in the magnetic field due to the strong downward attraction of the surface of the modified MNP.

Referring to FIG. 4 (J), attraction is exerted on the surface of the MNP modified in the magnetic field, and the adsorbed nanoparticles are pushed to the outer circumferential region of the transverse polymer chain to reduce the increase in cell size and spreading region .

Referring to FIG. 6C, the surface roughness of the cells treated with pMNP and aMNP were 130 nm and 140 nm, respectively, and increased to 174 nm and 176 nm when the magnetic field was formed.

The results demonstrate that pMNP and aMNP alter the cell roughness by the attachment of magnetic field-induced nanoparticles to the cells.

In Fig. 4 (J), the Young's modulus of cells treated with pMNP and aMNP was slightly higher than that of cells not treated with 3.5 kPa and 3.1 kPa, respectively. The above results support the modification of the cell surface by the polymer.

6 (D), the magnetic field further increased cell membrane elasticity to 4.5 kPa and 4.8 kPa for cells treated with pMNP and aMNP.

 Referring to Figures 4 (K) and 6 (D), the magnetic field increases intracellular uptake of pMNP and aMNP, and consequently also the Young's modulus.

The polymeric layer of PLGA and alginate can be functionalized on the surface of the cells, which explains the increase in Young's modulus.

< Experimental Example  6> Statistical processing

All data presented were obtained from three independent samples and analyzed three times each. One-way analysis of variance (ANOVA) was performed using commercial software (GraphPad Prism 5, San Diego, USA) with Tukey's multiple comparison post hoc test.

(P <0.05), ** (p <0.01), and *** (p <0.001), respectively, and there were significant differences.

Accordingly, the present invention provides a magnetic nanoparticle modified with a surface modified with iron oxide, poly (lacticco-co-glycolic acid), and sodium alginate by coprecipitation, emulsification, to provide.

The nanoparticles formed in the nanoparticle size range and were negatively charged.

The surface morphology and structural analysis of the nanoparticles confirmed that the surface of the magnetic nanoparticles was modified. The surface modified magnetic nanoparticles were biocompatible and showed very improved intracellular delivery capability in the magnetic field.

Claims (15)

Magnetic nanoparticles of a core-shell structure; And
And a polymer layer attached to the outer periphery of the magnetic nanoparticles,
The polymer layer is adsorbed on the surface of the magnetic nanoparticles to show a negative charge of -14 to -8 mV, and the degree of magnetization is reduced by the polymer layer,
Wherein the cytotoxic and physiological properties are changed by the magnetic field to increase the cell viability.
The method according to claim 1,
The magnetic nanoparticles
Fe ²⁺ and Fe ³⁺ ions are co-precipitated to form core-shell structure nanoparticles.
3. The method of claim 2,
Wherein the molar ratio of Fe ^{2 +} and Fe ^{3 +} ions is 1: 2.
The method according to claim 1,
The polymer layer
Characterized in that it is formed of poly (lactic-co-glycolic acid) or alginate.
delete delete The method according to claim 1,
The change in the physics &lt; RTI ID = 0.0 &gt;
Cell size, Young's modulus and cell roughness.
(a) preparing magnetic nanoparticles;
(b) adding a polymer to the magnetic nanoparticles to form a polymer layer on the surface of the magnetic nanoparticles; And
(c) recovering, washing and drying the modified magnetic nanoparticles,
When the polymer layer is poly (ricic-co-glycolic acid), chloroform and the poly (ricic-co-glycolic acid) are mixed to form a poly (vinyl alcohol) and a duplicate emulsion by ultrasonic treatment. In the case of alginate, sodium alginate is mixed with ethylene glycol and formed by electric spraying,
The polymer layer is adsorbed on the surface of the magnetic nanoparticles to exhibit a negative charge of -14 to -8 mV and the degree of magnetization is reduced by the polymer layer,
Wherein the cytotoxic and physiochemical properties of the modified nanoparticles are changed to increase the cell viability.
9. The method of claim 8,
The step (a)
Fe ²⁺ and Fe ³⁺ ions are co-precipitated to form magnetic nanoparticles in the form of a core-shell.
10. The method of claim 9,
The Fe ²⁺ and Fe ³⁺ ions
Wherein the molar ratio is 1: 2.
delete delete 9. The method of claim 8,
The ultrasonic treatment
Wherein the step of forming the modified magnetic nanoparticles is performed with a pulse at intervals of 5 seconds for 10 minutes.
delete 9. The method of claim 8,
Wherein the electric injection is performed at a flow rate of 45 to 50 μL min ^-1 and an applied voltage of 15 to 19 kV.

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