KR102201471B1 - Manufacturing device of nanocomposites, method of preparing nanocomposites and nanocomposite prepared therefrom - Google Patents

Abstract

The nanocomposite manufacturing apparatus and manufacturing method of the present application, and the nanocomposite manufactured by using the same, use a gas single-pass configuration that does not require hydrothermal reaction, separation purification, or post-treatment for binding of a physiologically active substance and a support to metal. Multifunctional nanocomposites can be continuously manufactured through reassembly of aggregates.

Description

Manufacturing device of nanocomposites, method of manufacturing nanocomposites, and nanocomposites prepared using the same {Manufacturing device of nanocomposites, method of preparing nanocomposites and nanocomposite prepared therefrom}

The present application relates to an apparatus for manufacturing a nanocomposite, a method for manufacturing a nanocomposite, and a nanocomposite manufactured using the same.

The plasmonic gold (Au)-based nanostructure is chemically inert and can be easily prepared. In addition, since the plasmonic gold (Au)-based nanostructure has high biocompatibility and can control optical properties, it is often used for treatment and diagnosis. In particular, the strong light absorption of the nanostructure in the near infrared (NIR) region causes the loss of non-radiative energy through Landau damping and generates sufficient heat for photothermal therapy. Accordingly, nanostructures have been widely used as such treatments using various treatment modes and different bioactive molecules such as drugs and targeting agents.

It was confirmed that the shape of the Au nanostructure was changed by manipulating the surface plasmon resonance wavelength from visible light to near infrared light. Compared to the very limited red-shifting of localized surface plasmon resonance (LSPR) absorption due to the increase in Au size, the shape change of the Au nanostructures is a stronger optical behavior in photothermal therapy and optical diagnosis. To induce. Au nanorods are often used to achieve red shifting or expansion, and the absorption spectrum can be adjusted by controlling the aspect ratio of Au.

However, recently, Au nanorods have been reported to limit cell viability due to physicochemical properties such as sharp edges, tips and residues, for example, cetyl trimethylammonium bromide, and due to anisotropic properties, residence time in blood vessels according to high aspect ratio There is this low problem.

Korean Patent Registration 10-1707468 Korean Patent Registration 10-1705033

The present application is a nanocomposite capable of continuously manufacturing a multifunctional nanocomposite through reassembly of metal aggregates using a gas single-pass configuration that does not require hydrothermal reaction, separation purification, or post-treatment for binding of a physiologically active material and a support It provides an apparatus for manufacturing a composite, a method for producing a nanocomposite, and a nanocomposite prepared using the same.

The present application relates to an apparatus for manufacturing a nanocomposite. According to an exemplary manufacturing apparatus of the present application, a spray unit for forming droplets by spraying a solution containing an organic material having an electron donating group or negatively charged and a metal nanoparticle aggregate; A light irradiation unit configured to form a nanostructure by irradiating light onto the droplets formed from the spray unit to rearrange the metal nanoparticles and organic substances; And a solvent removal unit configured to remove a solvent from the nanostructure formed from the light irradiation unit to generate a nanocomposite.

The present application relates to a method of manufacturing a nanocomposite. According to an exemplary manufacturing method of the present application, forming droplets by spraying a solution containing an organic material having an electron donating group or negatively charged and a metal nanoparticle aggregate; Irradiating light to the droplets formed in the step to rearrange the metal nanoparticles and the organic material to form a nanostructure; And removing the solvent from the nanostructure formed in the above step to generate a nanocomposite.

The present application relates to a nanocomposite. According to the nanocomposite according to the exemplary embodiment of the present application, a plurality of metal nanoparticles and an organic material having an electron donating group or negatively charged are included, and the organic material is electrostatically arranged between the metal nanoparticles. It is characterized.

The present application relates to a nanocomposite. According to the nanocomposite according to another embodiment of the present application, a plurality of gold (Au) nanoparticles, a surfactant having an electron donating group or or a negatively charged surfactant, and a drug, and the surfactant and the drug It is characterized in that the metal nanoparticles are electrostatically arranged.

The nanocomposite manufacturing apparatus and manufacturing method of the present application, and the nanocomposite manufactured by using the same, use a gas single-pass configuration that does not require hydrothermal reaction, separation purification, or post-treatment for binding of a physiologically active substance and a support to metal. Multifunctional nanocomposites can be continuously manufactured through reassembly of aggregates.

1 is a schematic diagram of an exemplary apparatus for manufacturing a nanocomposite of the present application.
2 is a view showing a solvent removal unit in the apparatus for manufacturing an exemplary nanocomposite of the present application.
3 is a view showing a cross-section of a droplet transfer unit in which an electric field application unit is formed in the apparatus for manufacturing an exemplary nanocomposite of the present application.
4 is a view showing a collection unit of an exemplary apparatus for manufacturing a nanocomposite of the present application.
5 is a diagram schematically illustrating an exemplary apparatus for manufacturing a nanocomposite, a method for manufacturing a nanocomposite, and a nanocomposite manufactured using the apparatus of the present application.
6 is a size distribution diagram of Au AG (spark elution of Au rods), TD droplets (TD dissolved in ethanol) and hybrid droplets (TD droplets including Au AG) under UV irradiation at 185 nm before solvent extraction through a diffusion dryer. .
7 is a low-magnification and high-magnification TEM image of (a) Au AGs and (b) Example 1 (TAuD) after passing through a diffusion dryer.
FIG. 8 is a graph of a UV-vis spectrum including (a) particle size distribution, zeta potential, and (b) TD of Example 1 (TAuD) nanocomposite dispersed in PBS using a dynamic light scattering (DLS) method.
9 is a TEM image of reassembled Au AG containing different T solutions.
Figure 10 is (a) XPS Au spectrum of Example 1 (TAuD), Au and Comparative Example 1 (TAu), (b) Example 1 (TAuD), Comparative Example 1 (TAu) and Comparative Example 2 (FT of AuD -IR) spectrum, (c) Raman spectrum graphs of Example 1 (TAuD), Comparative Example 3 (TD) and Au AG.
Figure 11 is from Example 1 (TAuD) (a) pH triggering (5.5 and 7.4) (24 hours) and (b) near-infrared (NIR) triggering (6 hours and 808 nm) (irradiation for 5 minutes after 2 hours) drug (DOX , D) is a graph of the in vitro release profile.
12 shows the intensity-dependence of (a) concentration dependence (2.5 W cm ^-2 ) and (b) Comparative Example 1 (TAu) and RPMI (control) for 6 minutes according to 808 nm near-infrared (NIR) irradiation (150 μg/ mL) temperature rise profile.
13 shows the intensity-dependence of (a) concentration dependence (2.5 W cm ^-2 ) and (b) Comparative Example 1 (TAu) and RPMI (control) for 6 minutes according to 632 nm near-infrared (NIR) irradiation (150 μg/ mL) temperature rise profile.
14 shows Comparative Examples 1 (TAu) and Example 1 (TAuD) in the absence and presence of near-infrared (NIR) irradiation including a single D in (a) MDA-MB-231 and (b) MCF-7 cells. In vitro anticancer effect graph, (c) CLSM image of Comparative Example 1 (TAu) loaded on coumarin-6 in MDA-MB-231 and MCF cells, (d) MDA-MB-231 and (e) MCF- 7 FACS analysis results showing cellular uptake of Example 1 (TAuD) by cells, (f) Western blot analysis of p53 in cells treated with Comparative Example 1 (TAu) with or without 808 nm near-infrared (NIR) irradiation It is the result.
15 shows MCF-7 after treatment with (i) control, (ii) single D group, (iii) Comparative Example 1 (TAu) + near-infrared (NIR) group and (iv) Example 1 (TAuD) + cell group. Top) and MDA-MB-231 (bottom) are FACS results to determine cell death.
16 is an inverse fluorescence microscopy image in the survival/death analysis.
17 is an optical microscope image measuring cell cycle analysis.
18 is an in vivo anti-tumor evaluation, MDA-MB- with xenograft mice for Comparative Example 1 (TAu) and Example 1 (TAuD) according to the presence or absence of near-infrared (NIR) irradiation including single D (A) tumor growth and (b) weight-change profile graph of 231 and (c) major organs (liver (i), lung (ii), heart (iii), spleen) collected from euthanized mice for 24 hours after injection. (iv) and kidney (v)] and in vitro fluorescence images of Cy5.5 alone (middle) and Comparative Example 1 (TAu) (right) combined with Cy5.5 against tumors (vi).
FIG. 19 is an image of in vivo photothermal imaging during near-infrared (NIR) irradiation on a tumor of a mouse pretreated with physiological saline and Example 1 (TAuD).
Figure 20 is another combination of using the lipid molecule LAu instead of T to prepare different types of nanocomposites, (a) low and high magnification TEM images of LAu from a droplet flow (FD) reaction, (b) photothermal activity of LAu From LAuD to confirm the near-infrared (NIR) (632 and 808 nm, 5 min)-trigger D emission profile and (c) pure DNA and compared to commercially available lipofectamine, LAu, that is, pDNA bound to LAuG (EGFP Including) is a graph of gene transfer performance.

In the present application, the term "nano" may mean a size in a nanometer (nm) unit, for example, it may mean a size of 1 to 1,000 nm, but is not limited thereto. In addition, in the present specification, the term "nanoparticle" may mean a particle having an average particle diameter in a nanometer (nm) unit, and for example, it may mean a particle having an average particle diameter of 1 to 1,000 nm. It is not limited.

Hereinafter, an apparatus for manufacturing a nanocomposite of the present application, a method of manufacturing a nanocomposite, and a nanocomposite manufactured using the same will be described with reference to the accompanying drawings, and the accompanying drawings are exemplary, and the apparatus for manufacturing a nanocomposite of the present application , The manufacturing method of the nanocomposite and the scope of the nanocomposite prepared using the same are not limited by the accompanying drawings.

1 is a schematic diagram of an exemplary apparatus for manufacturing a nanocomposite of the present application.

As shown in FIG. 1, the apparatus for manufacturing the nanocomposite includes a spray unit 13, a light irradiation unit 14, and a solvent removal unit 15.

The atomizing unit 13 may include an atomizing device and a transport gas injection port, and may be, for example, a collision atomizer. The spray unit 13 may form droplets by spraying a solution containing an organic material having an electron donating group or negatively charged and a metal nanoparticle aggregate.

In one example, the spray device may include a spray nozzle. The spray nozzle may be composed of an upper spray nozzle and a lower spray nozzle. The particle diameter of the nozzle is not particularly limited, but may be 0.05 to 2.0 mm. In the spraying unit, a solution containing an organic material having the electron donating group and a metal nanoparticle aggregate may be sprayed into the spraying unit 13 through the spray nozzle in the form of droplets.

The spraying unit 13 may further include a stirrer for preparing a solution by mixing a solution containing an organic material having an electron donating group and a metal nanoparticle aggregate. The stirrer is not particularly limited as long as it is a device capable of high-speed stirring, and is, for example, 200 to 4000 rpm, and may be used without limitation as long as it is a device capable of stirring by applying ultrasonic waves.

The transfer gas injection unit may inject a transfer gas into the droplet transfer unit 17. The transport gas may be an inert gas, and the inert gas may be at least one selected from the group consisting of nitrogen (N ₂ ), helium (He), neon (Ne), and argon (Ar), but is not limited thereto. The pressure of the gas injected into the droplet transfer unit may be 1.5 atmospheres or less, but is not limited thereto. When the pressure of the gas exceeds 1.5 atm, it may be difficult to generate reassembled nano vesicles, and vesicle formation may be improved by forming a swirl.

In one example, the apparatus for manufacturing the nanocomposite may further include a discharge unit 11. The discharge unit 11 may form metal particles from the metal electrode by discharge. The discharging part 11 is a part for generating metal nanoparticles by discharging, and includes a pair of conductive rods disposed at predetermined intervals and a power supply for applying voltage to the conductive rods.

The discharge may use spark discharge or arc discharge, but is not limited thereto. In one example, spark discharge may be used as the discharge. The "spark discharge" refers to a high-frequency discharge method performed in a kV-mA mode at normal pressure, and the "arc discharge" refers to a high current discharge method performed in a V-A mode in a vacuum.

The pair of conductive rods are spaced apart from each other to form a gap. For example, spark discharge occurs in the discharge unit, and metal nanoparticles are generated due to a high temperature locally generated between the conductive rods by the spark discharge. The term "gap" or "gap" used in this application means a gap between two moving or fixed parts, for example, the gap refers to a gap between a pair of conductive rods that are spaced apart from each other. . In addition, in the present application, the term “nano” is a size in a nanometer (nm) unit, and may mean, for example, a size of 1 nm to 1000 nm, but is not limited thereto. In addition, in the present application, the term "nanoparticle" may mean a particle having a size in a nanometer (nm) unit, for example, an average particle diameter of 1 nm to 1000 nm, but is not limited thereto.

The material constituting the conductive rod is not particularly limited as long as it is a metal having a work function of 6.0 eV or less, and for example, a metal having a work function of 5.7 eV or less, 5.0 eV or less, 4.6 eV or less, or 4.2 eV or less can be used. However, it is not limited thereto. In one example, the metal having a work function of 6.0 eV or less is gold, barium, silver, cadmium, aluminum, beryllium, cerium, cesium, cobalt, chromium, iron, gallium, gadolinium, hafnium, mercury, indium, magnesium, manganese, Molybdenum, lead, niobium, neodymium, rubidium, rhenium, rhodium, ruthenium, scandium, tin, strontium, tantalum, terbium, tellurium, thorium, titanium, uranium, vanadium, yttrium, thallium, ytterbium, zinc, palladium, iridium, platinum And it may include one or more selected from the group consisting of zirconium, preferably, gold may be used, but is not limited thereto.

In addition, the distance between the conductive rods, for example, the electrode gap, which is the shortest distance between the conductive rods, decreases the ignition required voltage as the distance decreases, and the higher the distance, the higher voltage is required. In addition, if the electrode gap is narrow, the voltage required to generate the spark decreases, but a short spark can cause misfire by transferring the minimum ignition energy to the mixer, so it is necessary to set an appropriate distance by experiment. In one example, the gap between the electrodes may be 0.1 to 10 mm, but is not limited thereto.

The power supply is a part for applying a voltage to each of the conductive rods, and in one example, the voltage applied from the power supply to the conductive load is 2 to 5 kV, and the amount of current may be 0.5 to 5 mA, but is limited thereto. It does not become. For example, in the power supply unit, the voltage applied to the pair of conductive rods may be constantly adjusted. Accordingly, by quantitatively supplying the metal nanoparticles, it is possible to manufacture the metal nanoparticles with excellent supply stability.

In one example, the power supply unit may include an electric circuit for applying a high voltage to the conductive rod. The electric circuit has a structure of a constant high voltage source composed of a high voltage supply source, an external capacitor, and a resistor, and the size of the metal nanoparticles is reduced by using a plurality of resistors, a plurality of capacitors, and a circuit capable of high-speed switching of circuit current. Can be adjusted.

Although not shown, the nanocomposite manufacturing apparatus of the present application may include a gas supply device such as a carrier air supply system, and a flow meter such as a mass flow controller (MFC). . In addition, an inert gas, oxygen or nitrogen may be quantitatively supplied at intervals between the conductive rods by the gas supply device and the flow meter.

When a high voltage is applied to the conductive rod, at least one selected from the group consisting of an inert gas, oxygen, and nitrogen flowing through the gap between the conductive rods by vaporizing or granulating the metal with the work function of 6.0 eV or less by spark discharge It may flow out to the atomizing part along the gas flow. For example, when a voltage is applied to the conductive rod of the discharge unit, the metal is vaporized in the gap between the pair of conductive rods of the discharge unit, and the vaporized metal moving along a carrier gas such as an inert gas or nitrogen is , As outside the gap, condensed, and thus, metal nanoparticles are formed.

The particle diameter of the metal nanoparticles generated from the discharge unit 11 may be widely adjusted from several nanometers to hundreds of nanometers depending on the flow rate or flow rate of the inert gas or nitrogen. For example, when the flow rate or flow rate of the supplied inert gas or nitrogen increases, as the concentration of the metal nanoparticles decreases, the aggregation phenomenon between particles also decreases, and the size of the metal nanoparticles decreases through this process. Can be. In addition, the particle diameter, shape, and density of the metal nanoparticles may include spark generation conditions such as applied voltage, frequency, current, resistance, and capacitance values; The type and flow rate of the inert gas; Alternatively, it may be changed by the shape of the spark electrode.

Argon (Ar) or helium (He) may be exemplified as the inert gas, but is not limited thereto.

In one example, the apparatus for manufacturing the nanocomposite may further include a solution generating unit 12. The solution generating unit 12 may be operated continuously. Although not shown in the drawings, the solution generating unit 12 may be connected to a pump and a stirrer.

The pump may quantitatively supply a solvent or one or more precursors to the solution generating unit 12. The pump is not particularly limited as long as it is a device capable of continuously or intermittently supplying the solvent or one or more precursors, and may be, for example, a metering pump or a syringe pump, but is not limited thereto.

The stirrer may uniformly mix the solvent or one or more precursors to prepare a solution. The stirrer is not particularly limited as long as it is a device capable of stirring, and the stirring speed may be changed according to the type, viscosity or content of a solvent and a precursor, and ultrasonic waves may be applied for more uniform stirring.

The light irradiation unit 14 is a part that irradiates light to the metal nanoparticles, and may irradiate light having a photon energy greater than a work function of the metal nanoparticles. The light may be ultraviolet rays in a wavelength range of 200 nm or less.

In one example, the light irradiation unit 14 may include a light source that irradiates light to the metal nanoparticles. The type of the light source is not particularly limited, and for example, a device capable of irradiating light having photon energy exceeding the work function of a metal, for example, light having a short wavelength of 200 nm or less Ramen can be used without restrictions. For example, a known light source such as a high-pressure mercury lamp, an ultra-high pressure mercury lamp, a halogen lamp, a black light lamp, a microwave-excited mercury lamp, various lasers or X-rays may be used, or an inert gas may be used at room temperature. Similar reactions can also be induced by irradiation with soft X-rays during the flow. By using a light source capable of irradiating light having a short wavelength of 200 nm or less, electrons on the surface of the transition metal nanoparticles can be released, and electric charges on the metal surface can be induced to a positive charge.

2 is a view showing a solvent removal unit in the apparatus for manufacturing an exemplary nanocomposite of the present application.

As shown in FIG. 2, the solvent removal unit 15 is a part for removing the solvent of the droplets transferred from the droplet transfer unit, and passes through the solvent removal unit 15 to include the nanocomposite. The solvent can be removed from.

The solvent removal unit 15 may include an inner tube 153 and a through passage 152, and may be a diffusion dryer or a denuder in which an adsorbent 151 is provided therein.

The solvent of the nanocomposite may be extracted while the droplet passes through the through passage 152. The through passage 152 may be formed between a plurality of inner tubes 153 including the adsorbent 151. The inner tube 153 is not limited as long as it has one or more hollows such as stainless mesh and has durability and heat resistance. The adsorbent 151 provided inside the inner tube 153 may be filled with an absorption-adsorption type extraction bed, for example, commercially available such as activated carbon, silica gel, zeolite, or activated alumina. It may be one or more of the available adsorbents, but is not limited thereto.

Although not shown in the drawing, the solvent removal means may include an extraction means, and the extraction means may be an extraction furnace including an inlet and an outlet, and the extraction solvent is extracted through the inlet. It may be introduced into a means, and a mixture from which the solvent is extracted by the extraction solvent, that is, the nanocomposite from which the solvent is extracted may be discharged to the outlet.

In one example, the apparatus for manufacturing the nanocomposite may further include a droplet transfer unit 17. 3 is a view showing a cross-section of a droplet transfer unit 17 in which the electric field application unit 18 is formed in the apparatus for manufacturing an exemplary nanocomposite of the present application.

As shown in FIG. 3, the solvent removal unit 15 may further include a droplet transfer unit 17, and the droplet transfer unit 17 may further include an electric field application unit 18. The electric field applying unit 18 is a part that applies an electric field to the nanocomposite formed from the solvent removing unit 15, and the electric field applying unit 18 is an electric field from the center of the droplet transfer unit 17 to be described later. It may be formed in a shape such as a probe or a needle to generate a, and the shape in which the electric field applying unit 18 is formed is limited as long as it is a shape capable of generating an electric field from the center of the droplet transfer unit 17 to the edge direction. It is not. The strength of the electric field generated by the electric field applying unit 18 may be -50 kV to +50 kV, but is not limited thereto. Aggregation of droplets can be prevented by applying an electric field having the same polarity as the formed droplets to the droplets.

The droplet transfer unit 17 is a part for transferring the nanocomposite formed from the solvent removal unit 18 and may transfer the formed nanocomposite to the collection unit 16 to be described later.

4 is a view showing a collection unit of an exemplary apparatus for manufacturing a nanocomposite of the present application. As shown in FIG. 4, the collection unit 16 is a part for collecting the nanocomposite formed from the solvent removal unit 15, and the collection unit 16 may include a metal unit and a voltage applicator. .

The metal part 161 of the collection part 16 may be electrostatically coupled to the nanocomposite, and the metal part 161 may have a shape such as a plate or a rod.

The voltage applicator 162 may be connected to the metal part 161. A voltage may be applied to the metal part 161 so that the metal part 161 and the nanocomposite may be coupled, and the voltage may be -10 to +10 kV, but is not limited thereto. When the generated nanocomposite has a positive charge, a negative voltage is applied, and when the nanocomposite has a negative charge, a positive voltage may be applied to collect the nanocomposite.

The present application also relates to a method of manufacturing a nanocomposite. The manufacturing method of the nanocomposite of the present application may be performed using the apparatus for manufacturing the nanocomposite described above, and accordingly, contents overlapping with the contents described in the apparatus for manufacturing the nanocomposite will be omitted. According to an exemplary method of manufacturing a nanocomposite of the present application, a droplet forming step, a nanostructure forming step, and a nanocomposite forming step are included.

The droplet forming step may be performed in the spray unit 13 of the above-described nanocomposite manufacturing apparatus, and in one example, the droplet forming step includes an organic material having an electron donating group or negatively charged. And spraying the solution and metal nanoparticle aggregates.

The electron donating group means a substituted or unsubstituted C1 to C24 alkyl, alkoxy, thioalkoxy, amine group, imine group, carboxyl group, phosphoric acid group, sulfonic acid group or a combination thereof, and "substituted" for a chemical species It means substituted by a group that does not interfere with the desired product or method, for example, the substituent may be alkyl, alkoxy, and the like. Specifically, the electron donating group is -NR ₂ , -NH ₂ , -OH, -OR, -NHC(=O)R, -OC(=O)R, -R, -CH=CH ₂ and -CH= Any one selected from the group consisting of CR ₂ may be mentioned, but is not limited thereto.

The surfactant may be a cationic surfactant, an anionic surfactant, an amphoteric surfactant and a nonionic surfactant, and specifically, may be a nonionic surfactant, but is not limited thereto.

The cationic surfactant is dodecyl trimethyl ammonium bromide, quaternary ammonium compound, benzalkonium chloride, cetyltrimethylammonium bromide, chitosan, lauryldimethylbenzylammonium chloride, acyl carnitine hydrochloride, alkylpyridinium halide, cetyl pyridinium chloride , Cationic lipids, polymethylmethacrylate trimethylammonium bromide, sulfonium compounds, polyvinylpyrrolidone-2-dimethylaminoethyl methacrylate dimethyl sulfate, hexadecyltrimethyl ammonium bromide, phosphonium compounds, quaternary ammonium compounds , Benzyl-di (2-chloroethyl) ethyl ammonium bromide, coconut trimethyl ammonium chloride, coconut trimethyl ammonium bromide, coconut methyl dihydroxyethyl ammonium chloride, coconut methyl dihydroxyethyl ammonium bromide, decyl triethyl ammonium chloride, decyl dimethyl Hydroxyethyl ammonium chloride, decyl dimethyl hydroxyethyl ammonium chloride bromide, (C12-15) dimethyl hydroxyethyl ammonium chloride, (C12-15) dimethyl hydroxyethyl ammonium chloride bromide, coconut dimethyl hydroxyethyl ammonium chloride, coconut dimethyl Hydroxyethyl ammonium bromide, myristyl trimethyl ammonium methylsulfate, lauryl dimethyl benzyl ammonium chloride, lauryl dimethyl benzyl ammonium bromide, lauryl dimethyl (ethenoxy)4 ammonium chloride, lauryl dimethyl (ethenoxy)4 ammonium Bromide, N-alkyl (C12-18)dimethylbenzyl ammonium chloride, N-alkyl (C14-18)dimethyl-benzyl ammonium chloride, N-tetradecyldimethylbenzyl ammonium chloride monohydrate, dimethyl didecyl ammonium chloride, trimethylammonium halide alkyl -Trimethylammonium salt, dialkyl-dimethylammonium salt, lauryl trimethyl ammonium chloride, ethoxylated alkylamidoalkyldialkylammonium salt, ethoxylated trialkyl ammonium salt, dialkylbenzene dialkylammonium chloride, N-didecyldimethyl ammonium chloride Chloride, N-tetradecyldimethylbenzyl ammonium chloride monohydrate, N-alkyl (C12-14) dimethyl 1- Naphthylmethyl ammonium chloride, dodecyldimethylbenzyl ammonium chloride, dialkyl benzenealkyl ammonium chloride, lauryl trimethyl ammonium chloride, alkylbenzyl methyl ammonium chloride, alkyl benzyl dimethyl ammonium bromide, C12 trimethyl ammonium bromide, C15 trimethyl ammonium bromide, C17 trimethyl Ammonium bromide, dodecylbenzyl triethyl ammonium chloride, polydiallyldimethylammonium chloride, dimethyl ammonium chloride, alkyldimethylammonium halogenide, tricetyl methyl ammonium chloride, decyltrimethylammonium bromide, dodecyltriethylammonium bromide, tetradecyltrimethyl Ammonium bromide, methyl trioctylammonium chloride, POLYQUAT 10, tetrabutylammonium bromide, benzyl trimethylammonium bromide, choline ester, benzalkonium chloride, stearalkonium chloride, cetyl pyridinium bromide, cetyl pyridinium chloride, 4 Halide salt of quenched polyoxyethylalkylamine, MIRAPOL, Alkaquat, alkyl pyridinium salt, amine, amine salt, imide azolinium salt, protonated quaternary acrylamide, methylated quaternary polymer , Cationic guar gum, benzalkonium chloride, triethanolamine, and may be one or more selected from the group consisting of poloxamine, but is not limited thereto.

The anionic surfactants are potassium laurate, triethanolamine stearate, sodium lauryl sulfate, sodium dodecyl sulfate, alkyl polyoxyethylene sulfate, sodium alginate, dioctyl sodium sulfosuccinate, phosphatidyl glycerol, phosphatidyl inositol , Phosphatidylserine, phosphatidic acid or salt thereof, glyceryl ester, sodium carboxymethylcellulose, bile acid or salt thereof, cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glycodeoxycholic acid, alkyl sulfonate, aryl sulfonate , Alkyl phosphate, alkyl phosphonate, stearic acid or salt thereof, calcium stearate, phosphate, carboxymethylcellulose sodium, dioctyl sulfosuccinate, dialkyl ester of sodium sulfosuccinic acid, phospholipids and calcium carboxymethylcellulose It may be one or more selected from, but is not limited thereto.

The nonionic surfactant is triton, SPAN 60, polyoxyethylene fatty alcohol ether, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene castor oil derivative, sor Non-ester, glyceryl ester, glycerol monostearate, polyethylene glycol, polypropylene glycol, polypropylene glycol ester, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, arylalkylpolyether alcohol, polyoxyethylenepolyoxypropylene copolymer , Poloxamer, poloxamine, methylcellulose, hydroxycellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, amorphous cellulose, polysaccharide, starch, Starch derivatives, hydroxyethyl starch, polyvinyl alcohol, triethanolamine stearate, amine oxide, dextran, glycerol, gum acacia, cholesterol, tragacanth, and polyvinylpyrrolidone, can be one or more selected from the group consisting of There may be, in detail, Triton-based, but is not limited thereto.

The drug may be an anticancer agent, and the types of the anticancer agent include, for example, doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actino Mycin-D (actinomycin-D), docetaxel (docetaxel), etoposide (etoposide), teniposide (teniposide), bisantrene (bisantrene), imatinib (imatinib), cisplatin (cisplatin), 5-fluorouracil (5- fluorouracil), adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, small molecule drugs, nitro Gen mustard (nitrogen mustard) and nitrosourea (nitrosourea) may include one or more selected from the group consisting of, but is not limited thereto.

The drug may be dispersed in a dispersion, and the dispersion may be as long as it is not harmful to the human body, and physiological saline, phosphate buffered saline (PBS), bovine serum albumin, fetal bovine serum, FBS), dichloromethane, dimethyl sulfoxide, ethanol, or the like may be used, but is not limited thereto.

The drug may be supplied at 0.1 mg to 3 mg per 1 mL of solvent, for example, 0.3 mg to 2.5 mg, 0.6 mg to 2 mg, or 0.9 mg to 1.5 mg, but is not limited thereto, and disease Depending on the patient's condition, it may be appropriately supplied within the above-described range.

The metal nanoparticles may include a metal having a work function of 2.0 eV or more, for example, 3.0 eV or more, 4.0 eV or more, 4.6 eV or more, or 5.0 eV or more, but are not particularly limited. By controlling the work function of the metal nanoparticles in the above-described range, the light having a photon energy greater than or equal to the work function of the metal nanoparticles, for example, ultraviolet rays, etc. Electrons on the metal surface can be released, and electric charges on the metal surface can be induced into positive charges. In addition, upon irradiation with ultraviolet light having a short wavelength of 200 nm or less, the anion of the group in which the surfactant and the drug are bound electrochemically binds with the positively charged metal nanoparticles, and thus, electrostatically ionized metal nanoparticles The nanoparticles of the present application having a structure in which an organic group is attached to the particles can be formed through a simple process such as irradiation with ultraviolet rays. In the present application, the term "electrochemical bond" means a covalent bond, an ionic bond, or a bond by physical adsorption.

The metal nanoparticles may be, for example, one or more selected from the group consisting of gold, silver, iron, platinum, copper, zinc, tin, ruthenium, rhodium, gadolinium, dysprosium, manganese, titanium, palladium, and chromium, and specifically It may be gold, but is not limited thereto.

The nanostructure formation step may be performed in the light irradiation unit 14 of the above-described nanocomposite manufacturing apparatus, and in one example, the nanostructure formation step may be performed by irradiating light onto the droplet formed in the droplet formation step. The metal nanoparticles and organic materials may be rearranged to form a nanostructure.

The nanocomposite generation step may be performed in the solvent removal unit 15 of the above-described nanocomposite manufacturing apparatus, and in one example, the nanocomposite generation step includes a solvent from the nanostructure formed in the nanostructure formation step. Can be removed to form a nanocomposite.

In one example, the manufacturing method of the nanocomposite of the present application may further include a nanocomposite collecting step. The nanocomposite collecting step may be performed in the collection unit 16 of the above-described nanocomposite manufacturing apparatus, and in one example, the nanocomposite collecting step is, after the step of generating the nanocomposite, the generated nanocomposite The nanocomposite can be collected by applying an electric field of opposite polarity to and

In one example, the method of manufacturing the nanocomposite of the present application may further include dispersing the collected nanocomposite in a dispersion. The step of dispersing the collected nanocomposites may be performed in a dispersing device of the above-described nanocomposite manufacturing apparatus, and the dispersion is possible as long as it is not harmful to the human body, physiological saline, phosphate buffered saline (PBS), Serum albumin (bovine serum albumin), fetal bovine serum (FBS), dichloromethane, dimethyl sulfoxide or ethanol may be used, but is not limited thereto.

The present application also relates to nanocomposites. The nanocomposite of the present application may be performed using the above-described nanocomposite manufacturing apparatus and nanocomposite manufacturing method, and accordingly, contents overlapping with the contents described in the nanocomposite manufacturing apparatus and nanocomposite manufacturing method are I will omit it.

According to the exemplary nanocomposite of the present application, an organic material having a plurality of metal nanoparticles and an electron donating group may be included.

The organic material having the electron donating group or negatively charged may be a surfactant, a lipid, a drug, or a mixture thereof, and the organic material may be electrostatically arranged between metal nanoparticles, but is not limited thereto. .

In the nanocomposite, which is another embodiment of the present application, contents overlapping with those described in the above-described nanocomposite manufacturing apparatus, nanocomposite manufacturing method, and nanocomposite will be omitted.

According to the nanocomposite, which is another embodiment of the present application, a plurality of gold (Au) nanoparticles, a surfactant having an electron donating group or negatively charged, and a drug may be included, and an interface having the electron donating group The active agent and the drug may be electrostatically arranged between the metal nanoparticles, but are not limited thereto.

In the nanocomposite of the present application, the term "nano" may mean a size in a nanometer (nm) unit, for example, it may mean a size of 1 to 1,000 nm, but is not limited thereto. In addition, in the present specification, the term "nanoparticle" may mean a particle having an average particle diameter in a nanometer (nm) unit, and for example, it may mean a particle having an average particle diameter of 20 to 200 nm. It is not limited.

The nanocomposite may have a d-space value, and in the present application, the term “d-space” may mean a gap between crystal planes of an additive nanocomposite. The D-space may be measured by a high-resolution transmission electron microscope (HR-TEM), and the D-space value may be 0.20 to 0.50 nm, 0.25 to 0.40 nm, or 0.27 to 0.29 nm, but is not limited thereto. When the nanostructure is formed by irradiating light to droplets formed from the spraying unit in the light irradiation unit of the above-described nanocomposite manufacturing apparatus, the D-space value of the nanocomposite is greater than the D-space value of the metal nanoparticles. Rearrangements can occur in increasing directions.

When the nanocomposite is measured by UV-vis spectroscopy, the nanocomposite may have a broadband absorption peak, and the metal nanoparticles and the metal nanoparticles and the The surfactant may react and exhibit a broadband absorption peak higher than that of the surfactant alone. The broadband absorption peak may have a wavelength of 350 to 1,000 nm, but is not limited thereto.

The concentration (content) of the drug in the above-described nanocomposite is not particularly limited, and may be, for example, 1 to 50 w/w%. If the concentration of the drug is less than 1 w/w%, the drug supply may not be sufficient, and if it exceeds 50 w/w%, a high concentration supply that may cause side effects may be obtained.

Hereinafter, the above-described contents will be described in more detail through Examples and Comparative Examples, but the scope of the present application is not limited by the contents presented below.

Example 1

A gold nanomaterial was manufactured using a pair of electrode rods made of gold. Specific conditions for manufacturing the gold nanomaterial are as follows. Air was injected into the discharge unit provided with a pair of electrode rods made of gold (Au-172561). The flow rate of the air was controlled to 0.9 L/min through a mass flow meter (MFC; Tylan, USA). The length of the electrode rod was 100 mm, the diameter was 8 mm, and the electrode spacing was 1 mm. Gold lumps (Au agglomerates, Au AG) were prepared by applying a voltage of 3.4 kV (resistance 0.5 MΩ, electric capacity 4.7 nF, current 2.8 mA, and frequency 580 Hz) to the electrode.

A solution containing Triton X-100 (T) and doxorubicin (DOX, D) was injected into a Collison nebulizer, and the solution was continuously delivered using nitrogen gas (99.999% purity; 3 L/min). do. The hybrid droplets from the nebulizer were irradiated with light at a wavelength of 185 nm for 7.8 seconds (E = 6.2 eV, I = 0.14 J m-2 s-1; 3SC-9-A0, UVP, UK) and the primary gold particles in Au AG ( Electrons were emitted from the work function, 5.1 eV). The positively charged Au surface is electrostatically conjugated with negatively charged groups in TD to induce reassembly of Au AG, which can form TAuD nanocomposites.

The hybrid water droplets were extracted by passing through a denuder in which silica gel and pellet-type activated carbon were mixed in a mass ratio of 5:5. The nanocomposite was charged with gaseous cations in an electric field forming configuration (pin (+4 kV)-ring (ground)), and then collected using an aluminum rod polished under an electric field (-2.7 kV/cm) through electrostatic attraction. .

The aluminum collection rod was sonicated at 40 kHz for 5 minutes, immersed in phosphate buffered saline (PBS, pH 7.4), and the nanocomposite was released from the rod to form a nanocomposite dispersion used for bioanalysis.

Comparative Example 1

It was prepared in the same manner as in Example 1 except that doxorubicin was not bound.

Comparative Example 2

It was prepared in the same manner as in Example 1, except that Triton X-100 was not combined.

Comparative Example 3

It was prepared in the same manner as in Example 1 except that Au AG was not combined.

Experimental Example 1-Analysis of particle properties of nanocomposite

5 is a diagram schematically illustrating an exemplary apparatus for manufacturing a nanocomposite, a method for manufacturing a nanocomposite, and a nanocomposite manufactured using the apparatus of the present application. Hybrid water droplets were formed through a flowing drop (FD) reaction using a scanning mobility particle sizer (SMPS, 3936, TSI, USA) prior to solvent extraction from the denuder, using Au AGs, TD water droplets, and nanocomposites (hybrid). The size distribution of water droplets) was measured. The size distribution of the prepared TAuD nanocomposite, Au AG and TD was determined using SMPS (3936, TSI, USA) to confirm the quantitative interaction between Au AG and TD. Sampling for SMPS measurement and flow rates of the exterior were 0.1 and 1.0 L/min, respectively, and the scan time was 135 seconds. Dynamic light scattering (DLS; Nano-ZS, Malvern Instruments, UK) was used to measure the size distribution of the TAuD nanocomposite dispersed in PBS.

Au AG was formed by Brownian motion (thermal collision behavior) of primary Au particles in the state of flowing compressed nitrogen gas. The TD droplets produced an ethanol TD solution by the Collison spray apparatus by injecting another compressed nitrogen gas. Hybrid droplets were prepared by injecting an Au AG-containing gas stream directly into the Collison atomizing apparatus as a working fluid, where the AG is incorporated into the TD solution to be sprayed as a hybrid droplet. Hybrid droplets were directed to a tubular reactor under 185 nm UV irradiation.

6 is a size distribution diagram of Au AG (spark elution of Au rods), TD droplets (TD dissolved in ethanol) and hybrid droplets (TD droplets including Au AG) under UV irradiation at 185 nm before solvent extraction through a diffusion dryer. . A summary of the distribution is shown in Table 1 below.

The size distribution of AG, TD and hybrid droplets measured using MPS is shown in FIG. 6. Results were expressed as geometric mean diameter (GMD), geometric standard deviation (GSD) and total number concentration (TNC). In the case of AG and TD, there was a significant difference, but the size of the hybrid droplets also showed a single shape (located between the distributions of AG and TD) similar to that of each AG and TD. This result means that almost all of the particles were contained within the droplets during the spraying process. Thus, this product was suitable for use in the subsequent FD reaction. Interestingly, the GSD of the hybrid droplets was significantly lower than that of the TD droplets, which can be caused by reassembly with the TD component (intervening and subsequent conjugation) AG under light irradiation, including fragments of AG during cleavage. This result suggests that the photophysicochemical recombination of Au AG is an appropriate route to fabricate functionalized Au nanostructures.

Au Ag TD droplet Hybrid droplet GMD 45.2 153.2 80.1 GSD 1.49 1.92 1.57 TNC 4.4x10 ⁶ 8.2x10 ⁶ 6.7x10 ⁶

7 is a low and high magnification TEM images of (a) Au AGs and (b) Example 1 (TAuD) after passing through a diffusion dryer. The images on the right of the two samples contain the interplanar distance and plausible microsurface structures (yellow, gold, blue, T and red, D). Au AG consists of dark spots (primary Au particles, ~6 nm), TAuD nanocomposites consist of a control element, dark spots (reassembled primary Au particles, ~4 nm) and a brighter layer (labeled TD). . In the case of TAuD, the position of the dot is confined in a brighter layer in the form of a nanocomposite.

The reassembly was also verified by transmission electron microscopy (TEM, CM-100, FEI / Philips, USA) measurements. The prepared nanocomposite was captured in a gas single-pass configuration without direct pretreatment in a carbon-coated copper grid (Tedpella, USA) through TEM grid capture (Ineris, France). The grid was transferred to a holder for TEM analysis (CM-100, FEI/Philips, USA) at increasing voltages ranging from 46-180 kV.

As shown in Fig. 7A, the particles resulting from spark exfoliation are AGs of primary Au particles (about 6 nm in side dimension) caused by Brownian motion during suspension in gas flow. Image analysis of 400 AG shows that the average size of only one isolated AG is about 48.8 nm, which is consistent with the size obtained from SMPS measurements. The primary particles are spherical, have a smooth surface, and have clear voids between the particles, which means that the AG contains only Au particles. The high magnification TEM showed a d spacing of 0.233 nm for the adjacent lattice corresponding to the Miller (111) face-centered cubic plane. Thus, this result further supports the fact that AG is derived from pure primary Au particles. As shown in Fig. 7b, in AG photoionization using TD (after solvent extraction in Denuder) the particle morphology was remarkably changed, resulting in dark spots tightly surrounded by a brighter contrast layer. The dark and light components correspond to Au particles and TD, respectively, and the dense structure within different materials (i.e., vesicle structures) is due to the electrostatic interaction between the positively charged Au surface and the negatively charged functional groups of the TD during light irradiation. Can be caused by. Image analysis of the 400 nanocomposite shows that the average lateral dimension of the isolated TAuD nanocomposite is 88.2 nm, which is consistent with the SMPS measurement.

FIG. 8 is a graph of a UV-vis spectrum including (a) particle size distribution, zeta potential, and (b) TD of Example 1 (TAuD) nanocomposite dispersed in PBS using a dynamic light scattering (DLS) method.

In order to measure the hydrodynamic size distribution of the nanocomposite, the collected nanocomposite was dispersed in phosphate buffered saline (PBS) of 0.01M, pH 7.4, and then a dynamic light scattering (DLS, Nano-ZS, Malvern Instruments, UK) method was used. . The average size was found to be 138 nm (Figure 8A), which is larger than that obtained from the SMPS measurement. The size difference between these two methods may have been due to the significantly high NV concentration used for DLS measurements. Nevertheless, the average hydrodynamic size was ≦200 nm, probably due to the overall positive potential (+7.8 mV). Thus, the produced NV is suitable for improved permeability and passive accumulation into tumor tissues through wound and subsequent internalization through non-specific adsorbent endocytosis. Analysis of the high-magnification image revealed particles of various sizes (about 4 nm) and d-spacing (0.288 nm), and the result was that Au on the surface of Au particles by other elements of TD during light irradiation recombining AG into TAuD NVs. Coincides with atomic substitution. UV-vis (T60, PG Instruments, UK) spectrum of the processed NV (Fig. 8b) dispersed in PBS (Fig. 8b, inset) showed a small peak around 550 nm, which can be attributed to the LSPR effect of Au NP (absorption Wavelength, 520-530 nm). TD shows an absorption peak only at about 470 nm, whereas NV shows a high broadband absorption spectrum after Au AG reacts with TD under light irradiation. This result may be caused by rearrangement of the primary Au particles in the hybrid droplets, thereby promoting plasmon bonding between particles of adjacent Au particles. This result also pointed out the feasibility of broadband NIR-induced photo-conversion activity in theranostic applications.

9 is a TEM image of a reassembled Comparative Example 1 (Tau) containing another T solution. The concentration of the T solution is 5.35 (0.5 vT/v·ethanol%), 10.70 (1.0 v/v%), 26.75 (2.5 v/v%) and 53.50 (5.0 v/v%) mg·T/mL· ethanol . Increasing the T concentration creates a nanocomposite that assumes a more detailed composition.

As shown in Figure 9, a T concentration of 2.5 v/v% (in ethanol) to avoid unwanted increases in size and cytotoxicity to prepare TAu and to prevent the formation of loose nano vesicle structures in undefined AGs. I chose.

Figure 10 is (a) XPS Au spectrum of Example 1 (TAuD), Au and Comparative Example 1 (TAu), (b) Example 1 (TAuD), Comparative Example 1 (TAu) and Comparative Example 2 (FT of AuD -IR) spectrum, (c) Raman spectrum graphs of Example 1 (TAuD), Comparative Example 3 (TD) and Au AG. The inset shows the optical microscope specimen for measurement on a glass disk.

The surface chemistry of the nanocomposite is determined by Fourier transform infrared (FT) in absorbance mode (1450-2300 cm ^{- 1} ) after depositing the nanocomposite on a polytetrafluoroethylene (PTFE) substrate (11807-47-N, Sartorius, Germany). -IR, iS-10, Thermo Electron, USA) analysis was used to evaluate. The difference in surface structure between Au AG and TAuD was confirmed using XPS (Axis-HIS, Kratos Analytical, Japan). The integration of TD on the gold surface was evaluated using Raman spectroscopy (T64000, HORIBA Jobin Yvon, Japan). The light absorption spectrum of the nanocomposite dispersed with PBS and TD alone was measured using UV-vis spectroscopy (T60, PG Instruments, UK).

The surface chemistry of the prepared nanocomposite was analyzed to confirm the bonding induced by light irradiation between Au and TD. As shown in Fig. 10(a), the results of Au AG and TAuD measured using Au 4f X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Scientific, USA). AG has two distinct peaks at 89.2 and 85.5 eV, Au 4f _{5 /2} and Au 4f _{7 /2} (separated by 3.7 eV in spin-orbit splitting), respectively. This peak coincides with the peak of Au ⁰ . Similar data for the TAuD sample were separated by 86.6 and 83.2 eV (3.4 eV) with negative shifts of 2.6 and 2.3 eV for Au 4f _{5 /2} and Au 4f _{7 /2} , respectively, due to charge transfer between Au and TD. And Au ^{3 +} ), which means that the light irradiation results in electrostatic conjugation between Au and TD.

As shown in FIG. 10(b), in the Fourier transform infrared (FTIR, iS-10, Thermo Electron, USA) analysis, the junction between Au and TD was further verified by comparing TAu and AuD with TAuD. The TAu spectrum showed a characteristic band at 1512 cm ^-1 corresponding to the aromatic CC stretching of T in TAu and the coupling of the CH-modification mode. In addition, this band exists in the TAuD spectrum but not in the AuD spectrum. In AuD and TAuD, a broadband of about 1610cm ^-1 can be imparted to the bending of the NH group in DOX, indicating that TD was successfully bonded to the Au surface by light-induced electrostatic bonding.

As shown in FIG. 10(c), the Raman spectrum of TAuD showed a sharp peak that was not present in the Au AG and TD spectrum at 1648 cm ^-1 . This result means that the Raman signal of D (corresponding to the hydrogen bonding of the C = O group of ring B) is increased by Au photoionization in TAuD. Such surface chemistry analysis can provide a feasible scientific possibility based on photo-ionization for reconstitution, recombination and functionalization of Au particles under ambient conditions, i.e. simplicity, environmentally friendly and gas flow in a continuous manufacturing platform.

Figure 11 is from Example 1 (TAuD) (a) pH triggering (5.5 and 7.4) (24 hours) and (b) near-infrared (NIR) triggering (6 hours and 808 nm) (irradiation for 5 minutes after 2 hours) drug (DOX , D) is a graph of the in vitro release profile.

The release of D in vitro of the TAuD nanocomposite was evaluated in ABS (pH 5.5, 0.14 M NaCl) and PBS (pH 7.4, 0.14 M NaCl). TAuD dispersion sample (1 mL, 1 mg/mL) (equivalent to 0.3 mg D) (MWCO, 4000-6000 Da) (Spectra/Por®, USA) was placed in a dialysis bag and 20 mL of ABS (or PBS) was added. It was continuously immersed in the contained 50 mL tube and placed in a water bath shaker (HST-205 SW, Hanbaek ST Co., Korea) operating at 37° C. with 100 strokes per minute. Aliquots of release medium (0.5 mL) were sampled at predetermined time intervals and then supplemented with an equal volume of fresh medium. The concentration of D in the medium was measured using UV-vis spectrophotometry (U-2800, PerkinElmer, USA). In order to investigate D emission under laser irradiation, 1 mL of TAuD was placed in a vial equipped with a stirrer (400 RPM), diluted to 3 mL with PBS, and then irradiated with a laser for 5 minutes. Aliquots (0.5 mL) were taken at regular time intervals and analyzed to determine the D concentration.

As shown in Fig. 11(a), since the multifunctional application of the nanocomposite processed to cancer tissue by chemo-photothermal therapy was selected, the drug release properties of the nanocomposite were varied in various buffer solutions (PBS, pH 7.4; acetate-buffered Brine (ABS), pH 5.5) and exposed to different near infrared wavelengths (808 and 632 nm). The release of D from the nanocomposite was based on the leakage of 50% in PBS at pH 7.4 and 70% in ABS at pH 5.5. Fraction) was found to be pH dependent. It is believed that higher water solubility of D (pKa ~ 8.3) at pH 5.5 contributed to the faster release. This result is highly related to the acidic environment of the endo-lysosomal compartment (pH 4.5-5.0) in which the nanocomposite is embedded in cancer cells.

As shown in FIG. 11(b), the generation of heat by near-infrared light indicates that the emission of D increases rapidly when exposed to the nanocomposite for 5 minutes to NIR (4W/cm ² ), and the cumulative D emission is from 16% to about 60%. It was confirmed that it increased to.

12 shows the intensity-dependence of (a) concentration dependence (2.5 W/cm ² ) and (b) Comparative Example 1 (TAu) and RPMI (control) for 6 minutes according to 808 nm near-infrared (NIR) irradiation (150 μg/ mL) temperature rise profile. The temperature profile for each condition was included, and in response to irradiation, the temperature near the laser beam spot increased significantly.

13 shows the intensity-dependence of (a) concentration dependence (2.5 W/cm ² ) and (b) Comparative Example 1 (TAu) and RPMI (control) for 6 minutes according to 632 nm near-infrared (NIR) irradiation (150 μg/ mL) temperature rise profile. The temperature profile for each condition was included, and in response to irradiation, the temperature near the laser beam spot increased significantly.

As shown in Figs. 12 and 13, the increase in light heat temperature during near-infrared irradiation was investigated. When the near-infrared rays were irradiated at 808 and 632 nm wavelengths, where there was no significant difference between the wavelengths, heat generation was found to be dependent on the concentration and irradiation intensity.

The results shown in FIGS. 11 to 13 mean that the nanocomposite fabricated in the present application can be used in a photo-triggered controlled release application.

14 shows Comparative Examples 1 (TAu) and Example 1 (TAuD) in the absence and presence of near-infrared (NIR) irradiation including a single D in (a) MDA-MB-231 and (b) MCF-7 cells. In vitro anticancer effect graph, (c) CLSM image of Comparative Example 1 (TAu) loaded on coumarin-6 in MDA-MB-231 and MCF cells, (d) MDA-MB-231 and (e) MCF- 7 FACS analysis results showing cellular uptake of Example 1 (TAuD) by cells, (f) Western blot analysis results of p53 in cells treated with Comparative Example 1 (TAu) with or without NIR (808 nm) irradiation to be.

As shown in Figure 14 (a) and (b), the anticancer effect in vitro is 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After culturing for 24 hours, cytotoxicity was measured and evaluated in MDA-MB-231 (FIG. 14(a)) and MCF-7 (FIG. 14(b)) cells.

After 48 hours of incubation, cytotoxicity measurements for single D, TAu and TAuD samples were measured in MDA-MB-231 and MCF-7 cells (ie, human breast adenocarcinoma) using MTT analysis with or without NIR irradiation. That is, 1 × 10 ⁴ cells per well were coated (sprayed) on a 96-well microtiter plate (Becton Dickinson Labware, USA), and cultured for 12 hours for cell adhesion. After 48 hours, the cells were washed, and 100 μL MTT solution (1.25 mg/mL) was added to each well. During incubation in the dark for 4 hours, the living cells produced purple formazan crystals, a metabolite of MTT. The crystals were dissolved in 100 μl dimethyl sulfoxide (DMSO), and absorbance was measured at 570 nm using a microplate reader (Multiskan EX, Thermo Scientific, USA). Cell viability was calculated as A _sample / A _control × 100%, where A is the absorbance at 570 nm.

The survival rate of cells treated with TAu was over 75% in both cell types, and TAu before D binding showed biocompatibility. T is known as a membrane-lytic agent, but is present in a glue concentration (<0.01 w/w%). TD incorporation into Au under light irradiation and subsequent solvent extraction was limited by the thermally stable amorphous T in the nanometer space. Upon TAuD administration, the IC50 values of MDA-MB-231 and MCF-7 cells were 0.24 and 0.27 μg/mL, respectively. This value was significantly lower than that of single D, which resulted in TAuD increasing cellular uptake and maintenance of D (MDA-MB-231: 0.48 μg/mL, MCF-7: 0.77 μg/mL), and replacement of D Means to increase the transmitted carrier-mediated outflow. When the cells treated with TAuD were irradiated with near-infrared rays, IC50 values were further reduced in both cell types (MDA-MB-231: 0.13 μg/mL, MCF-7: 0.15 μg/mL). This result can be inferred due to high heat-induced rupture drug release, chemical sensitization, and thermal damage during irradiation.

14(c) to 14(e), cell adsorption of TAu was performed by confocal laser scanning microscopy (CLSM, Leica Microsystems, Germany) (FIG. 14(c)) and fluorescently active cell selection (FACS, BD Biosciences, USA) (Fig. 14(d) and Fig. 14(e)).

Internalization of the nanocomposite prepared by the cells was observed using CLSM (TCS SP2, Leica Microsystems, Germany). MDA-MB-231 and MCF-7 cells in 2 mL medium were seeded on coverslips in 12-well plates at a density of 5 x 10 ⁴ cells/mL. The cells were cultured for 24 hours to attach the cells, and then TAu nanocomposites loaded in 5 μg/mL coumarin-6 and 100 ng LysoTracker Red were added to each well. After incubation for 10 minutes and subsequent removal of the medium, the cover slip was gently washed with PBS, fixed with 4% paraformaldehyde solution in the dark, placed on a glass slide, and sealed with glycerin. To confirm intracellular uptake, MDA-MB-231 or MCF-7 cells (1×10 ⁵ ) in 2 mL medium were seeded in 12-well plates. After incubation for 12 hours, TAu nanocomposites loaded on coumarin-6 with TAuD were cultured for a specific time. Cells were then washed with PBS and harvested by trypsinization and resuspended in 1 mL PBS containing binding buffer for flow cytometry using a FACS flow cytometer (BD FACS Verse, BD Biosciences, USA). Automatic fluorescence of untreated cells was used as an internal control.

The green fluorescent signal of TAu bound to coumarin-6 is isotopically atomized with a red lysosomal dye indicating lysosomal absorption through endocytosis. The red fluorescence of D was not used because of its relatively weak fluorescence, but fluorescence may occur by quenching due to interaction and/or aggregation between Au primary particles in the presence of T. The D of intracellular accumulation was confirmed by FACS, indicating that uptake was time dependent.

As shown in FIG. 14(f), Western blot analysis was used to investigate cell death through necrosis induced by NIR irradiation, and expression of the cell death marker p53 was measured.

Western blot analysis was performed to investigate the expression of apoptosis marker p53. MDA-MB-231 or MCF-7 cells (4 × 10 ⁵ cells) were cultured for 12 hours and treated with TAu for 24 hours. The cells were then harvested, proteins were extracted from the lysed cells and quantified using a bovine serum albumin protein assay kit. Proteins were separated on 10% polyacrylamide and transferred by electrophoresis to a PVDF membrane (Millipore, USA). After blocking the membrane with 5% skim milk in Tris buffered saline containing 1% Tween 20 (TBST; tris buffered saline with Tween), the membrane was then blocked with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and p53 (Santa Cruz Biotechnology, Inc., for 12 hours). USA) and incubated at 4°C with the primary antibody. The membrane was washed with TBST and incubated with secondary antibody for 1 hour. Proteins were detected using an improved chemiluminescent agent in a luminescence image analyzer (LAS-4000 mini, Fujifilm, Japan).

These results indicate that no serious necrosis occurs in TAu-treated cells exposed to NIR because cell death by photothermal effects is more immediate than in conventional chemotherapy or radiation therapy. Therefore, the nanocomposite prepared in the present application is an effective anticancer chemo-photothermal than coagulation necrosis controlled by adjusting the dosage and composition of NIR irradiation (intensity and duration) and light inducers (eg, nanocomposite). Therapy can be provided and the inflammatory response can be minimized.

15 shows MCF-7 after treatment with (i) control, (ii) single D group, (iii) Comparative Example 1 (TAu) + near-infrared (NIR) group and (iv) Example 1 (TAuD) + cell group. Top) and MDA-MB-231 (bottom) are FACS results to determine cell death.

To compare fractions containing pre-apoptosis, apoptosis, necrosis and living cells, 2 mL media containing MDA-MB-231 or MCF-7 cells (2×10 ⁵ ) were placed in a 12-well plate and 12 h Oriented. TAuD nanocomposite and single D were added with or without near-infrared irradiation. After 48 hours, cells were harvested by scraping, washed with PBS, and mixed with binding buffer. PE-annexin-V and 7-amino actinomycin D were added at 2 μL each, mixed gently, and left for 10 minutes in a dark place. The treated cells were finally diluted with binding buffer to a final volume of 1 mL, and cell death was analyzed using a FACS flow cytometer (BD FACS Verse, BD Bioscience, USA).

As shown in Fig. 15, it was confirmed by measuring the fraction of cells treated with TAuD (with or without NIR irradiation) at different cell death stages. After incubation with TAuD, it was confirmed that the proportion of cells in the early and late apoptosis stages was significantly greater than that of cells treated with a single D. It was confirmed that this ratio increased further with near-infrared irradiation in both cell types.

16 is an inverse fluorescence microscopy image in the survival/death analysis. The displayed image shows live cells (green, stained with calcein-AM) and dead cells (stained with EthD-1) for 5 minutes after NIR irradiation (4 W/cm ² ). The dotted line separates the irradiated area.

MDA-MB-231 or MCF-7 cells (3 x 10 ⁵ in 2 mL) were spread on 12-well dishes and cultured overnight for cell attachment. After addition of 0.1 mg TAu, the cells were incubated for 3 hours. After washing, the plate was placed at 14 cm below the laser focus (spot size, 2 mm) and irradiated for 4 W/cm ² and 3 minutes. After removing the PBS, the cells were supplemented with fresh medium and cultured for 3 hours. Finally, cells were stained with 2 μm of calcein-AM (viable cells, green fluorescence) and EthD-1 (dead cells, red fluorescence) in PBS and observed using an inverted fluorescence microscope (Eclipse Ti, Nikon, Japan). .

A survival/death analysis was performed in MDA-MB-231 cells treated with TAu exposed to NIR irradiation (beam diameter of about 2 mm, 4W/cm ² ). As shown in Figure 16, the light-exposed region showed apoptosis in the cells treated with TAu, but cytotoxicity was not observed in the control without NIR exposure (NIR alone) or in the cells treated with TAu, and photothermal cells It means that death has site specificity.

17 is an optical microscope image measuring cell cycle analysis. Cell clock labeling in MDA-MB-231 cells was performed after incubation with (a) control, (b) single D, (c) Example 1 (TAuD) for 24 hours.

The major cell cycle stages of MDA-MB-231 cells after treatment with a single D and TAuD nanocomposite were analyzed using a cell clock assay (Biocolor Ltd., UK). Cells were seeded on 12-well cell culture plates (2×10 ⁵ cells/well) and cultured overnight to allow cell adhesion. The cells were then treated with 0.1 μg/mL single D or TAuD (equal concentration) for 24 hours. Cells were washed, supplemented with fresh medium, and 150 μL cell clock dye reagent was added. After 1 hour, the cells were washed and images were obtained using an optical microscope (Eclipse Ti, Nikon, Japan). Cells in the G2/M, S and G0/G1 stages showed color changes in dark blue, green and yellow, respectively. The color pixels of each step were quantified by micrograph using ImageJ software.

Cell cycle analysis of MDA-MB-231 cells after single D and TAuD treatment was performed using a redox dye. As shown in Figure 17, most of the control cells were in the S and G0/G1 stages, and a single D treatment induced G2/M growth arrest in 53% of the cells. 67% of TAuD-treated cells reached the G2/M checkpoint. These observations may lead to increased TAuD internalization in endo-lysosomes and D release into the nucleus with a decrease in D outflow through cell membrane transporters.

18 is an in vivo anti-tumor evaluation, MDA-MB- with xenograft mice for Comparative Example 1 (TAu) and Example 1 (TAuD) according to the presence or absence of near-infrared (NIR) irradiation including single D (A) tumor growth and (b) weight-change profile graph of 231 and (c) major organs (liver (i), lung (ii), heart (iii), spleen) collected from euthanized mice for 24 hours after injection. (iv) and kidney (v)] and in vitro fluorescence images of Cy5.5 alone (middle) and Comparative Example 1 (TAu) (right) combined with Cy5.5 against tumors (vi).

MDA-MB-231 xenograft tumors were generated in 6 week old female BALB/c nude mice by subcutaneous injection of 1×10 ⁷ MDA-MB-231 cells mixed with Corning Matrigel matrix. When the tumor reached a volume of approximately 100 mm ³ , mice were divided into 5 groups of 4 to 6 mice. Four of these groups received intravenous administration of free D (saline), TAu (near-infrared irradiation), or TAuD (with or without near-infrared irradiation). Samples were administered at a dose corresponding to 5 mg drug per kg body weight. Mice were exposed to NIR through the skin 24 hours after injection at an intensity of 3 W/cm ² for 3 minutes. The size of the tumor was measured with a digital caliper, and the volume of the tumor was calculated as volume = 1/2×(longest dimension)×(shortest dimension). All animal handling procedures were performed in accordance with procedures approved by the institutional animal ethics committee of Yeungnam University.

As shown in Fig. 18(a), the antitumor efficacy of TAu and TAuD by NIR irradiation was investigated in vivo using female BALB/c nude mice carrying MDA-MB-231. Compared to the untreated control group, mice treated with single D or TAu or TAuD and irradiated with NIR significantly reduced tumor volume at the end of the experimental period. Mice treated with TAu and irradiated with NIR also showed significant tumor regression due to hyperthermia induction.

As shown in FIG. 18(b), the weight change profile did not cause a significant change in the weight of mice until the end of the treatment with TAuD, but single treatment D induced weight loss from day 7 of treatment. This means that TAuD treatment does not induce significant systemic cytotoxicity with respect to D.

As shown in FIG. 18(c), the biodistribution of TAu 24 hours after injection was evaluated using in vitro fluorescence imaging (FOBI, NeoScience Co., Ltd, Korea).

The in vivo distribution of TAu in MDA-MB-231 xenograft BALB/c mice was investigated using a fluorescence in vivo imaging system (FOBITM, Neoscience, Korea). The xenograft mice were injected intravenously with TAu conjugated to Cy5.5. After 24 hours, the mice were sacrificed by CO ₂ asphyxiation. Tumors and major organs were excised and scanned using FOBITM in the red channel. Fluorescence intensity was determined using NEOimage software.

TAu was found to accumulate in tumor, liver, lung and kidney, but accumulation in heart and spleen was insignificant. The average fluorescence intensity of TAu bound to Cy5.5 in tumors was doubled in the liver, indicating preferential and passive accumulation of TAu in tumor tissues due to increased transmission and retention by TAu size distribution.

FIG. 19 is an image of in vivo photothermal imaging during near-infrared (NIR) irradiation on a tumor of a mouse pretreated with physiological saline and Example 1 (TAuD).

When TAuD was administered, in vivo photothermal imaging was performed using a digital thermal imaging camera (Therm-App TH, Israel). Briefly, mice were treated with saline or TAuD. After 24 hours, the tumors were exposed to NIR irradiation (4.0 W/cm ² ) for up to 5 minutes. Then digital imaging was performed to determine the photothermal effect of the treatment.

As shown in Fig. 19, in vivo imaging was performed to investigate the photothermal effect of TAuD treatment in xenograft mice with MDA-MB-231. 24 hours after injection through the tail vein, the tumor was exposed to NIR irradiation (4.0 W/cm ₂ ) for up to 5 minutes. There was no significant increase in heat in the rats treated with physiological saline. Mice treated with TAuD showed significant heat rise from 34.8 °C to 43.4 °C in the focal area. No apparent temperature changes were observed in areas of the body that were not exposed to NIR. Near-infrared light can penetrate only to a depth of several millimeters in an organism, but exhibits a clear anticancer effect due to effective targeting of tumors by TAuD.

Figure 20 is another combination of using the lipid molecule LAu instead of T to prepare different types of nanocomposites, (a) low and high magnification TEM images of LAu from a drop drop (FD) reaction, (b) photothermal activity of LAu From LAuD to confirm the near-infrared (NIR) (632 and 808 nm, 5 min)-trigger D emission profile and (c) removed DNA and pDNA bound to LAuG compared to commercially available lipofectamine ( EGFP) is a graph of gene transfer performance. Insertion shows fluorescence images of HeLa cells cultured for 24 and 48 hours with LAuG.

HeLa cells were seeded in 24-well plates at a density of 1×10 ⁵ cells/well. The cells were treated with a complex solution containing 2 mg pDNA (NV to pDNA, 10: 1, w/w) at 37° C. for 4 hours. Luciferase activity was measured with a luminometer (TD-20/20, Promega, US). The final luciferase activity was expressed as RLU/mg of the protein. Green fluorescent protein expression was observed in cells using an inverted fluorescence microscope (DMI 4000 B, Leica Microsystems, Germany).

Other nanocomposites including lipid (L-1118, Echelon Biosciences, USA) molecule-bound Au (LAuD) and gene complex LAu (LAuG) were prepared through the FD reaction. As shown in Fig. 20(a), it shows the shape of the LAu and shows that the dark part (Au) is trapped within the brighter contrast material (L), and the FD reaction can be performed without modifying the complex system. It indicates that it can be extended to produce composites.

As shown in Fig. 20(b), the emission of NIR trigger D was tested for LAuD, and the results are similar to those of TAuD. The gene transfer performance of LAu to HeLa cells is further evaluated using plasmid DNA (pDNA) containing the gene for luciferase and enhanced green fluorescent protein (EGFP) as an application for another treatment of the FD response. And it is shown in Fig. 20(c). After culturing for 24 hours and 48 hours, the amount of LAu-pDNA complex, that is, the amount of LAuG, transfected into cells [per LA (relative luminescence unit; RLU)] for 24 hours and 48 hours, is comparable to the amount of lipofectamine and removed It was much larger than the amount of DNA. The gene transfer of pDNA including EGFP was compared with the removed DNA and commercially available lipofectamine to form a complex with LAu, that is, LAuG. The top image of FIG. 20(c) shows the corresponding fluorescence images (derived from EGFP expression) after 24 hours and 48 hours incubation to further confirm gene transfer performance. Results of NIR triggered D release and gene transfer with LAu demonstrated the potential of the FD response. Thus, nanocomposites with various components can be efficiently manufactured for a wide range of nanomedical applications.

11: Discharge part
12: solution generation unit
13: spray part
14: light irradiation unit
15: solvent removal unit
151: adsorbent
152: through euro
153: inner tube
16: collection unit
161: metal part
162: voltage applicator
17: droplet transfer unit
18: electric field application unit

Claims (23)

A spray unit for forming droplets by spraying a solution containing an organic material having an electron donating group or negatively charged and agglomerates of metal nanoparticles;
A light irradiation unit configured to form a nanostructure by irradiating light onto the droplets formed from the spray unit to rearrange the metal nanoparticles and organic substances; And
A solvent removal unit configured to remove a solvent from the nanostructure formed from the light irradiation unit to generate a nanocomposite,
The organic material is a surfactant, a lipid, a drug, or a mixture thereof,
The metal nanoparticles contain a metal having a work function of 6.0 eV or less,
The light irradiation unit irradiates light having a photon energy greater than the work function of the metal nanoparticles,
An apparatus for producing a nanocomposite in which metal nanoparticles and organic substances are electrostatically bonded by the light irradiation. The apparatus for manufacturing a nanocomposite according to claim 1, wherein the atomizing unit is a collision atomizer. The apparatus for manufacturing a nanocomposite according to claim 1, wherein the spray unit includes a transport gas inlet. The apparatus for manufacturing a nanocomposite according to claim 1, further comprising a discharge unit for forming metal particles from a metal electrode by discharge. delete The apparatus of claim 1, wherein the solvent removal unit is a diffusion dryer in which an adsorbent is provided. The apparatus of claim 1, further comprising an electric field application unit for applying an electric field to the nanocomposite formed from the solvent removal unit. The apparatus of claim 1, wherein the electric field application unit is provided to generate an electric field from the center of the droplet transfer unit toward the edge. The apparatus of claim 1, further comprising a collection unit for collecting the nanocomposite formed from the solvent removal unit. Forming droplets by spraying a solution containing an organic material having an electron donating group or negatively charged, and a metal nanoparticle aggregate;
Irradiating light to the droplets formed in the step to rearrange the metal nanoparticles and the organic material to form a nanostructure; And
Including the step of generating a nanocomposite by removing the solvent from the nanostructure formed in the step,
The organic material is a surfactant, a lipid, a drug, or a mixture thereof,
The metal nanoparticles are metals having a work function of 6.0 eV or less,
In the step of forming the nanostructure, irradiating light having a photon energy greater than the work function of the metal nanoparticles,
The method of manufacturing a nanocomposite using the device of claim 1, wherein the metal nanoparticles and the organic material are electrostatically bonded to each other by the light irradiation. The method of claim 10 wherein the electron donating group is -O ^-, -OH, ether, ester, amide, or a production method of an amine of the nanocomposite. delete The method of claim 10, wherein the metal nanoparticles are gold (Au) nanoparticles. The method of claim 10, further comprising collecting the nanocomposite by applying an electric field of opposite polarity to the generated nanocomposite after the step of generating the nanocomposite. The method of claim 10, further comprising dispersing the collected nanocomposite in a dispersion. The nanocomposite manufactured by the device of claim 1 or the method of claim 10.
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