KR20190035383A - Manufacturing device of nanocomposites, method of preparing nanocomposites and nanocomposite prepared therefrom - Google Patents

Abstract

A manufacturing device of a nanocomposite, a manufacturing method thereof, and the nanocomposite manufactured by using the same do not require a purification or separation process according to a hydrothermal reaction and exhibit effects of pH-sensitive drug release, excellent cell penetration, and minimized opsonization. In addition, according to the manufacturing device of the nanocomposite and the method for manufacturing the same, it is possible to exhibit a great possibility of efficiently ordering and producing a multifunctional nanotherapy application product.

Description

Technical Field [0001] The present invention relates to a nanocomposite manufacturing apparatus, a nanocomposite manufacturing method, and a nanocomposite fabricated using the nanocomposite.

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

The therapeutic suitability of anticancer agents is attenuated by limitations such as biopharmaceutical properties, drug resistance, unidentified toxicity, and limited concentration at the tumor site. Complex chemotherapy or better drug delivery systems are being developed to address these problems.

The nano-sized carrier carrying the drug may be useful for drug delivery through the leaky tumor vasculature of the leaky tumor which is enhanced by the permeation and maintenance effect. In addition, nanoparticles exhibiting external stimulus-responsive behavior are likely to be used in hybrid cancer therapy. One of these possibilities is to use a NIR-sensitive nano platform that is capable of transporting anticancer drugs and inducing photothermal effects to induce tumor degeneration.

Chemotherapeutic chemotherapy has been intensively studied in which a toxin (DOX) is combined with a gold nanoparticle (AuNP) - graphene oxide as a photothermal agent. Most of these studies use multi-step hydrothermal reaction, separation and purification steps to functionalize the surface of gold and graphene oxide to couple DOX. Nanocomposites with cationic surface charge (eg, Au-GO @ DOX) can easily penetrate cells by adsorption interaction with the cell membrane. However, these nanocomposites are easily removed from the body through the reticuloendothelial system, which results in poor drug delivery to the target site. Often, the nanocomposite is stealth coated with polyethylene glycol (PEG) to avoid this problem. However, there is a report that the PEG of the nanocomposite limits the interaction between the carrier and the target cell. Thus, there is a need for the development of stealth coated nanocomposites that can be removed when they reach the target site without being damaged during circulation.

Korean Patent Publication No. 10-2017-0025855

 Nanoscale, 2013, 5, 10591-10598, Zhijun Zhang et al.  Polymer Science and Technology Vol. 15, No. 4, August 2004,

The present application is directed to a nanocomposite manufacturing apparatus that can exhibit the effect of pH-sensitive drug release, good cell penetration and minimized opsonization, without the need for purification or separation processes according to a hydrothermal reaction, To provide a nanocomposite.

The present application relates to an apparatus for producing nanocomposites. According to an exemplary aspect of the present invention, there is provided a manufacturing apparatus comprising: a solution storage portion for containing a solution containing a precursor of a solvent and a nanocomposite; A droplet forming unit fluidly connected to the solution reservoir and including a vibrator having at least one perforation to form a droplet including a precursor of the nanocomposite through the perforations of the vibrator; A droplet transporting unit for transporting droplets formed from the droplet forming unit; And a solvent removing unit for removing the solvent from the droplets transferred from the transferring unit to produce a nanocomposite.

The present application relates to a method for producing a nanocomposite. According to an exemplary method of the present invention, a solution containing a precursor of a solvent and a nanocomposite is passed through a vibrator having at least one perforation to form a droplet including a precursor of the nanocomposite; And removing the solvent from the formed droplet to produce a nanocomposite.

The present application relates to nanocomposites. According to the exemplary nanocomposite of the present application, a sheet-like nanomaterial having a nano-size in the thickness direction; Metal nanoparticles physically bonded to the sheet-like nanomaterial; And an organic material electrostatically bonded to the sheet-like nanomaterial.

The nanocomposite manufacturing method and the nanocomposite fabricated using the nanocomposite of the present application do not require the purification or separation process according to the hydrothermal reaction, and the effect of pH-sensitive drug release, excellent cell penetration ability and minimized opsonization Lt; / RTI > In addition, according to the apparatus and the method for producing a nanocomposite of the present application, multifunctional nanotherapy applications can be efficiently ordered and produced.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of an exemplary nanocomposite manufacturing apparatus, a method of manufacturing a nanocomposite, and a nanocomposite manufactured using the same according to the present application.
Fig. 2 is a schematic diagram of an exemplary nanocomposite manufacturing apparatus of the present application.
3 is a view showing a droplet forming unit in an exemplary nanocomposite production apparatus of the present application.
Fig. 4 is a cross-sectional view of a droplet transporting unit in an exemplary nanocomposite production apparatus of the present application. Fig.
FIG. 5 is a view showing a solvent removing unit in an exemplary nanocomposite manufacturing apparatus of the present application.
6 is a view showing a collecting portion of an exemplary nanocomposite manufacturing apparatus of the present application.
7 is a diagrammatic illustration of an exemplary nanocomposite of the present application.
8 is a transmission electron microscope (TEM) image of the particle structure generated from the vibration nozzle (left side) and the spray nozzle (right side).
FIG. 9 is a size distribution diagram of (A) Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 2 (Au- (C) Example 1 (F) of Fucoidan (Fucoidan) -GO @ ZC-DOX, Comparative Example 1 (Au-GO), Comparative Example 2 (Au-GO @ DOX), Doxorubicin (DOX) and Amphiphilic Chitosan Low-power and high-power transmission electron microscope (TEM) images of Comparative Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 2 (Au-GO @ DOX).
10 is a digital image of (A) Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 2 (Au-GO @ DOX) and UV- ) Spectrum, (B) the in vitro drug release profile of DOX from Example 1 (Au-GO @ ZC-DOX) in phosphate buffered saline and acetate buffered saline, and (C) (NIR) of Comparative Example 1 (Au-GO) and Comparative Example 3 (Au-GO @ ZC).
Figure 11 shows the results of cell uptake of PANC-1 and MIA PaCa-2 cells according to Example 1 (Au-GO @ ZC-DOX) (A) confocal images and (B) fluorescence activated cell sorting (FACS) Graph.
(Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO), Comparative Example 2 (Au-GO @ DOX) and Comparative Example 3 (Au- GO < / RTI > ZC) in PANC-1 and MIA PaCa-2 cells.
(Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO), Comparative Example 2 (Au-GO @ DOX) and Comparative Example 3 (A) PANC-1 and (B) MIA PaCa-2 cells which had been treated with the anti-apoptotic agent.
FIG. 14 is a graph showing the results of Transwell analysis for measuring cell migration by treating DOX, Comparative Example 2 (Au-GO @ DOX) and Example 1 (Au-GO @ ZC-DOX).
FIG. 15 is a graph (A) confocal image and (B) fluorescence-activated cell sorting (FACS) analysis results for the cytoplasmic uptake of Au-GO @ FITC and Au-GO @ ZC-FITC in RAW 264.7 macrophages .
Fig. 16 is a graph showing the results obtained by comparing the results of (A) Comparative Example 2 (Au-GO @ DOX) carrying the phosphor Cy5.5 and Example 1 (Au-GO @ ZC-DOX) in vivo upon intravenous administration of PANC-1 tumor xenograft (B) a distribution image of Comparative Example 2 (Au-GO @ DOX) and Example 1 (Au-GO @ ZC-DOX) carrying a phosphor Cy5.5 in another organ, (C) (Au-GO @ ZC-DOX) and Comparative Example 2 (Au-GO @ ZC-DOX) in which the phosphor Cy5.5 was carried in another organ (Au-GO @ ZO-DOX) and Example 1 (Au-GO @ ZC-DOX) treated xenograft tumor.
FIG. 17 is a graph showing changes in tumor volume of PANC-1 xenografted mice (A) after in vivo antitumor treatment, (B) a graph of weight change of PANC-1 xenografted mice, and (C) 1 < / RTI > xenograft tumor.
Figure 18 is an image showing representative histopathological conditions (hematoxylin and eosin staining) of rats in different treatment groups.

The term " nano " in this application may refer to a size in nanometers (nm), for example, but is not limited to, a size of 1 to 1,000 nm. As used herein, the term " nanoparticle " may mean particles having an average particle size in nanometers (nm), for example, particles having an average particle size of 1 to 1,000 nm, But is not limited to.

Hereinafter, a nanocomposite manufacturing apparatus, a nanocomposite manufacturing method, and a nanocomposite manufactured by the same will be described with reference to the accompanying drawings, and the accompanying drawings are illustrative, and the nanocomposite manufacturing apparatus , The method of preparing the nanocomposite and the range of the nanocomposite produced by the method are not limited by the attached drawings.

FIGS. 1 to 6 are schematic diagrams of an apparatus for producing a nanocomposite according to an embodiment of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of an exemplary nanocomposite manufacturing apparatus, a method of manufacturing a nanocomposite, and a nanocomposite manufactured using the same according to the present application.

2 is a schematic view of an exemplary nanocomposite manufacturing apparatus 1 of the present application.

1 and 2, the apparatus 1 for producing a nanocomposite includes a solution storage part 11, a droplet forming part 12, a droplet transfer part 13 and a solvent removing part 16 .

The solution reservoir 11 can receive a solution containing a precursor of a solvent and a nanocomposite, and the solution reservoir 11 can operate continuously. Although not shown in the drawing, the solution reservoir 11 may be connected to the pump and the agitator.

The pump can quantitatively supply the solvent or one or more precursors to the solution reservoir 11. 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 agitator can prepare the solution by homogeneously mixing the solvent or the at least one precursor. The stirrer is not particularly limited as long as it is an apparatus capable of stirring, and the stirring speed may be changed according to the kind, viscosity or content of the solvent and the precursor, and ultrasonic waves may be applied for more uniform stirring.

Fig. 3 is a diagram showing the droplet forming portion 12 in the exemplary nanocomposite production apparatus 1 of the present application.

3, the droplet forming portion 12 may include a vibrator 124 and a vibrator 123 as a portion that is in fluid connection with the solution reservoir 11. The droplet forming unit 12 includes a vibrator 124 having one or more apertures 121 and a droplet containing the precursor of the nanocomposite may be formed through the perforations 121 of the vibrator 124 .

The vibrator 124 may include a perforation 121 and a disc 122. One or more perforations 121 may be formed on the disk 122 and the average size of the perforations 121 may range from 10 to 1,000 占 퐉, 10 to 800 占 퐉, 10 to 600 占 퐉, 10 to 400 占 퐉, or 10 But it is not limited thereto. If the average size of the pores exceeds the above range, the bonds between the precursors of the nanocomposite may be broken and the nanocomposite may not be formed.

The vibration applying unit 123 applies a predetermined frequency to the vibrator 124 so that the solution of the solution storing unit 11 passes through the perforations 121 of the vibrator 124 to form a precursor of the nanocomposite Droplets can be formed. Accordingly, the number of vibrations applied to the vibrator 124 may be 10 to 10 ⁴ kHz, 10 to 10 ³ kHz, 10 to 500 kHz, or 10 to 250 kHz, but is not limited thereto. The specific range of the frequency for forming droplets may vary depending on the kind of solvent, kind of precursor, and content. Nitrogen (N ₂ ) gas and a pressure of about 3 atmospheres are typically used to make a spray nozzle to form droplets. The nozzle moves up and down, and ΔP rises. That is, when such a spray nozzle is used, it is difficult to generate self-assembly type nano-cells. However, unlike the spray nozzle, the vibrator does not require a spray gas for forming droplets, has no AP, and forms droplets by self weight at atmospheric pressure.

In one example, the apparatus 1 for producing the nanocomposite may further include a transfer gas injection unit 14. [ The transfer gas injecting unit 14 can inject the transfer gas into the droplet transferring unit 13. In the vibration nozzle of the manufacturing apparatus of the present invention, since there is no fluid flow, a method of injecting the transfer gas before the droplet generated in the vibration nozzle comes down in the direction of gravity and before the droplet hits the bottom wall, But may be located in the middle rather than the bottom of the device. The transfer 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), argon (Ar), krypton (Kr), xenon But are not limited thereto. The pressure of the gas injected into the droplet conveyance unit 13 may be 1.5 atm or less, preferably atmospheric pressure, but is not limited thereto. When the pressure of the gas exceeds 1.5 atm, it may be difficult to generate self-assembled nanofibers, and vesicles may be formed by forming a swirl.

4 is a cross-sectional view of the droplet transporting unit 13 in the exemplary nanocomposite production apparatus 1 of the present application.

As shown in Fig. 4, the droplet transporting section 13 is a part for transporting droplets formed from the droplet forming section 12, and can transfer the formed droplet to the solvent removing section 16, which will be described later.

The droplet transporting unit 13 may further include an electric field applying unit 15. The electric field applying unit 15 may be formed in the shape of a probe or a needle so as to generate an electric field in the edge direction from the center of the liquid transferring unit 13, but is not limited thereto. The electric field applying unit 15 may apply an electric field to the droplet formed from the droplet forming unit 12. [ The intensity of the electric field generated by the electric field application unit 15 may be -50 kV to +50 kV, but is not limited thereto. An electric field having the same polarity as that of the formed droplet can be applied to the droplet to prevent agglomeration of the droplet.

5 is a view showing a solvent remover 16 in an exemplary nanocomposite manufacturing apparatus 1 of the present application.

5, the solvent removing unit 16 is a part for removing the solvent of the droplets transferred from the droplet transferring unit 13, and passes through the solvent removing unit 16 to remove the nanocomposite The solvent can be removed from the containing droplets.

The solvent removal unit 16 may include an inlet unit 164, an internal tube 163, a through passage 162 and an outlet unit 165. The solvent removal unit 16 may include a diffusion drier ).

The droplet introduced from the inflow portion 164 passes through the through passage 162 and the solvent-removed nanocomposite can be discharged to the outflow portion 165. The through-flow passage 162 may be formed between the inner tube 163 including the adsorbent 161. The inner tube 163 is not limited as long as it has one or more hollows, such as a stainless steel mesh, and has durability and heat resistance. The adsorbent 161 provided in the inner tube 163 may be at least one of commercially available adsorbents such as activated carbon, silica gel, zeolite or activated alumina, but is not limited thereto.

In one example, the apparatus 1 for producing the nanocomposite may further comprise a collecting part 17. [ Fig. 6 is a view showing the collection unit 17 among the exemplary nanocomposite production apparatus of the present application.

6, the collecting unit 17 collects the nanocomposite formed from the solvent removing unit. The collecting unit 17 includes a metal part 171 and a voltage source 172 can do.

The metal part 171 of the collecting part 17 may be electrostatically coupled with the nanocomposite, and the metal part 171 may be in the form of a plate or a rod.

The voltage source terminal 172 may be connected to the metal part 171. A voltage may be applied to the metal portion 171 so that the metal portion 171 and the nanocomposite can be coupled to each other. The voltage may be 0.5 to 5.0 kV, but is not limited thereto. When the generated nanocomposite is positive, a negative voltage is applied. When the nanocomposite is negatively charged, a positive voltage may be applied to collect the nanocomposite.

The present application also relates to a method for producing nanocomposites. The method of manufacturing the nanocomposite of the present application can be performed by using the above-described apparatus for producing a nanocomposite. Accordingly, the description of the method for manufacturing the nanocomposite will be omitted. Exemplary methods of making the nanocomposites of the present application include a droplet formation step and a nanocomposite production step.

The droplet forming step may be performed in the droplet forming section 12 of the above-described apparatus for producing a nanocomposite. In one example, the droplet forming step may include a step of forming a solution containing the precursor of the solvent and the nanocomposite And passing the vibrator 124 having the nanocomposite layer 121 to form a droplet including the precursor of the nanocomposite.

The vibrator 124 is in fluid communication with a solution reservoir 11 that contains a solution comprising the precursor of the solvent and the nanocomposite and is in fluid communication with the precursor of the nanocomposite through at least one To form droplets. When the liquid containing the solvent and the precursor of the nanocomposite contained in the solution reservoir 11 is transferred in the step of forming the droplet, vibration of 10 to 10 ⁴ kHz is generated by the vibration applicator 123 Is applied to the vibrator 124 and can be transported to the droplet conveyance unit by forming a droplet including the precursor of the nanocomposite through one or more apertures 121 of the vibrator 124. The kind of the solvent can be determined according to the drug to be described later. In one embodiment, water, phosphate buffered saline (PBS), bovine serum albumin, fetal bovine serum (FBS), dichloromethane, dimethylsulfoxide or an alcohol may be used, It is not.

The precursor of the nanocomposite may include, but is not limited to, at least two precursors selected from the group consisting of a cationic precursor, an anionic precursor, and a zwitterionic precursor.

The cationic precursor may be, but is not limited to, an anionic polymer, a cationic polymer, or a drug.

The anionic polymer may be, for example, polyglutamic acid, polyacrylic acid, alginate, carrageenan, hyaluronic acid, poly (styrene sulfonate) Carboxymethylcellulose, cellulose sulfate, dextran sulfate, heparin, heparin sulfate, poly (methylene-co-guanidine), and the like. Condroitin sulfate, or a copolymer thereof.

The cationic polymer may be selected from, for example, chitosan, gelatin, collagen, poly-L-lysine, poly-L-histidine, poly-L- arginine, hyaluronic acid, polygamargamic acid, alginate, carboxyalkylcellulose, glycogen, (Alkyl) cellulose, polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, polyvinyl alkyl ether, polyaminoalkyl (meth) acrylate, poly ) Acrylate, polylactic acid, polyglycolic acid, polylactic glycolic acid, polycaprolactone, polyethyleneimine, or copolymers thereof, but is not limited thereto.

The drug may be an anti-cancer agent, and examples of the anti-cancer agent include doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, But are not limited to, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, imatinib, cisplatin, 5-fluorouracil (5- fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, small molecule drugs, nitro But are not limited to, one or more selected from the group consisting of nitrogen mustard and nitrosourea.

The anionic precursor may be inorganic nanoparticles, and the inorganic nanoparticles may be metal nanoparticles, sheet-like nanomaterials, and mixtures thereof, but are not limited thereto.

The metal nanoparticles may include a metal having a work function of 2.0 eV or more and may be a metal such as gold, silver, iron, platinum, copper, zinc, tin, ruthenium, rhodium, gadolinium, dysprosium, manganese, Chromium, and the like. However, the present invention is not limited thereto.

The sheet-like nano material may be at least one selected from the group consisting of graphene oxide, iron sulfide, molybdenum sulfide, black phosphorus, graphene and reduced graphene oxide, It is not.

The amphoteric precursor may be an amphiphilic polymer, but is not limited thereto.

The amphiphilic polymer may be a complex in which a hydrophilic polymer and a hydrophobic polymer are combined, and the hydrophilic polymer is composed of, for example, chitosan, dextran, glycol chitosan, hyaluronic acid, poly-L-lysine and polyaspartic acid (PEI), poly (N-2- (hydroxypropyl) methacrylamide), poly (divinyl ether-co-maleic anhydride), poly (styrene- Maleic anhydride) and poly (ethylene glycol), and the hydrophobic polymer may be at least one selected from the group consisting of deoxycholic acid, taurodeoxycholic acid ), Taurocholic acid, glycochenodeoxyhoclic acid, or a steric acid or a derivative thereof. But may be at least one kind of fatty acid derivatives selected from oleic acid, but is not limited thereto. Specifically, it may be, but is not limited to, amphoteric chitosan. The amphiphilic chitosan can form a nano-sized self-assembly through a balance of hydrophobicity and hydrophilicity.

The precursor of the nanocomposite may be at least one selected from the group consisting of inorganic nanoparticles, organic nanoparticles, and a drug, but is not limited thereto. In one example, the inorganic nanoparticles may be metal nanoparticles, sheet-like nanomaterials, and mixtures thereof, but are not limited thereto. The metal nanoparticles 22 may include a metal having a work function of 2.0 eV or more and may include a metal such as gold, silver, iron, platinum, copper, zinc, tin, ruthenium, rhodium, gadolinium, dysprosium, , Palladium, and chromium, but is not limited thereto.

The sheet-like nanomaterial may have a size of 1 nm to 1000 nm in the thickness direction, and may have a size of, for example, 1 nm to 100 nm, 1 nm to 80 nm, or 1 nm to 60 nm But is not limited thereto. The nanomaterial has a size in the thickness direction within the above-mentioned range, so that it can have a large specific surface area and excellent drug loading capacity. The term " thickness direction " in this application means a direction perpendicular to the surface of the nanomaterial.

In one example, the nanomaterial may have a specific surface area of from 50 m ² / g to 4000 m ² / g, for example from 100 m ² / g to 3000 m ² / g, from 200 m ² / g to 2000 ^{m 2 / g, 300 m 2} / g to ^{1000 m 2 / g, 400 m} 2 / g to ^{800 m 2 / g, 500 m} 2 / g to 600 m ² / g or 550 m ² / g to 600 m ² / g. < / RTI > The " specific surface area " is the surface area of the pores measured by the BET (Brunauer-Emmett-Teller) method and means the total surface area per unit mass of the pores. The nanomaterial has a specific surface area within the above-mentioned range, so that it can have excellent drug loading capacity.

The sheet-like nano material 21 having a nano-size in the thickness direction may be negatively charged or positively charged, and the sheet-like nano material having a nano-size in the thickness direction may be graphene oxide, iron sulfide, molybdenum sulfide, But is not limited to, one or more selected from the group consisting of black phosphorus, graphene and reduced graphene oxide.

The organic nanoparticles may be at least one selected from the group consisting of an anionic polymer, a cationic polymer, and an amphipathic polymer, but is not limited thereto.

The term " amphiphilic polymer " in the present application means a polymer which simultaneously contains regions having different physical properties, for example, solubility parameters different from each other, for example, a hydrophilic region and a hydrophobic region May refer to a polymer that is simultaneously included.

The anionic polymer may be, for example, polyglutamic acid, polyacrylic acid, alginate, carrageenan, hyaluronic acid, poly (styrene sulfonate) Carboxymethylcellulose, cellulose sulfate, dextran sulfate, heparin, heparin sulfate, poly (methylene-co-guanidine), and the like. Condroitin sulfate, or a copolymer thereof.

The cationic polymer may be selected from, for example, chitosan, gelatin, collagen, poly-L-lysine, poly-L-histidine, poly-L- arginine, hyaluronic acid, polygamargamic acid, alginate, carboxyalkylcellulose, glycogen, (Alkyl) cellulose, polyvinyl pyrrolidone, polyethylene glycol, polyvinyl alcohol, polyvinyl alkyl ether, polyaminoalkyl (meth) acrylate, poly ) Acrylate, polylactic acid, polyglycolic acid, polylactic glycolic acid, polycaprolactone, polyethyleneimine, or copolymers thereof, but is not limited thereto.

The amphiphilic polymer may be a complex in which a hydrophilic polymer and a hydrophobic polymer are combined, and the hydrophilic polymer is composed of, for example, chitosan, dextran, glycol chitosan, hyaluronic acid, poly-L-lysine and polyaspartic acid (PEI), poly (N-2- (hydroxypropyl) methacrylamide), poly (divinyl ether-co-maleic anhydride), poly (styrene- Maleic anhydride) and poly (ethylene glycol), and the hydrophobic polymer may be at least one selected from the group consisting of deoxycholic acid, taurodeoxycholic acid ), Taurocholic acid, glycochenodeoxyhoclic acid, or a steric acid or a derivative thereof. But may be at least one kind of fatty acid derivatives selected from oleic acid, but is not limited thereto. Specifically, it may be, but is not limited to, amphoteric chitosan. The amphiphilic chitosan can form a nano-sized self-assembly through a balance of hydrophobicity and hydrophilicity.

Furthermore, the drug may be an anticancer agent, and examples of the anticancer agent include doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, But are not limited to, actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, imatinib, cisplatin, 5-fluorouracil 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, small molecule drugs, , Nitrogen mustard, and nitrosourea. However, the present invention is not limited thereto.

The nanocomposite producing step may be performed in the solvent removing unit 16 of the apparatus 1 for producing a nanocomposite as described above. In one example, the nanocomposite producing step is carried out through the droplet conveying unit 13 And removing the solvent from the resulting droplets by a diffusion drier having an adsorbent inside thereof to produce the nanocomposite.

In one example, the manufacturing method of the present application may further include an electric field application step, prior to the step of producing the nanocomposite. The electric field application step may be performed in the droplet transporting part, and the electric field application step may include applying an electric field having the same polarity as the droplet formed in the electric field application part of the droplet transporting part to the droplet to prevent aggregation of droplets, The nanocomposite can be produced by transferring the droplet to the removal part 16.

The method of producing the nanocomposite may further comprise the step of collecting the nanocomposite after the step of producing the complex. The step of collecting the nanocomposite may be performed in a collecting part of the apparatus for producing a nanocomposite as described above and the step of collecting the nanocomposite may include collecting the nanocomposite from the collector 172, An electric field of a polarity opposite to that of the nanocomposite from which the solvent has been removed from the rejection 16 is applied and electrostatically combined with the metal portion 171 of the collecting portion 17 in the above-described nanocomposite production apparatus 1 Nanocomposites can be collected.

In one example, the method of manufacture of the present application may further comprise dispersing the collected nanocomposite in a dispersion. The step of dispersing the collected nanocomposite may be performed in a dispersing device of the apparatus for producing a nanocomposite as described above. The dispersion may be any material that is not harmful to human body, and may be physiological saline, phosphate buffered saline (PBS) But are not limited to, bovine serum albumin, fetal bovine serum (FBS), dichloromethane, dimethylsulfoxide or ethanol.

The present application also relates to nanocomposites. Exemplary nanocomposites of the present application can be prepared by the above-described apparatus for producing a nanocomposite and a method for producing a nanocomposite. According to the nanocomposite, a sheet-like nanomaterial having a nano-size in a thickness direction; Metal nanoparticles physically bonded to the sheet-like nanomaterial; And an organic material electrostatically bonded to the sheet-like nanomaterial.

7 is a schematic diagram of an exemplary nanocomposite 21 of the present application.

As shown in FIG. 7, the sheet-like nanomaterial may have a size of 1 nm to 1000 nm in the thickness direction, for example, 1 nm to 100 nm, 1 nm to 80 nm or 1 nm to 60 nm, but is not limited thereto. The nanomaterial has a size in the thickness direction within the above-mentioned range, so that it can have a large specific surface area and excellent drug loading capacity. The term " thickness direction " in this application means a direction perpendicular to the surface of the nanomaterial.

In one example, the nanomaterial may have a specific surface area of from 50 m ² / g to 4000 m ² / g, for example from 100 m ² / g to 3000 m ² / g, from 200 m ² / g to 2000 ^{m 2 / g, 300 m 2} / g to ^{1000 m 2 / g, 400 m} 2 / g to ^{800 m 2 / g, 500 m} 2 / g to 600 m ² / g or 550 m ² / g to 600 m ² / g. < / RTI > The " specific surface area " is the surface area of the pores measured by the BET (Brunauer-Emmett-Teller) method and means the total surface area per unit mass of the pores. The nanomaterial has a specific surface area within the above-mentioned range, so that it can have excellent drug loading capacity.

The sheet-like nano material 21 having a nano-size in the thickness direction may be negatively charged or positively charged, and the sheet-like nano material having a nano-size in the thickness direction may be graphene oxide, iron sulfide, molybdenum sulfide, But is not limited to, one or more selected from the group consisting of black phosphorus, graphene and reduced graphene oxide.

The sheet-like nanomaterial 21 may be physically bonded to the metal nanoparticles 22 to be described later, and may be bonded by, for example, a capillary force or the like, but is not limited thereto. The metal nanoparticles 22 may include a metal having a work function of 2.0 eV or more and may include a metal such as gold, silver, iron, platinum, copper, zinc, tin, ruthenium, rhodium, gadolinium, dysprosium, , Palladium, and chromium, but is not limited thereto.

In one example, the organic material may be electrostatically bonded to the sheet-like nanomaterial 21, and the organic material may be a polarly charged organic material different from the sheet-like nanomaterial 21. [

The organic material may include a polymer 23 and a drug 24. The polymer 34 may be an amphipatic compound having a molecular weight of 10,000 to 200,000 and the amphipatic compound may be, May be a complex in which a hydrophilic polymer and a hydrophobic polymer are combined.

The hydrophilic polymer may be, for example, a biopolymer selected from the group consisting of chitosan, dextran, glycol chitosan, hyaluronic acid, poly-L-lysine and polyaspartic acid or polyethyleneimine (PEI) (Ethylene-co-maleic anhydride), poly (ethylene-co-maleic anhydride) and poly (ethylene glycol) But are not limited to, at least one of the synthetic polymers selected.

Wherein the hydrophobic polymer is selected from the group consisting of deoxycholic acid, taurodeoxycholic acid, taurocholic acid or glycochenodeoxyhoclic acid, Or a fatty acid derivative selected from steric acid or oleic acid, but the present invention is not limited thereto.

Specifically, it may be, but is not limited to, amphoteric chitosan. The amphiphilic chitosan can form a nano-sized self-assembly through a balance of hydrophobicity and hydrophilicity.

The term " amphiphilic polymer " in the present application means a polymer which simultaneously contains regions having different physical properties, for example, solubility parameters different from each other, for example, a hydrophilic region and a hydrophobic region May refer to a polymer that is simultaneously included.

The drug 24 may be an anticancer drug. Examples of the anticancer drug include doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, , Actinomycin-D, docetaxel, etoposide, teniposide, bisantrene, imatinib, cisplatin, 5-fluorouracil But are not limited to, 5-fluorouracil, adriamycin, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, small molecule drugs ), Nitrogen mustard, and nitrosourea. However, the present invention is not limited thereto.

The drug (24) may be dispersed in a dispersion, and the dispersion is not harmful to the human body. It may be physiological saline, phosphate buffered saline (PBS), bovine serum albumin, fetal serum bovine serum, FBS), dichloromethane, dimethylsulfoxide or ethanol, but the present invention is not limited thereto.

The drug 24 may be supplied at a dose of 0.1 mg to 3 mg per 1 mL of the solvent and may be, for example, 0.3 mg to 2.5 mg, 0.6 mg to 2 mg or 0.9 mg to 1.5 mg, And may be properly supplied within the above range depending on the condition of the patient having the disease.

The nanocomposite may have an absorption rate of 5 to 95 at a wavelength of 1,000 nm, but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples. However, the scope of the present application is not limited by the following description.

material

Graphite (C-072561) and gold (AU-172561) bars were purchased from Nilaco Corporation (Tokyo, Japan). Doxorubicin (DOX) was obtained from Dong-A Pharmaceutical (Yongin, Korea). (KMnO4) and fluorescein-5 (6) -isothiocyanate (FITC) were purchased from Sigma Aldrich (St Louis, MO, USA). Lysotracker green was purchased from Thermo Fisher Scientific (Waltham, MA, USA). PANC-1 and MIA PaCa-2 cancer cells were purchased from Korean Cell Line Bank (Seoul, Korea). All other chemicals were reagent grade and were used without further purification.

Example 1

A sheet-like gold-graphene oxide (Au-GO) nano material was prepared using a pair of electrode rods made of graphite and gold. Specific conditions for producing the sheet-like gold-graphene oxide (Au-GO) nanomaterial are as follows. Air was injected into a discharge portion provided with a pair of electrode rods made of graphite (C-072561) and gold (Au-172561). The flow rate of the air was controlled to 0.9 L / min through a flow controller (MFC; Tylan, USA). The length of the electrode rod was 100 mm, the diameter was 8 mm, and the interval between the electrodes was 1 mm. A graphene oxide nanoflake (GO nano flake) was prepared by applying a voltage of 3.4 kV (resistance of 0.5 M ?, electric capacity of 4.7 nF, current of 2.8 mA and frequency of 580 Hz) to the electrode.

0.4 g of the cationic polymer chitosan and 0.14 g of the hydrophobic polymeric dioxycholic acid were mixed and then 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (1-ethyl-3- dimethylamino) propyl) carbodiimide, EDC) was added dropwise. After 24 hours, the reaction mixture was poured into a methanol / ammonia solution mixed at a ratio of 7: 3, and the precipitate was filtered and washed, and vacuum-dried at room temperature to prepare zwitterionic chitosan (ZC).

Dispersion was prepared by dispersing 0.7 mg / mL of Au-GO, 0.1 mg / mL of ZC and 0.2 mg / mL of drug doxorubicin (DOX) in deionized water of 0.7 mg / mL, A hybrid droplet of 2 탆 in size was prepared through a vibrator having a diameter of 50 탆.

In order to remove the solvent of the hybrid droplet, a nanocomposite Au-GO @ ZC-DOX was prepared by passing the solution through a diffusion drier provided with an adsorbent inside a solvent removal portion of the production apparatus of the present application. The adsorbent was prepared by mixing silica gel and pellet type activated carbon in a mass ratio of 5: 5. The prepared Au-GO @ ZC-DOX was collected using a cylindrical aluminum rod with an electric field of 3 kV, and the Au-GO @ ZC-DOX was immersed in phosphate buffered saline (PBS, pH 7.4).

Comparative Example 1

In Example 1, Au-GO was prepared in the same manner except for the amphiphilic chitosan and the drug doxorubicin.

Comparative Example 2

Au-GO @ DOX was prepared in the same manner as in Example 1 except for the amphiphilic chitosan.

Comparative Example 3

Au-GO @ ZC was prepared in the same manner as in Example 1 except for doxorubicin.

Comparative Example 4

The Au-GO @ ZC-DOX of Example 1 was prepared using a spray nozzle rather than the production apparatus of the present application.

EXPERIMENTAL EXAMPLE 1 - Analysis of particle characteristics of nanocomposite

8 is a transmission electron microscope (TEM) image of the particle structure generated from the vibration nozzle (left side) and the spray nozzle (right side). 8, the vibration nozzle of the left image produces the vesicle structure, i.e., Example 1 (Au-GO @ ZC-DOX), while the spray nozzle of the right image produces the separation structure of Comparative Example 4, Point-gold nanoparticles and thin film-GO @ ZC-DOX. The main part of the experimental setup used to produce the Au-GO @ ZC-DOX is shown in the photo on the left. That is, the dispersion liquid containing Comparative Example 1 (Au-GO), amphoteric chitosan (ZC) and doxorubicin (DOX) passes through the oscillator of the droplet forming portion to form a hybrid droplet. The formed droplets are transferred to a diffusion dryer by an N ₂ gas stream, which removes the solvent by controlling ambient pressure and temperature conditions to produce a nanocomposite.

FIG. 9 is a size distribution diagram of (A) Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 2 (Au- (C) Example 1 (F) of Fucoidan (Fucoidan) -GO @ ZC-DOX, Comparative Example 1 (Au-GO), Comparative Example 2 (Au-GO @ DOX), Doxorubicin (DOX) and Amphiphilic Chitosan Low-power and high-power transmission electron microscope (TEM) images of Comparative Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 2 (Au-GO @ DOX).

As shown in FIG. 9A, the size distributions of Au-GO, Au-GO @ DOX and Au-GO @ ZC-DOX were measured using a scanning mobility particle sizer (SMPS, 3936, TSI, USA) Respectively. The possibility of the nanocomposite manufacturing apparatus and manufacturing method of the present invention for producing nanocomposite by measuring the size distribution in an aerosol state after solvent extraction was first verified. The geometric mean diameters of Au-GO, Au-GO @ DOX and Au-GO @ ZC-DOX were 27.5 ± 1.4 nm, 39.6 ± 1.5 nm and 53.6 ± 1.6 nm, respectively. It was confirmed that the size of Au-GO @ DOX was increased compared to that of Au-GO by bonding DOX to Au-GO. Au-GO @ DOX and Au-GO @ ZC-DOX showed a singular distribution similar to that of Au-GO alone because almost all Au-GOs were coupled with DOX or ZC-DOX during the vibratory droplet generation and diffusion drying process it means. The average zeta potentials of Au-GO, Au-GO @ DOX and Au-GO @ ZC-DOX were -5.4, +10.2 and + 1.4mV, respectively. The change in the average zeta potential is interpreted as a positive charge when DOX bonds to the Au-GO surface, and a neutralization due to the amphipathic nature of ZC when ZC is further coupled.

(FTIR, iS-10, Thermo Electron, USA) analysis was performed using a polytetrafluoroethylene substrate as a reference material for measurement, and this is shown in FIG. 9 (B). Characteristic peaks at 1400 and 1735 cm ^-1 correspond to CO bending and C = O stretching of carboxyl groups in graphene oxide (GO), respectively. DOX exhibited characteristic peaks at 1600 and 1735 cm ^-1 , while ZC exhibited broad peaks at 1500 to 1750 cm ^-1 . That is, when DOX was combined with Au-GO, the characteristic peak of DOX became prominent, which means the successful production of Au-GO @ DOX. In addition, it was confirmed that the Au-GO @ ZC-DOX was successfully produced because the characteristic peak of DOX was decreased and the characteristic peak of ZC was prominent when Au-GO @ DOX was combined with ZC.

As shown in FIG. 9 (C), morphological analysis of Au-GO, Au-GO @ DOX and Au-GO @ ZC-DOX was performed using a transmission electron microscope (TEM, CM-100, FEI / Philips, USA) Analysis was performed. As shown by particle size analysis, Au-GO particles were observed at uniform size. The high magnification TEM image showed AuNP deposition on the GO sheet. That is, lattice streaks appeared on the (111) plane of gold nanoparticles (AuNP), and it was confirmed that this had an interplanar distance of 0.23 nm. In addition, GO showed lattice stripes with interplanar spacing of 0.34 nm. TEM measurements showed clearly that the lateral dimensions of the Au-GO @ DOX and Au-GO @ ZC-DOX were increased, and Au-GO, DOX and ZC-DOX were successful without breaking the bonds of AuNP and GO. Respectively.

EXPERIMENTAL EXAMPLE 2 - Analysis of Drug Release Characteristics of Nanocomposite

10 is a digital image of (A) Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 2 (Au-GO @ DOX) and UV- ) Spectrum, (B) the in vitro drug release profile of DOX from Example 1 (Au-GO @ ZC-DOX) in phosphate buffered saline and acetate buffered saline (** P <0.01, *** P < (C) An image showing the light heat effect induced by near infrared (NIR) of Example 1 (Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO) and Comparative Example 3 (Au-GO @ ZC) .

10 (A) shows a digital image and ultraviolet-visible (UV-vis) spectrum of Au-GO, Au-GO @ DOX and Au-GO @ ZC-DOX. It was also measured using a UV-vis spectrophotometer (T60, PG Instruments, UK).

The single pass load from the Au-GO to the DOX was confirmed by the bright red color in the digital image. In Au-GO @ ZC-DOX, the color strength of Au-GO was reduced by bonding ZC to the nanocomposite. DOX or ZC-DOX induced a shoulder peak at about 500 nm, the UV-vis spectra for Au-GO @ DOX and Au-GO @ ZC-DOX showed broadband light absorption properties suitable for near-IR induced light heat effects .

As shown in FIG. 10 (B), the extracorporeal drug release profile of DOX from Au-GO @ ZC-DOX was determined under different pH conditions. The in vitro release profile of DOX from Au-GO @ ZC-DOX was measured using dialysis. The appropriate amount of Au-GO @ ZC-DOX was added to the membrane tube (Spectra / Por, MWCO 3500 Da, USA) and 30 mL of phosphate buffered saline (PBS, pH 7.4) or acetate buffered saline (ABS, pH 5.0) was injected. The tube was maintained at 37 DEG C and 100 rpm, and the sample was collected at predetermined time intervals. Performance liquid chromatography (HPLC) system (Hitachi, Japan) including an L-2130 pump, an L-2200 autosampler, an L-2420 UV-vis detector and an L-2350 column oven with Ezchrom Elite software (318a, Japan) Was used to quantify the amount of DOX released. Acetonitrile: acetic acid (1%) in a ratio of 50: 49: 1 (v / v / v) using an Inertsil C18 column (150 mm x 4.6 mm, particle size 5 μm, Cosmosil, NacalaiTesque Inc., USA) ) Was subjected to isocratic elution with a mobile phase composed of a flow rate of 1.0 mL / min and a column temperature of 25 ° C. Each 20 μL sample was injected for analysis and the UV absorbance at 254 nm was measured.

A significant increase in DOX release under acidic pH was evident. In other words, it can be seen that the microenvironment of the tumor has an acidic condition that promotes the release of DOX in cancer cells. Chitosan dissolves very well at acidic pH, which can enhance the exposure of the ZC layer in Au-GO @ ZC-DOX and subsequent DOX release. In addition, DOX protonation in an acidic microenvironment can lead to a hydrophilic form that enhances the drug release profile. The amount of DOX released from Au-GO @ ZC-DOX under NIR laser irradiation was measured at 1 hour, 3 hours and 6 hours, respectively. The steep DOX emission profile confirmed that the solubility of ZC and DOX in the acidic medium could be increased due to the NIR laser irradiation and the temperature rise by Au-GO.

As shown in FIG. 10 (C), the photothermal effects of Au-GO, Au-GO @ ZC and Au-GO @ ZC-DOX were evaluated using a thermal camera. The NIR exposure conditions are a wavelength of 808 nm, an intensity of 3.0 W / cm ² and a time of 5 minutes. After exposing Au-GO, Au-GO @ ZC and Au-GO @ ZC-DOX under the 808 nm near-infrared laser irradiation, the increasing temperature was digitally evaluated using a thermal camera (Therm-App TH, Israel) .

When irradiated with 808-nm NIR laser on GO, the optical absorption of heat induced temperature rise by energy transfer from pi-type network recovery. In addition, AuNP can generate heat when exposed to near-infrared rays through surface plasmon resonance (SPR) effect or electron oscillation. Under NIR laser irradiation, the heat rise observed at Au-GO showed a local temperature of about 57 ° C. Coupling of DOX slightly reduced the photothermal capacity from the NIR laser irradiation range on the Au-GO surface. A similar pattern of photothermal height was observed in Au-GO @ ZC-DOX, suggesting that the photothermal effect is well-coupled to DOX to induce potent anti-cancer effects.

Figure 11 shows the results of cell uptake of PANC-1 and MIA PaCa-2 cells according to Example 1 (Au-GO @ ZC-DOX) (A) confocal images and (B) fluorescence activated cell sorting (FACS) Graph.

Lysotracker green was purchased from Thermo Scientific (Waltham, Mass., USA). PANC-1 and MIA PaCa-2 cancer cells were purchased from Korean Cell Line Bank (Seoul, Korea).

PANC-1 and MIA PaCa-2 cells (2 x 10 ⁴ cells / well) were inoculated on a cover slip placed in a 12-well plate, cultured for 24 hours and treated with Au-GO @ ZC-DOX for 1 hour Respectively. LysoTracker green was used to track lysosomes in cells. The final concentrations of DOX and LysoTracker green were 1 μg / mL and 100 ng / mL, respectively. Finally, the cells were washed with PBS, fixed in 4% paraformaldehyde and placed on a glass slide for observation with a confocal laser scanning microscope (CLSM, Leica Microsystems, Wetzlar, Germany).

PANC-1 and MIA PaCa-2 cells (1 x 10 ⁵ cells / well) were inoculated into 6-well plates, incubated overnight at 37 ° C, and the concentration and time- Au-GO @ ZC-DOX was treated as a replicate to determine absorption. After incubation for an appropriate period of time, cells were washed twice with cold PBS and detached using trypsin. This was centrifuged and washed to obtain cell pellet and redispersed in 1 mL cold PBS. FACS analysis was performed for quantitative analysis of cellular uptake in various groups using BD FACS Verse flow cytometry (BD Biosciences, San Jose, CA, USA), and untreated cells were used as a control.

Cellular uptake of Au-GO @ ZC-DOX was qualitatively and quantitatively assessed in PANC-1 and MIA PaCa-2 cells. As shown in Fig. 11 (A), a confocal image of treated cells was shown. Cellular uptake was high and clear in both cell lines and intense red staining in the cells. The ingestion mechanism became more apparent using LysoTracker green. According to the results, nanocomposites were mainly enriched in cellular lysosomes after endocytosis. Figure 11 (B) shows a quantitative assessment of cell uptake in pancreatic cancer cells. Both cell lines showed concentration and time - dependent cellular uptake.

(Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO), Comparative Example 2 (Au-GO @ DOX) and Comparative Example 3 (Au- GO &lt; / RTI &gt; ZC) in PANC-1 and MIA PaCa-2 cells. The NIR exposure conditions are a wavelength of 808 nm, an intensity of 3.0 W / cm ² and a time of 5 minutes.

(MTS) analysis method (Promega, USA) was used in place of 3- (4,5-dimethylthiazol-2-yl) Were used to measure cell viability in vitro of PANC-1 and MIA PaCa-2 cells. Pancreatic cancer cells (1 x 10 ⁴ cells / well) were seeded in 96-well plates and seeded with DOX, Au-GO, Au-GO @ ZC, Au- Treated with ZC-DOX. After 24 hours of treatment, the MTS solution was added to each well to allow color development. Subsequently, the cell viability was determined by measuring the absorbance at 493 nm using a microplate reader (Multiskan EX, Thermo Scientific, USA).

As shown in FIG. 12, the treatment using DOX is inserted into DNA and exhibits a concentration-dependent anticancer effect by its ability to interfere with DNA repair mediated by topoisomerase-II. In NIR laser irradiation, Au-GO and Au-GO @ ZC slightly enhanced the enhanced cytotoxicity due to the photothermal effect of Au-GO. There was no significant difference in cell survival between Au-GO @ DOX and Au-GO @ ZC-DOX therapy. The nanocomposite was significantly absorbed by lysosomes (acidic pH) and allows the ZC layer to easily detoxify effective drug release and cytotoxic effects in the cell, as evidenced by confocal imaging analysis.

(Au-GO @ ZC-DOX), Comparative Example 1 (Au-GO), Comparative Example 2 (Au-GO @ DOX) and Comparative Example 3 (A) PANC-1 and (B) MIA PaCa-2 cells which had been treated with the anti-apoptotic agent.

The induction of apoptosis in cells of PANC-1 and MIA PaCa-2 was evaluated using fluorescence-activated cell sorting (FACS). The NIR exposure conditions are a wavelength of 808 nm, an intensity of 3.0 W / cm ² and a time of 5 minutes.

Au-GO and Au-GO @ ZC did not induce apoptosis significantly. However, when NIR laser was irradiated at 808 nm, the number of apoptotic cells was increased in PANC-1 and MIA PaCa-2 cells. A similar pattern of apoptosis was observed by successfully receiving intracellular nanocomplexes in cells treated with DOX, Au-GO @ DOX and Au-GO @ ZC-DOX. NIR laser irradiation further increased the number of apoptotic cells according to the merge effect, that is, chemotherapy and photothermal effect.

FIG. 14 is a graph showing the results of transwell analysis for measuring cell migration by treating DOX, Comparative Example 2 (Au-GO @ DOX) and Example 1 (Au-GO @ ZC-DOX). ** P < 0.01, *** P < 0.001, and scale bar represents 250 mu m.

Cell migration was analyzed in pancreatic cancer cells using transwell assay. PANC-1 and MIA PaCa-2 cells were separately treated with DOX, Au-GO @ DOX and Au-GO @ ZC-DOX for 6 hours. The cells were then harvested and the cell suspension (5 x 10 ⁵ cells / mL, prepared with serum-free medium) was sprayed onto collagen-coated inserts (0.8 μm, BD Bioscience). Cells were placed in a well containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) to determine the cell migration. After 12 hours of incubation, For dyeing, the dye from the intercalation membrane was dissolved in acetic acid and the absorbance at λ _max = 470 nm was measured using a UV-vis spectrophotometer. The inhibition of migration was calculated using the following general formula 1:

[Formula 1]

Where OD _control and OD _sample represent the optical density of the control and sample, respectively.

As shown in FIG. 14, since the possibility of cell migration in vivo by the epithelial mesenchymal transition is high, both of the pancreatic cancer cell lines have a higher rate of metastasis. After treatment with DOX, Au-GO @ DOX and Au-GO @ ZC-DOX, the ability of pancreatic cancer cells to migrate was significantly reduced. The final formulation, Au-GO @ ZC-DOX, offers the best anti-migratory effect that can be of great help in preventing metastases and extending life.

FIG. 15 is a graph (A) confocal image and (B) fluorescence-activated cell sorting (FACS) analysis results for the cytoplasmic uptake of Au-GO @ FITC and Au-GO @ ZC-FITC in RAW 264.7 macrophages . And a differential interference contrast (DIC) device. The scale bar represents 50 占 퐉.

Intracellular uptake of Au-GO @ FITC and Au-GO @ ZC-FITC was evaluated in RAW 264.7 macrophages. Macrophages were inoculated into 12-well plates and treated with Au-GO @ FITC and Au-GO @ ZC-FITC for 1 hour. Quantitative ingestion was confirmed by confocal microscopy and quantitative absorption was determined by FACS analysis as described above. FITC was purchased from Sigma Aldrich (St Louis, MO, USA).

The protective capacity of ZC to increase the blood circulation time of nanocomposites and prevent opsonization was measured using RAW 264.7 macrophages. (Au-GO @ FITC) supplemented with fluorescein-5 (6) -isothiocyanate (FITC) was widely absorbed by macrophages while Au The absorption of -GO @ ZC-FITC was not significant. Both confocal imaging and FACS analysis were consistent with these results. The addition of a ZC layer can maintain the neutral charge of the nanocomposite and prevent opsonis. This effect means that the Au-GO @ ZC-DOX is able to reach the tumor site more efficiently by extending the blood circulation time.

Fig. 16 is a graph showing the results obtained by comparing the results of (A) Comparative Example 2 (Au-GO @ DOX) carrying the phosphor Cy5.5 and Example 1 (Au-GO @ ZC-DOX) in vivo upon intravenous administration of PANC-1 tumor xenograft (B) a distribution image of Comparative Example 2 (Au-GO @ DOX) and Example 1 (Au-GO @ ZC-DOX) carrying a phosphor Cy5.5 in another organ, (C) (Au-GO @ ZC-DOX) and Comparative Example 2 (Au-GO @ ZC-DOX) in which the phosphor Cy5.5 was carried in another organ (Au-GO @ ZO-DOX) and Example 1 (Au-GO @ ZC-DOX) treated xenograft tumor. In vivo biodistribution of Au-GO @ DOX and Au-GO @ ZC-DOX was evaluated using intravenously injected phosphor Cy5.5.

16 (A) is a biodistribution image of a Au-GO @ DOX and Au-GO @ ZC-DOX nanocomposite carrying a phosphor Cy5.5 with or without ZC in mice transplanted with PANC-1 tumor.

FIG. 16 (B) shows the distribution image of Au-GO @ DOX and Au-GO @ ZC-DOX carrying the phosphor Cy5.5 in another organ after intravenous administration, and Fig. 5 (Au-GO @ DOX) and Example 1 (Au-GO @ ZC-DOX). * P <0.05; ** P < 0.01.

16 (D) shows NIR laser induced in vivo photothermal effect images in tumors pre-treated with Au-GO @ DOX and Au-GO @ ZC-DOX. The NIR exposure conditions are a wavelength of 808 nm, an intensity of 3.0 W / cm ² and a time of 5 minutes.

We identified significant differences between Au-GO @ DOX and Au-GO @ ZC-DOX in tumors and organs. The absorption of Au-GO @ ZC-DOX in PANC-1 tumors treated with Au-GO @ ZC-DOX was significantly higher than that of Au-GO @ DOX.

In addition, as shown in Figs. 16B and 16C, the absorption of Au-GO @ ZC-DOX in other organs is attributed to the weakened opsonin action of ZC, and Au-GO @ ZC-DOX The absorption of Au-GO @ ZC-DOX was significantly lower than that of Au-GO @ DOX, which takes longer cycle time to deliver. Macrophages are abundant in the liver, lungs, spleen and kidneys. Thus, opsonization by macrophages in these organs has led to extensive accumulation of Au-GO @ DOX. In contrast, Au-GO @ ZC-DOX was released to a minimum.

As shown in Fig. 16 (D), the photothermal effects of Au-GO @ DOX and Au-GO @ ZC-DOX treated xenograft tumors were confirmed in vivo. Biodistribution studies have shown that the accumulation of Au-GO @ ZC-DOX is higher in PANC-1 xenograft tumors, which has a better photothermal effect than Au-GO @ DOX. Elevated temperatures of 41 ° C promote blood perfusion and vasodilation, leading to thermal shock responses that repair and protect cells from heat injury. In addition, the high temperature of 46 to 52 ° C implies that induction of apoptosis through ischemia, microvascular thrombosis and hypoxia may control the photothermal resection of Au-GO @ ZC-DOX and may be suitable for enhanced anti-cancer effects .

FIG. 17 is a graph showing changes in tumor volume of PANC-1 xenografted mice (A) after in vivo antitumor treatment, (B) a graph of weight change of PANC-1 xenografted mice, and (C) 1 &lt; / RTI &gt; xenograft tumor.

18 is an image showing representative histopathological images (hematoxylin and eosin staining) of rats in different treatment groups.

Group 1 is a control group, Group 2 is DOX, Group 3 is Au-GO @ DOX, Group 4 is Au-GO @ DOX + NIR, Group 5 is Au-GO @ ZC- DOX + NIR. The NIR exposure conditions are a wavelength of 808 nm, an intensity of 3.0 W / cm ² and a time of 5 minutes. The scale bar is 120 탆.

1 x 10 ⁷ PANC-1 cells dispersed in 100 μl serum-free DMEM were subcutaneously injected to the right thigh of 6-week-old female BALB / c nude mice. When tumor size reached ~ 100 mm ³ , mice were randomly divided into 6 groups (n = 6 / group). All animal handling procedures followed procedures approved by Yeungnam University's Institutional Animal Ethics Committee.

The model of xenotransplantation mice received intravenous administration of DOX, Au-GO @ DOX, Au-GO @ DOX + NIR, Au-GO @ ZC-DOX and Au-GO @ ZC-DOX + NIR via tail vein. One group was used as a control without treatment. Treatment doses equivalent to 2.5 mg drug / kg mouse body weight were administered at days 0, 4, 8, and 12. The weight and tumor dimensions of the mice were recorded. Tumor volume was calculated using the following formula 2.

[Formula 2]

As shown in Figure 17 (A), in vivo antitumor effects were evaluated in PANC-1 xenografted rats. Treatment with DOX showed better antitumor effect than Au-GO @ DOX, suggesting that Au-GO @ DOX is easily opsonized by macrophages. In Group 3, NIR-irradiated mice treated with Au-GO @ DOX, the antitumor effect was improved by combination therapy for chemotherapy and phototherapy for tumor resection. The PANC-1 tumor volume was significantly reduced after treatment with reduced Au-GO @ ZC-DOX by irradiating the tumor with a NIR laser.

As shown in Fig. 17 (B), changes in body weight were measured for toxicity evaluation. After treatment with DOX, a significant decrease in body weight was observed in rats. Weight loss can occur because DOX is easily distributed throughout the body and can cause severe side effects, including cardiac toxicity. However, at the end of the measurement period, we found that the weight returned to normal due to the reversal of side effects. There was no significant change in body weight of all other treatment groups, which implies fitness for in vivo studies.

As shown in Fig. 17 (C), immunohistochemical analysis of tumors was performed. The expression of caspase-3, poly ADP-ribose polymerase (PARP), CD-31 and Ki-67 in tumor cells of the treatment group can be seen. (Ki-67) and angiogenesis (CD-31) markers were significantly decreased in group 6 Au-GO @ ZC-DOX + NIR constructs, but the apoptosis markers (caspase-3 and PARP) , Indicating an effective anti-cancer effect on pancreatic cancer.

Figure 18 is an image showing representative histopathological conditions (hematoxylin and eosin staining) of rats in different treatment groups.

As shown in Fig. 18, the suitability of Example 1 (Au-GO @ ZC-DOX) for in vivo antitumor studies was confirmed. The organ toxicity after treatment in the tested organs, namely heart, liver, lung, kidney and spleen for all treatments did not show abnormal findings similar to the results of FIG. 17 (B).

1: Nanocomposite manufacturing device
11: Solution storage part
12:
13:
14: Feed gas injection unit
15:
16: Solvent removal
17: collecting section
20: Nanocomposite
21: Sheet-like nanomaterial
22: metal nanoparticles
23: Polymer
24: Drugs
121: Perforation
122: disk
123: Vibrator
124: oscillator
161: Absorbent
162: Through-
163: Internal tube
171: metal part
172: Going to the voltage

Claims (29)

A solution reservoir for containing a solution containing a precursor of a solvent and a nanocomposite;
A droplet forming unit fluidly connected to the solution reservoir and including a vibrator having at least one perforation to form a droplet including a precursor of the nanocomposite through the perforations of the vibrator;
A droplet transporting unit for transporting droplets formed from the droplet forming unit; And
And a solvent removing unit for removing the solvent from the droplets transferred from the transferring unit to produce a nanocomposite. The apparatus for producing a nanocomposite according to claim 1, wherein the average size of the perforations is 10 to 1,000 μm. The apparatus for producing a nanocomposite according to claim 1, wherein the oscillator has a frequency of 10 to 10 ⁴ kHz. The apparatus for producing a nanocomposite material according to claim 1, further comprising a transfer gas injection unit for injecting a transfer gas into the droplet transfer unit. The apparatus for producing a nanocomposite according to claim 4, wherein the pressure of the gas injected into the droplet conveyance unit is 1.5 atm or less. The apparatus for manufacturing a nanocomposite material according to claim 1, wherein the liquid crystal transfer section further comprises an electric field applying section for applying an electric field to the droplet to be transferred. The apparatus for manufacturing a nanocomposite material according to claim 1, wherein the electric field applying unit is arranged to generate an electric field toward the edge side from a central portion of the droplet conveyance unit. The apparatus for manufacturing a nanocomposite according to claim 1, wherein the solvent removing unit is a diffusion dryer having an adsorbent therein. The apparatus for producing a nanocomposite according to claim 1, further comprising a collector for collecting the nanocomposite formed from the solvent removal. 10. The apparatus of claim 9, wherein the collector comprises a metal electrostatically bonded to the nanocomposite, and a voltage applied to the metal to allow the metal and the nanocomposite to bond. Passing a solution containing a precursor of a solvent and a nanocomposite through a vibrator having at least one perforation to form a droplet including a precursor of the nanocomposite; And
And removing the solvent from the formed droplet to produce a nanocomposite. 12. The method of claim 11, wherein the precursor of the nanocomposite comprises at least two precursors selected from the group consisting of a cationic precursor, an anionic precursor, and a zwitterionic precursor. 12. The method of claim 11, wherein the precursor of the nanocomposite is at least one selected from the group consisting of inorganic nanoparticles, organic nanoparticles, and a drug. 14. The method of claim 13, wherein the inorganic nanoparticles are metal nanoparticles, sheet-shaped nanomaterials, and mixtures thereof. 14. The method of claim 13, wherein the organic nanoparticles are at least one selected from the group consisting of an anionic polymer, a cationic polymer, or an amphipathic polymer. 14. The method of claim 13, wherein the drug is selected from the group consisting of doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, but are not limited to, docetaxel, etoposide, teniposide, bisantrene, imatinib, cisplatin, 5-fluorouracil, adriamycin, methotrexate, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, small molecule drugs, nitrogen mustard, and nitrosourea. and at least one anticancer agent selected from the group consisting of nitrosourea. 12. The method of claim 11, further comprising applying an electric field of the same polarity as the droplet formed to prevent aggregation of droplets prior to the step of producing the nanocomposite. 18. The method of claim 17, further comprising, after the step of producing the nanocomposite, adding the nanocomposite by applying an electric field of opposite polarity to the nanocomposite. 19. The method of claim 18, further comprising dispersing the collected nanocomposite in a dispersion. A sheet-like nano material having a nano-size in the thickness direction;
Metal nanoparticles physically bonded to the sheet-like nanomaterial; And
Wherein the nanocomposite comprises an organic material electrostatically bonded to the sheet-like nanomaterial. The nanocomposite according to claim 20, wherein the sheet-like nanomaterial having a nano-size in the thickness direction is negatively or positively charged. The method according to claim 21, wherein the sheet-like nano material having a nano-size in the thickness direction is at least one selected from the group consisting of graphene oxide, iron sulfide, molybdenum sulfide, black phosphorus, graphene and reduced graphene oxide &Lt; / RTI &gt; 22. The nanocomposite of claim 21, wherein the metal nanoparticle comprises a metal having a work function of at least 2.0 eV. 24. The nanocomposite according to claim 23, wherein the metal is at least one selected from the group consisting of gold, silver, iron, platinum, copper, zinc, tin, ruthenium, rhodium, gadolinium, dysprosium, manganese, titanium, palladium and chromium. 21. The nanocomposite according to claim 20, wherein the organic material is an organic material charged in a polarity different from that of the sheet-like nanomaterial. 26. The nanocomposite of claim 25, wherein the organic material comprises a polymer and a drug. 27. The nanocomposite according to claim 26, wherein the polymer is an amphiphilic compound having a molecular weight of 10,000 to 200,000. 27. The method of claim 26, wherein the drug is selected from the group consisting of doxorubicin, paclitaxel, vincristine, daunorubicin, vinblastine, actinomycin-D, but are not limited to, docetaxel, etoposide, teniposide, bisantrene, imatinib, cisplatin, 5-fluorouracil, adriamycin, methotrexate, methotrexate, busulfan, chlorambucil, cyclophosphamide, melphalan, small molecule drugs, nitrogen mustard, and nitrosourea. wherein the nanocomposite comprises at least one anticancer agent selected from the group consisting of nitrosourea. 21. The nanocomposite according to claim 20, wherein the nanocomposite has an absorption rate of 5 to 95 at a wavelength of 1,000 nm.

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