WO2014109459A1 - Method for preparing gold nanoparticles using micelles as mold - Google Patents

Abstract

The present invention relates to a method for preparing gold nanoparticles by forming nuclei of gold nanoparticles on the shell of micelles using nano-scale reducing micelles which include an antioxidant, as a mold, and then growing the nuclei of the gold nanoparticles. The gold nanoparticles prepared according to the present invention have a cog structure imitating the shape of a shell and surface of the micelles by forming and then growing the nuclei of the gold nanoparticles according to a high-arborescent surface structure of the micelles. In addition, the gold nanoparticles prepared according to the preparation method for the gold nanoparticles of the present invention has the same surface structure as that of the micelles and can be applied to the visualization of a surface structure of the micelles. Furthermore, the gold nanoparticles having a cog structure prepared according to the preparation method for the gold nanoparticles of the present invention can be applied as a probe particle for a drug delivery system and Raman spectrophotometry.

Description

Manufacturing method of gold nanoparticles using micelle as template

The present invention relates to a method of preparing gold nanoparticles by forming nuclei of gold particles on the outer shell of micelles and growing them using nano-scale reducing micelles containing an antioxidant as a template.

Gold nanoparticles (AuNP _S ) have attracted high interest due to their excellent chemical stability, biocompatibility, optical, electrical and catalytic properties, and have wide application of gold nanoparticles in drug delivery, electronics, sensing devices and catalysts. Due to the higher interest (1) P. Ghosh, G. Han, M. et al, Adv Drug Deliv Rev 60 (2008) 1307; 2) RJ Tseng, JX Huang, J. et al, Nano Letters 5 (2005) 1077; 3) GA Baker, DS Moore, Analytical and Bioanalytical Chemistry 382 (2005) 1751; 4) DT Thompson, Nano Today 2 (2007) 40).

Because topological factors, such as shape and size, have a significant impact on the properties of gold nanoparticles (AuNP _S ), controlling the phase in the nanoscale range is one of the most effective in the application of gold nanoparticles (AuNP _S ). It is considered important for improvement. To date, various methods have been developed to synthesize gold nanoparticles (AuNPs) with various shapes including spherical, rod, branched, polyhedral and hollow forms. (1) TK Sau, CJ Murphy, Journal of the American Chemical Society 126 (2004) 8648; 2) SF Pang, T. Kondo, T. Kawai, Chemistry of Materials 17 (2005) 3636; 3) BL Sanchez-Gaytan, SJ Park, Langmuir 26 (2010) 19170; 4) BE Brinson, JB Lassiter, CS Levin, R. Bardhan, N. Mirin, NJ Halas, Langmuir 24 (2008) 14166; 5) J. Perez-Juste, I. Pastoriza-Santos, LM Liz-Marzan, P. Mulvaney, Coordination Chemistry Reviews 249 (2005) 1870).

Among these spherical, rod, branched, polyhedral and hollow gold nanoparticles (AuNP _S ), anisotropic gold nanoparticles such as polyhedral and branched gold nanoparticles having a high surface-to-volume ratio (AuNPs) are of high interest because of their amazing catalytic activity and local surface plasmon resonance excitation for enhanced signals in surface enhanced Raman scattering (SERS). (1) HG Liao, YX Jiang, ZY Zhou, SP Chen, SG Sun, Angew Chem Int Ed Engl 47 (2008) 9100; 2) MK Hossain, Y. Kitahama, GG Huang, X. Han, Y. Ozaki, Anal Bioanal Chem 394 (2009) 1747).

To date, polyhedral, star-shaped and branched gold nanoparticles (AuNPs) have been reported capping surfactant cetyltrimethylammonium bromide (CTAB), deep eutectic solvents, and hydroxyphenols. Each derivative was synthesized using (1) S. Chen, ZL Wang, J. Ballato, S.H. Foulger, D.L. Carroll, J Am Chem Soc 125 (2003) 16186; 2) H.G. Liao, Y.X. Jiang, Z.Y. Zhou, S.P. Chen, S.G. Sun, Angew Chem Int Ed Engl 47 (2008) 9100; 3) Y. Lee, T.G. Park, Langmuir 27 (2011) 2965). In addition, high-branched dendritic gold nanoparticles (AuNPs) were used as surfactants, 3-heptadecafluorooctyl-sulfonyl aminopropyltrimethyl-ammonium iodide (HFOTAI) was used as a reducing agent (SF Pang, T. Kondo, T. Kawai, Chemistry of Materials 17 (2005) 3636).

Polymers comprising poly [1-methyl-3- (4-vinylbenzyl) -imidazolium chloride] (Poly [1-methyl-3- (4-vinylbenzyl) -imidazolium chloride]) in water-ethanol double solvent mixtures Polymeric ionic liquids (PILs) have also been used as stabilizers for the preparation of dendritic gold nanoparticles (AuNPs) (J. Zhang, L. Meng, D. Zhao, Z. Fei, Q. Lu, PJ Dyson, Langmuir 24 (2008) 2699).

However, according to the above-described method for producing dendritic gold nanoparticles (AuNPs), it took one week to one month and five to ten days to form dendritic nanoparticle structures in HFOTAI- and PIL-based systems, respectively. In addition, there is still a need for developing a new method for synthesizing dendritic gold nanoparticles (AuNPs), since the mechanism of formation of highly branched nanoparticle structures is not clear.

Accordingly, the present inventors have completed the present invention by confirming that dendritic gold nanoparticles (AuNPs) can be easily prepared using high-branched reducing micelles prepared using antioxidants and amphiphilic copolymers as templates. It was.

It is an object of the present invention to provide a method for preparing gold nanoparticles by forming nuclei of gold particles on the outer shell of micelles using nano-scale reducible micelles as templates and growing them.

In order to solve the above problems, the present invention comprises the steps of preparing a reducing micelle containing the antioxidant material by self-assembly using an amphiphilic copolymer and antioxidant (step 1), the micelle surface prepared in step 1 It provides a method for producing gold nanoparticles comprising the step of reducing the gold ions to form a nucleus according to the micellar shell structure (step 2) and growing the nucleus formed on the shell of the micelle (step 3). do.

In the method for preparing gold nanoparticles, step 1) is to prepare a reducing micelle containing an antioxidant, and the reducing micelle of the present invention is an amphiphilic block copolymer or graft copolymer having a hydrophilic polymer block and a hydrophobic polymer block. The coalescence of micelles takes the form of micelles or the amphiphilic lipids take the form of micelles by self-assembly, which encloses antioxidants therein.

The term "polymer" used in the present invention is used as a concept including an oligomer, and the "micelle form" refers to a core composed of a hydrophobic core and a hydrophilic shell by self-assembly of an amphiphilic polymer or lipid. It is used as a concept to refer to a nano-structure in the form of a shell (core-shell).

The term "self-assembly" used in the present invention is a spontaneous formation of a complex structure from the basic molecule without the help of a specific enzyme or factor, in the present invention, an amphiphilic block copolymer having a hydrophilic polymer block and a hydrophobic polymer block Graft copolymers or amphiphilic lipids form micelle structures in aqueous solution by self-assembly.

The amphiphilic block copolymer or graft copolymer of the present invention is composed of a hydrophilic polymer block (X) composed of chains of monomer X and a hydrophobic polymer block (Y) composed of chains of monomer Y. In this case, the monomer X may include ethyleneimine, alkylene oxide, oxazoline, vinylpyrrolidone, acrylamide, vinyl alcohol, amino acid, sacharide, and the like. Include. On the other hand, these polymers can be used regardless of the molecular weight, it is more preferable to use those having a weight average molecular weight of 1,000 to 300,000.

Meanwhile, according to the structure of the polyethyleneimine mentioned in the examples, there are linear polymers and branched polymers, and the preparation of amphiphilic copolymers is possible regardless of the structure, and the weight average molecular weight is generally 25,000 or less. It is preferable to use.

The hydrophobic polymer block (Y) may be a biodegradable hydrophobic aliphatic polyester-based polymer. Wherein monomer Y is D-lactic acid, L-lactic acid, DL-lactic acid, glycolic acid, caprolactone, amino acid, valerolactone, hydroxy butyrate, alkyl (meth) acrylate, (meth) acrylonitrile, (meth) acrylic acid Unsaturated carboxylic acids such as maleic acid, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl ether, styrene, p- or m-alkylstyrene, p- or m-chlorostyrene, p- or m-chloromethylstyrene, styrene Sulfonic acid and the like, and one or more selected from these monomers or a copolymer obtained by copolymerizing them are included. On the other hand, these polymers can be used regardless of the molecular weight, it is more preferable to use those having a weight average molecular weight of 1,000 to 300,000.

In the case of poly-D, L-lactic-co-glycolic acid (hereinafter referred to as "PLGA") in aliphatic polyester, the ratio of lactic acid and glycolic acid monomer is controlled, By modifying the polymer synthesis process, a biodegradable polymer having various decomposition lifetimes can be obtained, and its properties are excellent.

On the other hand, the block copolymer is a hydrophilic polymer block and a hydrophobic polymer block ester bonds, unhydride bonds, carbamate bonds, carbonate bonds, imine or amide bonds, secondary amine bonds, urethane bonds, phosphodiester bonds or hydra It may be covalently bonded through zone bonding. In addition, the polyethyleneimine (X) as the hydrophilic polymer and the aliphatic polyester polymer (Y) as the hydrophobic polymer may be a double block type of X-Y form or a triple block type composed of X-Y-X and Y-X-Y.

In the present invention, the amphiphilic lipid may be any kind of lipid capable of encapsulating an antioxidant, and dihexanoylphosphatidylcholine, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N- Distearyl-N, N-dimethylammonium bromide (DDAB), N- (1- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium chloride (DOTAP), N, N-dimethyl -(2,3-dioleoyloxy) propylamine (DODMA), N, N, N-trimethyl- (2,3-dioleoyloxy) propylamine (DOTMA), 1,2-diacyl-3- Trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP), 3beta- [N- (N ', N', N'-trimethylaminoethane) carbamoyl] cholesterol ( TC-cholesterol), 3beta [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol), 3beta [N- (N'-monomethylaminoethane) carbamoyl] cholesterol ( MC-cholesterol), 3beta [N- (aminoethane) carbamoyl] cholesterol (AC-cholesterol), choles Reyloxypropan-1-amine (COPA), N- (N'-aminoethane) carbamoylpropanoic tocopherol (AC-tocopherol) and N- (N'-methylaminoethane) carbamoylpropanoic tocopherol (MC Tocopherol), phosphatidylethanolamine, phosphatidylcholine and phosphatidic acid, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine Amine, dilinoleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1,2-dipitanoyl-3-sn-phosphatidylethanolamine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine , Dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dirinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1 , 2-dipitanoyl-3-sn-phosphatidylcholine, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid , Dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid, 1,2-dipitanoyl-3-sn-phosphatidic acid, or one or two or more combinations thereof. .

More preferably, the amphiphilic copolymer of the present invention is polyethyleneimine-g-poly (lactic-co-glycolic acid) (PEI-g-PLGA), poly (ethylene glycol) -g-polycaprolactone (PCL- g-PEG), PEG-PPG -PEG triblock copolymer (Pluronic ^® F-127), die hexanoyl phosphatidylcholine (DHPC) lipids can be used, but the shape of the bi-cell or albumin -PEG, but is not limited thereto.

The weight ratio of the hydrophobic polymer block (Y) and the hydrophilic polymer block (X) in the block copolymer or the graft copolymer is preferably 100: 1 to 1:10. If the amount of the hydrophobic polymer block (Y) increases, there is a problem that it does not form a stable nano micelles and precipitates, and if the amount of the hydrophilic polymer block (X) is too large, there is a problem that the core portion decreases, so the ratio is increased. It is preferable to adjust as described above. In addition, the graft copolymer is preferably an amphiphilic graft copolymer made of a hydrophobic polyester polymer in a polyethyleneimine main chain which is a hydrophilic polymer.

As the hydrophilic polymer block mentioned above, for example, polyethyleneimine has a linear polymer and a branched polymer according to its structure, and the formation of polymer nano micelles can be formed regardless of its structure. In the case of polyethyleneimine, the range which can be used depending on molecular weight is determined, and it is more preferable to use what is generally 10,000 or less.

Antioxidants of the present invention are α-tocopherol, polyphenols, retinol, retinyl palmitate, thioctic acid, carotenoids, vitamin C, vitamin C derivatives, caffeic acid, gallic acid and scopoletin At least one selected from the group consisting of, but is not limited thereto. In addition, the vitamin C derivative of the present invention may be ethyl ascorbyl ether or potassium ascorbyl tocopheryl phosphate, but is not limited thereto.

The polyethyleneimine and the biodegradable polyester copolymer (PEI-g-PLGA) of the present invention forms the structure of the cationic micelle in an aqueous solution, and the size of the cationic micelle formed is measured using a light scattering method (DLS), The nano size is about 150 nm to 200 nm.

According to an embodiment of the present invention is shown in Figure 1 the formation of dendritic AuNPs. PEI-g-PLGA micelles containing α-tocopherol using PEI-g-PLGA as the micelle-forming amphiphilic copolymer and α-tocopherol as antioxidants were hydrated with distilled water after co-solvent evaporation to film-casting It was prepared by the method.

In the method for preparing gold nanoparticles, step 2) is a step of forming gold nuclei along the micelle outer structure by reducing gold ions on the surface of the micelle prepared in step 1). At this stage Au ³⁺ ions along the surface of the micelles begin to reduce to Au °

According to one embodiment of the present invention, dendritic gold nanoparticles (AuNPs) may be prepared by adding HAuCl ₄ solution by the reduction gradient of the micelle surface formed by the antioxidant contained in the micelle without any additional reducing agent. The formation of nuclei of gold particles can be initiated by the reduction of Au ³⁺ ions after the aqueous HAuCl ₄ becomes AuCl ₄ ⁻ through electrostatic interchange with the protonated functional groups of the cationic micelles. Step 2) of the present invention is preferably carried out at room temperature.

In the method for preparing gold nanoparticles, step 3) is a step in which nuclei formed on the outer shell of the micelle are grown to generate gold nanoparticles. Once the nuclei of gold particles are formed on the micelle surface, large dendritic shapes may be formed by Au ° cluster formation and autocatalytic processes and / or growth of particle size by Oswald ripening. Can be. The dendritic form is a structure in which a protrusion structure extending from the center of the gold nanoparticles is formed.

Steps 2 to 3 of the present invention are preferably made in an aqueous solution, and thus formation and growth of gold nanoparticle nuclei may occur at the surface of the micelle.

Gold nanoparticles prepared in the present invention is formed by a cationic micelle prepared by self-assembly using an amphiphilic copolymer as a template, the shape is formed in a structure that mimics the shell and surface shape of the micelle. The structure of gold nanoparticles is a structure in which branches are developed in a dendric form from the center of nanoparticles. The degree of branching and the size of the gold nanoparticles can be controlled by varying the concentration of HAuCl ₄ (cyclolo (III) acid). According to one embodiment of the present invention, as the concentration of gold ions increases, the size of the gold nanoparticles increases. In addition, the amount of HAuCl ₄ used in the present invention is preferably 60 uM to 120 uM. When the amount of HAuCl ₄ is less than 60 uM, the structure of the gold nanoparticles is difficult to be formed, and when the amount of HAuCl ₄ is 120 uM or more, there is a problem that a dendritic structure in the form of a protrusion is formed.

In addition, the gold nanoparticles prepared by the present invention grow after the nucleus of the gold particles are formed along the high-branched surface structure of the micelles to have a protrusion structure that mimics the shell and surface shape of the micelles. Therefore, the gold nanoparticles prepared by the method for producing gold nanoparticles of the present invention are the same as the surface structure of micelles, and thus can be applied to visualization of micelle surface structures. In addition, the gold nanoparticles having a dendritic protrusion structure prepared by the method for preparing gold nanoparticles of the present invention can be applied as probe particles for drug carriers and Raman spectrophotometers.

According to the method for preparing gold nanoparticles using a reducing micelle containing the antioxidant substance of the present invention as a template, the shape and structure of the gold nanoparticles can be controlled by simple changes of micelle templates having various shapes and surface structures. Can be. In addition, since the structure of the gold nanoparticles is formed directly from the surface structure of micelles, it can be used as an alternative method of visualizing the surface structure of various micelles without the help of a special staining or a technique using a complicated microscope.

FIG. 1 shows a process of preparing gold nanoparticles using PEI-g-PLGA micelles containing α-tocopherol and a TEM image thereof.

FIG. 2A shows a TEM image showing the formation of dendritic AuNPs by PEI-g-PLGA micelles carrying α-tocopherol at various concentrations of HAuCl ₄ and a size distribution graph of AuNPs ((a) 30uM HAuCl ₄ ( b) 60 μM HAuCl ₄ (c) 120 μM HAuCl ₄ (d) 240 μM HAuCl ₄ ).

FIG. 2B shows the gold nanoparticle shape of the TEM image of FIG. 2A observed at low magnification.

Figure 3 shows the value of the zeta-potential with AuCl ₄ ^- concentration.

Figure 4A is a synthesis for 24 hours at room temperature, the gold particles by (a) using a _{30 uM HAuCl 4 (b) 60μM} HAuCl 4 (c) 120μM HAuCl 4 (d) 240μM HAuCl 4 in accordance with one embodiment of the present invention The absorption spectrum of subsequent gold nanoparticles is shown.

Figure 4B shows the time-dependent absorption spectrum of gold nanoparticles after (b) synthesis of gold nanoparticles at room temperature using (b) 60μM HAuCl ₄ according to an embodiment of the present invention.

5 shows EDS analysis images of gold nanoparticles (AuNP _S ) prepared using (b) 60 μM HAuCl ₄ according to one embodiment of the present invention. Regions of interest for elemental analysis are shown as red squares (internal, bars = 20 nm). Arrows indicate nitrogen (A) and oxygen (B) peaks.

6A shows TEM images and absorption spectra of gold nanoparticles formed using PEI-g-PLGA copolymer micelles.

FIG. 6B shows TEM images and absorption spectra of gold nanoparticles formed using PEI-g-PLGA copolymer micelles when α-tocopherol was added externally.

7A is a TEM image of gold nanoparticles formed using PEG-g-PCL copolymer micelles carrying α-tocopherol.

FIG. 7B shows the region of interest in red rectangles for elemental analysis of gold nanoparticles using a PEG-g-PCL copolymer micelle loaded with α-tocopherol (internal bar = 20 nm). Arrows indicate oxygen peaks.

FIG. 7C is a photodispersion analysis of the size distribution of gold nanoparticles formed using α-tocopherol-supported PEG-g-PCL copolymer micelles.

7D shows an absorption spectrum graph of gold nanoparticles formed using α-tocopherol-supported PEG-g-PCL copolymer micelles.

8A is a TEM image of gold nanoparticles formed using α-tocopherol-supported PEG-PPG-PEG triblock copolymer micelles. (a) 480 uM HAuCl ₄ (b) 960 μM HAuCl ₄

FIG. 8B is a photodispersion analysis of the size distribution of gold nanoparticles formed using α-tocopherol-supported PEG-PPG-PEG triblock copolymer micelles.

8C shows an absorption spectrum graph of gold nanoparticles formed using PEG-PPG-PEG triblock copolymer micelle loaded with α-tocopherol.

9 is a TEM image of gold nanoparticles formed using dihexanoylphosphatidylcholine (DHPC) lipid bicell loaded with α-tocopherol.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited by these examples.

material

Branched polyethyleneimine (PEI, Mw 10 KDa) was purchased from Polysciences (Warrington, PA). Poly (D, L-lactic-co-glycolic acid), Mw 14KDa, lactic acid, glycolic acid molar ratio = 50 / 50) was purchased from Boeringer Ingelheim (Ingelheim, Germany). Dichlorohexyl carbodiimide (DCC), N-hydroxyl succinimide (NHS), α-tocopherol and cichlorogold (III) acid (HAuCl ₄ ) were purchased from Sigma (St Louis, MO). The organic solvent was purchased from Merck (Darmstadt, Germany) and used without further purification. In addition, the method of preparing micelles was prepared according to a method known from MS Lee, MG Kim, YL Jang, K. Lee, TG Kim, SH Kim, TG Park, HT Kim, JH Jeong, Macromolecular Research 19 (2011) 688. .

Preparation Example 1 Preparation of PEI-g-PLGA Micelle

Poly (lactic-co-glycolic acid, PLGA, Mw 14,000) is dissolved at a concentration of 0.11 mM tetrahydrofuran, which is dichlorohexyl carbodiimide (DCC) and N-hydroxyl. The mixture was activated by mixing with succinimide (NHS), where the molar ratio of PLGA: DCC: NHS was 1: 6: 6. The activated PLGA was precipitated in hexane and dried under reduced pressure.

The activated PLGA was dissolved at a concentration of 0.02 mM on a co-solvent mixed with methylene chloride / DMSO in a 1: 1 ratio. On the other hand, branched PEI (bPEI, Mw 10,000) was prepared by dissolving at 0.01 mM in DMSO. The thus prepared PLGA and bPEI were mixed in a molar ratio of 2: 1 and stirred with a stirrer at room temperature.

The produced PEI-g-PLGA was dialyzed in deionized water (MWCO 10,000, Spectrun, Rancho Dominguez, Calif.), And passed through a filter to freeze-dry the undissolved solution to synthesize PEI-g-PLGA. Synthesis scheme was as follows, and the copolymer synthesis was confirmed using 1H-NMR and FT-IR spectroscopy.

Scheme 1

Preparation Example 2: Preparation of PEI-g-PLGA micelles carrying α-tocopherol

The micelle containing α-tocopherol was prepared by a film casting method using the PEI-g-PLGA copolymer prepared in Preparation Example 1. More specifically, 2 mg of α-tocopherol and 10 mg of PEI-g-PLGA were dissolved in dichloromethane (DCM) and stirred at room temperature for 30 minutes. After the film was formed in the flask using a rotary evaporator under reduced pressure, distilled water was added and dissolved in an ultrasonic bath. At this time, a transparent solution without a precipitate was obtained, and the solution remained transparent even after passing through a 0.80 μm filtration filter.

The micelle solution of non-ionized water was placed on a 300-mesh carbon-coated copper lattice and dried at ambient temperature. The lattice was stained with 2% uranyl acetic acid and observed under a transmission electron microscope (JEM-3010, ZEOL, Tokyo, Japan). Since the PEI-g-PLGA copolymer spontaneously forms cationic micelles in an aqueous medium, the water-insoluble antioxidant, α-tocopherol, could be loaded onto the hydrophobic core of the micelles. The micelle loaded with α-tocopherol (PEI-g-PLGA / Toco) was confirmed by transmission electron microscopy (TEM) to form spherical nanoparticles with a diameter of about 120 nm.

To quantify α-tocopherol, the nanoparticles were lyophilized, dissolved in DMSO and quantified using HPLC. Quantification using HPLC showed a loading of 11% and a loading efficiency of 73.3%.

Preparation Example 3: Preparation of PEG-g-PCL micelles carrying α-tocopherol

Micelles containing α-tocopherol were prepared by the film casting method using PEG-g-PCL. More specifically, 2 mg of α-tocopherol and 10 mg of PEG-g-PCL were dissolved in dichloromethane (DCM) and stirred at room temperature for 30 minutes. After the film was formed in the flask using a rotary evaporator under reduced pressure, distilled water was added and dissolved in an ultrasonic bath. At this time, a transparent solution without a precipitate was obtained, and the solution remained transparent even after passing through a 0.80 μm filtration filter.

Preparation Example 4: Preparation of PEG-PPG-PEG micelles carrying α-tocopherol

Micelles, including α- tocopherol using a PEG-PPG-PEG triblock copolymer (Pluronic ^® F-127) were prepared by film casting (casting) method. More particularly, the dissolved 4mg α- tocopherol and PEG-PPG-PEG triblock copolymer (Pluronic ^® F-127) 10mg in dichloromethane (DCM), and the mixture was stirred at room temperature for 30 minutes. After the film was formed in the flask using a rotary evaporator under reduced pressure, distilled water was added and dissolved in an ultrasonic bath. At this time, a transparent solution without a precipitate was obtained, and the solution remained transparent even after passing through a 0.80 μm filtration filter.

Preparation Example 5 Preparation of dihexanoylphosphatidylcholine (DHPC) lipid bicelle micelles carrying α-tocopherol

A micelle containing α-tocopherol was prepared by a film casting method using DHPC lipid bicelle (dihexanoylphosphatidylcholine, DHPC). More specifically, 4 mg of α-tocopherol and 10 mg of DHPC lipid bicelle (dihexanoylphosphatidylcholine (DHPC)) were dissolved in dichloromethane (DCM) and stirred at room temperature for 30 minutes. After the film was formed in the flask using a rotary evaporator under reduced pressure, distilled water was added and dissolved in an ultrasonic bath. At this time, a transparent solution without a precipitate was obtained, and the solution remained transparent even after passing through a 0.80 μm filtration filter.

Example 1 Synthesis of Gold Nanoparticles Using PEI-g-PLGA Micelle Containing Antioxidant α-Tocopherol (AuNPs)

Gold nanoparticles were prepared by varying the concentration of HAuCl ₄ slowly to a predetermined HAuCl ₄ concentration (30 uM, 60 uM, 120 uM and 240 uM) by slowly dropping HAuCl ₄ solution into PEI-g-PLGA micelle solution loaded with α-tocopherol. . The color of the solution immediately changed from colorless to reddish violet after the addition of cychlorogold (III) acid (tetra-chloroaurate, HAuCl ₄ ). The reaction was carried out for 24 hours. The resulting solution was purified by repeated centrifugation (14,000 rpm, 10 minutes) and resuspended in distilled water to prepare gold nanoparticles. The prepared gold nanoparticles were confirmed by TEM, EDS, spectrophotometer and light dispersion method.

Example 2 Synthesis of Gold Nanoparticles Using PCL-g-PEG Micelle Containing Antioxidant α-Tocopherol (AuNPs)

1 ml of PCL-g-PEG micelles containing α-tocopherol dissolved in water at a concentration of 1 mg / ml were mixed with cychlorogold (III) acid (tetra-chloroaurate, HAuCl ₄ ) to a concentration of 30 μM. After reacting in a stirrer for one day, the excess reactant was removed using a centrifuge, and the remaining gold particles were dissolved in water and resuspended to prepare gold nanoparticles. The prepared gold nanoparticles were confirmed by TEM, EDS, spectrophotometer and light dispersion method.

Example 3 Synthesis of Gold Nanoparticles Using Pluronic F-127 Micelles Containing Antioxidant α-Tocopherol (AuNPs)

1 ml of PEG-PPG-PEG triblock copolymer (Pluronic ^® F-127) micelle containing α-tocopherol dissolved in water at a concentration of 4 mg / ml was dissolved in tetra-chloroaurate (HAuCl ₄ ). Mix by mixing to 480μM, 960μM concentration. After reacting in a stirrer for one day, the excess reactant was removed using a centrifuge, and the remaining gold particles were dissolved in water and resuspended to prepare gold nanoparticles. The prepared gold nanoparticles were confirmed by TEM, spectrophotometer and light dispersion method.

Example 4 Synthesis of Gold Nanoparticles Using Dihexanoylphosphatidylcholine (DHPC) Lipid Bischel Containing Antioxidant α-Tocopherol (AuNPs)

1 ml of dihexanoylphosphatidylcholine (DHPC) containing α-tocopherol dissolved in water at a concentration of 1 mg / ml was mixed with tetra-chloroaurate (HAuCl ₄ ) to a concentration of 240 μM. . After reacting in a stirrer for one day, the excess reactant was removed using a centrifuge, and the remaining gold particles were dissolved in water and resuspended to prepare gold nanoparticles. The prepared gold nanoparticles were confirmed by TEM.

Experimental Example 1 Characterization of Gold Nanoparticles (AuNPs)

TEM images of gold nanoparticles (AuNP _S ) were measured using a JEOL JEM-3010 equipped with an EDS unit (Tokyo, Japan) as a transmission electron microscope (TEM). Absorbance spectra were measured using an Agilent Cary 5000 UV-Vis-NIR spectrophotometer (Santa Clara, Calif.). The hydrodynamic magnitude and zeta-potential were measured by a light scattering method with a He-Ne laser at a wavelength of 632 nm at a 90 ° measurement angle (zeta plus, Brookhaven Instrument Co, New York).

result

2 shows that gold nanoparticles (AuNP _S ) are produced from a PEI-g-PLGA micelle template. Each of the main branches developed from the center of the nanoparticles. This meant the formation of dendritic gold nanoparticle branches that spread from the PLGA core. The degree of branching and the size of the gold nanoparticles could be controlled by varying the concentration of AuCl ₄ ⁻ . The size of the gold nanoparticles increased to 165 ± 16.1 nm as the gold ion concentration increased.

As observed by the light scattering method, no aggregate of gold nanoparticles having a large particle size was observed. TEM images with low magnification also showed the formation of individually isolated AuNPs regardless of the respective concentration of HAuCl ₄ (FIG. 2B). Individual separation of particles appears to be effective due to the electrical repulsive force between particles due to the positive charge on the surface of AuNPs derived from amine groups of PEI on the surface.

The zeta-potential of AuNPs reduced with gold ions decreased with increasing gold ion concentration. This means that gold was deposited on the surface of the micelle-casting particles (FIG. 3). Red-transition of the absorption band by surface plasmon resonance (SPR) of AuNPs was observed according to the degree of branching and particle size (FIG. 4A), respectively. In particular, the highly branched AuNP _S formed in 60 uM HAuCl ₄ (sample b) showed a maximum SPR absorption at 627 nm. This is due to the relatively low aspect ratio nanorods, tripods and star-shaped gold nanoparticles with the tip of the branch protruding ((1) J. Perez-Juste, I. Pastoriza-Santos, LM Liz-Marzan, P. Mulvaney, Coordination Chemistry Reviews 249 (2005) 1870. (2) E. Hao, RC Bailey, GC Schatz, JT Hupp, SY Li, Nano Letters 4 (2004) 327. (3) F. Hao, CL Nehl, JH Hafner , P. Nordlander, Nano Lett 7 (2007) 729) and dendritic gold nanoparticles prepared by the method according to the invention have shown that they can behave similarly to each other. In contrast, AuNPs (sample d) formed in 240 uM HAuCl ₄ showed SPR maximal absorption at 533 nm, similar to spherical AuNPs (S. Eustis, MA el-Sayed, Chem Soc Rev 35 (2006) 209).

A wide SPR band was observed in AuNPs prepared with 30 uM and 120 uM HAuCl ₄ (samples a and c). This resulted from the overlap of the major SPR bands between 530 nm and 630 nm. 4B shows the evolution of the time-dependent absorption spectrum of sample b. As the reaction progressed, the maximum SPR absorption shifted from 690 nm to 645 nm. This indicated that gold grew continuously for more than one day in branched PEI molds, resulting in AuNPs with a more spherical smooth surface morphology over time.

Elemental analysis of AuNPs was performed using energy-dispersion spectroscopy (EDS). EDS patterns for gold, nitrogen and oxygen are shown in FIG. 5. Nitrogen from PEI and oxygen from the ester group of PLGA coexisted with gold in the outer shell portion (FIG. 5A) and the inner nuclear portion (FIG. 5B), respectively. Through the above results, AuNP _S was able to confirm that gold particles having a surface mimicking the micelle surface shape were formed on the highly branched surface of the PEI-g-PLGA micelle.

PEI has been reported to have the ability to reduce AuCl ₄ ⁻ to form AuNPs (PL Kuo, CC Chen, MW Jao, J Phys Chem B 109 (2005) 9445). AuNPs prepared through PEI showed a spherical shape having a particle size of 4 nm to 25 nm. In contrast, PEI-g-PLGA micelles carrying α-tocopherol promoted the formation of dendritic structures from branched PEI.

This allowed antioxidants released from PEI-g-PLGA to provide a reducing environment around micelles, which accelerated nucleation by the production of AuCl ₄ ⁻ on the PEI chain. α-tocopherol has a very low solubility in water because of its strong hydrophobic nature, which results in very limited release of antioxidants from micelles. Thus, α-tocopherol creates a reducing gradient surface surrounding the micelle's hydrophilic PEI corona (FIG. 1 II).

The ability of α-tocopherol to induce the growth of AuNPs has already been reported (W. Sermsri, P. Jarujamrus, J. Shiowatana, A. Siripinyanond, Anal Bioanal Chem 396 3079). This is consistent with previous findings, and addition of α-tocopherol to reaction medium containing PEI-g-PLGA also formed small spherical AuNPs with an average diameter of 15 ± 2.25 nm (FIG. 6B).

AuNPs could also be formed by the PEI-g-PLGA copolymer itself. However, the resulting morphology of the nanoparticles was mostly spherical (Fig. 6A). The results indicate that PEI-g-PLGA may be involved in the reduction of Au ³⁺ to Au ^{0 with} or without α-tocopherol, but PEI-g-PLGA without an α-tocopherol template This means that it is possible to induce gold nanoparticle formation through only transfer of reducing power, not as a role.

Also, poly (ethylene oxide) -b-poly (propylene oxide) -b-poly (ethylene oxide) (poly (ethylene oxide) -b-poly (propylene oxide) -b-poly (ethylene oxide, PEO-PPO-PEO ) And polyether based block-copolymers such as poly (ethylene oxide) -b-poly (propylene oxide) from AuCl ₄ ^- without additional reducing agent AuNP _S has been reported (1) T. Sakai, P. Alexandridis, Langmuir 20 (2004) 8426. (2) S. Goy-Lopez, P. Taboada, A. Cambon, J. Juarez , C. Alvarez-Lorenzo, A. Concheiro, V. Mosquera, Journal of Physical Chemistry B 114 (2010) 66).

However, according to the existing methods using such polyether-based block copolymers, studies on additional conditions for controlling and maintaining parameters are required to obtain the shape of the nanoparticles of the desired structure during the reaction. This is because the shape and size of AuNPs are influenced by several factors including PEO and PPO block composition, concentration, solvent properties, reaction time and temperature ((1) P. Khullar, V. Singh, A. Mahal , H. Kaur, V. Singh, TS Banipal, G. Kaur, MS Bakshi, Journal of Physical Chemistry C 115 (2011) 10442. (2) S. Chen, C. Guo, GH Hu, J. Wang, JH Ma , XF Liang, L. Zheng, HZ Liu, Langmuir 22 (2006) 9704).

In contrast, the process according to the invention led to the preparation of AuNP _S with the same morphology at room temperature and the same morphology was formed uniformly.

The present invention was also tested using other polymeric micelles with neutral surfaces other than PEI-g-PLGA. Poly (ethyleneglycol) -g-polycaprolactone (PEG-g-PCL) micelles containing α-tocopherol could also form AuNPs (FIG. 7A). In addition, the EDS pattern of gold nanoparticles prepared using PEG-g-PCL as a template showed that gold or oxygen atoms on PEG or PCL blocks co-directed PEG-g-PCL (FIG. 7B).

The surface structure of AuNPs formed from PEG-g-PCL template is characterized by the surface morphology of PEG-g-PCL micelles predicted by computer-aided coarse-grain molecular simulation (G. Srinivas, DE Discher, ML Klein, Nat. Mater 3 (2004) 638.) showed great similarity. Therefore, according to the method for producing gold nanoparticles of the present invention, it can be applied to visualize the fine structure of the micelle surface.

In the present invention it was also tested using a PEG-PPG-PEG triblock copolymer (Pluronic ^® F-127) micelle. a PEG-PPG-PEG triblock copolymer (Pluronic ^® F-127) involve α- tocopherol micelles could also form a AuNPs (Fig. 8).

The present invention was also tested using dihexanoylphosphatidylcholine (DHPC) micelles. DHPC micelles containing α-tocopherol could also form AuNPs (FIG. 9).

Claims (14)

1. Preparing a reducing micelle containing an antioxidant by self-assembly using an amphiphilic copolymer and an antioxidant material (step 1);

   Reducing gold ions on the surface of the micelles prepared in step 1 to form a nucleus according to the micelle shell structure (step 2); And

   A method for producing gold nanoparticles, comprising the steps of preparing a gold nanoparticle by growing a nucleus formed on the outer shell of a micelle.
2. The method of claim 1, wherein the antioxidant is α-tocopherol, polyphenol, retinyl palmitate, thioctic acid, carotenoids, vitamin C, vitamin C derivatives, Caffeic acid, gallic acid and scopoline (Scopoletin) is a method for producing gold nanoparticles, characterized in that at least one selected from the group consisting of.
3. The method of claim 1, wherein the amphiphilic copolymer is an amphiphilic block copolymer having a hydrophilic polymer block and a hydrophobic polymer block, an amphiphilic graft copolymer having a hydrophilic polymer block and a hydrophobic polymer block, or amphiphilic lipids Method for producing gold nanoparticles.
4. According to claim 3, The hydrophilic polymer block is any one monomer or two or more monomers selected from the group consisting of ethyleneimine, alkylene oxide, oxazoline, vinylpyrrolidone, acrylamide, vinyl alcohol, amino acid and sugar Method for producing gold nanoparticles characterized in that the copolymer copolymerized.
5. The method of claim 3, wherein the hydrophobic polymer block is D-lactic acid, L-lactic acid, DL-lactic acid, glycolic acid, caprolactone, amino acids, valerolactone, hydroxy butyrate, alkyl (meth) acrylate, (meth) acrylic Ronitrile, (meth) acrylic acid, maleic acid, vinyl acetate, vinyl propionate, vinyl butyrate, vinyl ether, styrene, p-alkylstyrene, m-alkylstyrene, p-chlorostyrene, m-chlorostyrene, p-chloro Method for producing gold nanoparticles, characterized in that the copolymer copolymerized with any one monomer selected from the group consisting of methyl styrene, m-chloromethyl styrene, styrene sulfonic acid or two or more monomers.
6. The amphiphilic lipid according to claim 3, wherein the amphiphilic lipid is dihexanoylphosphatidylcholine, N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), N, N-distearyl-N, N-dimethylammonium bromide ( DDAB), N- (1- (2,3-dioleoyloxy) propyl-N, N, N-trimethylammonium chloride (DOTAP), N, N-dimethyl- (2,3-dioleoyloxy) propyl Amine (DODMA), N, N, N-trimethyl- (2,3-dioleoyloxy) propylamine (DOTMA), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2- Diacyl-3-dimethylammonium-propane (DAP), 3beta- [N- (N ', N', N'-trimethylaminoethane) carbamoyl] cholesterol (TC-cholesterol), 3beta [N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol), 3beta [N- (N'-monomethylaminoethane) carbamoyl] cholesterol (MC-cholesterol), 3beta [N- (amino Ethane) carbamoyl] cholesterol (AC-cholesterol), cholesteryloxypropane-1-amine (COPA), N- (N'-aminoethane) carbamoylprop Norick Tocopherol (AC-tocopherol) and N- (N'-methylaminoethane) carbamoylpropanoic Tocopherol (MC-tocopherol), phosphatidylethanolamine, phosphatidylcholine and phosphatidic acid, dilauroyl phosphatidylethanolamine, dimyri Stoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dilinoleyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1 , 2-dipitanoyl-3-sn-phosphatidylethanolamine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleyl phosphatidylcholine, 1 Palmitoyl-2-oleoyl phosphatidylcholine, 1,2-dipitanoyl-3-sn-phosphatidylcholine, dilauroyl phosphatidine , Dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid, dirinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatide A method for producing gold nanoparticles, characterized in that any one or a combination of two or more selected from the group consisting of diacid, 1,2-dipitanoyl-3-sn-phosphatidic acid.
7. According to claim 3, wherein the block copolymer is a hydrophilic polymer block and a hydrophobic polymer block ester bonds, unhydride bonds, carbamate bonds, carbonate bonds, imine or amide bonds, secondary amine bonds, urethane bonds, phosphodiester A method for producing gold nanoparticles, which is covalently bonded through a bond or a hydrazone bond.
8. The method of claim 3, wherein the graft copolymer is an amphiphilic graft copolymer made of a hydrophobic polyester polymer in a polyethyleneimine main chain which is a hydrophilic polymer.
9. The gold nanoparticles of claim 1, wherein the amphiphilic copolymer is PEI-g-PLGA, PCL-g-PEG, PEG-PPG-PEG triblock copolymer, or dihexanoylphosphatidylcholine lipid bicell. Manufacturing method.
10. The method of claim 1, wherein the gold nanoparticles are manufactured according to the outer shell and surface shape of the micelle.
11. The method of claim 1, wherein the prepared gold nanoparticles are in a dendritic form.
12. The method of claim 1, wherein the micelle is nano-scale in size.
13. The method of claim 1, wherein the gold ion is HAuCl ₄ .
14. The method of claim 13, wherein the HAuCl ₄ is used at 60 uM to 120 uM.

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