KR20130094975A - Composites of sio2 shell/methylene blue hybrided au nano element and preparing method thereof - Google Patents

Abstract

PURPOSE: A SiO2 shell/MB-Au nanocore complex is provided to apply as a biosensor and molecular image contrast agent by having an excellent surface enhanced raman scattering (SERS) effect with hybridizing methylene blue with Au nano material. CONSTITUTION: A SiO2 shell/MB-Au nanocore complex comprises a coating layer and a core. The coating layer contains silica, and the core contains a gold (Au) nano material in which methylene blue is hybridized. The gold nano material is multicore gold nanoparticle or a gold nanorod. An average thickness of the coating layer is 1-500 nm. The SiO2 shell/MB-Au nanocore complex is used for a biosensor, a molecular image contrast agent, a capsule type endoscope and a treatment of cancer. The SiO2 shell/MB-Au nanocore complex enable a diagnosis of cancer and treatment of cancer at the same time. A manufacturing method of the SiO2 shell/MB-Au nanocore complex comprises the following step. The methylene blue and the gold nano material are hybridized by using the silica coating method.

Description

SiO2 shell / ＭＢ―Ａｕ nanocore composites and method for manufacturing the same {Composites of SiO2 shell / Methylene blue hybrided Au nano element and Preparing method}

The present invention relates to a SiO ₂ shell / MB-Au nanocore composite and a method for manufacturing the same, more specifically, methylene blue (hereinafter referred to as Au (gold) nanoparticles containing Au nanoparticles or Au nanorods) In order to introduce (MB), the present invention relates to an Au nanobody in which methylene blue is combined and hybridized with a silica (SiO ₂ ) coating method and a method of manufacturing the same. The present invention has excellent surface enhanced raman scattering (SERS) effect, it can be applied to products such as biosensors, molecular imaging contrast agents, capsule endoscope nanoparticles. In addition, the present invention can also treat cancer through the hyperthermia effect of Au and photodynamic therapy of methylene blue.

Metal nanoparticles such as gold (Au, Gold) have surface plasmon resonance due to the periodic oscillation of the electron clouds through the free electrons on the surface and the interference of the electromagnetic waves of external light. Plasmon Resonance (SPR). In particular, gold nanoparticles having a particle size of about 10 nm satisfy the SPR condition in light of about 520 nm wavelength according to Mie theory and causes strong extinction (Stintering and Absorption) as shown in FIG. 1.

Spherical gold nanoparticles have the same SPR phenomenon in all directions, while Au nanorods have mutually different electron clouds in the longitudinal and transverse directions from external electromagnetic waves, and thus, as shown in FIG. The SPR condition occurring in the longitudinal direction has a longer wavelength than the SPR condition in the transverse direction.

At this time, the larger the aspect ratio, that is, the longer the length of the gold nanorod, the absorption peak in the longer wavelength becomes red-shifted, and is generally about 700 to 1,000 nm NIR depending on the aspect ratio. (Near infrared) causes strong absorption in the wavelength of the region.

The SERS effect of the metal nanoparticles has recently been of great interest in that the molecular imaging method using quantum dots can overcome the disadvantage that the quantum dots cannot be used clinically due to the toxicity of the heavy metals of the quantum dots. I am getting it.

In general, Raman signals of organic dyes are too small to be applied to biosensors even though they have more information than Fourier Transform Infrared (FT-IR) signals.

However, when the metal nanoparticles are combined with the organic dye, the surface plasmon resonance (SPR) electrons of the metal nanoparticles move to the organic dye, and the Raman signal is amplified to about 10 ⁶ to 10 ¹² , and the metal nanoparticles are in the aggregate form ( When formed in an aggregated form, SPR electrons are amplified in the center. As shown in FIG. 2, this part is called a hot-spot area, and the Raman signal of the organic dye is greatly amplified. This effect is called a Surface Enhanced Raman Scattering (SERS) effect.

On the other hand, gold nanoparticles have recently received a lot of attention as a CT contrast agent and a fever treatment for cancer. However, aggregated particles of gold nanoparticles, such as gold nanoparticles, have a very unstable problem, and in order to overcome them, D8-4,4'-dipyridyl (D8-4,4'-dipyridyl, which is a laser active dye, has been applied to gold nanoparticles. -4,4'-dipyridyl) coating material is commercially available, but the laser active dye is very expensive (D8-4,4'-dipyridyl price 886,000 won / g, Sigma Aldrich).

Therefore, the present inventors have studied to develop a new metal nano-body that can be used as a biosensor, contrast agent, etc., methylene blue (MB) is already used as a therapeutic in the clinical and very low price (820 won / g, Sigma Aldrich) As shown in FIG. 3, MB shows bright fluorescence at a wavelength of 750 nm at excitation of 671 to 705 nm, and methylene blue having a concentration of about 80 to 300 M passes through a 3.5 mm thick pig skin. It was found that the fluorescence properties are known to be excellent enough to come out, and to know how to introduce them into the gold nano-body was completed the present invention.

The present invention for solving the above problems is a coating layer containing silica; And to provide a SiO ₂ shell / MB-Au nanocore composite comprising a; core containing methylene blue hybridized (hybrided) gold nanobody (core).

In addition, the present invention is to provide a method for producing a SiO ₂ shell / MB-Au nanocore composite in which methylene blue is combined with a gold nanobody using a silica coating method.

In the above production method, when the gold nanobody is a gold nanoparticle, methylene blue, tetraethylorthosilicate (TEOS) and ammonium hydroxide (NH) in an ethanol solution in which the multinucleated gold nanoparticles of the aggregated form are dispersed. ₄ OH), and then stirred to provide a method for producing a SiO ₂ shell / MB-Au nanocore composite characterized in that the multi-nuclear gold nanoparticles methylene blue bond and silica coating.

In the above method, when the gold nano-body is a gold nano-rod, preparing a PEG-coated gold nano-rod by introducing a PEG (polyethylene glycol) functional group to the gold nano-rod; Coating the PEG-coated gold nanorods with trimethoxysilan (MPTS) to produce a PEG / MPTS-coated gold nanorod in which silane groups are introduced; And removing or replacing PEG and MPTS of the PEG / MPTS-coated gold nanorods to prepare gold nanorods in which silica coating and methylene blue are combined; preparing a SiO ₂ shell / MB-Au nanocore composite To provide a method.

In addition, the present invention is to provide a biosensor, molecular imaging contrast agent, capsule endoscope and / or cancer therapy using the SiO ₂ shell / MB-Au nanocore complex.

The SiO ₂ shell / MB-Au nanocore composite of the present invention is very excellent in the SERS effect, and can be used in biosensors, molecular imaging contrast agents, capsule endoscopes, and the like. One of the methylene blue (MB) can be used as a cancer treatment by using the effect of generating free radicals to destroy cancer cells (photodymic effect) and the hyperthermia effect of gold nano-body. Furthermore, the complex of the present invention can simultaneously identify and treat cancer by using a capsule endoscope and CT.

1 shows Extinction and Surface Plasmon Resonance (SPR) conditions of gold nanoparticles.
FIG. 2 shows the hot-spot area and SERS effect of aggregated metal nanoparticles.
Figure 3 shows the fluorescence characteristics of methylene blue according to the concentration.
Figure 4 is a schematic diagram of the mechanism of cancer cell destruction of methylene blue.
5 is a photograph showing a solution containing gold nanoparticles aggregated by removing the solution and CTAB before the CTAB is removed from the gold nanoparticles.
6A is a TEM photograph of the gold nanoparticles synthesized in (1) of Example 1, and B is a TEM photograph of the SiO ₂ shell / MB-Au nanocore composite synthesized in Example 1. FIG.
7 is a Raman spectrum measurement results for comparing the SERS effect of Experimental Example 1.
Each of A to D in FIG. 8 is a TEM photograph of the SiO ₂ shell / MB-Au nanorod core composite prepared in Examples 2 to 5.
9 is a SERS effect measurement experiment results carried out in Experimental Example 2, A is the Raman spectrum measurement results of the composite and methylene blue of Example 1 and Example 2, B is the Raman spectrum measurement results of Oxonica products, C and Each D is a TEM photograph of the composite of Example 2 and Example 1.
FIG. 10 is a schematic diagram of conjugation of EGFR artificial binding protein (Affibody) and NLS peptide on the surface of the complex for experiments on cancer treatment effect of the SiO ₂ shell / MB-Au nanorod core complex carried out in Experimental Example 3. FIG.
11 is a fluorescence microscope measurement picture for the cancer treatment effect experiment of the SiO ₂ shell / MB-Au nanorod core complex carried out in Experimental Example 3, A is a fluorescence microscope measurement picture before incubating A431 cells with the complex, B is It is a photograph of fluorescence microscopy after incubation. (A) and (b) are fluorescence micrographs of MB (methylene blue), and (C) are fluorescence micrographs of DAPI (4 ', 6-diamidino-2-phenylindole) which is a cell nuclear dye.
12 is a schematic diagram of an embodiment in which the SiO ₂ shell / MB-Au nanobody core composite of the present invention is applied to a capsule endoscope auto read system.

As used herein, the term “gold nanoparticles” refers to gold nanoparticles having spherical, irregular spherical, and polyhedral forms. “Multicore gold nanoparticles” refers to a plurality of single gold nanoparticles. Refers to an aggregated form of particles.

Hereinafter, the present invention will be described in more detail.

The present invention hybridizes the commercially available methylene blue (hereinafter referred to as MB) with gold nanoparticles and / or gold nanorods using silica coating to produce new nanoparticles with excellent SERS effect. We want to synthesize

The present invention relates to a SiO ₂ shell / MB-Au nanocore composite, comprising a coating layer containing silica; And a core containing a gold nanobody bonded to methylene blue; wherein the gold nanobody is multicore gold nanoparticles or gold nanorods. It features.

Specifically, since gold nanoparticles have a smaller surface plasmon resonance (SPR) effect than gold nanorods, multinucleated gold nanoparticles containing MB in agglomerated form are used as shown in FIG. 2. However, since the gold nanorods have excellent SPR effect on their own, the SERS effect is excellent even if they are not agglomerated forms, unlike gold nanoparticles.

The multinucleated gold nanoparticles and the gold nanorods have an average diameter of 1 to 300 nm, preferably 2 to 100 nm, and particularly, when the average diameter of the gold nanoparticles exceeds 300 nm, due to sufficient coagulation effect. There is a problem that can not see the SPR effect, less than 1 nm is difficult in terms of manufacturing cost.

In addition, the coating layer has an average thickness of 1 to 500 nm, preferably an average thickness of 5 to 100 nm. Here, when the thickness of the coating layer is less than 1 nm, the stability is poor, the SERS effect may be weak, and when the thickness exceeds 500 nm, the total diameter of the composite may be too large, so it is preferable to have a coating layer thickness within the above range.

[ SiO ₂  Shell / MB - Au Nano core  Manufacturing Method of Composite]

The SiO ₂ shell / MB-Au nanocore composite of the present invention may be prepared by hybridizing methylene blue with a gold nanobody by using a silica coating method in order to introduce methylene blue into the gold nanobody. Are gold nanoparticles or gold nanorods.

In the method for producing the SiO ₂ shell / MB-Au nanocore composite, the method for producing the gold nanoparticles, the gold nanoparticles are multinuclear gold nanoparticles, the multinuclear gold nanoparticles After the preparation of gold nanoparticles in a CTAB (cetyltrimethylammonium bromide) solution, characterized in that the gold nanoparticles in the form (aggregated) by removing CTAB. In addition, the multinuclear gold nanoparticles are in the form of aggregated 2 to 10, preferably 4 to 8 gold nanoparticles.

Specifically, the multinuclear gold nanoparticles are prepared by: 1) preparing a gold seed solution by mixing HAuCl ₄ 3H ₂ O, a CTAB solution, and a reducing agent; 2) adding HAuCl ₄ 3H ₂ O, CTAB solution and ascorbic acid to the Au seed solution, followed by stirring to prepare gold nanoparticles; And 3) removing the CTAB from the gold nanoparticles to prepare gold nanoparticles in agglomerated form. And, as shown in Figure 5 before removing the CTAB from the gold nanoparticles, the red color, but CTAB is removed and the gold nanoparticles are agglomerated to become a light purple.

In addition, 3) preparing the gold nanoparticles in agglomerated form is 2) centrifuging the gold nanoparticles prepared in the step of preparing the gold nanoparticles to separate the gold nanoparticles, washed and then dispersed in distilled water Making (3-1); And removing the CTAB of the gold nanoparticles by centrifuging the distilled water in which the gold nanoparticles are dispersed and replacing the solvent with ethanol (3-2).

Here, the reducing agent is not particularly limited, but NaBH ₄ is preferably used.

In addition, the SiO ₂ shell / MB-Au nanocore composite of the present invention is methylene blue, tetraethylorthosilicate (TEOS) and ammonium hydroxide (NH ₄ ) in an ethanol solution in which the multinucleated gold nanoparticles of the aggregated form are dispersed. OH), and then stirred, can be prepared by hybridization and silica coating of methylene blue on the multinuclear gold nanoparticles.

In the method for producing the SiO ₂ shell / MB-Au nanocore composite of the present invention, when the gold nano-body is a gold nano-rod described a method for producing a gold nano-rod, the gold nano-rod is HAuCl ₄ · Mixing and vortexing the 3H ₂ O and CTAB solutions, followed by vortexing with the addition of a reducing agent, to prepare a gold seed solution comprising gold nanoparticles; And after aging the gold seed solution, and mixing with a growth solution (growing solution) to perform a crystal growth reaction to prepare a gold nano-rod; can be prepared by performing. The gold nanorods are separated by centrifuging the solution containing the gold nanorods prepared by the crystal growth reaction, washed with distilled water, and then dispersed in distilled water to obtain gold nanorods.

The growth solution may include CTAB, HAuCl ₄ · 3H ₂ O, AgNO ₃ and ascorbic acid. When the growth solution and the gold seed solution are mixed and the crystal growth reaction is performed, silver nitrate (AgNO ₃ ) acts as a catalyst for the growth of gold nanorods, and the solution color is Au (III) due to the reducing action of ascorbic acid. It becomes (0) and becomes a transparent solution.

Referring to the method of manufacturing the SiO ₂ shell / MB-Au nanocore composite of the present invention using a gold nanorod,

1) preparing a PEG-coated gold nanorod by introducing a PEG (polyethylene glycol) functional group into the gold nanorod; 2) coating the PEG-coated gold nanorods with trimethoxysilan (MPTS) to prepare a gold nanorod coated with PEG / MPTS having a silane group introduced therein; And 3) removing or replacing PEG and MPTS of the PEG / MPTS coated gold nanorods to prepare a gold nanorod hybridized with silica coating and methylene blue; and performing SiO ₂ shell / MB- of the present invention. Au nanocore composites can be prepared.

1) The step of preparing a PEG-coated gold nanorods can be prepared by reacting the gold nanorods synthesized using CTAB with m-PEG thiols, and unlike CT gold nanoparticles substituted CTAB with PEG The reason for this is that since CTAB is easily removed from the ethanol solvent, the gold nanorods are aggregated to replace CTAB with a PEG functional group.

The step of preparing the 2G PEG / MPTS-coated gold nanorods is to introduce a silane group to the gold nanorods, so that silica coating is uniformly performed on the gold nanorods. To coat silica by means of a stober method.

3) preparing the silica nanorods hybridized with silica coating and methylene blue may include mixing the PEG / MPTS-coated gold nanorods, ethanol, distilled water, methylene blue, ammonium hydroxide and tetraethyl orthosilicate (TEOS); It can make it react.

All steps of the method for preparing the SiO ₂ shell / MB-Au nanocore composite of the present invention using the gold nanorods described above may be performed under argon gas, preferably under a glove box. .

As shown in FIG. 2, the SiO ₂ shell / MB-Au nanocore composite of the present invention is a surface enhanced Raman (SERS) in which a Raman signal of methylene blue is greatly amplified in a hot-spot region of a gold nanobody or MB-Au nanocore. It has excellent scattering effect and can be used as a contrast agent for biosensor and molecular imaging.

And, conventional endoscopes have to endure pain or sleep anesthesia, but capsule endoscopes are based on the image data transmitted from the capsule endoscope to the receiver by simply swallowing and discharging after attaching the image data receiver to the body. As a result, the presence or absence of lesions can be analyzed, and the daily life is possible. However, the medical staff cannot control the endoscope directly, the accuracy is low due to the low magnification, and the cost of the procedure is very high due to the disadvantage that a large number of hundreds of thousands of image data measured by the capsule endoscope must be manually analyzed by a specialist. Therefore, no matter how convenient and safe capsule endoscopy for the patient's point of view, medical professionals and patients are reluctant to use the technology because the barriers to entry of the endoscopic endoscope are very high. In order to solve this problem, if the SiO ₂ shell / MB-Au nanocore complex of the present invention is used, nanoparticles may be selectively introduced into colon cancer cells or tissues through target orientation, thereby easily detecting the presence of cancer with methylene blue fluorescent light. Not only can it be confirmed, it is expected to be an encapsulated endoscope nanoparticle that can treat cancer cells by using the anticancer treatment effect of methylene blue, and is also expected to be used for the development of an automatic phantom system of the capsule endoscope.

In addition, cancer can be treated by the hyperthermia effect of the gold nano-body and the photodymic effect of methylene blue, and furthermore, cancer diagnosis can be performed simultaneously to use the SERS effect, the pyrogenic effect and the photodynamic effect. And cancer treatment is expected to be performed simultaneously.

Hereinafter, the present invention will be described in more detail with reference to preferred examples. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. It will be clear to those who have knowledge.

[Example]

Example 1 SiO ₂  Synthesis of Shell / MB-Au Nanoparticle Core Composites

(One) Au  Synthesis of nanoparticles

Au nanoparticles were synthesized in CTAB solution as follows.

First, 5mL of 0.1M CTAB solution and 0.0005M HAuCl ₄ 3H ₂ O 5mL were mixed, and then 0.6M of 0.01M NaBH ₄ was added to synthesize gold nanoparticle seeds. Next, 0.5 ml of the synthesized gold nanoparticle seed solution was added to 50 ml of 0.2 M CTAB, 50 ml of 0.001 M HAuCl ₄ ㆍ 3H ₂ O, and 0.7 ml of 0.0079 M Ascorbic acid. By stirring, gold nanoparticles having a uniform particle size of about 20 nm were synthesized, and a transmission electron microscopy (TEM) photograph thereof is shown in FIG. 6A.

Next, the synthesized gold nanoparticles were washed by centrifugation and then dispersed in distilled water.

(2) SiO ₂  Synthesis of Shell / MB-Au Nanoparticle Core Composites

20 mL of the gold nanoparticles dispersed in the distilled water was replaced with 20 mL of ethanol (EtOH) through centrifugation.

Gold nanoparticles dispersed in the ethanol was changed from the original red color to pale purple as shown in Figure 5 as the CTAB is removed and aggregated.

100 μl of 0.005 g methylene blue (MB), tetraethylorthosilicate (TEOS) and 2 ml of NH ₄ OH (29.4 wt.%) Were added to the ethanol in which the gold nanoparticles were dispersed, followed by 600 at 27 ° C. for about 5 hours. A strong stirring at rpm (Stirring) to give a silica (SiO ₂ ) coating.

In addition, the excess MB was removed by centrifugation, and the synthesized final product was dispersed in ethanol and the average particle size was about 185 nm as shown in the TEM photograph of FIG. 6-B. Looking at Figure 6-B it can be seen that the SiO ₂ shell / MB-Au nanoparticle core composite containing 4 to 8 gold nanoparticles inside the silica coating layer was synthesized.

Experimental Example 1 SiO ₂  SERS Effect Measurement of Shell / MB-Au Nanoparticle Core Composites

Raman was measured to examine the SERS effect of the SiO ₂ shell / MB-Au nanoparticle core composite prepared in Example 1, and the results are shown in FIG. 7. In the measurement conditions, the wavelength of the laser excitation (Excitation laser) was 785 nm, and the exposure time was 10 seconds.

As can be seen from the Raman spectrum shown in A of FIG. 7, the Raman signal of pure MB is very weak. However, looking at B of Figure 7, it can be seen that the Raman signal of the silica-coated MB-multicore gold nanoparticles is very strongly amplified compared to MB.

This Raman signal difference can be confirmed that the SERS effect is amplified by MB entering the hot-spot area of the multicore where gold nanoparticles are aggregated.

Example 2 SiO ₂  Synthesis of Shell / MB-Au Nanorod Core Composites

(1) Synthesis of Gold Nanorod Seeds

(1-1) gold Nano rod Seed's  synthesis

Gold nanorod seed was synthesized using NaBH ₄ which is a strong reducing agent in CTAB solution as follows.

5 ml of 0.02M CTAB and 5 ml of 0.005M HAuCl _{4 were} added to the vial and vigorously vortexed for about 1 minute, followed by 900 μl of ice cold 0.01M NaBH ₄ and vortexed again for 3 minutes. .

The color of the solution was brown with the formation of gold nanoparticles of about 3 to 5 nm, and the gold nanorod seeds thus prepared were used for gold nanorod synthesis after 3 hours of aging.

(1-2) Synthesis of Growing Solution

100 ml of 0.2 M CTAB, 100 ml of 0.001 M HAuCl ₄ , 0.001 M AgNO _{3 in} 250 ml Erlenmeyer flask A growth solution was prepared by mixing 5 ml and 1.4 ml of 0.0788 M ascorbic acid.

The AgNO ₃ serves as a catalyst for the growth of gold nanorods, and the color of the solution becomes transparent as Au (III) becomes Au (0) due to the reduction action of ascorbic acid.

(2) Synthesis of Gold Nanorods

The mixed solution containing 240 μl of the gold nanorod seeds in the growth solution was stirred for at least 12 hours to complete the crystal growth reaction of the gold nanorods.

The reaction solution was centrifuged at 15,000 rpm for 30 minutes for washing, washed twice with distilled water, and then dispersed in distilled water again. The distilled water (or solution) in which the gold nanorods were dispersed was burgundy.

(3) SiO ₂  Shell / MB - Au Nano rod  Synthesis of Core Complexes

(3-1) Polyethylene glycol (PEG) coating

The gold nanorod synthesized using CTAB solution has a problem that the gold nanorods are agglomerated easily because CTAB is easily removed from the ethanol solvent when silica is coated.The m-PEG thiol is used to convert the CTAB to a PEG functional group ( functional group), and the Stober method was carried out as follows.

0.01 g of m-PEG thiol was added to 40 ml of the distilled water in which the gold nanorods were dispersed and reacted for 3 hours to coat or replace CTAB on the surface of the gold nanorods with PEG, and then, the solvent was centrifuged. Substituted with ethanol.

The PEG-coated gold nanorods had no agglomeration at all and maintained a burgundy color that was the color of the original gold nanorod solution.

(3-2) MPTS (methacryloxypropyltrimethoxysilane) coating

When the silica coated silica coated nanorods by the stove method, the silica is not uniformly coated due to the PEG attached to the surface of the gold nanorods. Therefore, by introducing a silane group (Silane group) to the gold nanorods, MPTS was first coated on the gold nanorods as follows in order to achieve a uniform silica coating.

500 μl of MPTS was added to 40 mL of ethanol in which gold nanorods were dispersed, followed by stirring for 12 hours, followed by PEG and MPTS coated gold nanorods (hereinafter referred to as PEG / MPTS-gold nanoparticles). Called a rod). And, excess MPTS not bound with the gold nanorods was removed by centrifugation.

(4) SiO ₂  Synthesis of Shell / MB-Au Nanorod Core Composites

Into a solution of 20 ml of PEG / MPTS-gold nanorod, 64 ml of ethanol, 16 ml of distilled water and 0.05 g methylene blue, 2 ml of NH ₄ OH and 5 µl of TEOS (injected 5 times at 1 µl in 30 minutes) The SiO ₂ shell / MB-Au nanorod core composite was synthesized by stirring for 4 hours 40 minutes.

All reactions were carried out under argon gas in a glove box, and after the reaction was completed, the synthesized SiO ₂ shell / MB-Au nanorod core complex was washed with ethanol through centrifugation and dispersed in 20 ml ethanol. It was.

The TEM photograph of the synthesized SiO ₂ shell / MB-Au nanorod core composite is shown in FIG. 8, and the average thickness of the synthesized SiO ₂ shell (or silica coating layer) was 20 nm.

Examples 3 to 5: SiO ₂  Synthesis of Shell / MB-Au Nanorod Core Composites

Synthesis of the SiO ₂ shell / MB-Au nano-rod core composite in the same manner as in Example 1, when synthesizing the SiO ₂ shell / MB-Au nano-rod core composite of (4), as shown in Table 1 By adjusting the amount of use, the SiO ₂ shell / MB-Au nanorod core composite having an average thickness of 20 nm, 5 nm, and 1 nm of silica coating layer was prepared, and Examples 3 to 5 were prepared, respectively. It is shown to B-D.

division PEG / MPTS-
Gold nanorod ethanol Distilled water Methylene blue NH ₄ OH TEOS Example 2 20 ml 64 ml 16 ml 0.05g 2 ml 5 μl Example 3 20 ml 64 ml 16 ml 0.05g 2 ml 4 μl Example 4 20 ml 64 ml 16 ml 0.05g 2 ml 3 μl Example 5 20 ml 64 ml 16 ml 0.05g 2 ml 2 μl

Experimental Example  2 : SiO ₂  Shell / MB - Au Nano rod  Core composite SERS  Effect measurement experiment

SERS effect measurement experiment of the SiO ₂ shell / MB-Au nano-rod core composite prepared in Example 2 was carried out in the same manner as in Experimental Example 1, the results are shown in FIG. In addition, the Raman spectra of the SiO ₂ shell / MB-Au nanoparticle core composite prepared in Example 1 and MB itself are shown in FIG. 9A, and in FIG. 9B, D8-4,4′- is a Raman active dye. The Raman spectrum measurement result of Oxonica (Comparative Example 1) using dipyridyl (D8-4,4'-dipyridyl) is shown.

Looking at C and D of Figure 9, despite the particle size (C of Figure 9) of the composite of Example 2 is smaller than the particle size (D of Figure 9) of the composite of gold nanoparticles of Example 1, It can be seen from A of FIG. 9 that the Raman signal was amplified more largely due to the strong SERS effect of the rod.

More importantly, the Raman signal of the complex of the present invention using the MBs of Examples 1 and 2 was almost similar or rather greater in intensity of the Raman signal even when compared to Comparative Example 1 (B in Fig. 9). From this, it can be confirmed the superiority of the SERS effect of the present invention.

Experimental Example 3: SiO ₂  Experimental Effect of Cancer on Shell / MB-Au Nanorod Core Complex

A synthetic body was synthesized in Example 2 to target an epidermal growth factor receptor (EGFR) in the cytoplasm of cancer cells, and a NLS peptide (nuclear localization signal peptide) to target the nucleus was synthesized in Example 2. Conjugation was performed on the surface of the SiO ₂ shell / MB-Au nanorod core composite, and a schematic diagram thereof is shown in FIG. 10.

After incubating the complex prepared in Example 2 with 100 μg / ml of _{distilled water} for 24 hours with A431 cells, the result was confirmed by fluorescence microscopy. Surprisingly, even though no treatment was performed on A431 cells, all cell walls were destroyed and cancer cells were killed. It was confirmed through a fluorescence microscope, and the results are shown in FIG.

Looking at the fluorescence expression picture of B of Figure 11 it can be seen that the complex of Example 2 is present in a large amount in the A431 cell nucleus effectively due to the NLS peptide. DAPI (4 ', 6-diamidino-2-phenylindole) of FIG. 11 is a dye that stains the nucleus of a cell, and in this study, DAPI is used to determine the nucleus position of cancer cells in order to find out whether the NLS peptide is attached to the nanoparticles. As confirmed through the comparison of (a), (b) and (c) of FIG. 11B, the locations of MB and DAPI are almost identical. In other words, it can be seen that the nanoparticles to which the NLS peptide is attached enter the nucleus of cancer cells.

The reason why A431 cells are killed is that MB is reduced by NAD (P) H: quinone oxidoreductase (NQO1) and oxygen is reduced to generate reactive oxygen species (ROS) to stimulate mitochondria and caspase- It is believed that cancer cells were effectively killed by destroying DNA by activating 3 (Caspase-3).

In addition, such a cancer therapeutic effect is also a SiO ₂ shell / MB-Au nano Czech complex as well as, SiO ₂ shell / MB-Au nano Czech composite manufactured using the gold nano-particles prepared by using the gold nano-rods It can be seen that the cancer cell treatment effect.

At present, the organic phosphor is used for mapping the sentinel lymphnude to confirm whether cancer cells have metastasized after the third stage of cancer. Therefore, the SiO ₂ shell / MB-Au nanorod core complex incorporating the methylene blue of the present invention provides lymphoid mapping. Through cancer can be confirmed not only the presence of cancer cells can be destroyed.

That is, not only can be used for the biosensor, molecular imaging contrast agent, capsule endoscope of the present invention, as shown in the schematic diagram of the embodiment applied to the automatic capsule endoscope reading system of the present invention of Figure 12, capsule endoscope and CT Simultaneously, the presence and / or treatment of cancer may be simultaneously used.

Claims (21)

A coating layer containing silica; And a core containing a gold (Au) nanobody hybridized with methylene blue; SiO ₂ shell / MB-Au nanocore composite. The method of claim 1,
The gold nanobody is a SiO ₂ shell / MB-Au nanocore composite, characterized in that the multicore gold nanoparticles or gold nanorods. The method of claim 2, wherein the multinuclear gold nanoparticles and gold nanorods are
SiO ₂ shell / MB-Au nanocore composite, characterized in that the average diameter is 1 ~ 300 nm. The SiO ₂ shell / MB-Au nanocore composite according to claim 1, wherein the coating layer has an average thickness of 1 to 500 nm. A biosensor comprising the SiO ₂ shell / MB-Au nanocore composite of any one of claims 1 to 4. Molecular imaging contrast agent comprising the SiO ₂ shell / MB-Au nanocore composite of any one of claims 1 to 4. A capsule endoscope comprising the SiO ₂ shell / MB-Au nanocore composite according to any one of claims 1 to 4. Any one of claims 1 to cancer treatment, characterized in that includes any one of the SiO ₂ shell / MB-nm Au core composite selected from (4). The SiO ₂ shell / MB-Au nanocore complex according to any one of claims 1 to 4, wherein cancer diagnosis and cancer treatment can be performed simultaneously. A method for producing a SiO ₂ shell / MB-Au nanocore composite, characterized in that methylene blue is hybridized with a gold nanobody using a silica coating method. 11. The method of claim 10, wherein the gold nano-body method of producing a SiO ₂ shell / MB-nm Au core composite, characterized in that the gold nanoparticles or gold nanorods. The method according to claim 11, wherein the gold nanoparticles are multinuclear gold nanoparticles,
The multinuclear gold nanoparticles are
After preparing gold nanoparticles in a CTAB (cetyltrimethylammonium bromide) solution, the method for producing SiO ₂ shell / MB-Au nanocore composites characterized in that the gold nanoparticles in the form of agglomeration (aggregation) by removing CTAB. The method according to claim 11, wherein the gold nanoparticles are multinuclear gold nanoparticles,
The multinuclear gold nanoparticles are
Preparing a gold seed solution by mixing HAuCl ₄ 3H ₂ O, a CTAB solution, and a reducing agent;
Preparing gold nanoparticles by adding HAuCl ₄ · 3H ₂ O, CTAB solution and ascorbic acid to the Au seed solution, followed by stirring; And
Removing the CTAB from the gold nanoparticles to produce agglomerated gold nanoparticles;
Method for producing a SiO ₂ shell / MB-Au nanocore composite characterized in that it was prepared by performing. Methylene blue, tetraethylorthosilicate (TEOS) and ammonium hydroxide (NH ₄ OH) were added to an ethanol solution in which the multinucleated gold nanoparticles of the aggregated form were dispersed, and then stirred and methylene blue to the multinuclear gold nanoparticles. Method for producing a SiO ₂ shell / MB-Au nanocore composite characterized in that the hybrid and silica coating. Of claim 12 to claim 14 according to any one of claims, wherein the production of the multi-nuclear gold nanoparticles SiO ₂ shell / MB-Au nano-core, characterized in that 2 to 10 of gold particles in an aggregated form complexes . The method of claim 10, wherein the gold nanobody is a gold nanorod,
The gold nanorods
Mixing and vortexing HAuCl ₄ 3H ₂ O and CTAB solutions, followed by vortexing with the addition of a reducing agent, to prepare a gold seed solution comprising gold nanoparticles; And
Aging the gold seed solution, and then mixing with a growing solution to produce a gold nanorod by crystal growth reaction;
Method for producing a SiO ₂ shell / MB-Au nanocore composite characterized in that it was prepared by performing. 16. The method of claim 15,
The growth solution is a method of producing a SiO ₂ shell / MB-Au nanocore composite, characterized in that it comprises CTAB, HAuCl ₄ · 3H ₂ O, AgNO ₃ and ascorbic acid. Preparing a PEG-coated gold nanorod by introducing a PEG (polyethylene glycol) functional group into the gold nanorod;
Coating the PEG-coated gold nanorods with trimethoxysilan (MPTS) to produce a PEG / MPTS-coated gold nanorod in which silane groups are introduced; And
Preparing a gold nanorod in which silica coating and methylene blue are combined by removing or replacing PEG and MPTS of the PEG / MPTS-coated gold nanorod;
Method for producing a SiO ₂ shell / MB-Au nanocore composite comprising a. The method of claim 18, wherein the step of preparing the PEG coated gold nanorods
A method for producing a SiO ₂ shell / MB-Au nanocore composite, characterized in that the gold nanorod synthesized using CTAB was prepared by reacting with m-PEG thiol. The method of claim 18, wherein the step of preparing a gold nano-rod hybrid of the silica coating and methylene blue is
The SiO ₂ shell / MB-Au nanocore composites prepared by mixing and reacting the PEG / MPTS-coated gold nanorods, ethanol, distilled water, methylene blue, ammonium hydroxide and tetraethyl orthosilicate (TEOS) Manufacturing method. 21. The method of claim 20, wherein TEOS is process for producing a SiO ₂ shell / MB-nm Au core composite characterized in that the input divided.

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