WO2016072730A1 - Method for manufacturing plasmon particles containing nanopetal structures and use of particles manufactured thereby - Google Patents

Abstract

The present invention relates to a method for manufacturing metal nanostructures which are anisotropically branched and grown on a surface of a substrate having a first metal surface and, specifically, to a method for manufacturing branched nanostructures, the method comprising: a first step of forming a polydopamine coating layer on the substrate through a linkage of the first metal surface and a catechol group of polydopamine; and a second step of anisotropically branching and growing second metal nanostructures on the first metal substrate surface by oxidizing the catechol group of the polydopamine into quinone through a catalytic action of the metal surface to reduce a second metal precursor while inducing oxidative nanopeeling due to the oxidative disruption of the polydopamine coating layer, and to a use of plasmon particles comprising the thus manufactured metal nanostructures, as a photo-sensitizer for photodynamic and/or photothermal therapy.

Description

Method for producing plasmon particles comprising nano-petal structures and uses of the particles prepared accordingly

The present invention provides a method for producing a metal nanostructure anisotropically branched and grown on a substrate surface having at least a first metal surface, by combining the first metal surface with a catechol group of polydopamine, Forming a polydopamine coating layer; And oxidizing the catechol group of polydopamine to quinone through the catalysis of the metal surface, inducing oxidative nano peeling due to oxidative collapse of the polydopamine coating layer, and reducing the second metal precursor on the surface of the first metal substrate. A method for producing a branched nanostructures comprising a second step of anisotropically branching and growing a second metal nanostructure and photosensitive for photodynamic and / or photothermal treatment of plasmon particles comprising the metal nanostructures thus prepared. It relates to the use as a zero.

Plasmon metal nanostructures are attracting attention because of their strong and controllable optical properties and their potential for use in biosensing, bioimaging and therapeutic applications. Near-infrared (NIR) -mediated photo-therapeutic approaches using the plasmon nanostructures, such as photodynamic therapy (PDT) and photothermal therapy (PTT), provide high spatial resolution, improved target selectivity, It has a number of advantages, including reduced side effects, non-invasive, non-invasive, fast and effective treatment and low cost over conventional cancer treatment. PDT uses organic photosensitizer (PS) molecules to convert normal tissue oxygen ( ³ O ₂ ) into highly reactive cytotoxic singlet oxygen ( ¹ O ₂ ) using site-selective exposure to light of a specific wavelength. Is associated with. For highly effective PDT, the wavelength of light used should match the maximum absorption wavelength of the PS in the NIR region (700-1100 nm; phototherapeutic NIR window to avoid blood and tissue interference). Unfortunately, most therapeutic PS molecules absorb light in the visible range and tend to photolyze upon prolonged exposure to light, and continued PDT treatment can lead to severe local hypoxia due to tissue oxygen deficiency, which can inhibit further PDT performance. . PTT, on the other hand, is exposed to sustained or pulsed lasers, which induce hyperthermia and / or subsequent small shock waves, leading to local temperature rise dependent apoptotic or necrotic cancer cell death. Plasmon nanoparticles such as particles (AuNPs) are used. Even when the PTT method is applied, it is preferable to use light in the NIR region in order to overcome the disadvantage that the intensity of light absorbed by the nanoparticles may be reduced due to absorption by moisture or red blood cells in vivo. Effective hyperthermic necrotic destruction of cancer cells involves very high temperatures (> 70 ° C.) and can lead to secondary damage and unwanted modification of nanostructures in normal cells. For less invasive cancer cell death, a cold (<45 ° C.)-Based PTT strategy can be used. Furthermore, under continuous treatment conditions, cancer cells can acquire resistance to further treatment.

For this reason, there is an attempt to develop a PDT-PTT binding platform associated with various types of nanostructures that are being developed recently. These nanostructures allow the use of moderate hyperthermia with free radical species (ROS) -mediated intracellular damage. Challenges still to be addressed, including possible discrepancies between absorption wavelengths of PS and plasmon nanostructures, energy transitions between PS and nanostructures, the need for low operating temperatures or non-thermal treatments, toxicity of nanostructures, and complex conjugation chemistry All of these can be explained by the use of the hybrid nanostructure-based approach for practical applications. In particular, effective photothermal nanotransducers have high optical absorption cross-section, biocompatibility, easy synthesis, high structural accuracy and yield, and plasmon tunability in the NIR region. Must have To date, few have attempted to combine the ROS production of NIR-active plasmon nanostructures with their inherent hyperthermic effects for potential NIR laser-based cancer phototherapy that does not require organic PS molecules.

Branched plasmon nanostructures, such as nanostars, nanoflowers and nanolaces, can form strong electromagnetic fields inside particles by means of close and coupled sharp nanofeatures. Such strong plasmon coupling and large surface area are useful features for surface-enhanced Raman scattering (SERS), photothermal conversion and catalysis. Such branched plasmon nanostructures may be useful PDT and PTT substrates, but solution phase large-scale synthesis for growing anisotropically branched Au nanostructures with high structural accuracy and controllability may require high diffusion coefficients of Au atoms and Face-centered cubic faceting tendency is difficult to implement. Anisotropic growth of these nanobranches is dominant in kinetics and high-energy growth is rarely seen in reactions faster than low-energy growth. Furthermore, it is difficult to optimize these reaction conditions with varying surfactants in order to produce structurally reproducible uniform nanostructures in large quantities.

Thus, the inventors have made extensive research efforts to achieve controlled growth and / or synthesis of plasmon nanobranched structures with controllable structural properties and densities on metal surfaces using oxidative nanopeel chemistry of polydopamine (pdop). As a result, a polydopamine coating layer was formed on the surface of the substrate having a metal surface by bonding with a catechol group, and the catechol group was oxidized to quinone through catalysis of the metal surface to induce oxidative peeling of the polydopamine coating layer and at the same time a metal precursor. The nano-petal structure was prepared by reducing the anisotropically branched and / or grown metal nanostructure on the surface of the metal substrate, and the nano-petal structure has excellent light-heating effect and ROS generation ability. It can be used as an effective photosensitizer for the treatment of PTT / PDT The present invention has been completed.

As one aspect for achieving the above object, the present invention provides a method for producing a metal nanostructure anisotropically branched and grown on a substrate surface having at least a first metal surface, on the substrate, a first metal surface A first step of forming a polydopamine coating layer by combining with a catechol group of polydopamine; And oxidizing the catechol group of polydopamine to quinone through the catalysis of the metal surface, inducing oxidative nano peeling due to oxidative collapse of the polydopamine coating layer, and reducing the second metal precursor on the surface of the first metal substrate. Provided is a method for manufacturing a branched nanostructure comprising a second step of anisotropically branching and growing a second metal nanostructure.

In another aspect, the present invention provides plasmon particles comprising branched nanostructures prepared by the above method.

As another aspect, the present invention provides a photosensitive therapeutic composition for photodynamic therapy comprising the plasmon particles.

As another aspect, the present invention provides a photosensitive therapeutic composition for photothermal therapy comprising the plasmon particles.

In another aspect, the present invention provides a surface enhanced Raman scattering (SERS) substrate comprising the branched nanostructures.

In the method for producing a nano-petal structure comprising the step of introducing the polydopamine coating layer of the present invention, the polydopamine can be assembled stably and uniformly on the metal surface, the bond through the catechol group is Au (III) Induction oxidation may cause partial decomposition of the polydopamine coating layer and grow anisotropically by controlling the length and / or density of protrusions formed thereon. The absorption spectrum can be controlled through the control of such nanostructures, which can be usefully used for the preparation of probes useful for the treatment of PTT / PDT using near infrared rays.

Furthermore, the nano-petal structure of the present invention prepared by the above method does not exhibit toxicity by itself, but because of its synergistic effect, it is relatively low temperature (~ 42 ° C.) because it exhibits photothermal effect as well as excellent ROS generation ability by irradiating near infrared rays. ), It is possible to minimize the damage to the surrounding normal cells because of the effective chemotherapy.

1 is a view showing a method of synthesizing a core-petal structure and the shape of the synthesized structure according to the present invention. (a) is a schematic diagram illustrating the oxidative nano-peel chemistry of polydopamine (pdop) on gold nanoparticles (AuNPs) for the synthesis of plasmon core-petal nanostructures (CPNs). (b) shows a TEM image of pdop-AuNPs with 80-nm Au core and ˜5-nm pdop layer. (c) shows a TEM image of pdop-AuNPs immediately after HAuCl ₄ addition. (d) shows a TEM image 1 minute after adding HAuCl ₄ to pdop-AuNPs. (e) shows SEM images of CPNs. (f) shows a TEM image (left) of CPNs and an enlarged petal image (right) in a box. (g) shows TEM images of AuNP-CPN-1, AuNP-CPN-2, AuNP-CPN-3 and AuNP-CPN-4 synthesized with increasing amounts of HAuCl ₄ from left to right. CPN represents AuNP-CPN in the whole figure including FIG.

Figure 2 is a diagram showing the results of XPS analysis of pdop-AuNPs (80 nm Au core) according to the present invention. (a) is the XPS survey spectrum of pdop-AuNPs, (b) and (c) show deconvoluted high resolution C 1s and O 1s peaks respectively, and (d) shows high resolution N 1s peaks. (e) shows the comparison of Au 4f peaks of pdop-AuNPs and citrate-AuNPs.

3 shows Raman spectra (514 nm laser, 10 mW laser power) of pdop-AuNPs before (bottom spectrum) and after (top spectrum) HAuCl ₄ treatment.

FIG. 4 shows 13 C NMR (400 MHz in D ₂ O) of pdop-AuNPs before (bottom spectrum) and after (top spectrum) HAuCl ₄ treatment.

5 is a diagram showing the morphological and spectroscopic characteristics of the synthesized AuNP-CPNs. (a) is a TEM image and a visual representation of CPNs synthesized using 10, 50, 100 and 200 molar equivalents of amine hydroxides. (b) shows UV-Vis spectra corresponding to the TEM image shown in (a).

FIG. 6 shows TEM images of pdop-AuNPs (80 nm Au core) without amine hydroxide added after 1 hour of HAuCl ₄ addition at room temperature.

7 is a view showing the optical characteristics of the core-petal nanostructures according to the present invention. (a) shows UV / Vis spectra of AuNPs, pdop-AuNPs, AuNP-CPN-1, AuNP-CPN-2, AuNP-CPN-3 and AuNP-CPN-4. (b) shows dark field microscopic images of pdop-AuNPs (top) and CPN-4 (bottom). (c) shows a comparison of scattering intensity and color spectrum obtained from pdop-AuNPs (left) and AuNP-CPN-4 (right).

8 is a diagram showing the morphological and spectroscopic characteristics according to the core size of the synthesized AuNP-CPNs. (a) shows TEM images of AuNP-CPN with 10, 20, 30 and 60 nm core sizes, and (b) shows the corresponding UV-Vis spectra.

9 is a diagram showing the PDT-PTT effect of AuNP-CPN according to the present invention. (a) is a diagram schematically showing the laser-induced photothermal effect and ROS generation in AuNP-CPN. (b) shows the temperature rise for different gold nanoprobes as a function of laser irradiation time. (c) is a plot of temperature rise for different gold nanoprobes as a function of laser power. (d) shows reproducibility experiments on photothermal results in three consecutive cycles. (e) is a graph showing the concentration increase as a function of irradiation time for different concentrations of AuNP-CPN-4. (f) shows RNO-histidine colorimetric-based results for different nanostructures for quantifying laser-induced ¹ O ₂ production with increasing laser irradiation time. (g) shows RNO-histidine colorimetric-based results for different nanostructures for quantifying laser-induced ¹ O ₂ production with increasing laser power. All error bars were obtained from three replicate experiments.

10 is a diagram showing a low magnification (left) and a high magnification (right) TEM image of AuNP-CPN-4 after three cycles of 5-minute laser irradiation.

Fig. 11 shows the emission spectrum of the D ₂ O solution of AuNP-CPN-4 after laser irradiation (black) and after N ₂ gas purification (grey).

12 is a view showing a comparison between the photothermal effect and the production of singlet oxygen ( ¹ O ₂ ) of AuNP-CPNs and Au nanorods (AuNRs). For each nanostructure, ROS generation and photothermal temperature were measured three times to obtain error bars.

FIG. 13 is a diagram illustrating a comparison of diameters and petal lengths of AuNP-CPNs. FIG. 100 particles of each AuNP-CPN structure were analyzed by TEM image using ImageJ program.

14 is a diagram showing the calculated electromagnetic field distribution of AuNP-CPN and Au nanorods.

Figure 15 is a diagram showing the characteristics of AuNP-CPN-4 treated cells according to the present invention. (a) is a diagram showing dark field microscopic images of untreated HeLa cells. (b) is a diagram showing a dark field microscope image of AuNP-CPN-4-treated HeLa cells. (c) is a diagram showing low (i) and high (ii) fragment TEM images of immobilized HeLa cells treated with AuNP-CPN-4 probe. AuNP-CPN-4 particles were enclosed in endosome. (d) is a diagram showing the results of HeLa cell viability assay treated with AuNP-CPN-4 particles of different concentrations.

FIG. 16 is a plot of temperature rise in cell culture medium as a function of laser irradiation time for AuNP-CPN-4 probes.

17 is a diagram showing UV-Vis spectra of AuNP-CPN-4 deionized water solution and cell medium.

18 is a diagram showing the killing effect of AuNP-CPN treated cells by laser irradiation according to the present invention. (a) is a diagram showing superimposed fluorescence images from live / dead cell assays for HeLa cells. Light gray And dark gray spots represent living and dead cells, respectively. (b) is a diagram showing the results of HeLa cell death treated with nanoparticles with AuNPs, AuNP-CPN-1, AuNP-CPN-2, AuNP-CPN-3 and AuNP-CPN-4 in the presence or absence of laser irradiation. (c) is a diagram showing a high magnification truncated TEM image of AuNP-CPN-4 particles and fixed HeLa cells treated with laser irradiation.

19 is a digital photograph and fluorescence image of a cell culture dish after incubation with AuNP-CPN-4 particles. Black circles represent laser-irradiated areas. Low magnification fluorescence images from live / dead cell assays of HeLa cells showed exclusive cell death in the laser-irradiated area.

20 is a diagram showing the results of analysis of cells and DNA using AuNP-CPN-4 treated cells according to the present invention. (a) is a dark field microscopic image of AuNP-CPN-4-treated HeLa cells not exposed to live laser (inset TEM image shows intact cell membrane structure). (b) shows Amsia microscopic images of dead HeLa cells treated with AuNP-CPN-4 particles for PDT-PTT treatment and exposed to laser (inset TEM images show damaged cell membranes). (c) using AuNPs, AuNP-CPN-1, AuNP-CPN-2, AuNP-CPN-3, AuNP-CPN-4, particleless (negative control) and indocyanine green (ICG) (positive control) ROS assay results from HeLa cell lysates. (d) is a schematic diagram showing the process of DNA separation and SERS-based DNA characterization after PDT-PTT-ROS treatment. (e) is a diagram showing Raman spectra of DNA isolated from HeLa cells PDT-PTT-ROS-treated (0, 2, 4 or 6-minute laser exposure time) with AuNP-CPN-4.

FIG. 21 shows the results of ascorbic acid-based cell death experiments of AuNP-CPN-4 and polymer-coated AuNP-CPN-4 with various concentrations of ascorbic acid (AA), performed by irradiation for 6 minutes with a 785 nm laser. The figure shown.

FIG. 22 shows a low (left) and high (right) TEM image of polydopamine-coated CPN-4 (PD-CPN-4).

Figure 23 is a diagram showing the results of HeLa cell viability assay performed with H ₂ O ₂ .

FIG. 24 schematically shows a method for synthesizing nano-petal structures synthesized from AuNNP probes and an in vivo application method for PTT / PDT.

FIG. 25 shows (a) TEM, (b) SEM and (c) dark field microscope images of synthesized AuNNP-CPN. FIG.

Fig. 26 shows the comparison of AuNNP, AuNNP-CPN, AuNP-CPN-4 and pdop-AuNNP by (a) photothermal reaction and (b) histidine colorimetric method.

27 shows AuNNP-CPN induced PTT / PDT treatment in a tumor mouse model. (a) shows the PTT / PDT treatment process by laser irradiation, and (b) shows the comparison of PTT / PDT treatment efficiency in the control group (left) and the AuNNP-CPN-administered group (right) irradiated with laser only without AuNNP-CPN. It is also.

One aspect of the present invention is a method for producing a metal nanostructure anisotropically branched and grown on a substrate surface having at least a first metal surface, wherein the first metal surface and a catechol group of polydopamine are on the substrate. Bonding to form a polydopamine coating layer; And oxidizing the catechol group of polydopamine to quinone through the catalysis of the metal surface, inducing oxidative nano peeling due to oxidative collapse of the polydopamine coating layer, and reducing the second metal precursor on the surface of the first metal substrate. Provided is a method for manufacturing a branched nanostructure comprising a second step of anisotropically branching and growing a second metal nanostructure.

The present invention introduces a polydopamine coating layer on a substrate in order to produce branched and grown metal nanostructures that are anisotropically controlled in size and density on a substrate surface having a metal surface. The polydopamine coating layer is formed by binding the catechol group of the dopamine to the metal surface and induces oxidative nano peeling due to the collapse of the coating layer while the catechol group is oxidized to quinone through the catalytic action of the metal surface in the presence of a reducing agent. The second metal precursor may be reduced to form a branched nanostructure, and the rate and type of the reducing agent and the type and / or concentration of the metal precursor may be adjusted to form a nano peeling rate and a metal nanostructure of the polydopamine coating layer. It was confirmed for the first time that the size and / or density of the metal nanostructures formed by adjusting the reduction reaction rate of the second metal precursor can be controlled. In addition, by adjusting the size and / or density of the metal nanostructures as described above, it is possible to control the spectroscopic characteristics of the nanoparticles including the same, and to reduce or improve the photothermal effect and / or ROS generation ability.

For example, the second step may be performed by further including a reducing agent. As the reducing agent, amine hydroxide, ascorbic acid, hydroquinone, sodium borohydride, hydrazine and the like can be used. Preferably, amine hydroxide can be used, but is not limited thereto. The reducing agent may be used to a concentration of 0.2 to 10 mM in the final mixture, but is not limited thereto.

For example, the second step may be performed by further including a surfactant. Non-limiting examples of such surfactants include PVP, PEG, polyethyleneamine, CTAB, and the like. Preferably, the second step may be performed by including PVP as a surfactant, but is not limited thereto. In this case, the surfactant such as PVP may be used at a concentration of 0.01 to 0.5% (w / v) with respect to the final mixture, but is not limited thereto. By adding the surfactant, the surface of the nanostructures, which may occur during the nanostructure formation process, may be unstable to prevent aggregation of the nanostructures without being maintained as individual structures. For this purpose, a coating method using graphene oxide or silica may be used in addition to using the aforementioned surfactant.

For example, in order to determine the structure of the branched nanostructure by adjusting the reaction rate of the oxidative decay rate of the polydopamine and the reductive growth of the branched nanostructure through the collapsed polydopamine in the second step, Selecting the precursor type and concentration thereof, the reducing agent type and concentration thereof, or both. In the second step, it is possible to control the size and / or density of the branched nanostructures formed by controlling the oxidative decay rate of the polydopamine and the reaction rate of the reductive growth of the branched nanostructures through the collapsed polydopamine.

For example, the substrate having the first metal surface may be nanospheres, nanorods, core-shell nanoparticles, or core-gap-shell nanoparticles, but is not limited thereto. In a specific embodiment of the present invention, branched metal nanostructures were introduced on the surface of the substrate using gold nanospheres as well as core-gap-shell structured nanoparticles previously developed by the inventors. It is a substrate having a metal surface, for example, the substrate having the first metal surface is a type of metal so long as polydopamine can bind through its catechol group and exhibit nano-peelation by oxidative decay in the presence of a reducing agent. Materials known to those skilled in the art can be used without limitation in the form of the substrate.

For example, the second metal type of the first metal and the branched metal nanostructure forming the metal surface of the substrate may be the same or different from each other. Preferably, the first metal is gold, the second metal may be gold or silver, but is not limited thereto.

For example, when gold is used as the second metal, HAuCl ₄ may be used as the precursor. In this case, HAuCl ₄ used in the second step as the second metal precursor may be used to be 0.02 to 1 mM concentration in the final mixture, but is not limited thereto.

For example, when silver is used as the second metal, AgNO ₃ may be used as the precursor, but is not limited thereto.

Another aspect of the invention provides plasmon particles comprising branched nanostructures prepared by the above method.

The plasmon particles may have a photothermal effect, reactive oxygen species (ROS) generating ability or both.

Another aspect of the present invention provides a photosensitive therapeutic composition for photodynamic therapy comprising the plasmon particles.

Another aspect of the present invention provides a photosensitive therapeutic composition for photothermal therapy comprising the plasmon particles.

As used herein, photodynamic therapy (PDT or photochemotherapy) refers to a form of phototherapy that uses a non-toxic photosensitive material that is selectively exposed to light and is toxic (phototoxic) to targeted malignant and other diseased cells. Refers to. It is recognized as a relatively non-invasive, low toxicity treatment method and can be used, for example, in the treatment of wet age-related macular degeneration or malignant tumors. In such photodynamic therapy, three important factors are photosensitizer, light source and tissue oxygen. By the combination of these three elements, it can be achieved by selective delivery of the photosensitizer and by local light irradiation. The wavelength of the light source can be determined as an appropriate wavelength to excite the photosensitizer to generate reactive oxygen species. The active oxygen species may be free radicals (type 1 PDT) or singlet oxygen (type 2 PDT). Under normal conditions, most other molecules are in a singlet state, while oxygen is in a triplet state. From a quantum mechanics point of view, oxygen is relatively unreactive under physiological conditions because reactions between them, ie, triplet and singlet states, are not acceptable. As used herein, the term “photosensitive agent” may refer to a compound that absorbs light and transitions to an excited state, and then generates singlet oxygen by intersystem crossing with oxygen. However, the present invention is not limited thereto, and in the present invention, the photosensitizer absorbs light or electromagnetic waves and is converted into an excited state, and transfers energy to other molecules to switch its state or releases heat as a material to transfer to the surroundings. Broadly referred.

As used herein, photothermal therapy (PTT) refers to treating various diseases by mainly using electromagnetic radiation such as infrared rays. Examples of diseases that can be treated with PTT include cancer. This may be an extension of the aforementioned photodynamic therapy using photosensitizers excited with light of a particular wavelength. This is when the electromagnetic wave is irradiated to convert the photosensitizer into the excited state, the vibration energy emitted from it, that is, kills the targeted cells by heat. However, unlike photodynamic therapy, it does not require oxygen for interaction with the target cell or tissue. In addition, photothermal therapy utilizes longer wavelengths of light with lower energy and is therefore less harmful to other cells and tissues.

For example, the photosensitive agent composition may further include a tumor target material to quickly and accurately deliver the composition to the tumor site. For example, ligands, polypeptides, and the like that specifically bind to proteins or the like specifically expressed in tumor cells may be used on the surface of the branched nanostructures, but are not limited thereto.

The branched nanostructures of the present invention can be irradiated with near-infrared rays of 750 nm or more to exhibit a photothermal effect or to generate ROS, and thus are suitable for use as photosensitive agents in non-invasive photothermal and / or photodynamic therapy by in vivo injection. For example, particles exhibiting the photothermal effect and / or ROS generation ability using relatively short wavelengths of light such as visible light cannot be transmitted to the affected part of the body when irradiated with a light source outside the body, and thus should be irradiated to the affected area using an endoscope or an optical fiber. In the case of using near-infrared light of 750 nm or more, there is an advantage that non-invasive cancer treatment is possible because PTT / PDT can be performed by injecting a photosensitive agent into the affected area and irradiating a light source in vitro.

Another aspect of the invention provides a surface enhanced Raman scattering (SERS) substrate comprising the branched nanostructures.

For example, the SERS substrate may be used as a SERS probe further comprising a Raman active material on the surface or inside thereof. Non-limiting examples of such Raman actives include organic or inorganic molecules, atoms, complexes or synthetic molecules, dyes, naturally occurring dyes (such as picoeryrin), organic nanostructures such as C60, buckyballs, carbon nanotubes, quantum dots, Organic fluorescent molecules and the like. Specifically, examples of the Raman active substance include FAM, Dabcyl, TRITC (tetramethyl rhodamine-5-isothiocyanate), Rhodamine 6G, MGITC (malakit green isothiocyanate), XRITC (X-rhodamine- 5-isothiocyanate), DTDC (3,3-diethylthiadicarbocyanine iodide), TRIT (tetramethyl rhodamine isothiol), NBD (7-nitrobenz-2-1,3-diazole ), Phthalic acid, terephthalic acid, isophthalic acid, para-aminobenzoic acid, erythrosin, biotin, digoxigenin, 5-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxy, Fluorescein, 5-carboxy-2 ', 4', 5 ', 7'-tetrachlorofluorescein, 5-carboxyfluorescein, 5-carboxyrodamine, 6-carboxyrodamine, 6-carboxytetramethyl Amino phthalocyanine, azomethine, cyanine (Cy3, Cy3.5, Cy5), xanthine, succinyl fluorescein, aminoacridine, quantum dots, carbon allotropes, cyanide, thiols, chlorine, bromine, memethine Teal, phosphorus or sulfur, but is not limited thereto.

Preferably, when the SERS substrate is based on core-gap-shell particles, the nanogaps formed inside of the core-gap-shell particles, between branched metal nanostructures formed on the surface thereof, or nanogaps and It may include, but is not limited to, a Raman active material between all of the branched metal nanostructures. The core-gap-shell particles exhibit a significant electromagnetic field enhancement effect in the gap formed therein. Therefore, the inclusion of the Raman active material in this gap can exhibit an excellent surface enhancement Raman scattering effect. In addition, it was confirmed that the branched metal nanostructures formed on the surface thereof showed more enhanced photothermal effects and ROS generation ability than the case of forming similar branched metal nanostructures on general metal nanoparticles. It uses the core-gap-shell particles as a base material, and the particles containing the nanostructures branched on the surface prepared by including the Raman active material in the gap can be used as the SERS label as well as excellent photothermal effect and ROS Because of its ability to produce, it can be usefully used as a light sensitizer for PTT / PDT as well as a contrast agent.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example  1: materials and devices

All compounds were purchased and used as such without further purification. Dopamine-HCl was purchased from Sigma-Aldrich (USA). Spherical gold nanoparticles were purchased from BBInternational (USA). Hydrochloric acid and sodium hydroxide were purchased from Daejung Chemicals and Metals, Korea. Tris was purchased from USB Corporation (USA). Formvar / carbon-coated copper grids were purchased from Ted Pella, Inc. (USA). Nanopure water (Nanopure water, 18.0 MΩ-cm) was used for all experiments. UV-Vis spectra were acquired with a UV-Vis spectrophotometer (Agilent 8453 spectrophotometer, USA). Elemental analysis and binding energy measurements were performed using an X-ray photoelectron spectrometer (Axis HSi, KRATOS Analytical). Dynamic light scattering measurements were performed using Malvern Zetasizer (Nano ZS). TEM images were acquired using an Energy-Filtering Transmission Electron Microscope (LIBRA 120, Carl Zeiss) at an acceleration voltage of 120 kV. SEM images and EDS-element mapping data were obtained using a Field-Emission Scanning Electron Microscope (SUPRA 55VP, Carl Zeiss). Raman spectra were acquired using a Renishaw inVia Raman microscope with 514 nm, 633 nm, and 785 nm laser sources.

Example 2: Polydopamine (pdop) Coated Gold Nanoparticles

Commercially available 1 mL of 80-nm citrate-stabilized AuNPs colloidal solution (11 pM) was centrifuged and redispersed in Tris.HCl buffer (pH 8.5, 10 mM). Dopamine-HCl solution (5 mg / mL) dissolved in 5 μL of Tris-HCl buffer (pH 8.5, 10 mM) was added and the reaction mixture was vortexed at 25 ° C. for 4 hours. Finally, the reaction mixture was centrifuged at 8000 rpm for 5 minutes and the supernatant was removed. Precipitates containing pdop-AuNPs were redispersed in deionized water. The thickness of the pdop layer on the 80-nm Au core was found to be ~ 5 nm from TEM analysis.

Example  3: gold nano-petal structure nanostucture ; CPN ) Synthesis

To synthesize the nano-petal structures, 50 μL of HAuCl ₄ (5 mM), 100 μL in gold nanoparticles coated with polydopamine obtained from Example 2, pdop-AuNP solution (5 mL @ 1 nM) PVP (5% w / v, 10,000 MW) and 50 μL of amine hydroxide (50 mM) were added in succession and the reaction mixture was stirred vigorously for 5 minutes at 25 ° C. (hereinafter referred to as AuNP - CPN- 1 ). . Furthermore, except that 100 μL, 200 μL and 500 μL of HAuCl ₄ (5 mM) and amine hydroxide solution (50 mM) were used, AuNP-CPN-2, AuNP-CPN-3 and AuNP, respectively. -CPN-4 was synthesized.

Citrate-stabilized AuNPs (80 nm in diameter) were treated with dopamine-HCl dissolved in 10 mM Tris-HCl buffer (pH 8.5) for 4 hours at room temperature to form 5-nm pdop-coated AuNPs (pdop-AuNPs) (FIG. 1B). Localized surface plasmon resonance (LSPR) bands of pdop-AuNPs were slightly red-shifted (~ 9 nm) by charge transfer between AuNP and pdop (LSPR bands for citrate-AuNPs and pdop-AuNPs, respectively). Located at 522 nm and 531 nm). The pdop coating on AuNPs was also confirmed by X-ray photoelectron spectroscopy (XPS) (FIG. 2). Comparing high resolution Au 4f XPS spectra between pdop-AuNPs and citrate-AuNPs, 0.4 and 0.8 eV shifts in binding energy corresponding to Au 4f5 / 2 (83.6 eV) and Au 4f7 / 2 (87.1 eV), respectively, were observed. (FIG. 2). These results indicate that the catechol group is bonded to the AuNP surface. In the presence of a polymerization initiator (Tris) under basic conditions, dopamine is converted into its dione derivatives 5,6-dihydroxyindolines and other related molecules, and are transferred to charge transfer, π-π overlap and It was densely packed by strong supramolecular forces such as hydrogen bonds to form a pdop layer on AuNPs. As described above, HANCl ₄ , PVP and amine hydroxide were continuously added to the pdop-AuNP solution and reacted to synthesize CPNs from pdop-AuNPs. The role of HAuCl _{4 in} the reaction was confirmed by TEM, and an intermediate structure was observed (FIGS. 1C and 1D are images immediately after HAuCl ₄ addition and 1 minute respectively). Oxidative disruption and delamination of the pdop layer on the Au core and budding petal structures growing through the collapsed pdop layer were clearly observed (FIGS. 1C and 1D, respectively). The color of the solution gradually changed from red to blue, and the resulting blue solution was stable for several weeks without any aggregation or subsequent color change as confirmed by UV-vis spectroscopy and TEM images. Electron microscopic images of the samples confirmed the formation of highly branched AuNP-CPNs with plasmon petals located closely on the spherical Au cores (FIGS. 1E and 1F). Raman spectra of pdop-AuNPs before and after HAuCl ₄ treatment showed a decrease in catechol peak at 1617 cm ⁻¹ and an increase in quinone peak at 1651 cm ⁻¹ (FIG. 3). HAuCl ₄ -mediated oxidation of catechol to quinone was also confirmed by ¹³ C NMR, indicating an increase in quinone carbon signal intensity at 182.0 ppm and 183.7 ppm while decreasing 142.9 ppm and 143.7 ppm (catechol carbon peak) signals (Fig. 4). As shown in FIG. 1A, the oxidation of catechol to quinone causes disruption of the pdop assembly due to the absence of hydrogen bonds between the catechol and quinone groups, and the interaction between the quinone and AuNP cores between the catechol and Au cores. Weaker than binding (TEM image of collapsed pdop is shown in FIG. 1C). In the process, Au nanopetal structures budding from the Au core grow on an optionally oriented oxidized pdop-modified AuNP core, after which the reduction of extra HAuCl _{4 with} amine hydroxide results in anisotropic growth of Au petals. (Protruding Au petals are shown in FIG. 1D). Two major steps during the synthesis of AuNP-CPNs are the oxidative collapse of the pdop assembly on the Au core and the reductive growth of Au petals through the collapsed pdop layer on the Au core. The reaction rate of each of these two steps is important for the determination of the final AuNP-CPN structure. More degraded pdop units lead to high petal protrusions. As the amount of amine hydroxide increases, the reaction rate increases linearly, forming a smoother particle-surface (FIG. 5). Faster reduction of Au (III) by a large amount of amine hydroxide inhibits oxidative nano peeling of pdop, thereby inhibiting petal formation on the Au core. On the other hand, when no amine hydroxide was added, complete oxidative exfoliation of the pdop layer from the Au core was observed (FIG. 6). As a control, citrate-stabilized 80-nm AuNPs were reacted with HAuCl ₄ and NH ₂ OH under the same conditions as applied for synthesizing CPNs from pdop-AuNPs. At this time, no protruding nanobranches were observed, but the diameter of the spherical AuNPs increased from 80 nm to 83 nm. From this, we found that by simply changing the amount of HAuCl ₄ it is possible to control the formation of Au petals and the corresponding optical signal on the Au core (FIG. 1G). As the amount of HAuCl ₄ increases, the number and length of nanopetals increase, which shifts the LSPR peak from the visible region to the near infrared region (FIG. 7; AuNP-CPN-1 to AuNP-CPN-4). . In particular, AuNP-CPN-4 exhibited densely projected nanopetals on the Au cores that could generate strong plasmon coupling based optical signals. Broad and strong spectra from AuNP-CPN-4 particles are desirable for biological applications due to the deep transmission of near infrared light to biological samples. For the above reasons, AuNP-CPN-4 particles were used in subsequent experiments.

Example 4: Dark Field Microscopy of pdop-AuNPs and AuNP-CPN Probes

The washed glass slide was treated with 2% (v / v) 3-aminopropyl-trimethoxysilane (APTS) aqueous solution for 10 seconds, washed with deionized water and dried under nitrogen atmosphere. 10 μL of sample (pdop-AuNP or AuNP-CPN, 0.1 nM) was then loaded onto APTS-treated glass slides and covered with thinner glass slides. Dark field images were obtained with a Carl Zeiss (DE / Axiovert 200) microscope.

The UV-Vis spectrum of AuNP-CPNs covered a wide range (530 to 975 nm) from visible to near infrared by multimodal couplings of the nanopetals of AuNP-CPNs (FIG. 7A). 7b shows dark-field microscopic images of pdop-AuNPs and AuNP-CPN-4 particles, respectively. When plasma-coupled nanopetals were formed from pdop-AuNPs, strong color change and enhancement of scattering signal were observed. The red-to-green color ratio (R / G) varied from 0.84 for pdop-AuNPs to 1.62 for AuNP-CPN-4 particles. As shown in FIG. 7C, the change in Ray-leigh scattering may be due to extensive plasmon coupling between closely located metal nanobranches of AuNP-CPN particles. The synthesis method of the present invention could provide a powerful and versatile route to various branched nanostructures with different core sizes and branch shapes. We synthesized AuNP-CPNs with core sizes of 10 nm, 20 nm, 30 nm and 50 nm, respectively, all of which exhibited near-infrared activity, an important factor for in vivo applications (FIG. 8).

Example 5 RNO-Histidine Assay

2 mL of AuNP-CPN solution was added to a freshly prepared aqueous solution of RNO (50 μM) and 10 mM His (10 mL), and the mixed solution was transferred to a 1 mL quartz cuvette and laser (785 nm, 2 W / cm ² ). Was investigated. UV / Vis absorbance at 440 nm was recorded at a predetermined irradiation dosage.

Example 6: ^One O ₂  Emission detection

AuNP-CPNs (1 mL) dispersed in D ₂ O were irradiated with a laser and the emitted luminescence was recorded. Measurements were carried out at a constant temperature of 15 ° C. using a thermostat unit combined with a detector in the dark.

As described above, the inventors confirmed the possibility of using AuNP-CPNs as photothermal transducers for therapeutic applications (FIG. 9A). For this purpose, four different AuNP-CPNs (AuNP-CPN-1, AuNP-CPN-2, AuNP-CPN-3, and AuNP-CPN-4) with increasing density of nano petals were used. First, a quartz cuvette filled with AuNP-CPN solution (12 μg / mL of Au content measured by ICP-MS) was irradiated with a 785-nm laser (2W / cm ² ) from 0 to 10 minutes, and then thermocoupled. The temperature in the cuvette was measured. As shown in FIG. 9B, when the AuNP-CPN-4 solution was irradiated for 6 minutes, the temperature increased from 23 ° C. to 53.7 ° C. (after which no increase in temperature was observed). From these results, it was confirmed that photothermal conversion is dependent on the nano petal density of AuNP-CPNs. For 6 minutes of irradiation time the temperature of the solution was linearly proportional to the laser power (FIG. 9C). In the control experimental group using spherical AuNPs, poor photothermal reaction was observed under the same irradiation conditions (785 nm, 2 W / cm ² ). In order to confirm the safety of AuNP-CPNs against photothermal heating, the photothermal heat-cooling process was repeated several times. The nanoparticle solution was exposed to a 785-nm laser for 5 minutes and allowed to cool to room temperature for 30 minutes. The process was repeated three times. The results show that the photothermal heating process is fully repeatable with the same heat-cooling profile for all four different AuNP-CPNs (FIG. 9D). The inventors found that after three cycles of laser irradiation for 5 minutes, no structural change was seen from the AuNP-CPN structure (FIG. 10).

Furthermore, using AuNP-CPN-4, it was confirmed how the concentration of particles affects photothermal heating. 0.5 nM AuNP-CPN-4 solution irradiated for 4 minutes or more was heated to 50 ℃ or more, it was confirmed that 5 minutes of irradiation for 1 nM AuNP-CPN-4 solution is required to heat to 55 ℃ or more (Fig. 9e; 785 -nm laser, 2 W / cm ² ). In addition, the inventors have identified the ability of AuNP-CPNs for the generation of organic photosensitizer-free ROS and subsequent PDT applications. Gold nano structure is involved in electron transfer to the plasmon ³ O _2, and can be sensitized to light ³ O ₂ active for conversion to ¹ O _2. As a proof-of-concept study, AuNP-CPNs or AuNPs (1 nM concentration) were exposed to a 785-nm laser (2 W / cm ² ) for 5 minutes and N, N-dimethyl-4-nitrosoaniline (RNO)- Histidine colorimetry was used to monitor the presence of ¹ 0 ₂ (FIGS. 9F and 9G). In this assay, the imidazole moiety of histidine reacts with ¹ O ₂ and the resulting transient complex bleaches the RNO molecules. That is, the amount of ¹ O ₂ can be directly related to the reduction of the RNO band intensity in the UV-Vis spectrum, and the production of ¹ O ₂ is AuNP-CPN- in D ₂ O with light of wavelength matching the LSPR of the nanostructures. It was confirmed by analyzing the characteristic phosphorescence emission at ˜1268 nm when 4 nanoparticles were excited (FIG. 11). By thoroughly purging the solution with nitrogen, phosphorescence emission was reduced at 1268 nm, which further supports the presence of ¹ 0 ₂ (FIG. 11). As shown in FIGS. 9F and 9G, the amount of ¹ O ₂ produced was increased by longer laser exposure time and higher laser power. The maximum amount of ¹ O ₂ was achieved using AuNP-CPN-4, AuNPs was generated the least amount of _{^{1 O 2 (785-nm laser}} , 2 W / cm 2). This indicates that ¹ 0 ₂ production is highly nanopetal structure and density dependent. The pattern of ¹ O ₂ generation based on the laser irradiation was very similar to the pattern for AuNP-CPN based photothermal results. We also compared the performance of AuNP-CPNs with gold nanorods (AuNRs), which are widely used in photothermal and photodynamic therapy and plasmonics. AuNRs with absorption maximum wavelengths matching AuNP-CPN-4 were synthesized by known methods. As shown in FIG. 12, AuNP-CPN-4 probe (ΔT = 23.7 ° C.) showed better photothermal effects than AuNRs (ΔT = 9 ° C.) under the same laser irradiation conditions (785-nm source and 2 W / cm ² ). It was. In addition, AuNP-CPN-4 produced more than 2 times ¹ O ₂ than AuNRs (Fig. 12). The Au content in AuNR or AuNP-CPN-4 was the same in this experiment. AuNP-CPNs of different sizes and petal lengths are shown in FIG. 13. It is also well known that stronger plasmon coupling between metal nanostructures creates stronger electromagnetic fields and that the size and shape of the metal nanostructures affect the electromagnetic fields of the plasmon nanostructures. The inventors also performed a three-dimensional finite-element method (3D FEM) simulation (COMSOL, Stockholm, Sweden) to analyze the distribution and enhancement of electromagnetic fields (EM fields) of AuNP-CPN and AuNR. It was. As shown in FIG. 14, the electromagnetic field on AuNP-CPN is much stronger than that on AuNR, mainly due to the petal structure and significant plasmon coupling on AuNP-CPN. Finally, the photothermal conversion efficiency (η) of AuNP-CPN-4 was calculated to be 32%, which was a significant increase over AuNR (21%).

Example 7: Cell Culture, AuNP-CPN Probe Treatment, and Dark Field Imaging

Furthermore, the present inventors confirmed the applicability of AuNP-CPN to cancer treatment using live cervical cancer cells (HeLa cells). First, cell internalization of the nanoparticles was confirmed.

HeLa cells with 10% fetal bovine serum (fetal bovine serum; FBS) and 1% antibiotic solution 10 (GIBCO, Invitrogen, Karlsruhe, Germany ) Dulbecco's modified Eagle's medium (DMEM, 50 μL per well) containing ⁴ cells / Aliquots into 96-well plates at mL concentration were incubated overnight at 37 ° C. and 5% CO ₂ . Thereafter, the culture medium was exchanged with freshly prepared DMEM. AuNP-CPN solution dispersed in PBS was added at different concentrations and the cells were placed in the culture chamber for 2 hours.

Specifically, HeLa cells were cultured in 35 mm polylysine-modified glass bottom culture dishes (MatTek Corp., USA) and grown overnight in DMEM medium containing 10% FBS and 1% antibiotic (37 ° C., 5%). CO ₂ ). Next, the medium was exchanged with fresh culture medium containing AuNP-CPN nanoprobe (0.1 nM) and the cells were further incubated for 2 hours. The glass slides were then washed with PBS to remove excess AuNP-CPN nanoprobe and a dark field image was obtained with a Carl Zeiss (DE / Axiovert 200) microscope.

HeLa cells were treated with 1 nM nanoparticle solution, incubated at 37 ° C. for 2 hours, and the cells were washed with PBS to remove excess AuNP-CPNs. Darkfield light scattering images of cells treated with nanoparticles allow direct visualization of internalized AuNP-CPNs. As shown in Figure 15b, the internalization of AuNP-CPN-4 particles can identify the bright red-orange color in the cells. On the other hand, dark field images of untreated cells show much weaker nanoparticle-scattering signals (FIG. 15A). In order to confirm the exact position of AuNP-CPNs in the cells, AuNP-CPN-4-treated cells were fixed, cut and observed by TEM. TEM images show that AuNP-CPN-4 particles were internalized by the cells and distributed in the cytosol (FIG. 15C-i). High magnification TEM images showed that AuNP-CPN-4 particles were mainly located in the endosome, indicating endocytosis (FIG. 15c-ii). The average content of CPN-4 particles on the surface and inside of HeLa cells by ICP-MS analysis is ~ 1260 particles / cell, considering the size of relatively large AuNP-CPN-4 particles (diameter ~ 100 nm) That's a fairly high number. This may be due to the branched effect of promoting cell membrane permeation and the ionic screening effect by serum proteins and other cell membrane elements in cell growth media.

Example  8: Cell cross-sectional image using transmission electron microscope

To obtain cell cross-sectional images, cells incubated with AuNP-CPNs were first isolated from well plates. After washing with PBS solution,> 5 × 10 ⁵ cells were treated for 2 hours with modified Karnovsky's fixative (2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2). Fixed. After repeated washing with 0.05 M sodium cacodylate buffer (pH 7.2) at 4 ° C, the cells were fixed for 2 hours with 1% osmium tetrachloride dissolved in 0.05 M sodium cacodylate buffer (pH 7.2) and twice with distilled water. Washed. The immobilized cells were en bloc stained overnight at 4 ° C. with 0.5% uranyl acetate and dehydrated with a series of ethanol concentrations (30%, 50%, 70%, 80%, 90%, 100%). , 100%, and 100% ethanol; 10 minutes for each dehydration step). Infiltrated cells using propylene oxide and Spurr's resin were polymerized at 70 ° C. for 24 hours. Various sections of resin blocks were cut using ultramicrotome (MT-X, RMC, Tucson, AZ, USA) and stained for 7 minutes with 2% uranium acetate and Reynolds' lead citrate. The compartment of interest was then transferred onto a 300 mesh copper TEM grid.

Example 9: Cytotoxic Assays

Cytotoxicity of various concentrations of AuNP-CPN was confirmed using a Cell Counting Kit (Cell Counting Kit, CCK-8, Dojindo lab., Japan). Cells were grown in 96-well plates with DMEM containing 100 μL of FBS. From 24 hours after dispensing, cells were incubated with AuNP-CPN-4 probe at various concentrations (from 100 pM to 1 μM) for 48 hours and cell viability assays were performed. Cell metabolic activity was measured using CCK-8 (sensitive colorimetric assay to determine the number of viable cells after probe and incubation). 10 μL of CCK-8 solution was added directly to the incubated cells of each well. After incubation at 37 ° C. for 2 hours, the amount of formazan dye was measured with a microplate reader (Anthos 2010, Anthos Labtec, Eugendorf, Austria).

Example 10: Photothermal Therapy

After incubating HeLa cells with AuNP-CPNs, cell monolayers were washed three times with PBS buffer and irradiated with near infrared laser (spot size of 785 nm, 2 W cm ⁻² , 5 mm). Cells were incubated for 30 minutes with blocking of light with 200 μL of fresh LIVE / DEAD reagent solution (LIVE / DEAD Viability / Cytotoxicity Kit, Molecular Probes). Spots that appear green visually (light gray in the figure) indicate healthy cells, while red spots (dark gray in the figure) indicate dead cells.

Specifically, HeLa cells were incubated with different amounts of AuNP-CPN-4 particles (from 100 pM to 1 μM) for 24 hours, and cell viability results were obtained (FIG. 15D). The results show that AuNP-CPN-4 particles show little cytotoxicity for HeLa cells even at high probe concentrations (> 100 nM). Furthermore, we confirmed the possibility of using AuNP-CPN for dual PTT-PDT application using near infrared. First, the photothermal response of AuNP-CPN-4 was measured as a function of laser irradiation (785 nm, 2 W / cm ² ) time in cell growth medium (DMEM, 10% FBS, 1% antibiotic) for up to 15 minutes. It took about 10 minutes for the temperature to increase to 54 ° C. (FIG. 16). UV-Vis spectroscopy confirmed aggregation of only traces of AuNP-CPN-4 particles in cell medium (FIG. 17). Proteins abundantly present in cell media can interact strongly with nanoparticles (ACS Nano, 2010, 4 (1): pp 365-379) and form by laser-assisted photothermal heating Changes may appear (Nano Lett., 2014, 14 (1): pp 6-12). During this process, the heat from the AuNP-CPN probe may be partially dissipated. Such protein-based heat dissipation and aggregation of some nanoparticles can delay the heating process. A temperature increase of up to 45 ° C. to moderate near infrared laser power (˜2 W / cm ² ) is important to minimize unnecessary normal tissue heating in clinical applications of PDT-PTT treatment. The experiment was performed with AuNP-CPNs and spherical AuNPs. HeLa cells were incubated with 0.5 nM nanoparticle solution for 2 hours and irradiated with 785-nm laser (2 W / cm ² ) for 6 minutes, increasing the temperature to 42 ± 1 ° C. Cell viability after irradiation was determined using a colorimetric live / dead cell viability assay kit (Invitrogen). As shown in FIGS. 18A and 19, green (light gray in the figure) and red (dark gray in the figure) cells represent live and dead cells, respectively. The survival rate of the cells not treated with the nanoparticles was almost 100% even after 6 minutes of laser irradiation. This indicates that 6 minutes of laser irradiation (785-nm laser; 2 W / cm ² ) does not damage cells in the absence of plasmon nanoparticles. Nanostructures with more protruding nanobranches induced efficient cell death when the cells were exposed to a 785-nm laser (FIG. 18B). In particular, CPN-4 particles increased the temperature to ˜42 ° C. after 6 minutes of laser irradiation, nearly killing cancer cells. The cleaved TEM image of the cells fixed after exposure to 785-nm laser for different times is shown in FIG. 18C. This indicates that when no laser was irradiated (t = 0 min), AuNP-CPN-4 particles were enclosed in the endosome. However, after irradiation of the cells for 3 and 6 minutes with a 785-nm laser, the endosomes began to collapse, and endosomal escape of AuNP-CPN-4 particles was clearly observed (Fig. 18C-i-). iii). Next, we quantified the amount of ROS generated after laser irradiation from nanoparticle-treated HeLa cells using an OxiSelect assay (fluorescence signal based technique for measuring total ROS activity in cell lysates).

The above results showed that the amount of ROS is associated with the difference in AuNP-CPN structure (Fig. 20c). This also indicates that AuNP-CPN-4 particles can efficiently kill cancer cells by relatively moderate temperature increase (˜42 ° C.) through PTT-PDT dual treatment. To independently quantify the photothermal and photodynamic contributions, ascorbic acid (AA), a well-known antioxidant, was added at different concentrations during the laser induced cancer cell killing experiment. After AA addition, cell viability increased by 82% (for 500 μM AA) (FIG. 21). This indicated that ROS production could be stopped at AA concentrations >> 500 μM, in which case the photothermal contribution was ˜18%.

To make this even clearer, AuNP-CPN-4 was coated with a ~ 5-nm polydopamine layer to block ¹ 0 ₂ production on the Au surface (FIG. 22). Polydopamine-coated AuNP-CPN-4 was able to kill only ˜17% of cells (FIG. 21). The ROS levels produced by AuNP-CPN-4 were compared with the values for indocyanine green (ICG), a near infrared absorbing photosensitizer. As shown in FIG. 20C, ROS levels in the presence of AuNP-CPN-4 were 1.8-fold higher than in the case of ICG. We then identified the fate of cell elements affected by photo-induced PDT-PTT. Apoptotic cells could be directly monitored by fluorescence microscopy using ethidium bromide (EB), which stains the nuclei of dead cells due to leakage of the cytoskeleton and outwardly proliferating cell membranes. EB-treated HeLa cells showed a strong red color in the presence of laser-irradiated AuNP-CPN-4 particles. This indicates that blebbing of the cell membrane was induced by the AuNP-CPN-4-mediated PDT-PTT effect. After treatment with AuNP-CPN-4 particles and laser irradiation, changes in cell morphology and aggregation from irregular ellipses to circles from dark field microscopic images were observed (FIG. 20B), which indicates that cytoplasmic contraction and nucleation shrink under high oxidative stress. Caused by. The TEM image of FIG. 20B shows the formation of vesicles attached to the membrane in the cell membrane, which were formed by migration from the cytoplasmic side of the membrane to the extracellular side.

Example  11: Genetic DNA Isolation

After applying photothermal therapy for 0, 2, 4, and 6 minutes using AuNP-CPN-4 nanoprobe, cells were treated with 0.5 M Tris-HCl (pH 8.0), 20 mM EDTA, 10 mM NaCl, 1% SDS, And 4 mL of lysis buffer containing 0.5 mg / mL proteinase K. The mixture was incubated overnight at 55 ° C. 2 mL of saturated NaCl (6 M) was added and the sample was incubated at 55 ° C. for 10 minutes. After centrifugation at 5000 rpm for 30 minutes, the supernatant containing DNA was mixed with chilled excess ethanol and the mixture was carefully inverted to spool the DNA. The tubes were incubated at room temperature for 15 minutes and centrifuged at 10,000 rpm for 10 minutes at room temperature to recover DNA. DNA was washed thoroughly three or four times with 70% ethanol and finally air dried at room temperature.

Example  12: AuNP - CPN -4 Probe of DNA Isolated SERS  Measure

In addition, the inventors have identified ROS-mediated changes in nucleic acids using AuNP-CPN-4 particles as surface-enhanced Raman scattering (SERS) probes. DNA (10 μL, 0.1 mg / mL) isolated from HeLa cells after PDT-PTT treatment for different time periods was mixed with AuNP-CPN-4 nanoprobe (10 μL, 5 nM), 785-nm laser and 50 × SERS spectra were recorded from the mixture on glass slides using a Reinshaw InVia Raman spectrometer with an objective lens.

In order to confirm that the highly branched plasmon AuNP-CPN-4 construct is an excellent SERS substrate, after treatment with HeLa cells with AuNP-CPN-4 particles, the samples were subjected to 785-nm laser for 2 minutes, 4 minutes, and After 6 minutes of exposure, the gene DNA was isolated by known extraction methods (FIG. 20D). The SERS signal was then used to study ROS-mediated primary and secondary structure changes (FIG. 20E). 10 μL of DNA solution (0.1 mg / mL) was mixed with 10 μL of 5 nM AuNP-CPN-4 solution for SERS spectral measurements (it is known that DNA base can interact with Au surface). ¹ O ₂ and other intracellular secondary ROS are known to be mutagenic, genotoxic, and involved in many biological processes. Excessive hydroxyl radicals from lipid oxidation by singlet oxygen can extract hydrogen atoms from regions exposed to solvents of the sugar-phosphate DNA backbone, thereby causing β-cleavage and DNA bases of DNA strands. May lead to unstacking. At 1082 cm ^-1 , the phosphate-backbone-characteristic Raman peak (PO ² -symmetric elongation) shifted to 1075 cm ^-1 , the intensity of which was 785-nm in AuNP-CPN-4 treated samples. It gradually increased with increasing exposure to the laser. This change was caused by DNA phosphate-skeletal damage and DNA aggregation. Another ROS-mediated chemical modification on DNA is the oxidation of DNA bases (eg, 8-oxoguanosine formation by oxidation of guanine). Radical of guanine-Raman peak at 662 cm ^-1 corresponding to the respiratory (radical-breathing) mode vibration after PDT-treatment PTT ~ 649 ^cm-1 was shifted to. The shifted Raman band may be due to a desirable conformational change from the anti-form of guanine to the syn-form of 8-oxoguanine and electronically due to the high hydrogen bond filling of 8-oxoguanine over guanine. It may also be due to changes in the environment. All of the above results indicate that when the cells were treated with AuNP-CPN-4 particles and irradiated with a 785-nm laser, there was a chemical structural change in the DNA in the cells.

Example 13: Synthesis of Nano-Petal Structures Using Core-Gap-Shell Structures (AuNNP)

In order to confirm the effect on PTT / PDT according to the morphology and / or modification of gold nanoparticles as cores, nano-petal structures were prepared using the core-gap-shell structured nanoparticles previously developed by the present inventors. .

Specifically, SJ Hurst et al., Anal. Chem., 2006, 78: According to the method disclosed in 8313 ', the surface of gold nanoparticles having a diameter of 20 nm was prepared by DNA (SEQ ID NO: 3'-HS- (CH ₂ ) ₃ -A ₁₀ -PEG ₁₈ -AAACTCTTTGCGCAC-5 Modified with '). In order to form the gold shell surrounding the DNA-modified gold nanoparticle core, the gold-modified gold nanoparticles were converted into gold precursor (HAuCl ₄ ), reducing agent (NH ₂ OH-HCl) and 1% poly- N-vinyl-2-pyrrolidone (poly-N-vinyl-2-pyrrolidone; PVP; MW 40,000) and phosphate-buffered solution (0.3 M NaCl; 10 mM PB; pH 7.4) Vortex lightly at room temperature for 30 minutes.

Specifically, a gold nanoparticle solution (100 μL; 1 nM concentration in 0.3 M PBS) modified with DNA was mixed with 50 μL of 1% PVP solution. The solution is mixed with 1.5 μL, 5.2 μL, 10.3 μL or 30.4 μL of hydroxylamine hydrochloride solution (10 mM), and then 1.5 μL, 5.2 μL, 10.3 μL or 30.4 μL of chloroauric acid, respectively. It was mixed with a solution (chloroauric acid solution; 5 mM). As a result of observing with high-resolution TEM, it was confirmed that the core-shell particles prepared as described above formed a nanogap of about 1.2 nm between the core and the shell, and the shell partially contacted the core surface to form nanobridges. . In addition, the average diameter of the gold core-gap-shell particles was about 46 nm.

Also for the AuNNP solution obtained above, the dopamine-HCl solution was used at a concentration of 0.1 mg / mL, and the reaction mixture was vortexed for 1 hour, except that the AuNPs colloidal solution was used in the same manner as in Example 2. The reaction was carried out in the same manner to coat the pdop layer. TEM analysis confirmed that the thickness of the pdop layer coated on AuNNP was also ~ 5 nm.

In addition, for the pdop-AuNNP, nano-petal structure was synthesized on the surface by the same manufacturing method as in AuNP-CPN-1 in the same manner as in Example 3 except that 500 μL of HAuCl ₄ was used (hereinafter, , Denoted AuNNP - CPN . The preparation method is schematically illustrated in FIG. 24, and the TEM, SEM, and dark field microscope images of the synthesized AuNNP-CPN are shown in FIG. 25. The TEM image of FIG. 25 showed the successful formation of highly branched nano-petal structures with plasmonic petals located close to the AuNNP core, indicating that there was a nanogap at about 1 nm intervals therein.

Example 14 In Vivo Application and Effects of AuNNP-CPN for PTT / PDT

AuNNP-CPN prepared in Example 13 was found to form a strong electromagnetic field in the nanogap region contained therein, by introducing the nano-branched structure of the present invention based on this, PTT / PDT efficient in nano-petal structure In order to confirm whether it is possible or can show a more synergistically increased effect by the modification of the core as described above, the photothermal effect and ROS generation was confirmed using AuNNP-CPN prepared according to Example 13. As shown in FIG. 26, when the gold content is the same, AuNNP-CPN showed much more increased photothermal reaction and ROS generation rate than AuNP-CPN-4 particles.

Furthermore, the tumor treatment effect of PTT / PDT in vivo was confirmed by administering AuNNP-CPN to tumor mice, which is expected to exhibit an efficient PTT / PDT treatment effect in vivo based on the increased photothermal effect and ROS production rate. It was. All experimental animals were performed according to animal care protocols. Specifically, tumor mice were produced by injecting HEK-293 cells, and then AuNNP-CPN was injected into the tumor and irradiated with a laser of 785 nm wavelength. Tumor size was measured with calipers after the PTT / PDT treatment (FIG. 4). In the AuNNP-CPN-administered group, the tumor was effectively removed after irradiation, and it was confirmed that black scars were left at the original tumor site without recurrence.

Claims (17)

A method of making a metal nanostructure anisotropically branched and grown on a substrate surface having at least a first metal surface, A first step of forming a polydopamine coating layer on the substrate by combining the first metal surface with a catechol group of polydopamine; And The cathodic group of polydopamine is oxidized to quinone through the catalysis of the metal surface to induce oxidative nano peeling due to oxidative collapse of the polydopamine coating layer, while reducing the second metal precursor on the surface of the first metal substrate. A method for producing a branched nanostructure comprising a second step of anisotropically branching and growing a second metal nanostructure. The method of claim 1, Wherein the second step is to perform a branched nanostructures manufacturing method further comprising a reducing agent. The method of claim 2, Wherein said reducing agent is selected from the group consisting of amine hydroxide, ascorbic acid, hydroquinone, sodium borohydride, and hydrazine. The method of claim 1, Wherein said second step is performed further comprising a surfactant. The method of claim 4, wherein Wherein the surfactant is selected from the group consisting of polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), polyethyleneamine, and cetyl trimethylammonium bromide (CTAB). The method of claim 1, In order to determine the structure of the branched nanostructures by adjusting the oxidative decay rate of the polydopamine and the reaction rate of the reductive growth of the branched nanostructures through the collapsed polydopamine in the second step, A method for producing a branched nanostructure, further comprising the step of selecting a second metal precursor type and its concentration, a reducing agent type and its concentration, or both. The method of claim 1, And wherein said substrate having said first metal surface is nanospheres, nanorods, core-shell nanoparticles or core-gap-shell nanoparticles. The method of claim 1, And wherein the second metal species of the first metal and the branched metal nanostructure forming the metal surface of the substrate are the same or different from each other. The method of claim 8, Wherein the first metal is gold and the second metal is gold or silver. 10. Plasmon particles comprising branched nanostructures prepared by the method of claim 1. The method of claim 10, The plasmon particles have a photothermal effect, reactive oxygen species (ROS) generating ability or both. The photosensitive therapeutic composition for photodynamic therapy containing the plasmon particle of Claim 10. The method of claim 12, Photosensitive therapeutic composition for photodynamic therapy, further comprising a tumor target material. The photosensitive therapeutic composition for photothermal therapy containing the plasmon particle of Claim 10. Surface enhanced Raman scattering (SERS) substrate comprising the branched nanostructures prepared by the method of claim 1. The method of claim 15, The SERS substrate is a surface-enhanced Raman scattering substrate that is used as a SERS probe further comprises a Raman active material on the surface or inside of the substrate. The method of claim 16, When the SERS substrate is based on core-gap-shell particles, the nanogap formed inside of the core-gap-shell particles, between branched metal nanostructures formed on its surface, or nanogap and branched metal Surface-enhanced Raman scattering substrate that comprises a Raman active material between all the nanostructures.

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